cyanine-dye-3 and Disease-Models--Animal

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

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

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

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

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

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

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

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

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

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