carbocyanines and Disease-Models--Animal

Macromolecular imaging probes (or sensors) of enzymatic activity have a unique place in the armamentarium of modern optical imaging techniques. Such probes were initially developed by attaching optically "silent" fluorophores via enzyme-sensitive linkers to large copolymers of biocompatible poly(ethylene glycol) and poly(amino acids). In diseased tissue, where the concentration of enzymes is high, the fluorophores are freed from the macromolecular carrier and regain their initial ability to fluoresce, thus allowing in vivo optical localization of the diseased tissue. This chapter describes the design and application of these probes and their alternatives in various areas of experimental medicine and gives an overview of currently available techniques that allow imaging of animals using visible and near-infrared light.

Topics: Amino Acids; Animals; Biosensing Techniques; Carbocyanines; Diagnostic Imaging; Disease Models, Animal; Enzymes; Fluorescence; Fluorescent Dyes; Humans; Macromolecular Substances; Mice; Neoplasm Transplantation; Polyethylene Glycols; Polymers; Spectroscopy, Near-Infrared

Accurate and rapid detection of tumors is of great importance for interrogating the molecular basis of cancer pathogenesis, preventing the onset of complications, and implementing a tailored therapeutic regimen. In this era of molecular medicine, molecular probes that respond to, or target molecular processes are indispensable. Although numerous imaging modalities have been developed for visualizing pathologic conditions, the high sensitivity and relatively innocuous low energy radiation of optical imaging method makes it attractive for molecular imaging. While many human diseases have been studied successfully by using intrinsic optical properties of normal and pathologic tissues, molecular imaging of the expression of aberrant genes, proteins, and other pathophysiologic processes would be enhanced by the use of highly specific exogenous molecular beacons. This review focuses on the development of receptor-specific molecular probes for optical imaging of tumors. Particularly, bioconjugates of probes that absorb and fluoresce in the near infrared wavelengths between 750 and 900 nm will be reviewed.

Topics: Animals; Bombesin; Carbocyanines; Carrier Proteins; Cell Membrane; Coloring Agents; Contrast Media; Diagnostic Imaging; Disease Models, Animal; Epidermal Growth Factor; Fluorescent Dyes; Folic Acid; Humans; Light; Models, Chemical; Models, Molecular; Molecular Probes; Neoplasms; Neurotensin; Peptides; Photosensitizing Agents; Somatostatin; Xanthenes

Cancer and atherosclerosis are chronic diseases that share common characteristics at both early and advanced stages and can arise from multiple factors. Both diseases are characterized by uncontrolled cell proliferation, inflammation, angiogenesis and apoptosis. Herein we investigated the ability of a peptide (CTHRSSVVC), that was previously reported to bind atherosclerotic lesions to home in the tumor microenvironment. The CTHRSSVVC peptide was synthesized on solid phase and N-terminally labeled with a sulfo-Cy5 dye. The specific binding to macrophage was evaluated in vitro with flow cytometry and immunofluorescence and in vivo for tumor targeting in BALB/c mice bearing a 4T1 tumor using optical imaging. The sulfo-Cy5-CTHRSSVVC peptide was synthesized in greater than 99% purity. No selective binding of the sulfo-Cy5-CTHRSSVVC peptide to macrophages in vitro was observed, however in vivo the sulfo-Cy5-CTHRSSVVC peptide accumulated in the 4T1 tumor, with a tumor-to-normal tissue ratio of 7.21 ± 1.44 at 2 h post injection. Ex vivo analysis of tumor tissue by confocal microscopy suggested that the sulfo-Cy5-CTHRSSVVC peptide had accumulated in the stroma of the tumor specifically, in regions of spindle shaped cells. In conclusion, although the target for the sulfo-Cy5-CTHRSSVVC peptide remains to be identified, the Cy5-CTHRSSVVC peptide warrants further investigation as a tumor imaging agent.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Carbocyanines; Disease Models, Animal; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Immunohistochemistry; Macrophages; Mice; Neoplasms; Optical Imaging; Peptides; Plaque, Atherosclerotic; Protein Binding; Receptors, Cell Surface; Receptors, Scavenger; THP-1 Cells

Fluorogenic probes in the near-infrared (NIR) region have the potential to provide stimuli-dependent information in living organisms. Here, we describe a new class of fluorogenic probes based on the heptamethine cyanine scaffold, the most broadly used NIR chromophore. These compounds result from modification of heptamethine norcyanines with stimuli-responsive carbamate linkers. The resulting cyanine carbamates (CyBams) exhibit exceptional turn-ON ratios (∼170×) due to dual requirements for NIR emission: carbamate cleavage through 1,6-elimination and chromophore protonation. Illustrating their utility in complex

Topics: Animals; Carbamates; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Female; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; gamma-Glutamyltransferase; Humans; Mice; Microscopy, Confocal; Neoplasm Metastasis; Ovarian Neoplasms; Spectroscopy, Near-Infrared; Transplantation, Heterologous

Despite the wide use of magnetic resonance imaging (MRI) as a clinical diagnostic tool, there are still no clinically approved MRI contrast agents that can be applied for cerebral Alzheimer's disease (AD) biomarker imaging. We report here the design and development of the first amyloid-β (Aβ)-targeted, blood-brain barrier (BBB) penetrable theranostic Gd(DOTA)-cyanine dyad, which was synthesized by the conjugation of Gd(DOTA) complex and carbazole-based cyanine dye by the copper(I)-catalyzed azide-alkyne cycloaddition click reaction for imaging of Aβ

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Carbocyanines; Coordination Complexes; Disease Models, Animal; Gadolinium; Magnetic Resonance Imaging; Mice

In recent years, there has been a progress in the study of glycation reaction which is one the possible reason for multiple metabolic disorders. Glycation is a nonenzymatic reaction between nucleic acids, lipids, and proteins resulting into the formation of early glycation products that may further lead to the accumulation of advanced glycation end products (AGEs). The precipitation of AGEs in various cells, tissues, and organs is one of the factors for the initiation and progression of various metabolic derangements including the cancer. The AGE interaction with its receptor "RAGE" activates the inflammatory pathway; yet, the downregulation of RAGE and its role in these pathways are not clear. We explore the effect of anticancer novel nanoassemblies on AGEs to determine its role in the regulation of the expression of RAGE, NFƙB, TNF-

Topics: A549 Cells; Animals; Benzothiazoles; Blood Glucose; Carbocyanines; Cell Proliferation; Disease Models, Animal; Glycosylation; Humans; Interferon-gamma; Lung Neoplasms; MCF-7 Cells; Mice; Models, Biological; Nanoparticles; NF-kappa B; Oxidative Stress; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptor for Advanced Glycation End Products; Serum Albumin, Human; Spectrophotometry; Trifluridine; Tumor Necrosis Factor-alpha

Most photoacoustic (PA) imaging agents are based on the repurposing of existing fluorescent dye platforms that exhibit non-optimal properties for PA applications. Herein, we introduce PA-HD, a new dye scaffold optimized for PA probe development that features a 4.8-fold increase in sensitivity and a red-shift of the λ

Topics: Alzheimer Disease; Animals; Brain; Carbocyanines; Disease Models, Animal; Fluorescent Dyes; Hydrogen Peroxide; Mice; Molecular Structure; Optical Imaging; Oxidative Stress; Photoacoustic Techniques

Idiopathic pulmonary fibrosis (IPF) is a devastating and fatal interstitial lung disease due to various challenges in diagnosis and treatment. Due to its complicated pathogenesis and difficulty in early diagnosis, there is no effective cure. Cyclooxygenase-2 (COX-2) is inextricably associated with pulmonary fibrosis. The abnormal level of COX-2 leads to extremely exacerbated pulmonary fibrosis. Therefore, we reported a near-infrared fluorescent probe Cy-COX to detect the fluctuation of COX-2 levels during pulmonary fibrosis and explain its important protective effect. The probe Cy-COX showed a significant enhancement of fluorescence signal to COX-2 with excellent selectivity and sensitivity. In order to clarify the relationship between COX-2 and pulmonary fibrosis, we used the probe Cy-COX to detect COX-2 fluctuation in organisms with pulmonary fibrosis. The results showed that the COX-2 level increased in the early stage and decreased in the late stage with the aggravation of pulmonary fibrosis. Furthermore, up-regulation of COX-2 levels can effectively alleviate the severity of pulmonary fibrosis. Therefore, Cy-COX is a fast and convenient imaging tool with great potential to predict the early stage of pulmonary fibrosis and evaluate the therapeutic effects.

Topics: Animals; Carbocyanines; Cells, Cultured; Cyclooxygenase 2; Disease Models, Animal; Fluorescent Dyes; Idiopathic Pulmonary Fibrosis; Infrared Rays; Mice; Mice, Inbred C57BL; Optical Imaging; Rats

Because of the high mortality of coronary atherosclerotic heart diseases, it is necessary to develop novel early detection methods for vulnerable atherosclerotic plaques. Phenotype transformation of vascular smooth muscle cells (VSMCs) plays a vital role in progressed atherosclerotic plaques. Osteopontin (OPN) is one of the biomarkers for phenotypic conversion of VSMCs. Significant higher OPN expression is found in foam cells along with the aggravating capacity of macrophage recruitment due to its arginine-glycine-aspartate sequence and interaction with CD44. Herein, a dual-modality imaging probe, OPN targeted nanoparticles (Cy5.5-anti-OPN-PEG-PLA-PFOB, denoted as COP-NPs), is constructed to identify the molecular characteristics of high-risk atherosclerosis by ultrasound and optical imaging. Characterization, biocompatibility, good binding sensibility, and specificity are evaluated in vitro. For in vivo study, apolipoprotein E deficien (ApoE

Topics: Animals; Carbocyanines; Contrast Media; Disease Models, Animal; Drug Delivery Systems; Mice; Mice, Knockout, ApoE; Myocytes, Smooth Muscle; Nanoparticles; Optical Imaging; Osteopontin; Plaque, Atherosclerotic

Aberrant brown adipose tissue (BAT) metabolism is linked to obesity as well as other metabolic disorders. However, the paucity of imaging tools limits the study of in vivo BAT metabolism in animal models. The current work evaluated a heptamethine dye (CyHF-8) in living mice as a dual-modality BAT-avid molecular probe for two imaging approaches, including near-infrared fluorescence imaging (NIRF) and photoacoustic imaging (PAI). CyHF-8 exhibited favorable spectral properties in the near-infrared window (786/787/805 nm) and accumulated in the subcellular mitochondria of brown adipocytes. After intravenous injection of CyHF-8, NIRF and PAI were both capable of noninvasively detecting interscapular BAT at early time points in living mice. Quantitative analysis of NIRF and PAI images showed that CyHF-8 signals respond to dynamic BAT changes in mice stimulated by norepinephrine (NE) and in diabetic mice induced by streptozotocin (STZ). In summary, dual-modality NIRF/PAI probe CyHF-8 can be used for both NIRF and PAI to noninvasively assess BAT metabolism in living animals.

Topics: Adipose Tissue, Brown; Animals; Carbocyanines; Cell Line, Tumor; Diabetes Mellitus, Experimental; Disease Models, Animal; Female; Fluorescent Dyes; Humans; Mice; Mice, Inbred C57BL; Mice, Nude; Microscopy, Fluorescence; Norepinephrine; Photoacoustic Techniques

Assessment of the coronary microcirculation remains challenging.. we explored the feasibility of evaluating the coronary microvasculature in rats with myocardial infarction (MI) using a three-dimensional visualization technique.. Animals were divided into the sham operation group (S), MI 45 min group (M45), and MI 180 min group (M180). Opened microvessels were labelled with the fluorescent dye DiI (1, 1'-dioctadecyl-3, 3, 3'3'-tetramethylindo carbocyanine perchlorate) using a heart perfusion method. The microvascular distribution and opening status were observed under laser scanning confocal microscopy, which was adjusted to facilitate evaluation of subjects around 6 to 20 μm.. Microvascular vessels (6-20 μm) were successfully labelled by DiI. Intact and clear three-dimensional microvascular structures were observed in myocardium of sham rats and remote non-infarct myocardial tissue of MI rats, while there was almost no microvascular structure in the infarct area of the M45 group, and only a small amount of microvascular visualization was visualized in the infarct area of the M180 group. The microvascular area and microvascular density in M45 group and M180 group in the infarct border zone were significantly lower than corresponding area in S group.. Three-dimensional visualization of opened coronary microvascular vessels is feasible in DiI-labelled myocardium in this rat MI model. This novel technique might be useful for defining the underlying mechanisms of coronary microvascular diseases and observe the efficacy of various therapy strategies on coronary microvessels.

Topics: Animals; Carbocyanines; Coronary Vessels; Disease Models, Animal; Feasibility Studies; Fluorescent Dyes; Imaging, Three-Dimensional; Microscopy, Confocal; Microscopy, Fluorescence; Microvessels; Myocardial Infarction; Predictive Value of Tests; Rats, Sprague-Dawley

Currently, there are no non-invasive tools to accurately diagnose wound and surgical site infections before they become systemic or cause significant anatomical damage. Fluorescence and photoacoustic imaging are cost-effective imaging modalities that can be used to noninvasively diagnose bacterial infections when paired with a molecularly targeted infection imaging agent. Here, we develop a fluorescent derivative of maltotriose (Cy7-1-maltotriose), which is shown to be taken up in a variety of gram-positive and gram-negative bacterial strains in vitro. In vivo fluorescence and photoacoustic imaging studies highlight the ability of this probe to detect infection, assess infection burden, and visualize the effectiveness of antibiotic treatment in E. coli-induced myositis and a clinically relevant S. aureus wound infection murine model. In addition, we show that maltotriose is an ideal scaffold for infection imaging agents encompassing better pharmacokinetic properties and in vivo stability than other maltodextrins (e.g. maltohexose).

Topics: Animals; Carbocyanines; Disease Models, Animal; Drug Stability; Escherichia coli; Female; Fluorescent Dyes; Humans; Injections, Intravenous; Luminescent Measurements; Mice; Microscopy, Fluorescence; Molecular Imaging; Molecular Probes; Myositis; Photoacoustic Techniques; Rats; Staphylococcus aureus; Surgical Wound Infection; Trisaccharides

Optical molecular imaging technology that indiscriminately detects intracranial glioblastoma (GBM) can help neurosurgeons effectively remove tumor masses. Transferrin receptor 1 (TfR 1) is a diagnostic and therapeutic target in GBM. A TfR 1-targeted peptide, CRTIGPSVC (CRT), was shown to cross the blood brain barrier (BBB) and accumulate at high levels in GBM tissues. In this study, we synthesized a TfR 1-targeted near-infrared fluorescent (NIRF) probe, Cy5-CRT, for identifying the GBM tissue margin in mouse models.. We initially confirmed the overexpression of TfR 1 in GBM and the tumor-specific homing ability of Cy5-CRT in subcutaneous and orthotopic GBM mouse models. We then examined the feasibility of Cy5-CRT for identifying the tumor margin in orthotopic GBM xenografts. Finally, we compared Cy5-CRT with the clinically used fluorescein sodium in identifying tumor margins.. Cy5-CRT specifically accumulated in GBM tissues and detected the tumor burden with exceptional contrast in mice with orthotopic GBM, enabling fluorescence-guided GBM resection under NIRF live imaging conditions. Importantly, Cy5-CRT recognized the GBM tissue margin more clearly than fluorescein sodium.. The TfR 1-targeted optical probe Cy5-CRT specifically differentiates tumor tissues from the surrounding normal brain with high sensitivity, indicating its potential application for the precise surgical removal of GBM.

Topics: Animals; Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Fluorescein; Fluorescent Dyes; Glioma; Mice; Microscopy, Fluorescence; Optical Imaging; Receptors, Transferrin; Xenograft Model Antitumor Assays

More than 2.8 million annually in the United States are afflicted with some form of traumatic brain injury (TBI), where 75% of victims have a mild form of TBI (MTBI). TBI risk is higher for individuals engaging in physical activities or involved in accidents. Although MTBI may not be initially life-threatening, a large number of these victims can develop cognitive and physical dysfunctions. These late clinical sequelae have been attributed to the development of secondary injuries that can occur minutes to days after the initial impact. To minimize brain damage from TBI, it is critical to diagnose and treat patients within the first or "golden" hour after TBI. Although it would be very helpful to quickly determine the TBI locations in the brain and direct the treatment selectively to the affected sites, this remains a challenge. Herein, we disclose our novel strategy to target cyclosporine A (CsA) into TBI sites, without the need to locate the exact location of the TBI lesion. Our approach is based on TBI treatment with a cyanine dye nanocage attached to CsA, a known therapeutic agent for TBI that is associated with unacceptable toxicities. In its caged form, CsA remains inactive, while after near-IR light photoactivation, the resulting fragmentation of the cyanine nanocage leads to the selective release of CsA at the TBI sites.

Topics: Animals; Brain Injuries, Traumatic; Carbocyanines; Cyclosporine; Disease Models, Animal; Drug Carriers; Drug Liberation; Humans; Infrared Rays; Nanoparticles; Neuroprotective Agents; Photochemotherapy; Rats

Tumor development and metastasis are dependent on tumor infiltrating immune cells which form a characteristic tumor microenvironment (TME). Activated monocytes secrete the protein heterodimer S100A8/A9 promoting TME formation. Monocyte-dependent proteases facilitate local tumor cell invasion by degradation of the extracellular matrix. We aimed for target specific in vivo imaging of S100A8 and proteases to provide differentiating biomarkers for local tumor growth and metastatic potential.. Murine breast cancer cells of the 4T1 model with graduated metastatic potential (4T1 and 4T07: both hematogenous metastasis > 168FAR: lymph-node metastasis > 67NR: no metastasis) were orthotopically implanted into female BALB/c mice. At 4 mm size, tumors were investigated by injecting the protease-specific probe ProSense 750EX (PerkinElmer, 4T1 n = 7, 4T07 n = 10, 168FAR n = 16, 67NR n = 15) and anti-S100A8-Cy5.5 (n = 6 each) and performing fluorescence reflectance imaging at 0 and 24 h after injection. In vivo imaging was validated with immunohistochemistry.. At 24 h, S100A8-specific signals in 4T1 and 4T07 were significantly higher (1714.05/1683.45 AU) as compared to 168FAR and 67NR (174.85/167.95 AU, p = 0.0012/p = 0.0003), reflecting the capability of hematogenous spread. Protease-specific signals were significantly higher in 4T1 and 4T07 (348.01/409.93 AU) as compared to 168FAR (214.91 AU) and 67NR (129.78 AU p < 0.0001 each), reflecting local vessel invasion and tumor cell shedding. Immunohistology supported the in vivo imaging results.. Non-invasive in vivo imaging of S100A8 and monocytic proteases allows for differentiation of the tumors' local invasive and systemic metastatic potential in reflecting the TME formation. While proteases augment local tumor cell invasion, solid metastases seem to be dependent on a pro-tumoral microenvironment.

Topics: Animals; Calgranulin A; Carbocyanines; Cathepsins; Cell Line, Tumor; Disease Models, Animal; Female; Immunohistochemistry; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Molecular Imaging; Neoplasm Staging; Optical Imaging; Tumor Microenvironment

To investigate whether new dyes and dye combinations can give equivalent or better staining in anterior capsule surgery than existing dyes with a low degree of toxicity on relevant cells.. University laboratory of Jacobs University Bremen, Germany.. Laboratory experimental study.. Pig eyes were collected post mortem. Cataract was induced by microwave irradiation. Access to the lens capsule was through open-sky surgery. Staining was performed and results were documented by photography. The toxicity of the dyes was evaluated in 3 different cell lines immediately after exposure and with a delay of 24 hours, with exposure in the dark or subsequent strong illumination.. A new cyanine dye, BIP (2-[5-[3,3-dimethyl-1-(4-sulfobutyl)-1,3-dihydro-indol-2-ylidene]-penta-1,3-dienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium sodium), was found to lead to green staining, with reduced toxicity on corneal endothelial cells. Staining could be further enhanced by combining it with trypan blue. Methylene blue was very toxic, whereas its combination with trypan blue was much less toxic.. With BIP alone or in combination with trypan blue, safe staining of the capsule can be achieved, resulting in a green color.

Topics: Animals; Anterior Capsule of the Lens; Capsulorhexis; Carbocyanines; Cataract; Coloring Agents; Disease Models, Animal; Swine; Trypan Blue

Idiopathic pulmonary fibrosis (IPF) is a lung-limited and progressive fibrotic disease. The early diagnosis and therapies of IPF are still full of clinical challenges. Glutathione S-transferase (GSTs) plays significant roles in promoting the formation of pulmonary fibrosis. Herein, we report a fluorescent probe (Cy-GST) for the detection of GSTs concentration fluctuations in cells and in mice models. The probe can selectively and sensitively respond to GSTs with an "off-on" type fluorescence switch. Our results demonstrated that the level of intracellular GSTs increase in the pulmonary fibrosis cells and mice models. And the IPF patients hold high levels of GSTs concentrations. Thus, GSTs are likely to play important roles in pulmonary fibrosis. The inhibitor of GSTs TLK117 can reduce the severity of pulmonary fibrosis. The synergistic treatment of TLK117 and pirfenidone have better therapeutic effects than only using pirfenidone in pulmonary fibrosis mice models. The level of GSTs in IPF may be a new potential marker for IPF diagnosis. And the inhibition of GSTs may be a new therapeutic strategy for IPF treatment.

Topics: Animals; Carbocyanines; Disease Models, Animal; Enzyme Inhibitors; Fluorescent Dyes; Glutathione; Glutathione Transferase; Idiopathic Pulmonary Fibrosis; Infrared Rays; Mice; Mice, Inbred C57BL; Optical Imaging; Particle Size; Surface Properties

Topics: Animals; Carbocyanines; Disease Models, Animal; Fluorescent Dyes; Hepatocytes; Humans; Malaria, Falciparum; Mice; Microscopy, Confocal; Microscopy, Video; Models, Theoretical; Plasmodium berghei; Plasmodium falciparum; Plasmodium yoelii; Skin; Sporozoites; Staining and Labeling

Intracerebral hemorrhage (ICH) is a cerebrovascular disease with high mortality and morbidity, and the effective treatment is still lacking. We designed this study to investigate the therapeutic effects and mechanisms of melatonin on the secondary brain injury (SBI) after ICH. An in vivo ICH model was induced via autologous whole blood injection into the right basal ganglia in Sprague-Dawley (SD) rats. Primary rat cortical neurons were treated with oxygen hemoglobin (OxyHb) as an in vitro ICH model. The results of the in vivo study showed that melatonin alleviated severe brain edema and behavior disorders induced by ICH. Indicators of blood-brain barrier (BBB) integrity, DNA damage, inflammation, oxidative stress, apoptosis, and mitochondria damage showed a significant increase after ICH, while melatonin reduced their levels. Meanwhile, melatonin promoted further increasing of expression levels of antioxidant indicators induced by ICH. Microscopically, TUNEL and Nissl staining showed that melatonin reduced the numbers of ICH-induced apoptotic cells. Inflammation and DNA damage indicators exhibited an identical pattern compared to those above. Additionally, the in vitro study demonstrated that melatonin reduced the apoptotic neurons induced by OxyHb and protected the mitochondrial membrane potential. Collectively, our investigation showed that melatonin ameliorated ICH-induced SBI by impacting apoptosis, inflammation, oxidative stress, DNA damage, brain edema, and BBB damage and reducing mitochondrial membrane permeability transition pore opening, and melatonin may be a potential therapeutic agent of ICH.

Topics: Animals; Annexin A5; Antioxidants; Apoptosis; Benzimidazoles; Brain Edema; Brain Injuries; Carbocyanines; Cerebral Cortex; Cerebral Hemorrhage; Disease Models, Animal; DNA Damage; In Situ Nick-End Labeling; Inflammation; Male; Melatonin; Mitochondrial Diseases; Neurons; Oxidative Stress; Rats; Rats, Sprague-Dawley; Rhodamines; Time Factors

Non-invasive assessment of inflammatory activity in the course of various diseases is a largely unmet clinical challenge. An early feature of inflammation is local secretion of the alarmin S100A8/A9 by activated phagocytes. We here evaluate a novel S100A9-targeted small molecule tracer Cy5.5-CES271 for in vivo optical imaging of inflammatory activity in exemplary disease models.. Dynamics of Cy5.5-CES271 was characterized in a model of irritant contact dermatitis by sequential fluorescence reflectance imaging (FRI) up to 24 h postinjection (p.i.). Specificity of Cy5.5-CES271 binding to S100A9 in vivo was examined by blocking studies and by employing S100A9. Cy5.5-CES271 shows significant accumulation in models of inflammatory diseases and specific binding to S100A9 in vivo. This study, for the first time, demonstrates the potential of a small molecule non-peptidic tracer enabling imaging of S100A9 as a marker of local phagocyte activity in inflammatory scenarios suggesting this compound class for translational attempts.

Topics: Animals; Calgranulin B; Carbocyanines; Dermatitis, Irritant; Disease Models, Animal; Fluorescence; Ligands; Lipopolysaccharides; Mice, Inbred BALB C; Optical Imaging; Peptides; Phagocytes; Pneumonia

Disturbance of intestinal wound closure leads to insufficient anastomotic healing and is associated with considerable morbidity following colorectal resections. Matrix metalloproteinases (MMPs) play a crucial role in regulation of wound closure. Here fluorescence endoscopy was evaluated for assessment of MMP-2/-9 expression during failed intestinal anastomotic healing.. Distal colonic anastomoses were performed as a model for disturbed healing in 36 Balb/c mice. Healing was evaluated endoscopically, macroscopically, and histologically after 1, 3 and 5 days. For detection of MMP-2/-9 expression fluorescence endoscopy (FE) was used following i.v.-administration of a Cy5.5-labeled MMP-2/-9 specific tracer. FE was complemented by quantification of the fluorescence signal using the MS-FX PRO Optical Imaging System. An overall leakage score was calculated and correlated with the results of FE.. With increasing incidence of anastomotic leakage from POD1 (17%) to POD5 (83%) the uptake of the MMP tracer gradually increased (signal-to-noise ratio (SNR), POD1: 17.91 ± 1.251 vs. POD3: 30.56 ± 3.03 vs. POD5: 44.8 ± 4.473, P<0.0001). Mice with defective anastomotic healing showed significantly higher uptake compared to non-defective (SNR: 37.37± 3.63 vs. 26.16± 3.635, P = 0.0369). White light endoscopy and FE allowed evaluation of anastomotic healing and visualization of mucosal MMPs in vivo. Using FE based detection of MMPs in the anastomosis, an overall positive predictive value of 71.4% and negative predictive value of 66.6% was calculated for detection of anastomotic leakage.. During disturbed anastomotic healing increased expression of MMP-2/-9 was observed in the anastomotic tissue. Fluorescence endoscopy for detection of MMP-2/-9 during the healing process might be a promising tool for early identification of anastomotic leakage.

Topics: Administration, Intravenous; Anastomosis, Surgical; Anastomotic Leak; Animals; Carbocyanines; Colon; Colonoscopy; Disease Models, Animal; Feasibility Studies; Fluorescence; Fluorescent Dyes; Humans; Intestinal Mucosa; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Staining and Labeling; Wound Healing

As atherosclerotic plaque ruptures are the primary cause of ischaemic events, their preventive identification by imaging remains a clinical challenge. Matrix metalloproteinases (MMP) are involved in plaque progression and destabilisation and are therefore promising targets to characterize rupture-prone unstable plaques. This study aims at evaluating MMP imaging to discriminate unstable from stable plaque phenotypes.. ApoE deficient mice (ApoE-/-) on a high cholesterol diet underwent implantation of a tapered cuff around the right common carotid artery (CCA) inducing a highly inflamed atherosclerotic plaque upstream (US) and a more stable plaque phenotype downstream (DS) of the cuff. 8 weeks after surgery, the MMP inhibitor-based photoprobe Cy5.5-AF443 was administered i.v. 3h prior to in situ and ex vivo fluorescence reflectance imaging of the CCAs. Thereafter, CCAs were analysed regarding plaque size, presence of macrophages, and MMP-2 and MMP-9 concentrations by immunohistochemistry and ELISA.. We found a significantly higher uptake of Cy5.5-AF443 in US as compared to DS plaques in situ (1.29 vs. 1.06 plaque-to-background ratio; p<0.001), which was confirmed by ex vivo measurements. Immunohistochemistry revealed a higher presence of macrophages, MMP-2 and MMP-9 in US compared to DS plaques. Accordingly, MMP-2 concentrations were significantly higher in US plaques (47.2±7.6 vs. 29.6±4.6 ng/mg; p<0.05).. In the ApoE-/- cuff model MMP-2 and MMP-9 activities are significantly higher in upstream low shear stress-induced unstable atherosclerotic plaques as compared to downstream more stable plaque phenotypes. MMP inhibitor-based fluorescence molecular imaging allows visualization of these differences in shear stress-induced atherosclerosis.

Topics: Animals; Apolipoproteins E; Carbocyanines; Disease Models, Animal; Disease Progression; Humans; Macrophages; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Molecular Imaging; Plaque, Atherosclerotic; Shear Strength; Stress, Physiological

Neural stem cells (NSCs) have been attractive donor sources for cell therapy in traumatic brain injuries (TBI). Monitoring the fate of transplanted cells, including the survival and differentiation, will provide vital information to assess the outcome during the therapy time course. However, the current labeling methods are based on the principles of cell endocytosis, demanding relatively high fluorescent probes concentration and long incubation time, which may affect the proliferation and differentiation of transplanted cells. In our study, an efficient and relatively fast labeling strategy for NSCs with Cy3 based on DNA hybridization was proposed for monitoring the fate of transplanted cells. The oligo[dA]

Topics: Animals; Brain Injuries, Traumatic; Carbocyanines; Cell Differentiation; Cell Survival; Disease Models, Animal; DNA; Mice; Neural Stem Cells; Nucleic Acid Hybridization; Stem Cell Transplantation

In the present study mechanical damage to the corneal endothelium was induced by elevation of intraocular pressure (IOP, 140 mmHg, 60 min) to one eye of rats, delivered either in complete darkness or in the presence of red light (16.5 W/m

Topics: Animals; Benzimidazoles; Blotting, Western; Carbocyanines; Cell Proliferation; Cell Survival; Cells, Cultured; Corneal Endothelial Cell Loss; Disease Models, Animal; Electron Transport Complex IV; Endothelium, Corneal; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Infrared Rays; Intraocular Pressure; Male; Mitochondria; Ocular Hypertension; Phototherapy; Rats; Rats, Wistar; Zonula Occludens-1 Protein

Functional deficits in sensory systems are commonly noted in neurodevelopmental disorders, such as the Rett syndrome (RTT). Defects in methyl CpG binding protein gene (MECP2) largely accounts for RTT. Manipulations of the Mecp2 gene in mice provide useful models to probe into various aspects of brain development associated with the RTT. In this study, we focused on the somatosensory cortical phenotype in the Bird mouse model of RTT. We used voltage-sensitive dye imaging to evaluate whisker sensory evoked activity in the barrel cortex of mice. We coupled this functional assay with morphological analyses in postnatal mice and investigated the dendritic differentiation of barrel neurons and individual thalamocortical axon (TCA) arbors that synapse with them. We show that in Mecp2-deficient male mice, whisker-evoked activity is roughly topographic but weak in the barrel cortex. At the morphological level, we find that TCA arbors fail to develop into discrete, concentrated patches in barrel hollows, and the complexity of the dendritic branches in layer IV spiny stellate neurons is reduced. Collectively, our results indicate significant structural and functional impairments in the barrel cortex of the Bird mouse line, a popular animal model for the RTT. Such structural and functional anomalies in the primary somatosensory cortex may underlie orofacial tactile sensitivity issues and sensorimotor stereotypies characteristic of RTT.

Topics: Afferent Pathways; Animals; Carbocyanines; Dendrites; Disease Models, Animal; Male; Methyl-CpG-Binding Protein 2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Rett Syndrome; Silver Staining; Somatosensory Cortex; Vibrissae; Voltage-Sensitive Dye Imaging

Cavitation-facilitated microbubble-mediated focused ultrasound therapy is a promising method of drug delivery across the blood-brain barrier (BBB) for treating many neurological disorders. Unlike ultrasound thermal therapies, during which magnetic resonance thermometry can serve as a reliable treatment control modality, real-time control of modulated BBB disruption with undetectable vascular damage remains a challenge. Here a closed-loop cavitation controlling paradigm that sustains stable cavitation while suppressing inertial cavitation behavior was designed and validated using a dual-transducer system operating at the clinically relevant ultrasound frequency of 274.3 kHz. Tests in the normal brain and in the F98 glioma model in vivo demonstrated that this controller enables reliable and damage-free delivery of a predetermined amount of the chemotherapeutic drug (liposomal doxorubicin) into the brain. The maximum concentration level of delivered doxorubicin exceeded levels previously shown (using uncontrolled sonication) to induce tumor regression and improve survival in rat glioma. These results confirmed the ability of the controller to modulate the drug delivery dosage within a therapeutically effective range, while improving safety control. It can be readily implemented clinically and potentially applied to other cavitation-enhanced ultrasound therapies.

Topics: Acoustics; Animals; Antibiotics, Antineoplastic; Blood-Brain Barrier; Brain Neoplasms; Carbocyanines; Corpus Striatum; Disease Models, Animal; Doxorubicin; Drug Delivery Systems; Fluorescent Dyes; Glioma; Hippocampus; Luminescent Proteins; Magnetic Resonance Imaging; Male; Microbubbles; Molecular Targeted Therapy; Polyethylene Glycols; Rats; Rats, Sprague-Dawley; Transducers; Ultrasonic Therapy; Ultrasonic Waves

Subretinal injections are used to deliver agents in experimental studies of retinal diseases, often through viral vectors. However, few studies have investigated the effects of subretinal injections alone on the structure and function of the healthy or diseased retina, particularly in models of oxygen-induced retinopathy (OIR). We report on the effects of subretinal injections in a rat OIR model, which is used to study mechanisms of retinopathy of prematurity.. Within 6 h of birth, neonatal rat pups were exposed to repeated cycles of oxygen between 50% and 10% O. Compared to untreated animals, the avascular area of the retina was greater in the pilot-treated (p<0.05) and sPBS-treated eyes (p<0.01), and the sPBS-treated eyes had a greater avascular retinal area compared to the pilot-treated eyes (p<0.01). The intravitreal neovascular area was larger in the sPBS-treated eyes compared to the untreated eyes (p<0.01). The outer nuclear and outer segment layers were thinner in the pilot- (p<0.01) and sPBS-treated eyes (p<0.05) compared to the untreated eyes as measured with OCT and immunohistochemical staining of the retinal cryosections. Compared to the untreated eyes, the amplitudes of the scotopic a- and b-waves in the Ganzfeld ERG were reduced in the pilot-treated eyes (p<0.001 and p<0.01, respectively), but only the a-wave was reduced in the sPBS-treated eyes (p<0.001). The a-wave amplitude in the focal ERG was reduced in the pilot- and sPBS-treated eyes, and no difference was seen in the b-wave amplitude between any of the groups. There was no difference between the light-treated and untreated eyes in the areas of the avascular retina or intravitreal neovascularization or Ganzfeld or focal ERG.. Pilot injections alone without injection into the subretinal space resulted in an increased avascular retinal area, reduced thickness of the photoreceptors, and reduced ERG function compared to the untreated animals. Although subretinal PBS injections further increased the areas of avascular retina and intravitreal neovascularization and resulted in similar retinal thinning compared to the pilot treatment, inner retinal function was improved, as evidenced by higher Ganzfeld b-wave amplitudes. Differences in the Ganzfeld and focal ERGs may indicate that the peripheral retina is more susceptible to remote beneficial effects from potential protective mechanisms induced by subretinal injection. This study stresses the importance of appropriate controls in experiments with subretinal delivery of agents.

Topics: Animals; Animals, Newborn; Carbocyanines; Disease Models, Animal; Electroretinography; Female; Immunohistochemistry; Injections, Intraocular; Male; Microscopy, Confocal; Opsins; Oxygen; Rats; Rats, Sprague-Dawley; Retina; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Tomography, Optical Coherence

Topics: Adenocarcinoma; Animals; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Ferrosoferric Oxide; Fluorescent Dyes; Humans; Injections, Subcutaneous; Magnetic Resonance Imaging; Magnetite Nanoparticles; Male; Mice; Mice, Nude; Molecular Probes; Neovascularization, Pathologic; Optical Imaging; Peptides, Cyclic; Polyethylene Glycols; Stomach Neoplasms

Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells and endothelial cells associated with obliteration of small pulmonary arterioles and formation of plexiform lesions. To date, no curative treatments have been identified for pulmonary arterial hypertension. There are various therapeutic options, including conventional medical therapies and oral, subcutaneous, intravenous, and inhalation delivery. We have previously shown that miR-143/145 knockout can prevent the development of chronic hypoxia-induced pulmonary hypertension (PH) in mice. Here, we use chronic hypoxia-induced PH as a disease model to evaluate miR-143/145 inhibition after delivery of antimiRNAs via the subcutaneous or intranasal routes. We use qRT-PCR and immunofluorescence to confirm that both delivery strategies efficiently inhibit miR-143/145 in lung tissue from mice with chronic hypoxia-induced PH.

Topics: Administration, Intranasal; Animals; Antagomirs; Carbocyanines; Chronic Disease; Disease Models, Animal; Female; Fluorescent Antibody Technique; Gene Transfer Techniques; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung; Mice, Inbred C57BL; MicroRNAs; Real-Time Polymerase Chain Reaction

Topics: Animals; Carbocyanines; Carcinoma, Squamous Cell; Cetuximab; Disease Models, Animal; Female; Humans; Hyaluronic Acid; Mice; Neoplasm Metastasis; Optical Imaging; Ovarian Neoplasms; Pathology, Surgical; Sentinel Lymph Node; Staining and Labeling; Trastuzumab

The potential benefits of high-density lipoproteins (HDL) against atherosclerosis are attributed to its major protein component, apolipoprotein A-I (apoA-I). Most of the apoA-I in the vascular wall appears to be in its lipid-poor form. The latter, however, is subjected to degradation by proteases localized in atherosclerotic plaques, which, in turn, has been shown to negatively impact its atheroprotective functions. Here, we report the development and in vivo use of a bioactivatable near-infrared full-length apoA-I-Cy5.5 fluorescent probe for the assessment of apoA-I-degrading proteolytic activities.. Fluorescence quenching was obtained by saturation of Cy5.5 fluorophore molecules on apoA-I protein. ApoA-I cleavage led to near-infrared fluorescence enhancement. In vitro proteolysis of the apoA-I probe by a variety of proteases including serine, cysteine, and metalloproteases resulted in an up to 11-fold increase in fluorescence (n = 5, p ≤ 0.05).. We detected activation of the probe in atherosclerotic mice aorta sections using in situ zymography and showed that broad-spectrum protease inhibitors protected the probe from degradation, resulting in decreased fluorescence (-54%, n = 6 per group, p ≤ 0.0001). In vivo, the injected probe showed stronger fluorescence emission in the aorta of human apoB transgenic Ldlr. The use of this probe in different applications may help to assess new molecular mechanisms of atherosclerosis and may improve current HDL-based therapies by enhancing apoA-I functionality.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Apolipoprotein A-I; Apolipoprotein B-100; Atherosclerosis; Carbocyanines; Cathepsins; Cell Line; Chymases; Disease Models, Animal; Fluorescent Dyes; Humans; Kinetics; Macrophages; Matrix Metalloproteinase 12; Mice, Inbred C57BL; Mice, Knockout; Molecular Imaging; Peptide Hydrolases; Protein Stability; Proteolysis; Receptors, LDL; Spectrometry, Fluorescence; Spectroscopy, Near-Infrared; Trypsin

Mitochondrial dysfunction has been implicated in various drug-induced toxicities and genetic disorders. Recently, the zebrafish has emerged as a versatile animal model for both chemical and genetic screenings. Taking advantage of its transparency, various in vivo fluorescent imaging methods have been developed to identify novel functions of chemicals and genes in zebrafish. However, there have not been fluorescent probes that can detect mitochondrial membrane potential in living zebrafish. In this study, we identified a novel cyanine dye called ZMJ214 that detects mitochondrial membrane potential in living zebrafish from 4 to 8 days post fertilization and is administered by simple immersion. The fluorescence intensity of ZMJ214 in zebrafish was increased and decreased by oligomycin and FCCP, respectively, suggesting a positive correlation between ZMJ214 fluorescence and mitochondrial membrane potential. In vivo imaging of zebrafish stained with ZMJ214 allowed for the detection of altered mitochondrial membrane potential induced by the antidiabetic drug troglitazone and the antiepileptic drug tolcapone, both of which have been withdrawn from the market due to mitochondrial toxicity. In contrast, pioglitazone and entacapone, which are similar to troglitazone and tolcapone, respectively, and have been used commercially, did not cause a change in mitochondrial membrane potential in zebrafish stained with ZMJ214. Live imaging of zebrafish stained with ZMJ214 also revealed that knock-down of slc25a12, a mitochondrial carrier protein associated with autism, dysregulated the mitochondrial membrane potential. These results suggest that ZMJ214 can be a useful tool to identify chemicals and genes that cause mitochondrial dysfunction in vivo.

Topics: Animals; Anti-Bacterial Agents; Anticonvulsants; Benzophenones; Carbocyanines; Chromans; Disease Models, Animal; Fluorescent Dyes; Hypoglycemic Agents; Membrane Potential, Mitochondrial; Mitochondria; Nitrophenols; Oligomycins; Optical Imaging; Pioglitazone; Thiazolidinediones; Tolcapone; Toxicity Tests; Troglitazone; Zebrafish; Zebrafish Proteins

Cerebral amyloid angiopathy (CAA) is a neurovascular disease that is strongly associated with an increase in the number and size of spontaneous microbleeds. Conventional methods of magnetic resonance imaging for detection of microbleeds, and positron emission tomography with Pittsburgh Compound B imaging for amyloid deposits, can separately demonstrate the presence of microbleeds and CAA in affected brains in vivo; however, there still is a critical need for strong evidence that shows involvement of CAA in microbleed formation. Here, we show in a Tg2576 mouse model of Alzheimer's disease, that the combination of histochemical staining and an optical clearing method called optical histology, enables simultaneous, co-registered three-dimensional visualization of cerebral microvasculature, microbleeds, and amyloid deposits. Our data suggest that microbleeds are localized within the brain regions affected by vascular amyloid deposits. All observed microhemorrhages (n=39) were in close proximity (0 to 144 μm) with vessels affected by CAA. Our data suggest that the predominant type of CAA-related microbleed is associated with leaky or ruptured hemorrhagic microvasculature. The proposed methodological and instrumental approach will allow future study of the relationship between CAA and microbleeds during disease development and in response to treatment strategies.

Topics: Animals; Benzothiazoles; Carbocyanines; Cerebral Amyloid Angiopathy; Cerebral Hemorrhage; Cerebrovascular Circulation; Disease Models, Animal; Fluorescent Dyes; Genetic Predisposition to Disease; Imaging, Three-Dimensional; Mice, Transgenic; Microcirculation; Microscopy, Confocal; Microvessels; Optical Imaging; Plaque, Amyloid; Staining and Labeling; Thiazoles

Drug penetration influences the efficacy of tumor therapy. Although the leaky vessels of tumors can improve the penetration of nanodrugs via the enhanced permeability and retention (EPR) effect, various aspects of the tumor microenvironment still restrict this process. This study investigated whether vascular disruption using the acoustic vaporization of micro- or nanoscale droplets (MDs or NDs) induced by ultrasound sonication can overcome the limitations of the EPR effect to allow drug diffusion into extensive regions. The intravital penetration of DiI-labeled liposomes (as a drug model with red fluorescence) was observed using an acousto-optical integrated system comprising a 2-MHz focused ultrasound transducer (transmitting a three-cycle single pulse and a peak negative pressure of 10 MPa) in a window-chamber mouse model. Histology images of the solid tumor were also used to quantify and demonstrate the locations where DiI-labeled liposomes accumulated. In the intravital image analyses, the cumulative diffusion area and fluorescence intensity at 180 min were 0.08±0.01 mm(2) (mean±standard deviation) and 8.5±0.4%, respectively, in the EPR group, 0.33±0.01 mm(2) and 13.1±0.4% in the MD group (p<0.01), and 0.63±0.01 mm(2) and 18.9±1.1% in the ND group (p<0.01). The intratumoral accumulations of DiI-labeled liposomes were 1.7- and 2.3-fold higher in the MD and ND groups, respectively, than in the EPR group. These results demonstrate that vascular disruption induced by acoustic droplet vaporization can improve drug penetration more than utilizing the EPR effect. The NDs showed longer lifetime in vivo than MDs and provided potential abilities of long periods of treatment, intertissue ND vaporization, and intertissue NDs-converted bubble cavitation to improve the drug penetration and transport distance.

Topics: Animals; Antineoplastic Agents; Blood Vessels; Carbocyanines; Disease Models, Animal; Histocytochemistry; Liposomes; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Optical Imaging; Sound; Staining and Labeling; Ultrasonography; Volatilization

Polyamidoamine dendrimer nanoparticles (~ 4 nanometers) are inert polymers that can be linked to biologically active compounds. These dendrimers selectively target and accumulate in inflammatory cells upon systemic administration. Dendrimer-linked compounds enable sustained release of therapeutic compounds directly at the site of damage. The purpose of this study was to determine if dendrimers can be used to target the optic nerve (ON) ischemic lesion in our rodent and nonhuman primate models of nonarteritic anterior ischemic optic neuropathy (NAION), a disease affecting >10,000 individuals in the US annually, and for which there currently is no effective treatment.. NAION was induced in male Long-Evans rats (rNAION) and in one adult male rhesus monkey (pNAION) using previously described procedures. Dendrimers were covalently linked to near-infrared cyanine-5 fluorescent dye (D-Cy5) and injected both intravitreally and systemically (in the rats) or just systemically (in the monkey) to evaluate D-Cy5 tissue accumulation in the eye and optic nerve following induction of NAION.. Following NAION induction, Cy-5 dendrimers selectively accumulated in astrocytes and circulating macrophages. Systemic dendrimer administration provided the best penetration of the ON lesion site when injected shortly after induction. Systemic administration 1 day post-induction in the pNAION model gave localization similar to that seen in the rats.. Dendrimers selectively target the ischemic ON lesion after induction of both rNAION and pNAION. Systemic nanoparticle-linked therapeutics thus may provide a powerful, targeted and safe approach to NAION treatment by providing sustained and focused treatment of the cells directly affected by ischemia.

Topics: Animals; Astrocytes; Carbocyanines; Dendrimers; Disease Models, Animal; Drug Carriers; Fluorescent Dyes; Humans; Injections, Intravenous; Intravitreal Injections; Macaca mulatta; Macrophages; Male; Molecular Targeted Therapy; Nanoparticles; Optic Nerve; Optic Neuropathy, Ischemic; Particle Size; Polyamines; Rats; Rats, Long-Evans

Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes.

Topics: Albendazole; Animals; Anthelmintics; Benzimidazoles; Carbocyanines; Disease Models, Animal; Echinococcosis; Echinococcosis, Hepatic; Echinococcus multilocularis; Gerbillinae; Humans; Male; Metformin; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Parasite Load

Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with. The necrosis avid properties of the radiotracer [. Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [. The radiotracer [

Topics: Animals; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Indium Radioisotopes; Mice, Inbred BALB C; Mice, Nude; Multimodal Imaging; Necrosis; Neoplasms; Optical Imaging; Pentetic Acid; Tissue Distribution; Tomography, Emission-Computed, Single-Photon

Topics: Animals; Arrhythmias, Cardiac; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Carbocyanines; Cell Differentiation; Coronary Vessels; Disease Models, Animal; Dogs; Myocardial Infarction; Myocardium; Transplantation, Autologous

We established a murine periodontitis model by local injection of lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS) into the gingival sulcus of mandibular left incisor four times with 48-h interval. The histological examination of the periodontal tissues demonstrated that significant loss of periodontal bone and ligaments was observed in the lesion side with abundant inflammatory cell infiltration. Two days after the last injection, Cy5-labelled siRNA/chitosan particles were injected intraperitoneally (ip). The chitosan/siRNA particles were taken up by peritoneal macrophages, which subsequently migrated to the inflamed gingival area evaluated by in vivo imaging. The localization of macrophages in the inflamed region was further confirmed by immunofluorescent staining. The present report demonstrates that intragingival injection of Pg-LPS can be used to create an experimental model of periodontal inflammation in mice and that recruitment of macrophages with chitosan/siRNA nanoparticles to the inflamed area opens the possibility of an RNAi-based therapeutic approach using chitosan as a carrier in periodontitis.

Topics: Adoptive Transfer; Alveolar Bone Loss; Animals; Carbocyanines; Chitosan; Disease Models, Animal; Female; Lipopolysaccharides; Macrophages; Mice; Nanoparticles; Periodontitis; Porphyromonas gingivalis; RNA, Small Interfering; RNAi Therapeutics; Tissue Distribution

Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their antiresorptive effects on the skeleton. However, because bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their antitumor action, particularly on cells outside of bone, remain unknown. Here, we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumors, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumor-associated macrophages (TAM), but not tumor cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumor of a patient and showed the resected tumor to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumors outside the skeleton and that their antitumor activity is likely to be mediated via TAMs.. Bisphosphonates are assumed to act solely in bone. However, mouse models and clinical trials show that they have surprising antitumor effects outside bone. We provide unequivocal evidence that bisphosphonates target TAMs, but not tumor cells, to exert their extraskeletal effects, offering a rationale for use in patients with early disease.

Topics: Animals; Bone Density Conservation Agents; Breast Neoplasms; Calcinosis; Carbocyanines; Diphosphonates; Disease Models, Animal; Female; Humans; Macrophages; Mice; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasms; Phagocytosis; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; Xenograft Model Antitumor Assays

Early detection of colonic polyps can prevent up to 90% of colorectal cancer deaths. Conventional colonoscopy readily detects the majority of premalignant lesions, which exhibit raised morphology. However, lesions that are flat and depressed are often undetected using this method. Therefore, there is a need for molecular-based contrast agents to improve detection rates over conventional colonoscopy. We evaluated a quenched fluorescent activity-based probe (qABP; BMV109) that targets multiple cysteine cathepsins that are overexpressed in intestinal dysplasia in a genetic model of spontaneous intestinal polyp formation and in a chemically induced model of colorectal carcinoma. We found that the qABP selectively targets cysteine cathepsins, resulting in high sensitivity and specificity for intestinal tumors in mice and humans. Additionally, the qABP can be administered by either intravenous injection or by local delivery to the colon, making it a highly valuable tool for improved detection of colorectal lesions using fluorescence-guided colonoscopy.

Topics: Adenomatous Polyposis Coli Protein; Animals; Carbocyanines; Catalytic Domain; Cathepsins; Colonoscopy; Disease Models, Animal; Fluorescent Dyes; Humans; Immunohistochemistry; Intestinal Neoplasms; Male; Mice; Mice, Inbred C57BL; Polyps; Sensitivity and Specificity

A new type of photodynamic therapy (PDT) agents using upconversion nanoparticles (UCNPs) with incorporated photosensitizers as the inner core and an erythrocyte membrane (RM) decorated with dual targeting moieties as the cloak is developed. Owing to the endogenous nature of RM, the RM-coating endows the PDT agents with perfect biocompatibility and stealth ability to escape from the entrapment by the reticulo-endothelial system (RES). More importantly, owing to the unique nature of erythrocyte as an oxygen carrier in the blood, the RM outer layer of the agents unequivocally facilitates the permeation of ground-state molecular oxygen ((3)O2) and the singlet oxygen ((1)O2) as compared to the previously developed PDT agents with other types of coating. Another salient feature of the as-prepared PDT platform is the decoration of RM with dual targeting moieties for selective recognition of cancer cells and mitochondrial targeting, respectively. The synergistic effect of RM coating and dual-targeting of such feature-packed agents are investigated in tumor-bearing mice and the improved PDT therapeutic efficacy is confirmed, which is the first paradigm where RM-coated NIR-triggered nanovectors with programmed delivery ability is applied in PDT of tumor in vivo.

Topics: Animals; Biomimetic Materials; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Erythrocyte Membrane; Infrared Rays; Melanoma; Mice; Microscopy, Confocal; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Singlet Oxygen; Skin Neoplasms; Spectroscopy, Near-Infrared; Survival Rate

Lipopolymer 49, a solid-phase synthesized T-shaped peptide-like oligoamide containing two central oleic acids, 20 aminoethane, and two terminal cysteine units, is identified as very potent and biocompatible small interfering RNA (siRNA) carrier for gene silencing in glioma cells. This carrier is combined with a novel targeting polymer 727, containing a precise sequence of Angiopep 2 targeting peptide, linked with 28 monomer units of ethylene glycol, 40 aminoethane, and two terminal cysteines in siRNA complex formation. Angiopep-polyethylene glycol (PEG)/siRNA polyplexes exhibit good nanoparticle features, effective glioma-targeting siRNA delivery, and intracellular siRNA release, resulting in an outstanding gene downregulation both in glioma cells and upon intravenous delivery in glioma model nude mice without significant biotoxicity. Therefore, this novel siRNA delivery system is expected to be a promising strategy for targeted and safe glioma therapy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; Brain; Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Down-Regulation; Drug Delivery Systems; Electrophoresis, Agar Gel; Endocytosis; Gene Silencing; Gene Transfer Techniques; Glioma; Humans; Mice, Nude; Nanoparticles; Oxidation-Reduction; Peptides; Polyethylene Glycols; Polymerase Chain Reaction; Polymers; RNA, Messenger; RNA, Small Interfering; Transfection

Sepsis causes mitochondrial oxidative injury and swelling. Ethyl pyruvate (EP) is a cytoprotective agent, while aquaporin-8 (AQP8) is a mitochondrial water channel that can induce mitochondrial swelling. We assessed whether EP protects mitochondria during sepsis, and whether AQP8 contributes to the underlying mechanisms. A cecal ligation and puncture (CLP) sepsis model was established in Sprague-Dawley rats, randomized to 3 groups: sham (n=20), CLP (n=59) and CLP+EP (n=51). All rats received postoperative intraperitoneal fluid resuscitation (30 ml/kg); the CLP+EP group also received intraperitoneal EP (100 mg/kg). Survival was assessed at 24 hours. Hepatic mitochondrial ultrastructure was characterized by electron microscopy. The membrane potential of isolated hepatic mitochondria was determined using JC-1 and flow cytometry. Mitochondrial AQP8 expression and cytochrome C (Cyt C) release were measured by Western blotting (values normalized to ß-actin). Survival in the sham, CLP and CLP+EP groups was 100%, 21% and 41%, respectively. Mitochondrial cross-sectional area was smaller in the CLP+EP group than in the CLP group (0.231±0.110 vs. 0.641±0.460 µm(2); P<0.001), with a tendency for a lower form factor (a measure of contour irregularity) in the CLP+EP group. Mitochondrial depolarization by CLP was inhibited by EP. Mitochondrial Cyt C release was higher in the CLP group than in the sham (1.211±0.24 vs. 0.48±0.03) or CLP+EP (0.35±0.39) groups. AQP8 expression was similar between groups, with a trend for lower expression in the CLP+EP group compared with the CLP group. EP improves sepsis outcome by targeting the mitochondrion, possibly through modulation of AQP8 expression.

Topics: Animals; Aquaporins; Benzimidazoles; Carbocyanines; Disease Models, Animal; Humans; Liver; Liver Diseases; Male; Mitochondria, Liver; Mitochondrial Swelling; Pyruvates; Random Allocation; Rats; Rats, Sprague-Dawley; Sepsis

Quantification of tumor necrosis in cancer patients is of diagnostic value as the amount of necrosis is correlated with disease prognosis and it could also be used to predict early efficacy of anti-cancer treatments. In the present study, we identified two near infrared fluorescent (NIRF) carboxylated cyanines, HQ5 and IRDye 800CW (800CW), which possess strong necrosis avidity. In vitro studies showed that both dyes selectively bind to cytoplasmic proteins of dead cells that have lost membrane integrity. Affinity for cytoplasmic proteins was confirmed using quantitative structure activity relations modeling. In vivo results, using NIRF and optoacoustic imaging, confirmed the necrosis avid properties of HQ5 and 800CW in a mouse 4T1 breast cancer tumor model of spontaneous necrosis. Finally, in a mouse EL4 lymphoma tumor model, already 24 h post chemotherapy, a significant increase in 800CW fluorescence intensity was observed in treated compared to untreated tumors. In conclusion, we show, for the first time, that the NIRF carboxylated cyanines HQ5 and 800CW possess strong necrosis avid properties in vitro and in vivo. When translated to the clinic, these dyes may be used for diagnostic or prognostic purposes and for monitoring in vivo tumor response early after the start of treatment.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carbocyanines; Cell Death; Cell Line, Tumor; Disease Models, Animal; Female; Fluorescent Dyes; Humans; Lymphoma; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Confocal; Necrosis; Quantitative Structure-Activity Relationship; Random Allocation

An embryo has the capability to accept allo- or xeno-geneic cells, which probably makes it an ideal candidate for stem cell transplantation of various cerebral cortex abnormalities, such as cortical dysplasia. The aim of this study was to determine hair follicle-associated pluripotent (HAP) stem cells homing into various organs of mother and fetus. Cells were obtained, analyzed for immunophenotypic features, and then labelled with CM-Dil; nestin(+)HAP stem cells or media phosphate-buffered saline (PBS) were intravenously delivered on day 16 of gestation in BALB/c mice, which intraperitoneally received methylazoxymethanol (MAM) one day in advance, and homing was assessed at 24 h after cell injection. Flow cytometry and immunocytochemistry manifested positive expression of nestin in HAP stem cells. For both mother and fetus, brain, lungs, liver, and spleen were the host organs for cell implants. For the brain, the figure was considerably higher in fetus, 4.05 ± 0.5% (p ≤ 0.05 vs. mother). MAM-injected mice had a downward trend for SDF-1α and CXCR4 (p ≤ 0.05 vs. control), but HAP stem cells group showed an upward trend for CXCR4 (p ≤ 0.05 vs. MAM). We conclude the HAP stem cells show homing potential in experimental cortical dysplasia, which may permit these cells to be a target in future work on prenatal therapy of neural disorders.

Topics: Animals; Brain; Carbocyanines; Disease Models, Animal; Female; Fetal Therapies; Flow Cytometry; Fluorescent Dyes; Hair Follicle; Immunohistochemistry; Malformations of Cortical Development; Maternal-Fetal Exchange; Mice, Inbred BALB C; Microscopy, Fluorescence; Nestin; Neurons; Pluripotent Stem Cells; Pregnancy; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction

Microgyria is associated with epilepsy and due to developmental disruption of neuronal migration. However, the role of endogenous subventricular zone-derived neural progenitors (SDNPs) in formation and hyperexcitability has not been fully elucidated. Here, we establish a neonatal cortex freeze-lesion (FL) model, which was considered as a model for focal microgyria, and simultaneously label SDNPs by CM-DiI. Morphological investigation showed that SDNPs migrated into FL and differentiated into neuronal and glia cell types, suggesting the involvement of endogenous SDNPs in the formation of FL-induced microgyria. Patch-clamp recordings in CM-DiI positive (CM-DiI(+)) pyramidal neurons within FL indicated an increase in frequency of spontaneous action potentials, while the resting membrane potential did not differ from the controls. We also found that spontaneous excitatory postsynaptic currents (EPSCs) increased in frequency but not in amplitude compared with controls. The evoked EPSCs showed a significant increase of 10-90% in rise time and decay time in the CM-DiI(+) neurons. Moreover, paired-pulse facilitation was dramatically larger in CM-DiI(+) pyramidal neurons. Western blotting data showed that AMPA and NMDA receptors were increased to some extent in the FL cortex compared with controls, and the NMDA/AMPA ratio of eEPSCs at CM-DiI(+) pyramidal neurons was significantly increased. Taken together, our findings provide novel evidence for the contribution of endogenous SDNPs in the formation and epileptogenicity of FL-induced focal microgyria.

Topics: Action Potentials; Animals; Animals, Newborn; Carbocyanines; Cell Movement; Disease Models, Animal; Excitatory Postsynaptic Potentials; Injections, Intraventricular; Malformations of Cortical Development; Neural Stem Cells; Organ Culture Techniques; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Stem Cell Niche

Vessel painting is a simple, cost-effective way to visualize the vascular architecture of the mouse brain and other organs. DiI is a lipophilic carbocyanine dye that binds to lipid membranes and is commonly used for tract tracing in the brain. After perfusion with PBS to remove the blood, perfusion with a special DiI solution allows direct staining of the vasculature. This step is followed by perfusion fixation and removal of the brain from the skull. Pial vessels can be directly imaged using a standard fluorescent microscope. To acquire images of the whole brain, a montage of images at different focal planes is assembled. Basic cerebral vascular anatomy is reviewed in the context of vessel painting, and examples are presented showing enhanced collateralization in a mouse model of metabolic syndrome. Vessel painting offers a cost-effective and efficient alternative to more complex approaches such as corrosion casting.

Topics: Animals; Carbocyanines; Cerebral Arteries; Cerebral Cortex; Cerebral Veins; Disease Models, Animal; Fluorescent Dyes; Mice; Mice, Inbred C57BL; Staining and Labeling

We sought to investigate the effects of co-grafting neural stem cells (NSCs) with olfactory ensheathing cells (OECs) on neurological behavior in rats subjected to traumatic brain injury (TBI) and explore underlying molecular mechanisms.. TBI was established by percussion device made through a weight drop (50 g) from a 30 cm height. Cultured NSCs and OECs isolated from rats were labeled by Hoechst 33342 (blue) and chloromethyl-benzamidodialkyl carbocyanine (CM-Dil) (red), respectively. Then, NSCs and/or OECs, separately or combined, were transplanted into the area surrounding the injury site. Fourteen days after transplantation, neurological severity score (NSS) were recorded. The brain tissue was harvested and processed for immunocytochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reverse transcription-polymerase chain reaction (RT-PCR).. Significant neurological function improvement was observed in the three transplant groups, compared to the TBI group, and co-transplantation gave rise to the best improvement. Morphological evaluation showed that the number of neurons in cortex from combination implantation was more than for other groups (P <0.05); conversely, the number of apoptotic cells showed a significant decrease by TUNEL staining. Transplanted NSCs and OECs could survive and migrate in the brain, and the number of neurons differentiating from NSCs in the co-transplantation group was significantly greater than in the NSCs group. At the molecular level, the expressions of IL-6 and BAD in the co-graft group were found to be down regulated significantly, when compared to either the NSC or OEC alone groups.. The present study demonstrates for the first time the optimal effects of co-grafting NSCs and OECs as a new strategy for the treatment of TBI via an anti-inflammation mechanism.

Topics: Animals; Apoptosis; Benzimidazoles; Brain Injuries; Carbocyanines; Cell Differentiation; Cell Transplantation; Cells, Cultured; Cytokines; Disease Models, Animal; Female; Neural Stem Cells; Neurologic Examination; Olfactory Bulb; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Schwann Cells

To establish red-green dual-color fluorescence glioma model in nude mice and to explore its practical values.. CM-DiI-stained rat glioma C6 cells (C6-CM- DiI cells) expressing red fluorescence were inoculated into the brain of athymic nude mice expressing green fluorescence protein (NC-C57BL/6J-EGFP). Then the whole-body dual-color fluorescence imaging was detected dynamically. Finally whole brains of the tumor-bearing mice were removed and 5 µm thick serial frozen slices were made. Light microscopy, fluorescence microscopy and confocal laser scanning microscopy were performed to observe the transplanted tumor tissue structure and fluorescent cells.. Tumor mass with red fluorescence increased gradually under continuous in-vivo fluorescence imaging monitoring. Under the fluorescence microscope, cells with red, green and yellow fluorescence were observed in the frozen sections of transplanted tumor tissue and the mutual structural relationship among them could be defined. The tumor cells migration, implantation and cell fusion between transplanted tumor cells and host cells could be observed. It could be distinguished according to the fluorescence, that blood vessels of tumor-origin displayed red fluorescence, blood vessels of host-origin displayed green fluorescence and mosaic blood vessels appeared yellow fluorescence. It was depicted that host innate astrocytes and oligodendrocytes in the microenvironment at the tumor periphery could be activated and dedifferentiated into nestin-positive cells.. In contrast to traditional animal model, the dual-color fluorescence imaging of nude mouse models of glioma possesses enormous advantages in investigating tumor mass in-vivo fluorescence imaging, tumor cells migration and metastasis, tumor angiogenesis and reactive activation of host innate cells in the microenvironment at tumor periphery, thus, has highly practical application value.

Topics: Animals; Astrocytes; Brain Neoplasms; Carbocyanines; Cell Fusion; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Fluorescent Dyes; Glioma; Green Fluorescent Proteins; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Nude; Microscopy, Confocal; Microscopy, Fluorescence; Neoplasm Transplantation; Neovascularization, Pathologic; Nestin; Oligodendroglia; Rats; Red Fluorescent Protein

Hydrogen sulfide (H2S) is an important gaseous signaling agent mediated by many physiological processes and diseases. In order to explore its role in biological signaling, much effort has been focused on developing organic fluorescent probes to image H2S. However, these downconversion H2S probes are impractical for bio-imaging beyond a certain depth because of the short tissue penetration of UV/visible light (as an excitation source). In most circumstance, these probes are also not suitable for long-term assay due to photo-bleaching. Herein, a new design to detect H2S based on the coumarin-hemicyanine (CHC1)-modified upconversion nanophosphors is reported. This inorganic-organic integrated nanoprobe is demonstrated to display a fast response time with a large ratiometric upconversion luminescence (UCL) enhancement, and extraordinary photo-stability. CHC1-UCNPs not only can be used for ratiometric UCL monitoring of pseudo-enzymatic H2S production in living cells, but can also be used to identify the risk of endotoxic shock through ratiometric UCL imaging of tissue and measurement of endogenous H2S levels in plasma. The first ratiometric UCL H2S nanoprobe reported here may be further developed as the next-generation diagnostic tool for the detection of inflammatory-related diseases.

Topics: Animals; Carbocyanines; Coumarins; Disease Models, Animal; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen Sulfide; Inflammation; Lipopolysaccharides; Luminescence; Magnetic Resonance Spectroscopy; Mice; Mice, Nude; Microscopy, Electron, Transmission; Nanoparticles; Nanostructures; Nanotechnology; Shock, Septic; Signal Transduction; Spectrophotometry, Ultraviolet; Spectroscopy, Near-Infrared

The aquaporin family comprises a large family of integral membrane proteins that enable the movement of water and other small, neutral solutes across plasma membranes. Although function and mechanism of aquaporins in central nervous system injury have been reported, the pathophysiologic role of aquaporin 1 (AQP1) in peripheral nerve has not been extensively documented. In the present study, we aimed to study the temporal and spatial distribution of AQP1 in spinal cord and dorsal root ganglia after sciatic nerve injury.. Forty-eight adult female mice were randomly divided into four groups (intact controls, sham operated, cut injury, and crush injury). Animals receiving cut or crush injuries were sacrificed at the 2nd, 24th, and 48th postoperative hours. Spinal cord samples at the level of lumbosacral intumescences and corresponding dorsal root ganglia on the experimental and contralateral side were dissected free and proceeded to AQP1 immunohistochemistry.. Our quantitative estimations revealed that a sharp increase in AQP1 immunoreactivity at the 24th postoperative hour was observed. This sharp increase was no more evident at 48 h after sciatic nerve injury. Identical peak was observed after both cut and crush injuries.. We demonstrated that there was a temporal relationship with an increased expression of AQP1 following injury sustained to the sciatic nerve that was significantly observed in dorsal root ganglia and spinal cord. Those expressions were also subsided over time.

Topics: Animals; Aquaporin 1; Carbocyanines; Disease Models, Animal; Female; Ganglia, Spinal; Gene Expression Regulation; Image Processing, Computer-Assisted; Mice; Mice, Inbred BALB C; Sciatic Neuropathy; Spinal Cord; Time Factors

The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo.

Topics: Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Membrane; Cell Survival; Cell Tracking; Disease Models, Animal; Female; Immunohistochemistry; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-2; Ionomycin; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Spectroscopy, Near-Infrared; T-Lymphocytes; Transplantation, Homologous

The helix-loop-helix transcription factor Olig2 is essential for lineage determination of oligodendrocytes. Differentiation of stem cells into oligodendrocytes and transplanting them is a novel strategy for the repair of different demyelination diseases. Dental pulp stem cells (DPSCs) are of great interest in regenerative medicine due to their potential for repairing damaged tissues. In this study, DPSCs were isolated from human third molars and transfected with the human Olig2 gene as a differentiation inducer for the oligodendrogenic pathway. Following the differentiation procedure, the expression of Sox2, NG2, PDGFRα, Nestin, MBP, Olig2, Oct4, glial fibrillary acidic protein and A2B5 as stage-specific markers was studied by real-time RT-qPCR, immunocytochemistry and Western blot analysis. The cells were transplanted into a mouse model of local sciatic damage by lysolecithin as a model for demyelination. Oligodendrocyte progenitor cells (OPCs) actively remyelinated and recovered the lysolecithin-induced damages in the sciatic nerve as revealed by treadmill exercise, the von Frey filament test and hind paw withdrawal in response to a thermal stimulus. Recovery of behavioral reflexes occurred 2-6 weeks after OPC transplantation. The results demonstrate that the expression of Olig2 in DPSCs reduces the expression of stem cell markers and induces the development of oligodendrocyte progenitors as revealed by the emergence of oligodendrocyte markers. DPSCs could be programmed into oligodendrocyte progenitors and considered as a simple and valuable source for the cell therapy of neurodegenerative diseases.

Topics: Adult; Animals; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Carbocyanines; Cell Differentiation; Cell Lineage; Cell Separation; Cell Shape; Demyelinating Diseases; Dental Pulp; Disease Models, Animal; Humans; Immunohistochemistry; Lysophosphatidylcholines; Mice; Nerve Tissue Proteins; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Real-Time Polymerase Chain Reaction; RNA, Messenger; Sciatic Nerve; Stem Cell Transplantation; Stem Cells; Touch; Young Adult

Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis.. Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent "smart" probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging.. We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio.. We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition.

Topics: Acute Disease; Animals; Carbocyanines; Disease Models, Animal; Endopeptidases; Enzyme Activation; Female; Fluorescent Dyes; Optical Imaging; Pancreatitis; Protease Inhibitors; Rats; Trypsin; Trypsin Inhibitors

Progressive dynamic, relative quantitative changes were compared in glycans associated with retinal proteins of wild type (wt) and retinal degeneration 1 (rd1) mice during neonatal development and degeneration of retinae.. Proteins extracted from retinae of postnatal days 2 (PN2), PN7, and PN14 wt and rd1 mice were labeled with Cy3-fluorescent dye. Glycome of these proteins was quantified relatively by lectin microarray technique. Net fluorescence emitted by individual complexes formed between 45 lectins and Cy3-labeled proteins was measured by evanescent-field fluorescence-assisted microarray reader.. GlcNAcβ1-oligomer and high-mannose/Manα1-6Man were major glycans associated with the proteins of PN2, PN7, and PN14 wt and rd1 mice retinae. Gal/GalNAc/Man3-core-bi-/tri-antennary-complex, Sia2-3Galβ1-4GlcNAc, and high-mannose glycans were conjugated mainly to proteins from PN7 rd1 and PN14 wt retinae, respectively. With increasing neonatal age, mannosylated, GlcNAcβ, and sialylated (minor component) glycans were increased, and fucosylated GlcNAc/Galβ glycans were decreased significantly in wt retinal proteins. This trend was less evident in PN14 rd1 retinal proteins. Mouse retina was almost devoid of Siaα2-6 (except WGA bound Sia), Fucα1-2, and Gal/GalNAc-containing glycans. STL reacting GlcNAc oligomers were high in PN2 rd1 retinae.. Quantitative dynamic, relative variation in high-mannose and GlcNAc glycans, Siaα2-3Galβ1-4GlcNAc associated with proteins from PN2, PN7, and PN14 wt and rd1 mice retinae suggested that these glycans participate in retinal development and degeneration, and may be used as markers for retinal electrophysiologic integrity during transplantation/therapy studies; Siaα2-3Galβ1-4GlcNAc-specific Agrocybe cylindracea lectin and other lectins may be used to enrich/purify retinal ribbon synapse glycoproteins and other glycoproteins including rhodopsin. Further investigations are required.

Topics: Animals; Animals, Newborn; Carbocyanines; Disease Models, Animal; Eye Proteins; Fluorescent Dyes; Glycomics; Glycoproteins; Lectins; Mice; Polysaccharides; Protein Array Analysis; Retina; Retinitis Pigmentosa

Alterations of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial respiration are possible triggers of pulmonary vascular remodeling in pulmonary hypertension (PH). We investigated the role of MMP in PH and hypothesized that deletion of the mitochondrial uncoupling protein 2 (UCP2) increases MMP, thus promoting pulmonary vascular remodeling and PH. MMP was measured by JC-1 in isolated pulmonary arterial smooth muscle cells (PASMCs) of patients with PH and animals with PH induced by exposure to monocrotaline (MCT) or chronic hypoxia. PH was quantified in vivo in UCP2-deficient (UCP2(-/-)) mice by hemodynamics, morphometry, and echocardiography. ROS were measured by electron spin resonance spectroscopy and proliferation by thymidine incorporation. Mitochondrial respiration was investigated by high-resolution respirometry. MMP was increased in PASMCs of patients and in animal models of PH. UCP2(-/-) mice exhibited pulmonary vascular remodeling and mild PH compared with wild-type (WT) mice. PASMCs of UCP2(-/-) mice showed increased proliferation, MMP, and ROS release. Increased proliferation of UCP2(-/-) PASMCs could be attenuated by ROS inhibitors and inhibited by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which decreased MMP to the level of WT mice. Mitochondrial respiration was altered in PASMCs from MCT rats and PASMCs exposed to hypoxia but not in isolated pulmonary mitochondria of UCP2(-/-) mice or PASMCs after treatment with small interfering RNA for UCP2. Our data suggest that increased MMP causes vascular remodeling in UCP2(-/-) mice partially via increased ROS. In chronic hypoxia and MCT-induced PH, additional pathomechanisms such as decreased respiration may play a role.

Topics: Animals; Benzimidazoles; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Disease Models, Animal; Fluorescent Dyes; Free Radical Scavengers; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Hypoxia; Ion Channels; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitochondrial Proteins; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Primary Cell Culture; Pulmonary Artery; Rats; Reactive Oxygen Species; RNA, Small Interfering; Uncoupling Protein 2

The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.

Topics: Algorithms; Alzheimer Disease; Animals; Biomarkers; Carbocyanines; Diagnosis, Differential; Disease Models, Animal; Electrophoresis, Gel, Two-Dimensional; Female; Mass Spectrometry; Mice; Mice, Inbred C57BL; Mice, Transgenic; Principal Component Analysis; Proteins; Proteome; Reproducibility of Results; Scrapie

Bone marrow stromal cells (BMSC) have been successfully employed for movement deficit recovery in spinal cord injury (SCI) rat models. One of the unsettled problems in cell transplantation is the relative high proportion of cell death, specifically after neural differentiation. According to our previous studies, p75 receptor, known as the death receptor, is only expressed in BMSC in a time window of 6-12 hours following neural induction. Moreover, we have recently reported a decreased level of apoptosis in p75-suppressed BMSC in vitro. Therefore, our objective in this research was to explore the functional effects of transplanting p75:siRNA expressing BMSC in SCI rats.. Laminectomy was performed at L1 vertebra level to expose spinal cord for contusion using weight-drop method. PBS-treated SCI rats (group one) were used as negative controls, in which cavitations were observed 10 weeks after SCI. pRNA-U6.1/Hygro- (group two, as a mock) and pRNA-U6.1/Hygro-p75 shRNA- (group three) transfected BMSC were labeled with a fluorescent dye, CM-DiI, and grafted into the lesion site 7 days after surgery. The Basso-Beattie-Bresnehan locomotor rating scale was performed weekly for 10 weeks.. There was a significant difference (P≤0.05) between all groups of treated rats regarding functional recovery. Specifically, the discrepancy among p75 siRNA and mock-transfected BMSC was statistically significant. P75 siRNA BMSC also revealed a higher level of in vivo survival compared to the mock BMSC.. Our data suggest that genetically modified BMSC that express p75:siRNA could be a more suitable source of cells for treatment of SCI.

Topics: Animals; Behavior, Animal; Carbocyanines; Cell Lineage; Cell Movement; Cell Survival; Disease Models, Animal; Female; Fluorescence; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Motor Activity; Rats; Rats, Sprague-Dawley; Receptor, Nerve Growth Factor; Recovery of Function; Spinal Cord Injuries; Staining and Labeling; Transfection

Tumors are characterized by a high degree of diversity and heterogeneity in receptor expression. Monoclonal antibodies (mAbs) are an established therapeutic method of targeting cell surface receptors. However, high affinity antibodies targeting highly expressed receptors are often prevented from distributing evenly throughout the tumor due to the "binding site barrier" whereby antibody is trapped peripherally before it can reach deeper into the tumor that leads inhomogeneous micro-distribution. When employing armed antibodies it is important that the toxin (in this case, phototoxin) be distributed evenly to more effectively treat the cancer. By adding an additional antibody conjugate, targeting a secondary, unsaturated receptor with lower expression, a more uniform distribution of the phototoxin can be achieved. In this study, panitumumab (Pan) and basiliximab (Bas) were conjugated with the phthalocyanine dye, IRDye700DX (IR700). Upon exposure to near infrared light, these armed antibodies produce rapid cell death only when bound to their respective receptors, a treatment termed photo-immunotherapy (PIT). ATAC4 cells which demonstrate high expression of human epidermal growth factor receptor (EGFR) and low expression of interleukin-2 receptor-alpha (CD25) were treated by PIT using a cocktail of Pan-IR700 and Bas-IR700. An in vivo study showed that the cocktail Pan-Bas-IR700 resulted in significantly reduced tumor growth and prolonged survival in ATAC4 tumor-bearing mice compared with either Pan-IR700 or Bas-IR700 alone. In conclusion, a cocktail injection of two different antibody-IR700 conjugates created a more homogeneous microdistribution of antibody-conjugates resulting in enhanced therapeutic effects after PIT, compared to the use of either antibody-IR700 conjugate.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Basiliximab; Carbocyanines; Cell Death; Cell Line, Tumor; Disease Models, Animal; Humans; Immunotherapy; Immunotoxins; Infrared Rays; Mice; Neoplasms; Organophosphorus Compounds; Panitumumab; Phototherapy; Recombinant Fusion Proteins; Treatment Outcome

Carcinomas make up the majority of cancers. Their accurate and specific diagnoses are of great significance for the improvement of patients' curability.. In this paper, we report an effectual example of the in vivo fluorescence molecular imaging of carcinomas with extremely high specificity based on whole cell-SELEX aptamers. Firstly, S6, an aptamer against A549 lung carcinoma cells, was adopted and labeled with Cy5 to serve as a molecular imaging probe. Flow cytometry assays revealed that Cy5-S6 could not only specifically label in vitro cultured A549 cells in buffer, but also successfully achieve the detection of ex vivo cultured target cells in serum. When applied to in vivo imaging, Cy5-S6 was demonstrated to possess high specificity in identifying A549 carcinoma through a systematic comparison investigation. Particularly, after Cy5-S6 was intravenously injected into nude mice which were simultaneously grafted with A549 lung carcinoma and Tca8113 tongue carcinoma, a much longer retention time of Cy5-S6 in A549 tumor was observed and a clear targeted cancer imaging result was presented. On this basis, to further promote the application to imaging other carcinomas, LS2 and ZY8, which are two aptamers selected by our group against Bel-7404 and SMMC-7721 liver carcinoma cells respectively, were tested in a similar way, both in vitro and in vivo. Results showed that these aptamers were even effective in differentiating liver carcinomas of different subtypes in the same body.. This work might greatly advance the application of whole cell-SELEX aptamers to carcinomas-related in vivo researches.

Topics: Animals; Aptamers, Nucleotide; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Fluorescent Dyes; Heterografts; Humans; Male; Mice; Mice, Nude; Molecular Imaging; Neoplasms; SELEX Aptamer Technique; Time-Lapse Imaging

Mesenchymal stem cells (MSCs) have been shown to improve cardiac electrophysiology when administered in the setting of acute myocardial infarction. However, the electrophysiological phenotype of MSCs in situ is not clear. We hypothesize that MSCs delivered intramyocardially to cryoinjured myocardium can engraft, but will not actively generate, action potentials. Cryoinjury-induced scar was created on the left ventricular epicardial surface of adult rat hearts. Within 30 min, hearts were injected with saline (sham, n = 11) or bone marrow-derived MSCs (2 × 10(6)) labeled with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine percholate (DiI; n = 16). At 3 wk, optical mapping and cell isolation were used to measure optical action potentials and calcium transients, respectively. Histological analysis confirmed subepicardial scar thickness and the presence of DiI-positive cells that express connexin-43. Optical action potential amplitude within the scar at MSC-positive sites (53.8 ± 14.3%) was larger compared with sites devoid of MSCs (35.3 ± 14.2%, P < 0.05) and sites within the scar of shams (33.5 ± 6.9%, P < 0.05). Evidence of simultaneous action potential upstroke, the loss of action potential activity following ablation of adjacent viable myocardium, and no rapid calcium transient response in isolated DiI+ cells suggest that the electrophysiological influence of engrafted MSCs is electrotonic. MSCs can engraft when directly injected into a cryoinjury and are associated with evidence of action potential activity. However, our results suggest that this activity is not due to generation of action potentials, but rather passive influence coupled from neighboring viable myocardium.

Topics: Action Potentials; Animals; Calcium Signaling; Carbocyanines; Cell Communication; Connexin 43; Cryosurgery; Disease Models, Animal; Fluorescent Dyes; Heart Conduction System; Heart Ventricles; Male; Mesenchymal Stem Cell Transplantation; Myocardial Infarction; Myocardium; Rats; Rats, Inbred Lew; Regeneration; Time Factors; Voltage-Sensitive Dye Imaging

To study the effectiveness of a peptide targeted nanoglobular Gd-DOTA complexes for MR molecular imaging of prostate cancer in a mouse orthotopic PC-3 prostate cancer model.. A CLT1 (CGLIIQKNEC) peptide-targeted generation 2 nanoglobular Gd-DOTA monoamide conjugate [CLT1-G2-(Gd-DOTA)] was used for imaging fibrin-fibronectin complexes in prostate tumor using a non-specific peptide KAREC modified conjugate, KAREC-G2-(Gd-DOTA) as a control. Cy5 conjugates of CLT1 and KAREC were synthesized for binding studies. Orthotopic PC-3 prostate tumors were established in the prostate of athymic male nude mice. MRI study was performed on a Bruker 7T small animal MRI system.. CLT1 peptide showed specific binding in the prostate tumor with no binding in normal tissues. The control peptide had little binding in normal and tumor tissues. CLT1-G2-(Gd-DOTA) resulted in stronger contrast enhancement in tumor tissue than KAREC-G2-(Gd-DOTA). CLT1-G2-(Gd-DOTA) generated ~100% increase in contrast-to-noise ratio (CNR) in the tumor compared to precontrast CNR at 1 min post-injection, while KAREC-G2-(Gd-DOTA) resulted in 8% increase.. CLT1-G2-(Gd-DOTA) is a promising molecular MRI contrast agent for fibrin-fibronectin complexes in tumor stroma. It has potential for diagnosis and assessing prognosis of malignant tumors with MRI.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Contrast Media; Disease Models, Animal; Fibrin; Fibronectins; Fluorescent Dyes; Heterocyclic Compounds; Humans; Magnetic Resonance Imaging; Male; Mice; Mice, Nude; Molecular Imaging; Nanoparticles; Organometallic Compounds; Peptides, Cyclic; Prostatic Neoplasms; Transplantation, Heterologous

Beta-amyloid (Aβ) peptides are considered to play a major role in the pathogenesis of Alzheimer's disease (AD) and molecules that can prevent pathways of Aβ toxicity may be potential therapeutic agents for treatment of AD. We have previously reported that NK-4, a cyanine photosensitizing dye, displays neurotrophic and antioxidant activities. In this study, we report the effects of NK-4 on the toxicity of Aβ and on cognitive function and Aβ concentration in a transgenic mouse model of AD (Tg2576). In vitro, NK-4 effectively protected neuronal cells from toxicity induced by Aβ. In addition, it displayed profound inhibitory activities on Aβ fibril formation. In vivo, Tg2576 mice received an intraperitoneal injection at 100 or 500 µg/kg of NK-4 once a day, five times a week for 9 months. Administration of NK-4 to the mice attenuated impairment of recognition memory, associative memory, and learning ability, as assessed by a novel object recognition test, a passive avoidance test, and a water maze test, respectively. NK-4 decreased the brain Aβ concentration while increasing the plasma amyloid level in a dose-dependent manner. NK-4 also improved memory impairments of ICR mice induced by direct intracerebroventricular administration of Aβ. These lines of evidence suggest that NK-4 may affect multiple pathways of amyloid pathogenesis and could be useful for treatment of AD.

Topics: Alzheimer Disease; Animals; Carbocyanines; Central Nervous System Agents; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Male; Maze Learning; Mice; Mice, Inbred ICR; Mice, Transgenic; PC12 Cells; Photosensitizing Agents; Quinolines; Rats; Recognition, Psychology; Swimming

Glioblastoma (GBM), the most common malignant brain tumor, is among the most lethal neoplasms, with a median survival of approximately 1 year. Prognosis is poor since GBMs possess a strong migratory and highly invasive potential, making complete surgical resection impossible. Reduced expression of carboxypeptidase E (CPE), a neuropeptide-processing enzyme, in a cell death-resistant glioma cell line and lower CPE expression levels in the cohort of GBM samples of The Cancer Genome Atlas compared to normal brain control specimens prompted us to analyze the function of CPE as a putative tumor suppressor gene. In our samples, CPE was also reduced in GBM compared to normal brain with the strongest loss in cells surrounding hypoxic tumor areas as well as in most glioma cell lines and primary glioma cells. In our cohort of glioma patients, loss of CPE predominantly occurred in glioblastomas and was associated with worse prognosis. In glioma cells, CPE overexpression was significantly reduced, whereas knockdown or inhibition enhanced glioma cell migration and invasion. The decreased migratory potential following CPE overexpression was paralleled by altered cellular morphology, promoting a transition to focal adhesions and associated stress fibers. In contrast to the decreased migration, high CPE levels were associated with higher proliferative rates. As microenvironmental regulation cues, we identified CPE as being downregulated upon hypoxia or glucose deprivation. Our findings indicate an oxygen- and nutrition-dependent anti-migratory, but pro-proliferative role of CPE in gliomas with prognostic impact for patient survival, thereby contributing to the understanding of the "go or grow" hypothesis in gliomas.

Topics: Animals; Brain Neoplasms; Carbocyanines; Carboxypeptidase H; Cell Adhesion; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioma; Glucose; Humans; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; RNA, Messenger; RNA, Small Interfering; Stress, Physiological; Succinates; Survival Analysis; Time Factors

Experiments in rodents revealed neuropeptide S (NPS) to constitute a potential novel treatment option for anxiety diseases such as panic and post-traumatic stress disorder. However, both its cerebral target sites and the molecular underpinnings of NPS-mediated effects still remain elusive. By administration of fluorophore-conjugated NPS, we pinpointed NPS target neurons in distinct regions throughout the entire brain. We demonstrated their functional relevance in the hippocampus. In the CA1 region, NPS modulates synaptic transmission and plasticity. NPS is taken up into NPS receptor-expressing neurons by internalization of the receptor-ligand complex as we confirmed by subsequent cell culture studies. Furthermore, we tracked internalization of intranasally applied NPS at the single-neuron level and additionally demonstrate that it is delivered into the mouse brain without losing its anxiolytic properties. Finally, we show that NPS differentially modulates the expression of proteins of the glutamatergic system involved inter alia in synaptic plasticity. These results not only enlighten the path of NPS in the brain, but also establish a non-invasive method for NPS administration in mice, thus strongly encouraging translation into a novel therapeutic approach for pathological anxiety in humans.

Topics: Analysis of Variance; Animals; Anti-Anxiety Agents; Anxiety; CA1 Region, Hippocampal; Carbocyanines; Cell Line, Transformed; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Electrophysiology; Evoked Potentials; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Humans; In Vitro Techniques; Intravitreal Injections; Male; Maze Learning; Mice; Mice, Inbred C57BL; Neurofilament Proteins; Neurons; Neuropeptides; Oxazolidinones; Protein Transport; Pyrazines; Receptors, AMPA; Receptors, Neuropeptide; Rhodamines; RNA, Messenger; Synapsins; Transfection

The purpose of this study was to use a near-infrared (NIR) fluorescent cyclic His-Try-Gly-Phe peptide to characterize and image the expressions of matrix metalloproteinases (MMPs), which are correlated with cancer promotion, in an inflammation-induced colorectal cancer (ICRC) model. We explored the relationship between the development of colon cancer and the expression of MMPs at the same colonic sites in ICRC models. To develop ICRC models, mice were administered a single intraperitoneal dose (10 mg/kg) of azoxymethane (AOM) and exposed orally to 2% dextran sodium sulfate (DSS) for one week. MMP-2 expression and β-catenin activation in colonic lesions were characterized by immunohistochemical (IHC) staining. After being treated with inducers for some time, cancerous lesions were found to express high β-catenin and MMP-2. The profiles of MMP expression were correlated with β-catenin activation in the colonic lesions. c(KAHWGFTLD)NH(2) (C6) peptide was prepared by standard Fmoc peptide synthesis to target MMPs. Molecular weight of Cy5.5-C6 was 1,954.78 g/mol (calculated MW = 1955.23 g/mol). The in vitro characterization of Cy5.5-C6 showed MMP binding specificity in a cell experiment. In vivo NIRF imaging showed high accumulation of Cy5.5-C6 in tumors with associated expression of MMP-2 in colonic lesions after intravenous injection. The MMP-2 specificity of Cy5.5-C6 was confirmed by successful inhibition of probe uptake in the tumor due to the presence of excess C6 peptide. The use of Cy5.5-C6 to target MMP-2 has the potential to be developed into an effective molecular imaging agent to monitor ICRC progress.

Topics: Animals; Azoxymethane; beta Catenin; Blotting, Western; Carbocyanines; Carcinogens; Colonic Neoplasms; Dextran Sulfate; Diagnostic Imaging; Disease Models, Animal; Disease Progression; Immunoenzyme Techniques; Inflammation; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Peptide Fragments

To investigate a dual labeling technique, which would enable real-time monitoring of transplanted embryonic stem cell (ESC) kinetics, as well as long-term tracking.. Liver damage was induced in C57/BL6 male mice (n = 40) by acetaminophen (APAP) 300 mg/kg administered intraperitoneally. Green fluorescence protein (GFP) positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) immediately before transplantation into the spleen. Each of the animals in the cell therapy group (n = 20) received 5 × 10(6) ESCs 4 h following treatment with APAP. The control group (n = 20) received the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS Lumina-2 at 30 min post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohistochemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine aminotransferase (ALT) was measured as an indication of liver damage.. DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradually moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imaging, and confirmed that the highest photon emission was in the liver (P < 0.0001). At 2 wk, the DiRsignal was no longer detectable in vivo; however, immunohistochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP +ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP +ve cells incorporated within the sinusoidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP +ve cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly (P = 0.016) lower and albumin (P = 0.009) was significantly higher when compared with the control group at the 2 wk time-point; however there was no difference in mortality between the two groups.. Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribution, survival and engraftment of transplanted cells, and could be used for cell therapy models.

Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Carbocyanines; Cells, Cultured; Disease Models, Animal; Embryonic Stem Cells; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Liver Failure, Acute; Liver Function Tests; Male; Mice; Mice, Inbred C57BL; Staining and Labeling; Stem Cell Transplantation

The early antecedents of cerebral palsy (CP) are unknown but are suspected to be due to hypoxia-ischemia (H-I). In our rabbit model of CP, the MRI biomarker, apparent diffusion coefficient (ADC) on diffusion-weighted imaging, predicted which fetuses will develop postnatal hypertonia. Surviving H-I fetuses experience reperfusion-reoxygenation but a subpopulation manifested a continued decline of ADC during early reperfusion-reoxygenation, which possibly represented greater brain injury (RepReOx). We hypothesized that oxidative stress in reperfusion-reoxygenation is a critical trigger for postnatal hypertonia. We investigated whether RepReOx predicted postnatal neurobehavior, indicated oxidative stress, and whether targeting antioxidants at RepReOx ameliorated motor deficits, which included testing of a new superoxide dismutase mimic (MnTnHex-2-PyP). Rabbit dams, 79% gestation (E25), were subjected to 40 min uterine ischemia. Fetal brain ADC was followed during H-I, immediate reperfusion-reoxygenation, and 4-72 h after H-I. Endpoints were postnatal neurological outcome at E32, ADC at end of H-I, ADC nadir during H-I and reperfusion-reoxygenation, and area under ADC curve during the first 20 min of reperfusion-reoxygenation. Antioxidants targeting RepReOx were administered before and/or after uterine ischemia. The new MRI-ADC biomarker for RepReOx improved prediction of postnatal hypertonia. Greater superoxide production, mitochondrial injury, and oligodendroglial loss occurred in fetal brains exhibiting RepReOx than in those without. The antioxidants, MnTnHex-2-PyP and Ascorbate and Trolox combination, significantly decreased postnatal motor deficits and extent of RepReOx. The etiological link between early injury and later motor deficits can thus be investigated by MRI, and allows us to distinguish between critical oxidative stress that causes motor deficits and noncritical oxidative stress that does not.

Topics: Age Factors; Animals; Animals, Newborn; Antioxidants; Ascorbic Acid; Benzimidazoles; Blood Flow Velocity; Brain; Brain Mapping; Carbocyanines; Chromans; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Embryo, Mammalian; Female; Flow Cytometry; Hypoxia-Ischemia, Brain; Ionophores; Laser-Doppler Flowmetry; Membrane Potential, Mitochondrial; Metalloporphyrins; Microvessels; Mitochondria; Movement Disorders; Muscle Hypertonia; O Antigens; Pregnancy; Rabbits; Reperfusion Injury; Superoxides; Time Factors; Valinomycin

Among the classical collagenases, matrix metalloproteinase-13 (called MMP-13, collagenase-3) is one of the most important components for cartilage destruction of osteoarthritis (OA) developments. Despite many efforts, the detection methods of MMP-13 activity have been met with limited success in vivo, in part, due to the low sensitivity and low selectivity by homology of MMP family. Previously, we demonstrated the use of strongly dark-quenched fluorogenic probe allowed for the visual detection of MMP-13 in vitro and in OA-induced rat models. In this study, we described the optimization of MMP-13 fluorogenic probe for OA detection in vivo. Three candidate probes demonstrated recovered fluorescent intensity proportional with MMP-13 concentrations, respectively; however, Probe 2 exhibited both high signal amplification and selective recognition for MMP-13, not MMP-2 and MMP-9 in vitro. When Probe 2 was applied to OA-induced rat models, clear visualization of MMP-13 activity in OA-induced cartilage was obtained. Optimized MMP-13 fluorogenic probe can be applied to detect and image OA and have potential for evaluating the in vivo efficacy of MMP-13 inhibitors which are being tested for therapeutic treatment of OA.

Topics: Animals; Carbocyanines; Disease Models, Animal; Fluorescent Dyes; Humans; Kinetics; Matrix Metalloproteinase 13; Molecular Imaging; Molecular Probes; Osteoarthritis; Peptides; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Spectroscopy, Near-Infrared

Myelination is one of the fundamental biological processes in the development of vertebrate nervous system. Disturbance of myelination is found to be associated with progression in many neurological diseases such as multiple sclerosis. Tremendous efforts have been made to develop novel therapeutic agents that prevent demyelination and/or promote remyelination. These efforts need to be accompanied by the development of imaging tools that permit direct quantification of myelination in vivo. In this work, we describe a novel near-infrared fluorescence imaging technique that is capable of direct quantification of myelination in vivo. This technique is developed based on a near-infrared fluorescent probe, 3,3'-diethylthiatricarbocyanine iodide (DBT) that readily enters the brain and specifically binds to myelinated fibers. In vivo imaging studies were first conducted in two animal models of hypermyelination and hypomyelination followed by longitudinal studies in the cuprizone-induced demyelination/remyelination mouse model. Quantitative analysis suggests that DBT is a sensitive and specific imaging probe of myelination, which complements other current myelin-imaging modalities and is of low cost.

Topics: Analysis of Variance; Animals; Benzothiazoles; Carbocyanines; Corpus Callosum; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Longitudinal Studies; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Mice, Transgenic; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Permeability; Protein Binding; Proto-Oncogene Proteins c-akt; Spectroscopy, Near-Infrared; Time Factors

Neurotrophic factors may be future therapeutic agents for neurodegenerative disease. In the screening of biologically active molecules for neurotrophic potency, we found that a photosensitizing cyanine dye, NK-4, had remarkable neurotrophic activities and was a potent radical scavenger.. In this study, we evaluated the effect of NK-4 on the protection of neurons against oxidative damage and investigated the associated intracellular signaling pathways. Subsequently, we evaluated the effect of NK-4 in an animal model of neurodegeneration. In vitro, NK-4 showed dose-dependent protection of PC12 cells from toxicity induced by oxidative stress caused by hydrogen peroxide (H(2)O(2)) or 6-hydroxydopamine (6-OHDA). Comparison of extracellular signal-regulated kinase signaling pathways between treatment with NK-4 and nerve growth factor (NGF) using K252a, an inhibitor of the NGF receptor TrkA, revealed that NK-4 activity occurs independently of NGF receptors. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the protective effect of NK-4, and NK-4 caused activation of Akt/protein kinase B, a downstream effector of PI3K. These results suggest that the neuroprotective effects of NK-4 are mediated by the PI3K-Akt signaling pathway. NK-4 treatment also attenuated stress-induced activation of SAPK/JNK, which suggests that NK-4 activates a survival signaling pathway and inhibits stress-activated apoptotic pathways independently of the TrkA receptor in neuronal cells. In vivo, administration of NK-4 improved motor coordination in genetic ataxic hamsters, as assessed by rota-rod testing. Histological analysis showed that cerebellar atrophy was significantly attenuated by NK-4 treatment. Notably, the Purkinje cell count in the treated group was threefold higher than that in the vehicle group.. These results suggest that NK-4 is a potential agent for therapy for neurodegenerative disorders based on the activation of survival signaling pathways.

Topics: Animals; Atrophy; Carbocyanines; Cell Proliferation; Cerebellar Ataxia; Coloring Agents; Cricetinae; Disease Models, Animal; Female; Intracellular Space; JNK Mitogen-Activated Protein Kinases; Male; Mutation; Neurites; Neurodegenerative Diseases; Neuroprotective Agents; Neurotoxins; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Psychomotor Performance; Purkinje Cells; Rats; Signal Transduction

To clarify whether use of angiogenic vessel-homing peptide, Ala-Pro-Arg-Pro-Gly (APRPG)-modified liposomes encapsulating 3-([2,4-dimethylpyrrhol-5-yl] methylidenyl)-indolin-2-one (SU5416), an angiogenesis inhibitor, can inhibit the development of experimental choroidal neovascularization (CNV) in rats.. Liposomes were prepared using the thin-film hydration method. To set up the rat experimental CNV model, intense fundus laser photocoagulation at 6 spots per eye was performed on pigmented rats. After photocoagulation, the rats were divided into 4 groups (6 rats in each group): an APRPG-liposomal SU5416 treatment group and control groups treated with a balanced salt solution, APRPG liposomes, or soluble SU5416. Each rat received a single intravitreal injection immediately after the injury. One week or 2 weeks after laser injury, the extent of CNV was evaluated by perfusion with high-molecular-weight fluorescein isothiocyanate-dextran.. Two weeks after injection, the CNV area was significantly (P < .05) smaller in the APRPG-liposomal SU5416-treated group compared with the CNV area in the balanced salt solution-and APRPG liposome-treated groups.. Liposomes modified by APRPG and encapsulating SU5416 constitute a potential drug formulation for CNV treatment that would require only a single intravitreal injection.. This liposomal delivery may enable the sustained release of small molecules and be a new treatment modality for CNV.

Topics: Angiogenesis Inhibitors; Animals; Carbocyanines; Choroidal Neovascularization; Dextrans; Disease Models, Animal; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Indoles; Intravitreal Injections; Liposomes; Male; Oligopeptides; Protein-Tyrosine Kinases; Pyrroles; Rats; Rats, Inbred BN

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34(-/-) mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34(-/-) mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34(-/-) mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.

Topics: Animals; Antigens, CD34; Capillary Permeability; Carbocyanines; Cell Proliferation; Disease Models, Animal; Hematopoietic System; Lymphocytes, Tumor-Infiltrating; Mast Cells; Melanoma, Experimental; Mice; Models, Biological; Neoplasm Metastasis; Neoplasm Staging; Subcutaneous Tissue; Time Factors; Tumor Microenvironment

Apolipoprotein A-I (ApoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum, and participates in the reverse transport of cholesterol from tissues to the liver for excretion. The natural HDL tropism to the liver and cancer cells has been used extensively to target encapsulated drugs. The alteration of the plasmatic isoforms of ApoA-I is a hallmark of chronic hepatitis and hepatocarcinoma in mice and humans. Woodchucks infected with the woodchuck hepatitis virus (WHV) represent the best animal model for the study of chronic viral hepatitis B and viral induced hepatocarcinoma (HCC). WHV-infected woodchuck represents a clinically relevant animal model under which new treatment strategies can be evaluated and optimized. Therapeutic efficacy in this model is likely to be translated into a successful therapy for patients infected with HBV. The present study describes, for the first time, the cloning and characterization of woodchuck ApoA-I. The open reading frame (ORF) of the woodchuck ApoA-I is 795 bp long, coding for 264 amino acids. Unexpectedly, phylogenetic analysis revealed that the closest sequences are those of human and macaque. Woodchuck HDLs were isolated successfully from sera by density gradient ultracentrifugation. A commercial antibody that recognized the woodchuck ApoA-I was also identified. Finally, taking advantage of the techniques and tools developed in this study, two potential applications of woodchuck HDLs are illustrated: drug delivery to a woodchuck hepatocarcinoma cell line and the use of isoelectrofocusing to identify ApoA-I isoforms.

Topics: Animals; Apolipoprotein A-I; Base Sequence; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cloning, Molecular; Disease Models, Animal; Drug Delivery Systems; Flow Cytometry; Hepatitis B Virus, Woodchuck; Hepatitis B, Chronic; Humans; Liver; Liver Neoplasms; Marmota; Mice; Molecular Sequence Data; Molecular Targeted Therapy; Protein Isoforms; Transfection; Virus Replication

Impairment of synaptic plasticity underlies memory dysfunction in Alzheimer's disease (AD). Molecules involved in this plasticity such as PSD-95, a major postsynaptic scaffold protein at excitatory synapses, may play an important role in AD pathogenesis. We examined the distribution of PSD-95 in transgenic mice of amyloidopathy (5XFAD) and tauopathy (JNPL3) as well as in AD brains using double-labeling immunofluorescence and confocal microscopy. In wild type control mice, PSD-95 primarily labeled neuropil with distinct distribution in hippocampal apical dendrites. In 3-month-old 5XFAD mice, PSD-95 distribution was similar to that of wild type mice despite significant Aβ deposition. However, in 6-month-old 5XFAD mice, PSD-95 immunoreactivity in apical dendrites markedly decreased and prominent immunoreactivity was noted in neuronal soma in CA1 neurons. Similarly, PSD-95 immunoreactivity disappeared from apical dendrites and accumulated in neuronal soma in 14-month-old, but not in 3-month-old, JNPL3 mice. In AD brains, PSD-95 accumulated in Hirano bodies in hippocampal neurons. Our findings support the notion that either Aβ or tau can induce reduction of PSD-95 in excitatory synapses in hippocampus. Furthermore, this PSD-95 reduction is not an early event but occurs as the pathologies advance. Thus, the time-dependent PSD-95 reduction from synapses and accumulation in neuronal soma in transgenic mice and Hirano bodies in AD may mark postsynaptic degeneration that underlies long-term functional deficits.

Topics: Age Factors; Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Analysis of Variance; Animals; Brain; Carbocyanines; Cell Count; Disease Models, Animal; Disks Large Homolog 4 Protein; Female; Gene Expression Regulation; Guanylate Kinases; Humans; Male; Membrane Proteins; Mice; Mice, Transgenic; Middle Aged; Synapses; tau Proteins; Tauopathies

In addition to its key role in complex motor function, the cerebellum is increasingly recognized to have a role in cognition. Thus, motor and cognitive deficits can be associated with cerebellar degeneration. After unilateral lesion in cerebellum (folia VI) was caused by Quinolinic acid, CM-DiI labeled mesenchymal stem cells (MSCs), which were isolated and purified from bone marrow, were transplanted into the damaged folium. Motor function was assessed using the cylinder test, rotarod, hanging wire and beam balance during 6 weeks after transplantation. Cognitive function was assessed using the Morris water maze learning paradigm in 3 weeks after transplantation. Six weeks after transplantation surviving MSCs were detectable in QA-treated animals. The MSC-transplanted group showed markedly improved functional performance in spatial memory, motor learning, locomotor asymmetry, dysmetria, abnormality in neuromuscular strength and equilibrium 2-6 weeks compared with the controls. We found that cerebellar lesions produced deficits (folia VI) in motor and cognitive aspects of a spatial task. The results indicate that transplantation of MSCs can significantly reduce the behavioral abnormalities of these animals during six weeks after engraftment. According to results of this assay, cell therapy by means of bone marrow derived adult stem cells promises for treatment of cerebellar diseases.

Topics: Analysis of Variance; Animals; Behavior, Animal; Carbocyanines; Cerebellum; Disease Models, Animal; Female; Hand Strength; Maze Learning; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Movement; Neurodegenerative Diseases; Organ Size; Psychomotor Performance; Rats; Rats, Wistar; Time Factors

The blood-brain barrier (BBB) disruption following cerebral ischemia (stroke) contributes to the development of life-threatening brain edema. Recent studies suggested that the ischemic BBB disruption is not uniform throughout the affected brain region. The aim of this study was to establish in vivo optical imaging methods to assess the size selectivity and spatial distribution of the BBB disruption after a focal cerebral ischemia. The BBB permeability was assessed in mice subjected to a 60-min middle cerebral artery occlusion and 24 h of reperfusion using in vivo time domain near-infrared optical imaging after contrast enhancement with two tracers of different molecular size, Cy5.5 (1 kDa) and Cy5.5 conjugated with bovine serum albumin (BSA) (67 kDa). Volumetric reconstruction of contrast-enhanced brain areas in vivo and ex vivo indicated that the BSA-Cy5.5-enhancement is identical to the volume of infarct determined by TTC staining, whereas the volume of enhancement with Cy5.5 was 40% greater. The volume differential between areas of BBB disruption for small and large-size molecules could be useful for determining the size of peri-infarct tissues (penumbra) that can respond to neuroprotective therapies.

Topics: Animals; Blood-Brain Barrier; Brain; Carbocyanines; Cattle; Cone-Beam Computed Tomography; Contrast Media; Disease Models, Animal; Fluorescence; Histocytochemistry; Humans; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Mice; Mice, Inbred Strains; Microtomy; Molecular Imaging; Reperfusion Injury; Serum Albumin; Tetrazolium Salts

This experiment was designed to assess the protective effect of betacyanins from Portulaca oleracea L. against the D-galactose (D-gal)-induced neurotoxicity in mice. Betacyanins from Portulaca oleracea markedly reversed the D-gal-induced learning and memory impairments, as measured by behavioral tests. The activities of superoxide dismutases (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in D-gal-treated mice were enhanced, while the content of the lipid peroxidation product malondialdehyde (MDA) was decreased by betacyanin administration. Furthermore, significant negative correlations were found between mouse latency in finding the platform and the activities of SOD, CAT GR and GPx in the mouse brain, but the level of MDA correlated positively with the latency. These results suggest that the neuroprotective effect of betacyanins against D-gal-induced neurotoxicity might be caused, at least in part, by an increase in the activities of antioxidant enzymes with a reduction in lipid peroxidation. In comparison with vitamin C (VC), the betacyanins had a more pronounced effect on ameliorating cognition deficits in mice.

Topics: Animals; Antioxidants; Ascorbic Acid; Brain; Carbocyanines; Cognition Disorders; Disease Models, Animal; Enzymes; Galactose; Lipid Peroxidation; Male; Malondialdehyde; Maze Learning; Memory; Mice; Mice, Inbred Strains; Neurotoxins; Oxidative Stress; Phytotherapy; Plant Extracts; Portulaca

Here we evaluate an improved orthotopic rat bladder tumor model, to be used for the evaluation of the therapeutic potential of novel cancer therapeutics. Before instilling AY-27 tumor cells into chemically denudated rat bladders, AY-27 cells were labeled with the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). We found that the presence of Dil did not alter the in vitro AY-27 cell proliferation and that the Dil label was strongly associated with the cells. We further provide evidence that the use of fluorescently labeled AY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inoculation process. Using this technique it was possible to track down the tumor cells after inoculation into the bladder, which makes it straightforward to distinguish tumor cells from remaining or regenerated normal urothelium over a period of 5 d. The results also demonstrated that malignant AY-27 tissue exists as an intact non-muscle invasive bladder tumor only for 1-3 d after cell implantation. Accordingly the AY-27 bladder tumor model was used to evaluate the antitumoral effect of a single intravesical MM-C instillation. All rats instilled with 1 mM MM-C survived the final endpoint of 30 d after intravesical MM-C. Moreover, 10 and 30 d after treatment the urothelium of the MM-C-treated animals was completely restored. Remarkably, after MM-C treatment distinct patchy fluorescent dots were found into the submucosa and the regenerated urothelium, suggesting that dye retention is secondary to the digestion of Dil-loaded AY-27 cells and cellular debris by macrophages and related immune cells.

Topics: Animals; Antibiotics, Antineoplastic; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Female; Fluorescence; Fluorescent Dyes; Mitomycin; Rats; Rats, Inbred F344; Staining and Labeling; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

To evaluate cetuximab treatment in head and neck squamous cell carcinoma xenografts and cell lines, we investigated a preclinical model of head and neck squamous cell carcinoma. Head and neck squamous cell carcinoma cell lines SCC-1, FaDu, CAL27, UM-SCC-5 and UM-SCC-22A were used to generate subcutaneous flank xenografts in SCID mice. Mice were divided into control and cetuximab treatment groups, mice in the latter group received 250 μg cetuximab once weekly for four weeks. After completion of therapy, SCC-1 (p < 0.001), UM-SCC-5 (p < 0.001), UM-SCC-22A (p = 0.016) and FaDu (p = 0.007) tumors were significantly smaller than control, while CAL27 tumors were not different from controls (p = 0.90). Mice were systemically injected with 50 μg of the Cy5.5-cetuximab bioconjugate and imaged by stereomicroscopy to determine if tumor fluorescence predicted tumor response. Intact tumor fluorescence did not predict response. Tissue was harvested from untreated xenografts to evaluate ex vivo imaging. Cell lines were then evaluated in vitro for fluorescence imaging after Cy5.5-cetuximab bioconjugate labeling. The location of fluorescence observed in labeled cells was significantly different for cell lines that responded to treatment, relative to unresponsive cells. Tumors from cell lines that showed low internalized signal in vitro responded best to treatment with cetuximab. This preclinical model may aid in determining which cancer patients are best suited for cetuximab therapy.

Topics: Analysis of Variance; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carbocyanines; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Disease Models, Animal; ErbB Receptors; Fluorescent Antibody Technique; Fluorescent Dyes; Head and Neck Neoplasms; Humans; Mice; Mice, SCID; Microscopy, Fluorescence; Statistics, Nonparametric; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) with a fluorescent probe, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolo-carbocyanine iodide), for the quantitative evaluation of neonatal hypoxic-ischemic brain injury. Seven-day-old rat pups were subjected to 2h of 8% oxygen following unilateral carotid artery ligation. Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI), and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI), terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c. Flow cytograms of JC-1 showed two distinct sub-populations with different ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V(+)/PI(+) necrotic cells, TUNEL-positive cells, TTC infarct area and western blot of cytosolic cytochrome c, and negative correlation with annexin V(-)/PI(-) live cells. In summary, low ΔΨ measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI. Therefore, fluorocytometric analysis of ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.

Topics: Animals; Animals, Newborn; Annexin A5; Benzimidazoles; Brain; Brain Infarction; Carbocyanines; Cell Death; Cytochromes c; Disease Models, Animal; Flow Cytometry; Fluorescent Dyes; Hypoxia-Ischemia, Brain; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Neurons; Propidium; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Time Factors

Bevacizumab is a potent recombinant humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF). The purpose of this study was to evaluate the therapeutic effect of subconjunctival injection of bevacizumab on corneal neovascularization (NV) in different rabbit models.. Several rabbit models of corneal NV were used, including (1) a corneal micropocket assay with VEGF pellet, (2) a corneal micropocket assay with basic fibroblast growth factor (b-FGF) pellets, (3) mechanical limbal injury-induced corneal NV, and (4) an alkali-induced model of corneal NV. Subconjunctival injections of bevacizumab (0.25-2.5 mg) were applied twice per week for 2 to 8 weeks. Digital photographs of the cornea were analyzed to determine the length of corneal NV and the area of cornea covered by NV as a percentage of the total corneal area. Immunohistochemical staining with anti-human IgG antibody labeled with Cy3 was used to determine the detection of intracorneal distribution of bevacizumab after injection.. Subconjunctival injection of bevacizumab caused significant inhibition of corneal NV formation as measured by length or surface area in all animal models (P<0.05). No significant ocular complications were found. Staining of bevacizumab was found in the corneal stroma for 3 to at least 14 days in the different rabbit models.. Subconjunctival injection of bevacizumab is effective in inhibiting corneal NV in several rabbit models. Bevacizumab may diffuse into the corneal stroma and persist for a few days after injection. It may be useful in preventing corneal NV in the acute phase of various kinds of corneal inflammation.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Blotting, Western; Carbocyanines; Conjunctiva; Corneal Neovascularization; Disease Models, Animal; Endothelium, Vascular; Female; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Injections; Platelet Endothelial Cell Adhesion Molecule-1; Rabbits; Vascular Endothelial Growth Factor A

Imaging with labeled monoclonal antibodies may be useful in detecting, staging, and monitoring tumors. Despite their high affinity and specificity, a critical limitation of antibody imaging is the high background signal due to prolonged clearance from the blood, which reduces the tumor-to-background ratio. To address this problem, we developed a molecular imaging probe consisting of multiple self-quenching fluorophores [Cy5.5 or Alexa Fluor 680 (Alexa680)] conjugated to a monoclonal antibody (trastuzumab) to synthesize Tra-Cy5.5(SQ) or Tra-Alexa680(SQ), respectively. This agent only becomes fluorescently "active" after cellular internalization but is quenched in the unbound state leading to high tumor-to-background ratios. The in vitro quenching capacity for both conjugates was approximately 9-fold. In vivo imaging experiments were done in mice bearing both 3T3/HER-2+ and BALB/3T3/ZsGreen/HER-2- xenografts. Tra-Alexa680(SQ) produced specific enhancement in the 3T3/HER-2+ tumors but not in the HER-2- control tumors. However, Tra-Cy5.5(SQ) produced nonspecific enhancement in both 3T3/HER-2+ and control tumors. In conclusion, whereas Cy5.5-conjugates produced nonspecific results as well as rapid liver accumulation, conjugating multiple Alexa680 molecules to a single monoclonal antibody resulted in a near-infrared optical agent that activated within specific target tumors with high tumor-to-background ratio with considerable potential for clinical translation.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibody Specificity; Carbocyanines; Cell Line; Disease Models, Animal; Hydrogen-Ion Concentration; Mice; Microscopy, Fluorescence; Neoplasm Transplantation; Neoplasms; Protein Binding; Recombinant Proteins; Staining and Labeling; Substrate Specificity; Trastuzumab

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene MECP2. Girls with RTT show dramatic changes in brain function, but relatively few studies have explored the structure of neural circuits. Examining two mouse models of RTT (Mecp2B and Mecp2J), we previously documented changes in brain anatomy. Herein, we use confocal microscopy to study the effects of MeCP2 deficiency on the morphology of dendrites and axons in the fascia dentata (FD), CA1 area of hippocampus, and motor cortex following Lucifer yellow microinjection or carbocyanine dye tracing. At 3 weeks of age, most (33 of 41) morphological parameters were significantly altered in Mecp2B mice; fewer (23 of 39) were abnormal in Mecp2J mice. There were striking changes in the density and size of the dendritic spines and density and orientation of axons. In Mecp2B mice, dendritic spine density was decreased in the FD (approximately 11%), CA1 (14-22%), and motor cortex (approximately 16%). A decreased spine head size (approximately 9%) and an increased spine neck length (approximately 12%) were found in Mecp2B FD. In addition, axons in the motor cortex were disorganized. In Mecp2J mice, spine density was significantly decreased in CA1 (14-26%). In both models, dendritic swelling and elongated spine necks were seen in all areas studied. Marked variation in the type and extent of changes was noted in dendrites of adjacent neurons. Electron microscopy confirmed abnormalities in dendrites and axons and showed abnormal mitochondria. Our findings document widespread abnormalities of dendrites and axons that recapitulate those seen in RTT.

Topics: Analysis of Variance; Animals; Axons; Carbocyanines; Dendritic Spines; Disease Models, Animal; Hippocampus; Isoquinolines; Male; Methyl-CpG-Binding Protein 2; Mice; Mice, Transgenic; Microinjections; Microscopy, Confocal; Microscopy, Electron; Motor Cortex; Neurons; Rett Syndrome

Using molecular imaging techniques, we examined interstitial alterations during postmyocardial infarction (MI) remodeling and assessed the efficacy of antiangiotensin and antimineralocorticoid intervention, alone and in combination.. The antagonists of the renin-angiotensin-aldosterone axis restrict myocardial fibrosis and cardiac remodeling after MI and contribute to improved survival. Radionuclide imaging with technetium-99m-labeled Cy5.5 RGD imaging peptide (CRIP) targets myofibroblasts and indirectly allows monitoring of the extent of collagen deposition post-MI.. CRIP was intravenously administered for gamma imaging after 4 weeks of MI in 63 Swiss-Webster mice and in 6 unmanipulated mice. Of 63 animals, 50 were treated with captopril (C), losartan (L), spironolactone (S) alone, or in combination (CL, SC, SL, and SCL), 8 mice received no treatment. Echocardiography was performed for assessment of cardiac remodeling. Hearts were characterized histopathologically for the presence of myofibroblasts and thick and thin collagen fiber deposition.. Acute MI size was similar in all groups. The quantitative CRIP percent injected dose per gram uptake was greatest in the infarct area of untreated control mice (2.30 +/- 0.14%) and decreased significantly in animals treated with 1 agent (C, L, or S; 1.71 +/- 0.35%; p = 0.0002). The addition of 2 (CL, SC, or SL 1.31 +/- 0.40%; p < 0.0001) or 3 agents (SCL; 1.16 +/- 0.26%; p < 0.0001) demonstrated further reduction in tracer uptake. The decrease in echocardiographic left ventricular function, strain and rotation parameters, as well as histologically verified deposition of thin collagen fibers, was significantly reduced in treatment groups and correlated with CRIP uptake.. Radiolabeled CRIP allows for the evaluation of the efficacy of neurohumoral antagonists after MI and reconfirms superiority of combination therapy. If proven clinically, molecular imaging of the myocardial healing process may help plan an optimal treatment for patients susceptible to heart failure.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Carbocyanines; Cardiovascular Agents; Disease Models, Animal; Drug Therapy, Combination; Echocardiography; Fibrillar Collagens; Fibroblasts; Fibrosis; Losartan; Mice; Mineralocorticoid Receptor Antagonists; Myocardial Infarction; Myocardium; Oligopeptides; Predictive Value of Tests; Spironolactone; Technetium; Tomography, Emission-Computed, Single-Photon; Ventricular Function, Left; Ventricular Remodeling

The aim of the current study is to test the ability to label and detect murine embryonic stem cell-derived cardiovascular progenitor cells (ES-CPC) with cardiac magnetic resonance (CMR) using the novel contrast agent Gadofluorine M-Cy3 (GdFM-Cy3).. Cell therapy shows great promise for the treatment of cardiovascular disease. An important limitation to previous clinical studies is the inability to accurately identify transplanted cells. GdFM-Cy3 is a lipophilic paramagnetic contrast agent that contains a perfluorinated side chain and an amphiphilic character that allows for micelle formation in an aqueous solution. Previous studies reported that it is easily taken up and stored within the cytosol of mesenchymal stem cells, thereby allowing for paramagnetic cell labeling. Investigators in our laboratory have recently developed techniques for the robust generation of ES-CPC. We reasoned that GdFM-Cy3 would be a promising agent for the in vivo detection of these cells after cardiac cell transplantation.. ES-CPC were labeled with GdFM-Cy3 by incubation. In vitro studies were performed to assess the impact of GdFM-Cy3 on cell function and survival. A total of 500,000 GdFM-Cy3-labeled ES-CPC or control ES-CPC were injected into the myocardium of mice with and without myocardial infarction. Mice were imaged (9.4-T) before and over a 2-week time interval after stem cell transplantation. Mice were then euthanized, and their hearts were sectioned for fluorescence microscopy.. In vitro studies demonstrated that GdFM-Cy3 was easily transfectable, nontoxic, stayed within cells after labeling, and could be visualized using CMR and fluorescence microscopy. In vivo studies confirmed the efficacy of the agent for the detection of cells transplanted into the hearts of mice after myocardial infarction. A correspondence between CMR and histology was observed.. The results of the current study suggest that it is possible to identify and potentially track GdFM-Cy3-labeled ES-CPC in murine infarct models via CMR.

Topics: Animals; Carbocyanines; Cell Line; Cell Proliferation; Cell Survival; Contrast Media; Disease Models, Animal; Embryonic Stem Cells; Female; Fluorescent Dyes; Fluorocarbons; Magnetic Resonance Imaging; Mesenchymal Stem Cell Transplantation; Mice; Mice, SCID; Microscopy, Fluorescence; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Organometallic Compounds; Staining and Labeling; Time Factors

Triple transgenic (3xTg-AD) mice harboring the presenilin 1, amyloid precursor protein, and tau transgenes (Oddo et al., 2003b) display prominent levels of amyloid-beta (Abeta) immunoreactivity in forebrain regions. The Abeta immunoreactivity is first seen intracellularly in neurons and later as extracellular plaque deposits. The present study examined Abeta immunoreactivity that occurs in layer III of the granular division of retrosplenial cortex (RSg). This pattern of Abeta immunoreactivity in layer III of RSg develops relatively late, and is seen in animals older than 14 months. The appearance of the Abeta immunoreactivity is similar to an axonal terminal field and thus may offer a unique opportunity to study the relationship between afferent projections and the formation of Abeta deposits. Axonal tract tracing techniques demonstrated that the pattern of axon terminal labeling in layer III of RSg, following placement of DiI in medial septum, is remarkably similar to the pattern of cholinergic axons in RSg, as detected by acetylcholinesterase histochemical staining, choline acetyltransferase immunoreactivity, or p75 receptor immunoreactivity; this pattern also is strikingly similar to the band of Abeta immunoreactivity. In animals sustaining early damage to the medial septal nucleus (prior to the advent of Abeta immunoreactivity), the band of Abeta in layer III of RSg does not develop; the corresponding band of cholinergic markers also is eliminated. In older animals (after the appearance of the Abeta immunoreactivity) damage to cholinergic afferents by electrolytic lesions, immunotoxin lesions, or cutting the cingulate bundle, result in a rapid loss of the cholinergic markers and a slower reduction of Abeta immunoreactivity. These results suggest that the septal cholinergic axonal projections transport Abeta or amyloid precursor protein (APP) to layer III of RSg.

Topics: Acetylcholine; Afferent Pathways; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Carbocyanines; Cholinergic Fibers; Disease Models, Animal; Gyrus Cinguli; Humans; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuroanatomical Tract-Tracing Techniques; Presenilin-1; Septal Nuclei; tau Proteins

Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial Na(+) channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-labeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)-anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the gamma-subunit, which releases an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 microg/ml). At a high plasmin concentration of 30 microg/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells, as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, gammaENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of gammaENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the gamma-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC.

Topics: Animals; Biotinylation; Carbocyanines; Cell Line; Disease Models, Animal; Epithelial Sodium Channels; Fibrinolysin; Fluorescent Antibody Technique; Fluorescent Dyes; Ion Channel Gating; Kidney Tubules, Collecting; Membrane Potentials; Mice; Nephrotic Syndrome; Oligopeptides; Patch-Clamp Techniques; Peptides; Phosphoinositide Phospholipase C; Protein Binding; Rats; Recombinant Fusion Proteins; RNA Interference; Serine Endopeptidases; Time Factors

The authors describe a method for percutaneous transplantation of human umbilical cord blood (hUCB)-derived multipotent stem cells (MSCs) under fluoroscopic guidance. The investigators then tested whether percutaneous transplantation of hUCB-derived MSCs improved neurological functional recovery after acute spinal cord injury (SCI).. The authors induced SCI in 10 dogs by percutaneous balloon compression. The 10 injured dogs were assigned randomly to the following groups (2 dogs each): Group 1, evaluated 2 weeks after sham transplantation; Group 2, evaluated 2 weeks after transplantation; Group 3, evaluated 4 weeks after sham transplantation; Group 4, evaluated 4 weeks after transplantation; and Group 5, evaluated 4 weeks after multispot transplantations. The dogs with sham transplantation (Groups 1 and 3) received the same volume of saline, as a control. A spinal needle was advanced into the spinal canal, and the investigators confirmed that the end of the spinal needle was located in the ventral part of spinal cord parenchyma by using contrast medium under fluoroscopic guidance. The hUCB-derived MSCs were transplanted into the cranial end of the injured segment in 6 injured dogs at 7 days after SCI.. Two dogs in Group 2 showed no improvement until 2 weeks after transplantation. Three of 4 dogs (Groups 4 and 5) that received cellular transplants exhibited gradual improvement in hindlimb locomotion from 3 weeks after cell transplantation. The CM-DiI-labeled hUCB-derived MSCs were observed in the spinal cord lesions at 4 weeks posttransplantation and exerted a significant beneficial effect by reducing cyst and injury size. The transplanted cells were positive for NeuN, glial fibrillary acidic protein, and von Willebrand factor.. The percutaneous transplantation technique described here can be easily performed, and it differs from previous techniques by avoiding surgical exposure and allowing cells to be more precisely transplanted into the spinal cord. This technique has many potential applications in the treatment of human SCI by cell transplantation. The results also suggest that transplantation of hUCB-derived MSCs may have therapeutic effects that decrease cavitation for acute SCI.

Topics: Animals; Carbocyanines; Contrast Media; Cord Blood Stem Cell Transplantation; Disease Models, Animal; Dogs; Fluorescent Dyes; Fluoroscopy; Humans; Immunoenzyme Techniques; Multipotent Stem Cells; Random Allocation; Spinal Cord Injuries; Staining and Labeling

Basic and clinical studies have shown that bone marrow cell therapy can improve cardiac function following infarction. In experimental animals, reported stem cell-mediated changes range from no measurable improvement to the complete restoration of function. In the clinic, however, the average improvement in left ventricular ejection fraction is around 2% to 3%. A possible explanation for the discrepancy between basic and clinical results is that few basic studies have used the magnetic resonance (MR) imaging (MRI) methods that were used in clinical trials for measuring cardiac function. Consequently, we employed cine-MR to determine the effect of bone marrow stromal cells (BMSCs) on cardiac function in rats. Cultured rat BMSCs were characterized using flow cytometry and labeled with iron oxide particles and a fluorescent marker to allow in vivo cell tracking and ex vivo cell identification, respectively. Neither label affected in vitro cell proliferation or differentiation. Rat hearts were infarcted, and BMSCs or control media were injected into the infarct periphery (n = 34) or infused systemically (n = 30). MRI was used to measure cardiac morphology and function and to determine cell distribution for 10 wk after infarction and cell therapy. In vivo MRI, histology, and cell reisolation confirmed successful BMSC delivery and retention within the myocardium throughout the experiment. However, no significant improvement in any measure of cardiac function was observed at any time. We conclude that cultured BMSCs are not the optimal cell population to treat the infarcted heart.

Topics: Animals; Bone Marrow Cells; Bone Marrow Transplantation; Carbocyanines; Cell Differentiation; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Ferric Compounds; Flow Cytometry; Fluorescent Dyes; Green Fluorescent Proteins; Immunohistochemistry; Magnetic Resonance Imaging, Cine; Male; Myocardial Contraction; Myocardial Infarction; Myocardium; Rats; Rats, Wistar; Staining and Labeling; Stroke Volume; Stromal Cells; Time Factors; Ventricular Function, Left

Glioblastoma multiforme (GBM) is a WHO grade IV malignant brain tumor with poor prognosis, despite advances in surgical and adjuvant therapy. GBM is characterized by areas of central necrosis and high levels of angiogenesis, during which increased vascular permeability allows for the extravasation of endothelial progenitor cells to support blood vessel and tumor growth. The purpose of this study was to characterize changes in tumor vascular permeability, vascular density and vessel morphology in vivo during angiogenesis.. An orthotropic murine (GL26) glioblastoma model was used in this study. in vivo serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in combination with histologic and molecular genetic analyses was performed to correlate in vivo imaging of vascular development.. DCE-MRI revealed a significant change in tumor vessel permeability dependent upon tumor progression and size. Time to max signal intensity displayed a stepwise increase between days 21 and 24 (p<0.05), a critical period before exponential tumor growth during which a significant increase in tumor vascular density and vessel caliber is observed on histology. Furthermore, quantitative real-time PCR revealed a corollary increase in angiogenic signaling molecules before the observed changes on DCE-MRI.. In vivo changes of orthotopic glioma blood vessel permeability as shown by DCE-MRI correlates with histologic quantification of vascular density and vessel caliber as well as with the molecular expression of angiogenic factors. DCE-MRI is a useful tool for non-invasive in vivo monitoring of angiogenesis in pre-clinical tumor models.

Topics: Analysis of Variance; Angiopoietin-1; Angiopoietin-2; Animals; Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Contrast Media; Disease Models, Animal; Disease Progression; Glioblastoma; Image Enhancement; Immunohistochemistry; Magnetic Resonance Imaging; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms, Experimental; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Vascular Endothelial Growth Factor A

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is commonly diagnosed late due to its deep location and vague symptoms. To identify biomarkers for improving NPC diagnosis, we established a proteomic platform for detecting aberrant serum proteins in nude mice bearing NPC xenografts. We first removed the three most abundant proteins from serum samples of tumor-bearing and control mice, and then labeled the samples with different fluorescent cyanine (Cy) dyes. The labeled serum proteins were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Differentially expressed proteins were identified by in-gel tryptic digestion and MALDI-TOF MS. We identified peroxiredoxin 2 (Prx-II) and carbonic anhydrase 2 (CA-II) as being elevated in the xenograft mouse model compared to controls. Western blot analysis confirmed up-regulation of Prx-II and CA-II in plasma from five NPC patients, and ELISA showed that plasma Prx-II levels were significantly higher in NPC patients (n = 84) versus healthy controls (n = 90) (3.03 +/- 4.47 versus 1.90 +/- 2.74 microg/mL, p = 0.047). In conclusion, Cy dye labeling combined with three-dimensional fractionation is a feasible strategy for identifying differentially expressed serum proteins in an NPC xenograft model, and Prx-II may represent a potential NPC biomarker.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Biomarkers, Tumor; Carbocyanines; Carbonic Anhydrase II; Child; Disease Models, Animal; Female; Fluorescent Dyes; Humans; Male; Mice; Mice, Nude; Middle Aged; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Peroxiredoxins; Proteomics; Transplantation, Heterologous; Up-Regulation

To visualize retinal ganglion cells (RGCs) and their gradual loss in the living mouse.. With the use of B6.Cg-Tg(Thy1-CFP)23Jrs/J mice, which express cyan fluorescent protein (CFP) in RGCs, and a commercially available mydriatic retinal camera attached with a 5 million-pixel digital camera to visualize RGCs in vivo, the authors recorded fundus photographs longitudinally in the ischemia reperfusion model group and the untreated group to evaluate longitudinal changes in the number of RGCs in experimental models. Moreover, RGCs expressing CFP were evaluated histologically by a retrograde-labeling method and retinal whole mount or sections.. The authors devised an in vivo imaging technique using a conventional retinal camera and visualized RGCs at the single-cell level. In the ischemia reperfusion model, a longitudinal reduction in the number of RGCs was demonstrated in each mouse eye. The number of RGCs and the fluorescence intensity of the nerve fiber decreased considerably during the first week. The percentages of RGCs decreased to 34.2% +/- 7.5%, 24.1% +/- 9.1%, 23.0% +/- 9.3%, and 22.2% +/- 8.4% (mean +/- SD, n = 5) of the percentages before injury at 1, 2, 3, and 4 weeks after injury, respectively (P < 0.001). In this transgenic mouse, 97% of CFP-expressing cells were RGCs and 73% of RGCs expressed CFP.. This in vivo technique allows noninvasive, repeated, and longitudinal evaluation of RGCs for investigation of retinal neurodegenerative diseases and new therapeutic modalities for them.

Topics: Animals; Carbocyanines; Cell Count; Disease Models, Animal; Female; Fluorescein Angiography; Fundus Oculi; Gene Expression; Green Fluorescent Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Photography; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells

DNA array analysis of dorsal root ganglion (DRG) using a rat model with nerve root constriction.. To determine the molecular changes in the DRG adjacent to the injured nerve root in a lumbar radiculopathy model.. DNA array analysis in lumbar radiculopathy model has so far focused on the spinal dorsal horn. The molecular changes in the DRG adjacent to the injured nerve root in lumbar radiculopathy remain to be determined.. Bilateral L5 DRGs were removed from 12 Sprague-Dawley rats on days 2, 7, 14, and 21 after nerve root ligation and on day 7 from 3 rats with sham operation. The aRNAs from the DRGs with nerve root ligation were labeled with Cy5 dye and those from the opposite side DRG (control) were labeled with Cy3 dye, and then hybridized to a 7793-spot Panorama Micro Array. It was considered to be significantly upregulated, when an average expression ratio of Cy5 to Cy3 was 2 or more. Genes upregulated were classified into early phase group (upregulated on day 2), midphase group (upregulated on days 7 and 14), and continuous group (upregulated from day 2 to 21). Seventeen genes were subjected to validation analysis with real-time quantitative PCR.. There were 16 upregulated genes in the early phase group, 56 genes in the midphase group, and 17 genes in the continuous group. Functional categorization revealed dominantly upregulated gene categories in each group; transcription/translation in the early phase group, enzyme/metabolism in the midphase group, and structure in the continuous group. Validation analysis of 17 genes demonstrated mean relative expression of 2.0 or more in all but 1 gene in the DRGs with nerve root ligation and none of them in the DRGs with sham operation.. The genes identified in this study, especially those involved in pain signaling and inflammation, serve as potential targets for molecular-based therapy for lumbar radiculopathy.

Topics: Animals; Carbocyanines; Disease Models, Animal; Fluorescent Dyes; Ganglia, Spinal; Gene Expression; Gene Expression Profiling; Lumbar Vertebrae; Male; Oligonucleotide Array Sequence Analysis; Pain; Radiculopathy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Nerve Roots; Time Factors; Up-Regulation

Removal of head and neck neoplasms, especially those of the parotid gland and those of the internal auditory canal and cerebellopontine angle, often requires microdissection of the facial nerve. Displacement or splaying of the nerve can make it difficult to identify facial nerve fibers and/or distinguish them from surrounding tissues. Here we tested a method of labeling the facial nerve with fluorescent lipophilic dyes as a method of providing intraoperative visual confirmation of nerve fibers.. The facial nerves of adult rats were retrogradely labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), or 3,3'-dilinoleyloxacarbocyanine perchlorate (Fast DiO) either by direct application to the nerve sheath or by microinjection into the facial muscles. The nerves were examined 30 days after dye application by means of a dissecting stereomicroscope equipped with epifluorescence filters.. Of the dyes tested, Fast DiO proved to be the most effective, providing labeling of the nerve sufficient to be seen with combined fluorescent and bright field stereomicroscopy. Nerve conduction studies indicated that fluorescent labeling did not adversely affect nerve function.. These results raise the possibility of using fluorescent lipophilic dyes to label nerves as a method of enhancing identification and distinguishing nerve fibers during surgery.

Topics: Animals; Carbocyanines; Disease Models, Animal; Electric Stimulation; Facial Nerve; Facial Nerve Injuries; Head and Neck Neoplasms; Humans; Monitoring, Intraoperative; Neural Conduction; Rats; Rats, Sprague-Dawley

Studies were performed to develop a prolonged antiangiogenesis therapy regimen based on theranostic alpha(nu)beta(3)-targeted nanoparticles.. Antiangiogenesis therapy may normalize atherosclerotic plaque vasculature and promote plaque stabilization. alpha(nu)beta(3)-targeted paramagnetic nanoparticles can quantify atherosclerotic angiogenesis and incorporate fumagillin to elicit acute antiangiogenic effects.. In the first experiment, hyperlipidemic rabbits received alpha(nu)beta(3)-targeted fumagillin nanoparticles (0, 30, or 90 microg/kg) with either a continued high fat diet or conversion to standard chow. The antiangiogenic response was followed for 4 weeks by cardiac magnetic resonance (CMR) molecular imaging with alpha(nu)beta(3)-targeted paramagnetic nanoparticles. In a second 8-week study, atherosclerotic rabbits received atorvastatin (0 or 44 mg/kg diet) alone or with alpha(nu)beta(3)-targeted fumagillin nanoparticles (only week 0 vs. weeks 0 and 4), and angiogenesis was monitored with CMR molecular imaging. Histology was performed to determine the location of bound nanoparticles and to correlate the level of CMR enhancement with the density of angiogenic vessels.. The alpha(nu)beta(3)-targeted fumagillin nanoparticles reduced the neovascular signal by 50% to 75% at 1 week and maintained this effect for 3 weeks regardless of diet and drug dose. In the second study, atherosclerotic rabbits receiving statin alone had no antineovascular benefit over 8 weeks. The alpha(nu)beta(3)-targeted fumagillin nanoparticles decreased aortic angiogenesis for 3 weeks as in study 1, and readministration on week 4 reproduced the 3-week antineovascular response with no carry-over benefit. However, atorvastatin and 2 doses of alpha(nu)beta(3)-targeted fumagillin nanoparticles (0 and 4 weeks) achieved marked and sustainable antiangiogenesis. Microscopic studies corroborated the high correlation between CMR signal and neovessel counts and confirmed that the alpha(nu)beta(3)-targeted nanoparticles were constrained to the vasculature of the aortic adventia.. The CMR molecular imaging with alpha(nu)beta(3)-targeted paramagnetic nanoparticles demonstrated that the acute antiangiogenic effects of alpha(nu)beta(3)-targeted fumagillin nanoparticles could be prolonged when combined with atorvastatin, representing a potential strategy to evaluate antiangiogenic treatment and plaque stability.

Topics: Angiogenesis Inhibitors; Animals; Aorta, Thoracic; Atherosclerosis; Atorvastatin; Carbocyanines; Cyclohexanes; Disease Models, Animal; Drug Carriers; Drug Synergism; Drug Therapy, Combination; Fatty Acids, Unsaturated; Heptanoic Acids; Heterocyclic Compounds, 1-Ring; Integrin alphaVbeta3; Liver; Magnetic Resonance Imaging; Nanoparticles; Neovascularization, Pathologic; Pyrroles; Rabbits; Sesquiterpenes; Time Factors

We studied sensitization of retrogradely labeled bladder sensory neurons and plasticity of P2X receptor function in a model of cystitis using patch-clamp techniques. Saline (control) or cyclophosphamide (CYP) was given intraperitoneally to rats on days 0, 2, and 4. On day 5, lumbosacral (LS, L6-S2) or thoracolumbar (TL, T12-L2) dorsal root ganglia were removed and dissociated. Bladders from CYP-treated rats showed partial loss of the urothelium and greater myeloperoxidase activity compared with controls. Bladder neurons from CYP-treated rats were increased in size (based on whole cell capacitance) compared with controls and exhibited lower activation threshold, increased action potential width, and greater number of action potentials in response to current injection or application of purinergic agonists. Most control LS bladder neurons (>85%) responded to ATP or alpha,beta-metATP with a slowly desensitizing current; these agonists affected only half of TL neurons, producing predominantly fast/mixed desensitizing currents. CYP treatment increased the fraction of TL bladder neurons sensitive to purinergic agonists (>80%) and significantly increased current density in both LS and TL bladder neurons compared with control. Importantly, LS and TL neurons from CYP-treated rats showed a selective increase in the functional expression of heteromeric P2X(2/3) and homomeric P2X(3) receptors, respectively. Although desensitizing kinetics were slower in LS neurons from CYP-treated compared with control rats, recovery kinetics were similar. The present results demonstrate that bladder inflammation sensitizes and increases P2X receptor expression and/or function for both pelvic and lumbar splanchnic pathways, which contribute, in part, to the hypersensitivity associated with cystitis.

Topics: Action Potentials; Adenosine Triphosphate; Animals; Carbocyanines; Cell Size; Cyclophosphamide; Cystitis, Interstitial; Disease Models, Animal; Ganglia, Spinal; Hypogastric Plexus; Inflammation Mediators; Male; Neurons, Afferent; Patch-Clamp Techniques; Purines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Splanchnic Nerves; Urinary Bladder; Urothelium; Visceral Afferents

We developed a new imaging probe for atherosclerotic lesion imaging by chemically conjugating an atherosclerotic plaque-homing peptide (termed the AP peptide) to hydrophobically modified glycol chitosan (HGC) nanoparticles. The AP peptide was previously discovered by using an in vivo phage display screening method. HGC nanoparticles were labeled with the near-infrared (NIR) fluorophore Cy5.5, yielding nanoparticles 314 nm in diameter. The binding characteristics of nanoparticles to cytokine (TNF-alpha)-activated bovine aortic endothelial cells (BAECs) were studied in vitro under static conditions and in a dynamic flow environment. AP-tagged HGC-Cy5.5 nanoparticles (100 microg/ml, 2 h incubation) bound more avidly to TNF-alpha-activated BAECs than to unactivated BAECs. Nanoparticles were mostly located in the membranes of BAECs, although some were taken up by the cells and were visible in the cytoplasm, suggesting that the AP peptides in HGC nanoparticles retained target selectivity for activated BAECs. Binding selectivity of AP-tagged HGC-Cy5.5 nanoparticles was also studied in vivo. NIR fluorescence imaging demonstrated that AP-tagged HGC-Cy5.5 nanoparticles bound better to atherosclerotic lesions in a low-density lipoprotein receptor-deficient (Ldlr(-/-)) atherosclerotic mouse than to such lesions in a normal mouse. These results suggest that the newly designed AP-tagged HGC-Cy5.5 nanoparticles may be useful for atherosclerotic lesion imaging, and may also be employed to elucidate pathophysiological changes, at the molecular level, on atherosclerotic endothelium.

Topics: Animals; Atherosclerosis; Binding Sites; Carbocyanines; Cattle; Cells, Cultured; Chitosan; Disease Models, Animal; Endothelial Cells; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Nanoparticles; Particle Size; Peptides; Protein Binding; Receptors, LDL; Spectroscopy, Near-Infrared; Staining and Labeling; Time Factors; Tumor Necrosis Factor-alpha

Fluorescence imaging of the heart is currently limited to invasive ex vivo or in vitro applications. We hypothesized that the adaptation of advanced transillumination and tomographic techniques would allow noninvasive fluorescence images of the heart to be acquired in vivo and be coregistered with in vivo cardiac magnetic resonance images.. The uptake of the magnetofluorescent nanoparticle CLIO-Cy5.5 by macrophages in infarcted myocardium was studied. Ligation of the left coronary artery was performed in 12 mice and sham surgery in 7. The mice were injected, 48 hours after surgery, with 3 to 20 mg of iron per kilogram of CLIO-Cy5.5. Magnetic resonance imaging and fluorescence molecular tomography were performed 48 hours later. An increase in magnetic resonance imaging contrast-to-noise ratio, indicative of myocardial probe accumulation, was seen in the anterolateral walls of the infarcted mice but not in the sham-operated mice (23.0+/-2.7 versus 5.43+/-2.4; P<0.01). Fluorescence intensity over the heart was also significantly greater in the fluorescence molecular tomography images of the infarcted mice (19.1+/-5.2 versus 5.3+/-1.4; P<0.05). The uptake of CLIO-Cy5.5 by macrophages infiltrating the infarcted myocardium was confirmed by fluorescence microscopy and immunohistochemistry.. Noninvasive imaging of myocardial macrophage infiltration has been shown to be possible by both fluorescence tomography and magnetic resonance imaging. This could be of significant value in both the research and clinical settings. The techniques developed could also be used to image other existing fluorescent and magnetofluorescent probes and could significantly expand the role of fluorescence imaging in the heart.

Topics: Animals; Carbocyanines; Disease Models, Animal; Macrophages; Magnetic Resonance Imaging; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Myocardial Infarction; Myocardium; Tomography, Optical

It has been shown that ED-B fibronectin (ED-B) is a potential target for plaque imaging. The aim of this study was to test a novel modified single chain anti-ED-B antibody (scFv) conjugated for near infrared fluorescence imaging (NIRF) with tetrasulfonated carbocyanine-maleimide (TSC-scFv) and to examine the association of ED-B with the presence of macrophages in a murine model of atherosclerosis. Expression of ED-B was observed in plaque areas in apolipoprotein E-deficient (apoE(-/-)) mice which increased with age and plaque load. Robust imaging was possible after explantation of the aorta and demonstrated a strong NIRF signal intensity in focal aortic and brachiocephalic plaque lesions, whereas no signals were found in undiseased areas. Plaque lesion ED-B was expressed by smooth muscle cell and was closely associated to macrophage infiltrates. Although not expressed by the same cell type, there was a significant correlation (p<0.01) between ED-B and macrophage immunoreactivity. In vitro human coronary and mouse smooth muscle cells significantly increased ED-B expression after angiotensin II and TNF-alpha treatment. This study demonstrates that plaque NIRF imaging is feasible with a novel single chain antibody and that ED-B expression is closely associated with inflammation in experimental atherosclerosis.

Topics: Animals; Antibodies; Aorta, Thoracic; Apolipoproteins E; Atherosclerosis; Carbocyanines; Cells, Cultured; Cholesterol, Dietary; Coronary Artery Disease; Disease Models, Animal; Feasibility Studies; Fibronectins; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Humans; Immunoglobulin Variable Region; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular

Multiple sclerosis is an inflammatory disease of the central nervous system characterized by inflammation, demyelination, axonal degeneration and accumulation of neurological disability. Previously, we demonstrated that stem cells constitute a possible endogenous source for remyelination. We now addressed the question of whether neurogenesis can occur in neuroinflammatory lesions. We demonstrated that, in experimental autoimmune encephalomyelitis, induced in rats 1,1'-dioctadecyl-6,6'-di(4sulphopentyl)-3,3,3',3'tetramethylindocarbocyanin(DiI)-labelled ependymal cells not only proliferated but descendants migrated to the area of neuroinflammation and differentiated into cells expressing the neuronal markers beta-III-tubulin and NeuN. Furthermore, these cells were immunoreactive for bromodeoxyuridine and PCNA, markers for cells undergoing cell proliferation. Using the whole-cell patch-clamp technique on freshly isolated 1, DiI-labelled cells from spinal cord lesions we demonstrated the ability of these cells to fire overshooting action potentials similar to those of immature neurones. We thus provide the first evidence for the initiation of neurogenesis in neuroinflammatory lesions in the adult spinal cord.

Topics: Animals; Bromodeoxyuridine; Carbocyanines; Cell Differentiation; Cell Enlargement; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Radiation; Female; Fluorescent Antibody Technique; In Vitro Techniques; Membrane Potentials; Microscopy, Confocal; Multiple Sclerosis; Neurons; Patch-Clamp Techniques; Phosphopyruvate Hydratase; Proliferating Cell Nuclear Antigen; Rats; Spinal Cord

Neuronal nitric oxide synthase (nNOS) regulates neurogenesis in normal developing brain, but the role of nNOS in neurogenesis in the ischemic brain remains unclear. To investigate the temporal and spatial relationship between cell proliferation of the ependymal/subventricular zone (SVZ), a principal neuroproliferative region in the adult brain, and nNOS expression, the male Sprague-Dawley rats weighing 250-350 g were used. The focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). 10 microl of 0.2% fluorescence dye DiI was injected into the right lateral ventricle to prelabel ependymal/subventricular zone cells before ischemia. The rats were killed immediately after ischemia and days 1, 3, 7, 11, 14, 21 and 28 after ischemia. DiI-labeled cell counting was employed to assess cell proliferation. Immunohistochemistry and grayscale analysis were performed to determine nNOS localization and its quantity in the specific regions. Compared with control, the density of DiI-labeled cells in the ipsilateral ependyma/SVZ was significantly higher at days 1, 3, 7 and 11 after ischemia, whereas the quantity of nNOS expression in the ependyma/SVZ adjacent regions was significantly lower at the above time points. Additionally, nNOS positive cells were largely excluded from SVZ, and their long processes did not enter the ependyma/SVZ. Our results indicate that after focal cerebral ischemia, decreased nNOS expression in the ipsilateral ependymal/SVZ adjacent regions might be related to cell proliferation in the ependymal/SVZ.

Topics: Animals; Brain Ischemia; Carbocyanines; Cell Count; Cell Proliferation; Cerebral Ventricles; Disease Models, Animal; Ependyma; Gene Expression; Immunohistochemistry; Male; Nitric Oxide Synthase Type I; Rats; Time Factors

Expansion of a polyglutamine tract in the huntingtin protein causes neuronal degeneration and death in Huntington's disease patients, but the molecular mechanisms underlying polyglutamine-mediated cell death remain unclear. Previous studies suggest that expanded polyglutamine tracts alter transcription by sequestering glutamine rich transcriptional regulatory proteins, thereby perturbing their function. We tested this hypothesis in Caenorhabditis elegans neurons expressing a human huntingtin fragment with an expanded polyglutamine tract (Htn-Q150). Loss of function alleles and RNA interference (RNAi) were used to examine contributions of C. elegans cAMP response element-binding protein (CREB), CREB binding protein (CBP), and histone deacetylases (HDACs) to polyglutamine-induced neurodegeneration. Deletion of CREB (crh-1) or loss of one copy of CBP (cbp-1) enhanced polyglutamine toxicity in C. elegans neurons. Loss of function alleles and RNAi were then used to systematically reduce function of each C. elegans HDAC. Generally, knockdown of individual C. elegans HDACs enhanced Htn-Q150 toxicity, but knockdown of C. elegans hda-3 suppressed toxicity. Neuronal expression of hda-3 restored Htn-Q150 toxicity and suggested that C. elegans HDAC3 (HDA-3) acts within neurons to promote degeneration in response to Htn-Q150. Genetic epistasis experiments suggested that HDA-3 and CRH-1 (C. elegans CREB homolog) directly oppose each other in regulating transcription of genes involved in polyglutamine toxicity. hda-3 loss of function failed to suppress increased neurodegeneration in hda-1/+;Htn-Q150 animals, indicating that HDA-1 and HDA-3 have different targets with opposing effects on polyglutamine toxicity. Our results suggest that polyglutamine expansions perturb transcription of CREB/CBP targets and that specific targeting of HDACs will be useful in reducing associated neurodegeneration.

Topics: Aging; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Carbocyanines; CREB-Binding Protein; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Enzyme Inhibitors; Gene Expression; Histone Deacetylases; Humans; Huntingtin Protein; Huntington Disease; Hydroxamic Acids; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Peptides; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger

It is well established that experimental testicular torsion induces germ cell specific apoptosis. Annexin V (BD Pharmingentrade mark) binds phosphatidylserine that becomes exposed on the cell membrane in apoptotic cells. In vivo detection of apoptotic cells with fluorescently labeled annexin V is an emerging technique that we evaluated for detecting apoptotic germ cells in a mouse model of testicular torsion.. Annexin V labeled with an indocyanine fluorophore (bisfunctional succinimidyl ester of cyanine 5.5) (Amersham, Little Chalfont, United Kingdom) was injected intravenously in mice 18 hours after the repair of unilateral 720-degree testicular torsion for 2 hours. Serial fluorescence images were obtained 21, 24, 28 and 42 hours after torsion repair. Relative fluorophore localization was visualized in vivo using an optical small animal imaging system mounted with a filter in near infrared light. Average fluorescence intensity in torsed and sham testes was quantified in images of testes in situ exposed through an abdominal incision and in ex vivo testes.. A significant increase in fluorescence intensity was found in images of torsed vs sham operated testes. This was seen in ex vivo, exposed and in vivo testes (215%, 250% and 161%, respectively, p <0.05). Bisfunctional succinimidyl ester of cyanine 5.5 conjugated to dehydrogenase, a protein with a size similar to that of annexin V, was used to assess for capillary leakage. It was also more localized to the torsed testis relative to its contralateral sham control whether exposed or ex vivo (174% and 176%, respectively).. To our knowledge this study demonstrates for the first time the possibility of in vivo near infrared fluorescence imaging of apoptotic germ cells after testicular torsion in mice. It shows important confounding factors that must be considered as this new imaging technique is developed for detecting apoptotic cells in vivo in testes or in any other organ.

Topics: Animals; Annexins; Apoptosis; Carbocyanines; Cytological Techniques; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Spermatic Cord Torsion

Glaucoma is a chronic neurodegenerative disease of the retina, characterized by the degeneration of axons in the optic nerve and retinal ganglion cell apoptosis. DBA/2J inbred mice develop chronic hereditary glaucoma and are an important model system to study the molecular mechanisms underlying this disease and novel therapeutic interventions designed to attenuate the loss of retinal ganglion cells. Although the genetics of this disease in these mice are well characterized, the etiology of its progression, particularly with respect to retinal degeneration, is not. We have used two separate labeling techniques, post-mortem DiI labeling of axons and ganglion cell-specific expression of the betaGeo reporter gene, to evaluate the time course of optic nerve degeneration and ganglion cell loss, respectively, in aging mice.. Optic nerve degeneration, characterized by axon loss and gliosis is first apparent in mice between 8 and 9 months of age. Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe. Although nerve damage is typically bilateral, the progression of disease is asymmetric between the eyes of individual mice. Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region. Ganglion cell loss, as a function of the loss of betaGeo expression, is evident in some mice between 8 and 10 months of age and is prevalent in the majority of mice older than 10.5 months. Most eyes display a uniform loss of ganglion cells throughout the retina, but many younger mice exhibit focal loss of cells in sectors extending from the optic nerve head to the retinal periphery. Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal.. A comparison of the data collected from the two cohorts of mice used for this study suggests that the initial site of damage in this disease is to the axons in the optic nerve, followed by the subsequent death of the ganglion cell soma.

Topics: Age Factors; Animals; beta-Glucosidase; Carbocyanines; Cell Count; Chi-Square Distribution; Disease Models, Animal; Disease Progression; DNA-Binding Proteins; Glaucoma; Mice; Mice, Inbred DBA; Mice, Transgenic; Optic Nerve Diseases; Retinal Degeneration; Retinal Ganglion Cells; Time Factors; Transcription Factors; Ubiquitin-Protein Ligase Complexes

Nerve injury represents a major cause of disability. In the peripheral nervous system, nerves have the capacity to regrow but within weeks after injury, it is impossible to clarify whether proper regeneration is under way or is failing. In this experimental study, we report on a novel tool to assess nerve outgrowth in vivo. After systemic application, the novel gadolinium-based magnetic resonance (MR) contrast agent Gadofluorine M (Gf) selectively accumulated and persisted in nerve fibers undergoing Wallerian degeneration causing bright contrast on T1-weighted MR images. Gf enhancement on MR imaging was present already at 48 hours within the entire nerve segments undergoing Wallerian degeneration, and subsequently disappeared from proximal to distal parts in parallel to regrowth of nerve fibers. Most importantly, Gf enhancement persisted in nonregenerating, permanently transected nerves. Our novel Gf-based MR imaging methodology holds promise for clinical use to bridge the diagnostic gap between nerve injury and completed nerve regeneration, and to determine the necessity for neurolysis and engraftment if spontaneous regeneration is not successful.

Topics: Animals; Carbocyanines; Disease Models, Animal; Ectodysplasins; Functional Laterality; Immunohistochemistry; Magnetic Resonance Imaging; Male; Membrane Proteins; Nerve Crush; Nerve Fibers; Nerve Regeneration; Organometallic Compounds; Rats; Rats, Wistar; Sciatic Neuropathy; Staining and Labeling; Time Factors; Tolonium Chloride; Wallerian Degeneration

The signaling of retinal ganglion cell (RGC) death after axotomy is partly dependent on the generation of reactive oxygen species. Shifting the RGC redox state toward reduction is protective in a dissociated mixed retinal culture model of axotomy. The hypothesis for the current study was that tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would protect RGCs in a rat optic nerve crush model of axotomy.. RGCs of postnatal day 4 to 5 Long-Evans rats were retrogradely labeled with the fluorescent tracer DiI. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 muL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 microM. The right eye served as the control. Eight or 14 days after the crush, the animals were killed, retinal wholemounts prepared, and DiI-labeled RGCs counted. Bandeiraea simplicifolia lectin (BSL-1) was used to identify microglia.. The mean number of surviving RGCs at 8 days in eyes treated with 60 microM TCEP was significantly greater than in the vehicle group (1250 +/- 156 vs. 669 +/- 109 cells/mm(2); P = 0.0082). Similar results were recorded at 14 days. Labeling was not a result of microglia phagocytosing dying RGCs. No toxic effect on RGC survival was observed with TCEP injection alone.. The sulfhydryl-reducing agent TCEP is neuroprotective of RGCs in an optic nerve crush model. Sulfhydryl oxidative modification may be a final common pathway for the signaling of RGC death by reactive oxygen species after axotomy.

Topics: Animals; Axotomy; Carbocyanines; Cell Count; Cell Survival; Disease Models, Animal; Fluorescent Dyes; Injections; Neuroglia; Neuroprotective Agents; Optic Nerve Injuries; Phosphines; Rats; Rats, Long-Evans; Retinal Ganglion Cells; Sulfhydryl Compounds; Vitreous Body

Synaptic damage and loss are factors that affect the degree of dementia experienced in Alzheimer disease (AD) patients. Multicolor DiOlistic labeling of the hippocampus has been undertaken which allows the full dendritic arbor of targeted neurons to be imaged. Using this labeling technique the neuronal morphology of two transgenic mouse lines (J20 and APP/PS1) expressing mutant forms of the Amyloid Precursor Protein (APP), at various ages, have been visualized and compared to Wild Type (WT) littermate controls. Swollen bulbous dystrophic neurites with loss of spines were apparent in the transgenic animals. Upon quantification, statistically significant reductions in the number of spines and total dendrite area was observed in both transgenic mouse lines at 11 months of age. Similar morphological abnormalities were seen in human AD hippocampal tissue both qualitatively and quantitatively. Immunohistochemistry and DiOlistic labeling was combined so that Abeta plaques were imaged in relation to the dendritic trees. No preferential localization of these abnormal dystrophic neurites was seen in regions with plaques. DiI labeled reative astrocytes were often apparent in close proximity to A beta plaques.

Topics: Aging; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Astrocytes; Biolistics; Carbocyanines; Dendrites; Dendritic Spines; Disease Models, Animal; Hippocampus; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Mutation; Plaque, Amyloid; Pyramidal Cells; Staining and Labeling

Mitochondrial dysfunction has long been implicated in the death of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) and its experimental models. Here we further analyzed changes in the mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) of striatal synaptosomes after the infusion of 1-methyl-4-phenylpyridinium (MPP+) into rat striatum. MPP+ (40 nmol) treatment produced decreases in mitochondrial REDOX activity and Deltapsi(m) at 18 h, as measured by fluorometric analysis with both Alamar blue and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) dyes. At this time point, tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels were not altered, but both decreased at 7 days after MPP+ (40 nmol) infusion. Both measures of mitochondrial dysfunction induced by MPP+ (40 nmol) at 18 h were attenuated, at least in part, by pretreatment with a selective dopamine uptake inhibitor GBR-12909 (1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl) piperazine). In addition, GBR-12909 partially attenuated MPP+ (40 nmol)-caused a loss of striatal nerve terminal as indicated by decreases in TH and DAT immunoreactivities as well as dopamine and its metabolites levels. The present study indicates that decreases in mitochondrial REDOX activity and Deltapsi(m) may play a role in MPP+ -induced dopaminergic neurotoxicity, and further provides that improvement of mitochondrial dysfunction may be a better way to slow progressive dopaminergic neurodegeneration commonly associated with PD.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Benzimidazoles; Carbocyanines; Coloring Agents; Corpus Striatum; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Fluorometry; Male; Membrane Glycoproteins; Membrane Potentials; Membrane Transport Proteins; Mitochondria; Nerve Tissue Proteins; Oxazines; Oxidation-Reduction; Parkinsonian Disorders; Piperazines; Presynaptic Terminals; Rats; Rats, Wistar; Sodium Cyanide; Synaptosomes; Tyrosine 3-Monooxygenase; Xanthenes

In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.

Topics: Animals; Animals, Newborn; Axotomy; Brain Tissue Transplantation; Carbocyanines; Cell Survival; Ciliary Neurotrophic Factor; Cyclic AMP; Disease Models, Animal; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Growth Cones; Nerve Growth Factors; Nerve Regeneration; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Retina; Retinal Ganglion Cells; Stilbamidines; Thionucleotides; Tubulin; Up-Regulation

In multiple sclerosis, the central nervous system is lesioned through invasion of plaque-forming inflammatory cells, primarily contributing to immune attack of myelin and oligodendrocytes. In this report we address the possible activation and differentiation of central nervous system stem cells following such immunological insults in a well-characterized rat model of multiple sclerosis characterised by spinal cord pathology. Dye-labeled central nervous system stem cells, residing within the ependymal layer of the central canal responded to the multiple sclerosis-like conditions by proliferation, while some of the migrating stem cell-derived cells expressed markers typical for oligodendrocytes (04) and astrocytes (glial fibrillary acidic protein, GFAP) in the demyelinated area. Our results indicate that regenerative stem cell activation following immunoactivity is different from that after trauma, exemplified by the slower time course of stem cell proliferation and migration of progeny, in addition to the ability of the stem cell-derived cells to express oligodendrocyte markers. Finally, deleterious effects of macrophages on the stem cell population were evident and may contribute to the depletion of the stem cell population in neuroinflammatory disorders.

Topics: Animals; Bromodeoxyuridine; Carbocyanines; Cell Count; Demyelinating Diseases; Disease Models, Animal; Ectodysplasins; Encephalomyelitis, Autoimmune, Experimental; Ependyma; Female; Fluorescent Dyes; Freund's Adjuvant; Glial Fibrillary Acidic Protein; Indoles; Macrophages; Membrane Proteins; Microscopy, Confocal; Myelin Proteins; Myelin Sheath; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; O Antigens; Rats; Rats, Inbred Strains; Spinal Cord; Stem Cells; Time Factors

Erythropoietin (Epo) has been shown to have potent anti-apoptotic activity in central nervous system neurons in animal models of ischaemic injury. Recently, Epo and its receptor (EpoR) have been identified in the peripheral nervous system [Campana & Myers (2001), FASEB J., 15, 1804-1806]. Herein, we demonstrate that in painful neuropathy caused by L5 spinal nerve crush (SNC), therapy with recombinant human Epo (rhEpo) reduced dorsal root ganglion (DRG) apoptosis and pain behaviours. Quantification of both DRG neurons and satellite cells revealed that vehicle-treated, crush-injured DRGs had 35.5 +/- 8.3% apoptotic neurons and 23.5 +/- 2.36% satellite cells compared with 7.5 +/- 6.3% apoptotic neurons and 6.4 +/- 3.94% satellite cells in rhEpo-treated, crush-injured DRGs (P < 0.05). While rhEpo-treated animals were not initially protected from mechanical allodynia associated with L5 SNC, rhEpo did significantly improve recovery rates compared to vehicle-treated animals (P < 0.01). Systemic rhEpo therapy increased JAK2 phosphorylation, a key anti-apoptotic signalling molecule for Epo-induced neuroprotection, in DRGs after crush. Dual immunofluorescence demonstrated Epo-induced JAK2-p was associated with both neuronal and glial cells. JAK2-p was associated with NF200-positive large neurons and with smaller neurons. This population of small neurons did not colocalize with IB4, a marker of nonpeptidergic, glial derived growth factor-responsive neurons. The findings link anti-apoptosis activities of Epo/EpoR/JAK2 in DRG neurons capable of inducing protracted pain states with reductions in pain behaviours, and therefore support a role for Epo therapy in the treatment of neuropathic pain.

Topics: Animals; Apoptosis; Behavior, Animal; Blotting, Western; Carbocyanines; Cell Count; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Female; Fluorescent Antibody Technique; Functional Laterality; Ganglia, Spinal; Glial Fibrillary Acidic Protein; Humans; Hyperalgesia; In Situ Nick-End Labeling; Janus Kinase 2; Microscopy, Confocal; Neurofilament Proteins; Pain; Pain Measurement; Pain Threshold; Peripheral Nervous System Diseases; Plant Lectins; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Ribosome Inactivating Proteins; Satellite Cells, Perineuronal; Time Factors

We describe the design and construction of a miniaturized multichannel near infrared (NIR) endoscopic imaging system developed for high-resolution imaging of mice. The device allows for simultaneous real-time video images in white light and two independent NIR channels. Testing demonstrated independent acquisition of nanomolar concentrations of fluorochromes Cy5.5 and Cy7. Cross-talk between the NIR channels, partially a result of broad tails in the spectra of commonly used organic fluorochromes, was assessed, modeled for the linear range of the concentration/signal intensity function, and compensated. The calculated compensation was 5.5% and 22% of the total signal intensity in the two channels NIR700 and NIR780, respectively, at equal concentrations of the two fluorochromes. Using a mouse model of colonic adenomatosis, we show that both perfusion and protease activity can be detected simultaneously, independently, and repeatedly in live mice. The developed device should be useful for in vivo imaging of diverse molecular targets.

Topics: Adenoma; Animals; Carbocyanines; Colonic Polyps; Colonoscopy; Disease Models, Animal; Endoscopes; Endoscopy; Image Processing, Computer-Assisted; Intestinal Neoplasms; Mice; Mice, Mutant Strains; Sensitivity and Specificity; Software; Spectroscopy, Near-Infrared

During development, the survival of spinal motoneurons depends on the integrity of the connection to their peripheral targets. Peripheral nerve axotomy induces apoptosis in neonatal neurons supplying axons to the nerve. Bax is known to promote apoptosis among developing neurons. To examine the effect of axotomy on spinal motoneurons in Bax-deficient (Bax-/-) and wild-type neonatal mice (Bax+/+), the sciatic nerve was axotomized on postnatal day (P) 0, and motoneurons in the fourth lumbar (L4) segment were visualized at P7 by acetylcholinesterase (AChE) histochemical staining. Presumably due to the reduction in naturally occurring cell death resulting from the deficiency of Bax, there were about 50% more AChE-positive cells in Bax-/- than in Bax+/+. Motoneurons in the dorsolateral motor pool of L4 project through the sciatic nerve. In Bax+/+, axotomy of the sciatic nerve induced significant cell loss in the pool. Most motoneurons survived such axotomy in Bax-/-, although they appeared atrophic and their AChE expression was decreased. Motoneurons may receive vital support retrogradely from their targets, and loss of such support may lead to hypofunction of spinal motoneurons, as indicated by the reduced production of AChE by axotomized motoneurons and their small size in Bax-/-.

Topics: Acetylcholinesterase; Animals; Animals, Newborn; Apoptosis; Axotomy; bcl-2-Associated X Protein; Carbocyanines; Cell Differentiation; Disease Models, Animal; Gene Expression Regulation, Developmental; Mice; Mice, Knockout; Motor Neurons; Muscular Atrophy, Spinal; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retrograde Degeneration; Sciatic Nerve; Spinal Cord

Fiber tract lesions in the central nervous system (CNS) often induce delayed retrograde neuronal degeneration, a phenomenon that represents an important therapeutic challenge in clinical neurotraumatology. In the present study, we report an in vivo trauma model of graded axonal lesion of CNS neurons. Controlled by a newtonmeter device, we induced retrograde degeneration of adult rat retinal ganglion cells (RGCs) by graded crush of the optic nerve. The extent of secondary RGC death increased linearly with the applied crush force. Moreover, visually evoked potentials were used to characterize the consequences of controlled optic nerve lesion on the functional integrity of the visual projection. The presented model of fiber tract lesion closely resembles the clinical conditions of traumatic brain injury and could prove useful to screen for neuroprotective drugs based on both a morphological and functional read-out.

Topics: Animals; Benzoxazines; Brain Injuries; Carbocyanines; Disease Models, Animal; Evoked Potentials, Visual; Female; Fluorescent Dyes; Nerve Crush; Neural Conduction; Neural Pathways; Neuroprotective Agents; Optic Nerve; Optic Nerve Injuries; Oxazines; Rats; Reaction Time; Retinal Ganglion Cells; Retrograde Degeneration

This research has examined the relationship between axonal regeneration and the return of normal movement following complete transection of the spinal cord. We made measurements of tail beat frequency and amplitude of the caudal body wave from video recordings of eels (Anguilla anguilla) swimming in a water tunnel at several speeds. Each eel was then anaesthetised and the spinal cord cut caudal to the anus; in some animals the resulting gap was filled with a rubber block. All animals were kept at 25 degrees C for recovery periods ranging from 7 to 128 days, during which their swimming performance was monitored regularly. Each fish was then re-anaesthetised and perfused with fixative and the regrowing descending axons labelled with 1,1'-diotadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. For all animals and at all speeds after surgery, tail beat frequency increased, while amplitude decreased. In non-blocked animals, an improvement in performance was first seen from 8 days following transection and thereafter tail beat frequency decreased progressively until it had returned to normal after 35 to 45 days, while amplitude remained below baseline until at least 45 days. In these animals, few axonal growth cones had penetrated the caudal stump by 7 days, but some had extended as much as 3 mm by 15 days. Many had reached as far as 6 mm between 25 and 36 days, while by 128 days they had progressed up to 10.5 mm. Contralateral crossing was never observed. Functional recovery was never witnessed in animals in which the cord had been blocked and these eels swam at all times with elevated tail beat frequency and reduced caudal amplitude. No labelled axons could be traced into the caudal spinal cord at any recovery stage in such animals. We conclude that re-innervation of only 1-2 segments caudal to the injury is necessary for functional recovery, although continued axonal growth may be important for the refinement of some aspects of movement.

Topics: Anguilla; Animals; Carbocyanines; Denervation; Disease Models, Animal; Efferent Pathways; Fluorescent Dyes; Growth Cones; Locomotion; Movement Disorders; Nerve Regeneration; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Swimming

Endometriosis is an adhesion disorder characterized by the presence of endometrial tissue in ectopic sites outside the uterus. The disease is associated with dysmenorrhea, pelvic pain and infertility. Although endometriosis is the most common gynecologic disorder, relatively little is known regarding its etiology, pathogenesis and the course of the disease. This situation is primarily due to the absence of experimental systems to examine the mechanism of endometrial cell adhesion, role of inflammatory cells and the interactions of epithelial, and stromal cells with the peritoneum and ovarian tissue leading to the development of this disorder. Dissociated human endometrial cells were suspended in peritoneal fluids of individuals with and without endometriosis and were injected into the peritoneal cavity of athymic mice. This led to development of ectopic adhesions of endometrial cells at the peritoneal and ovarian surfaces. Endometrial cells which were marked with fluorescent lipophylic dyes, prior to intraperitoneal injection, could be visualized without surgery at such sites. The studies demonstrate a model for endometriosis in athymic mice.

Topics: Adult; Animals; Ascitic Fluid; Carbocyanines; Disease Models, Animal; Endometriosis; Endometrium; Female; Fluorescent Dyes; Humans; Injections, Intraperitoneal; Leukocyte Transfusion; Mice; Mice, Nude; Microscopy, Fluorescence; Middle Aged

Stimulatory effects of a novel isobenzofranone, MD-700, on low density lipoprotein (LDL) receptor activity were investigated in vitro and in vivo. MD-700 at 0.03 microg/ml elevated the expression of LDL receptor in HepG2 cells within 4 h. Corresponding to this, uptake of fluorescent labeled-LDL (3,3'-dioctadecylindocarbocyanine-LDL) by the cells increased linearly in time- and dose-dependent manner by MD-700 for up to 12 h. In the experiment using HepG2 cells transiently transfected with promoter-luciferase gene constructs, MD-700 increased luciferase activity in a dose-dependent manner from 0.03 to 0.1 microg/ml. In contrast, luciferase activity was not stimulated by MD-700 in construct with a deleted sterol regulatory element (SRE)-1, suggesting importance of SRE-1 in stimulation of the LDL receptor gene promoter by MD-700. Binding experiments on liver membranes from MD-700-treated hamsters showed about a 60% increase in 125I-labeled LDL binding. A Scatchard plot revealed that MD-700 increased the maximal binding without affecting binding affinity. In contrast to findings with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, pravastatin, MD-700 had no effect on the sterol synthesis in hamster liver homogenates. These results suggest that MD-700 stimulates the expression of LDL receptor, presumably in a manner independent of change in sterol metabolism, and thereby promotes LDL clearance. Hypocholesterolemic actions of MD-700 in hamsters were then examined. MD-700 lowered serum cholesterol levels in hamsters fed normal chow or a high-fat diet. Fractionation of serum lipoproteins demonstrated that MD-700 selectively decreased LDL and very low density lipoprotein cholesterol. Dose-dependent decrease in serum cholesterol was also seen in hypercholesterolemic rats. Thus, the hypocholesterolemic action of MD-700 may be attributed to up-regulation of the LDL receptor, based on stimulation of the transcription of the LDL receptor gene. Although pravastatin stimulates LDL uptake and lowers serum cholesterol in a manner similar to that seen with MD-700, the mechanism responsible for hypocholesterolemic action appears to differ.

Topics: Animals; Benzofurans; Blotting, Northern; Carbocyanines; Carcinoma, Hepatocellular; Cell Membrane; Cholesterol; Cricetinae; Disease Models, Animal; DNA Primers; Fluorescent Dyes; Humans; Hypercholesterolemia; Lipoproteins, LDL; Lipoproteins, VLDL; Liver Neoplasms; Male; Promoter Regions, Genetic; Rats; Rats, Wistar; Receptors, LDL; RNA, Messenger; RNA, Neoplasm; Sterols; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation

Ganglion cells in the chicken retina fail to regenerate their axons upon mechanical injury. In order to determine whether this failure to regenerate axons is intrinsic to the neurons or is mediated by the environment, we asked whether ganglion cells possess an ability to regrow their injured axons in the absence of their natural environment, namely in vitro. Since the retina contains morphologically different types of ganglion cells, it became desirable to investigate whether all types of ganglion cells contribute to regeneration of axons. Ganglion cells were labelled post-mortem with the fluorescent dye DiI and described morphologically. Morphometric parameters like the sizes of their perikarya, their dendrites, and the patterns of dendritic ramification and stratification were considered for grouping cells. Although a strong classification of the cells could not be achieved because of the high diversity among this population of neurons, the chick retinal ganglion cells could be separated into seven regular groups which have their somata located within the ganglion cell layer and into one group with the somata located within the inner nuclear layer (displaced ganglion cells). The experimental procedure for regeneration combines crush injury of the chick optic nerve in situ with explanation of retinal pieces 1 week later for organ cultures in a serum-free medium. Under these conditions, the ganglion cells extended axons 1 day after explanation on polylysine/laminin. The densities of ganglion cells contributing to the axonal regrowth reached up to 1447 cells mm-2 (mean 1028 cells mm-2; S.D. 237). This density corresponds to 13% of the ganglion cell density in the normal retina, averaged across the total retina area. Although the dendrites of some cells whose axons had regenerated were altered in comparison with the normal ganglion cells, all morphological types of ganglion cells including those with displaced cell bodies contributed proportionally to the regrowth of axons from the explants.

Topics: Affinity Labels; Animals; Axons; Carbocyanines; Cell Count; Chickens; Dendrites; Disease Models, Animal; Fluorescent Dyes; Microscopy, Fluorescence; Nerve Regeneration; Optic Nerve; Organ Culture Techniques; Retinal Ganglion Cells