carbocyanines and Reperfusion-Injury

Retinal ischemia-reperfusion (I/R) injuries are involved in the universal pathological processes of many ophthalmic diseases, including glaucoma, diabetic retinopathy, and retinal arterial occlusion. The reason is that the ischemia-reperfusion injury is accompanied by the abnormal accumulation of reactive oxygen species (ROS), which can cause damage to retinal ganglion cells (RGCs), promote their apoptosis, and finally lead to the irreversible loss of the visual field. RGCs are specialized projection neurons that are situated in the inner retinal surface of the eye, and they transmit visual images into certain areas of the brain in the form of action potentials. Therefore, any damage that affects the viability of RGCs can cause visual field defects or even irreversible vision loss. There is no effective drug treatment in clinical practice for the loss of the visual field that is caused by the oxidation and apoptosis of RGCs. Hence, finding a drug with neuroprotective and antioxidant functions is urgently needed. As a new type of nanomaterial, tetrahedral framework nucleic acids (tFNAs) exhibit outstanding biocompatibility and have been shown in our previous studies to participate in the positive regulation of cell behavior. In this experiment, we first established a cellular model of oxidative stress in RGCs with tert-butyl peroxide (TBHP). Then, we primarily explored the antioxidant and neuroprotective effects of tFNAs after TBHP-induced oxidative stress and the main mechanisms by which the tFNAs function. Our research showed that tFNAs could reduce the production of reactive oxygen species (ROS) in cells and protect the cells from oxidative stress by regulating intracellular oxidation-related enzymes. In addition, tFNAs could simultaneously improve oxidative stress-induced apoptosis significantly via affecting the expression of apoptosis-related proteins. Finally, we confirmed by western blotting that the mechanism by which tFNAs prevent damage caused by oxidative stress involves activating the Akt/Nrf2 pathway. Our findings provide new ideas for the prevention and treatment of a series of diseases that are caused by oxidative stress to RGCs.

Topics: Animals; Antioxidants; Carbocyanines; Cell Line; Cell Survival; DNA, Single-Stranded; L-Lactate Dehydrogenase; Mice; Microscopy, Fluorescence; Nanostructures; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidative Stress; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Reperfusion Injury; Retinal Ganglion Cells; Signal Transduction; Superoxide Dismutase; tert-Butylhydroperoxide

Microglial activation and associated neuroinflammation play a key role in the pathogenesis of many diseases of the retina, including viral infection, diabetes, and retinal degeneration. Strategies to target activated microglia and macrophages and attenuate inflammation may be valuable in treating these diseases. We seek to develop dendrimer-based formulations that target retinal microglia and macrophages in a pathology-dependent manner, and deliver drugs, either intravenously or intravitreally.. Retinal uptake of cyanine dye (Cy5)-conjugated dendrimer (D-Cy5) was assessed in normal and ischemia/reperfusion (I/R) mouse eyes. Microglia/macrophage uptake of the dendrimer was assessed with immunofluorescence using rabbit Iba-1 antibody with Cy3-tagged secondary antibody (microglia/macrophage). Uptake in retina and other organs was quantified using fluorescence spectroscopy.. Clearance of D-Cy5 from normal eyes was almost complete by 72 hours after intravitreal injection and 24 hours after intravenous delivery. In eyes with activated microglia after I/R injury, D-Cy5 was retained by activated microglia/macrophage (Iba1+ cells) up to 21 days after intravitreal and intravenous administration. In I/R eyes, the relative retention of intravitreal and intravenous D-Cy5 was comparable, if a 30-fold higher intravenous dose was used.. Intravitreal and systemic dendrimers target activated microglia and show qualitatively similar retinal biodistribution when administered by either route. Results provide proof-of-concept insights for developing dendrimer drug formulations as treatment options for retinal diseases associated with microglia or macrophage activation such as age-related macular degeneration, diabetic retinopathy, and retinal degenerations.

Topics: Animals; Carbocyanines; Dendrimers; Immunohistochemistry; Injections, Intravenous; Intravitreal Injections; Macrophages; Male; Mice; Mice, Inbred BALB C; Microglia; Microscopy, Confocal; Nylons; Reperfusion Injury; Retinal Diseases; Retinal Neurons; Tissue Distribution

The diagnostic utility of the apoptosis-sensing nanoparticle (NP), AnxCLIO-Cy5.5, is well established. Here we sought to define the pathophysiological impact of the nanoparticle (NP) on apoptotic cells. Confocal microscopy showed that AnxCLIO-Cy5.5 remained bound to apoptotic cell membranes for 3 hours but by 7 hours had become completely internalized. AnxCLIO-Cy5.5 exposure did not impact energetics, metabolism or caspase-3 activity in apoptotic cells. Gene expression in cells exposed to AnxCLIO-Cy5.5 did not reveal upregulation of pro-inflammatory or cell-death pathways. Moreover, exposure to AnxCLIO-Cy5.5 decreased the frequency of membrane rupture of early apoptotic cells. Similarly, in mice exposed to 1 hour of ischemia -reperfusion, the injection of AnxCLIO-Cy5.5 at the onset of reperfusion reduced infarct size/area at risk by 16.2%. Our findings suggest that AnxCLIO-Cy5.5 may protect apoptotic cells by stabilizing their cell membranes and has the potential to become a theranostic agent, capable of both identifying and salvaging early apoptotic cells. From the Clinical Editor: This study demonstrates that AnxCLIO-Cy5.5 nanoparticles may protect apoptotic cells by cell membrane stabilization and have the potential to become a "theranostic agent" capable of identifying and salvaging early apoptotic cells.

Topics: Animals; Annexins; Apoptosis; Carbocyanines; Cell Membrane; CHO Cells; Cricetinae; Cricetulus; Energy Metabolism; Flow Cytometry; Gene Expression Profiling; Humans; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Nanoparticles; Protective Agents; Reperfusion Injury

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

Our recent studies have shown that ischemia/reperfusion (I/R) produces significant necrosis and apoptosis in the cells of skeletal muscle. Our previous studies also demonstrated that melatonin provides significant protection against superoxide generation, endothelial dysfunction, and cell death in the skeletal muscle after I/R. Mitochondria are essential for cell survival, because of their roles as ATP producers as well as regulators of cell death. However, the efficacy of melatonin on I/R-induced mitochondrial dysfunction in the skeletal muscle in vivo has not been demonstrated in the literature.. Vascular pedicle isolated rat gracilis muscle model was used. After 4 h of ischemia followed by 24 h of reperfusion, gracilis muscle was harvested, and mitochondrial as well as cytosolic fractions were isolated. Mitochondrial dysfunction was determined by the alteration of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c. Three groups were designed; sham I/R, I/R-V (I/R with vehicle), and I/R-Mel (I/R with melatonin). Melatonin or vehicle was given intravenously 10 min prior to reperfusion and 10 min after reperfusion.. We found that the capability of uptake of fluorescent JC-1 dye in skeletal muscle cells was substantially improved in I/R-Mel group compared with I/R-V group. Melatonin significantly inhibited the outflow of cytochrome c from mitochondria to cytoplasm, which was demonstrated in the I/R-V group.. Melatonin significantly attenuates I/R-induced mitochondrial dysfunction, such as the depolarization of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c, from the mitochondria.

Topics: Animals; Antioxidants; Benzimidazoles; Carbocyanines; Cytochromes c; Fluorescent Dyes; Male; Melatonin; Membrane Potential, Mitochondrial; Mitochondria, Muscle; Muscle, Skeletal; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxides

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

The objective of this study is to design a fluorescent imaging agent with R-Gel, one of the recombinant polymers (RCP), for renal inflammation. The R-Gel based on human type I collagen has multiple Arg-Gly-Asp (RGD) motifs which are ligands for some types of integrin receptors on the cell surface. After intravenous administration of R-Gel labeled by Cy7 of a fluorescent dye to three animal models of nephritis mousse, interstitial nephritis (by using UUO model mice), glomerulonephritis (HIGA mice), and ischemia-reperfusion injured kidney (I/R mice), the extent of fluorescent imaging at the renal inflammation was assessed. The Cy7-labeled R-Gel was accumulated in the inflammation site to a significantly greater extent than in the normal one at 24h after administration. The renal pattern of fluorescent imaging was similar to that of administration anti-Mac1 antibody. Taken together, it is conceivable that the R-Gel was targeted to macrophages infiltrated into the inflammation site of kidney.

Topics: Animals; Carbocyanines; Collagen Type I; Epithelial Cells; Fluorescence; Fluorescent Dyes; Humans; Kidney; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Nephritis; Oligopeptides; Reperfusion Injury

Some photosensitizing cyanine dyes act on the immune system to enhance the phagocytic capacity of macrophages. In this study, we examined whether these dyes have neurotrophin-like activities and neuroprotective effects in vitro and in vivo. By screening more than 250 cyanine dyes, we found that NK-4 and NK-150, which belong to a group of pentamethine trinuclear cyanine dyes, significantly potentiated nerve growth factor (NGF)-primed neurite outgrowth of PC12HS cells in nanomolar to micromolar concentrations. Both NK-4 and NK-150 showed a remarkable hydroxyl radical-scavenging activity using an in vitro electron spin resonance (ESR)-based technique. They also effectively scavenged peroxy radicals, and in addition, NK-4 acted on superoxides to a similar extent as ascorbate. In vivo, NK-4 and NK-150 prevented cerebral ischemic injury induced by 2 h middle cerebral artery occlusion (MCAO) and 24 h reperfusion in rats. Dyes were intravenously administrated twice 1 h after the occlusion and immediately after the start of reperfusion. NK-4 and NK-150 (100 µg/kg) reduced cerebral infarct volumes by 57.0% and 46.0%, respectively. Those dyes also decreased brain swelling in the ischemic semispheres. As a result, administration of NK-4 and NK-150 provided substantial improvements in MCAO-induced neurological deficits in a dose-dependent manner. These results suggest that NK-4 and NK-150 effectively prevented ischemia-induced brain injury through their potent neurotrophin-like activity as well as antioxidative activity.

Topics: Animals; Antioxidants; Brain; Brain Ischemia; Carbocyanines; Cell Line; Cerebral Infarction; Dose-Response Relationship, Drug; Edema; Electron Spin Resonance Spectroscopy; Infarction, Middle Cerebral Artery; Male; Nerve Growth Factor; Neurites; Neuroprotective Agents; Quinolines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

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

Ischemia-reperfusion injury is a major complication occurring in acute myocardial infarction, cardiopulmonary bypass surgery, and heart transplantation. The aim of this study was to identify proteins that were involved in ischemia-reperfusion injury using fluorescence two-dimensional difference gel electrophoresis. We compared the 100,000 x g precipitate fractions of normal, ischemic and ischemia-reperfused rat hearts and detected six spots which changed more than two-fold in expression level and two additional spots related to these spots. Using peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, we identified five of these spots as protein disulfide isomerase A3 (PDA3), one as 60 kDa heat shock protein (HSP60) and two as elongation factor Tu (EF-Tu). HSP60 was increased during ischemia and decreased to normal expression level after reperfusion. EF-Tu was increased in ischemia but not decreased by reperfusion. We also found that several protein spots of PDA3 shifted towards a higher isoelectric point in ischemia and ischemia-reperfusion. Our data strongly suggested that PDA3 underwent dephosphorylation during ischemia and reperfusion and serine 343 of PDA3 was one of the phosphorylation sites.

Topics: Animals; Binding Sites; Carbocyanines; Chaperonin 60; Databases as Topic; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Image Processing, Computer-Assisted; Ischemia; Male; Microscopy, Fluorescence; Myocardium; Peptide Elongation Factor Tu; Phosphorylation; Protein Disulfide-Isomerases; Proteome; Rats; Rats, Wistar; Reperfusion Injury; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions