carbocyanines and Nerve-Degeneration

Neurodegeneration and gliosis are prominent pathological features of subjects with human immunodeficiency virus (HIV) dementia complex (HAD). In these patients, neurodegeneration occurs in uninfected neurons. In addition, these patients develop sensory neuropathy despite the antiretroviral therapy. The HIV protein gp120, which mimics some of the pathological alterations seen in HAD, is retrogradely transported in rodent neurons. However, it is still unclear whether gp120 can also be transported anterogradely and whether axonal transport can occur in the peripheral nervous system (PNS). To determine whether gp120 is transported retrogradely and/or anterogradely, we injected gp120IIIB together with the retrograde tracer fluoro-ruby (FR) or the anterograde tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyamine perchlorate (DiI) into the rat superior colliculi. We discovered that gp120 is retrogradely transported with FR along a direct pathway from the superior colliculus to the retina and anterogradely transported with DiI to several areas of the occipital cortex. To determine whether gp120 is also axonally transported in the peripheral nerves, gp120 and FR were injected into the sciatic nerve. No gp120 immunoreactivity was found in the sciatic nerve or dorsal root ganglia, suggesting that gp120 axonal transport does not occur in the PNS. Gp120 axonal transport may play a role in neuronal injury. Therefore, we examined apoptosis at various time points after gp120 injection. Activated caspase-3 was evident within neurons transporting gp120. These results indicate that axonal transport of gp120 might exacerbate the pathogenesis of HIV-1.

Topics: Analysis of Variance; Animals; Apoptosis; Axonal Transport; Blotting, Western; Carbocyanines; Caspase 3; Dextrans; HIV Envelope Protein gp120; Immunohistochemistry; Male; Microscopy, Confocal; Nerve Degeneration; Nervous System; Neural Pathways; Occipital Lobe; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Rhodamines; Sciatic Nerve; Superior Colliculi

Accumulating evidence indicates that the polyphenol resveratrol (trans-3, 5, 4"-trihydroxystibene, RVT) potently protects against cerebral ischemia neuronal damage due to its oxygen free radicals scavenging and antioxidant properties. However, it is unknown whether RVT can attenuate ischemia-induced early impairment of neuronal excitability. To address this question, we simulated ischemic conditions by applying oxygen-glucose deprivation (OGD) to acute rat hippocampal slices and examined the effect of RVT on OGD-induced pyramidal neuron excitability impairment using whole-cell patch clamp recording. 100 microM RVT largely inhibited the 15 min OGD-induced progressive membrane potential (Vm) depolarization and the reduction in evoked action potential frequency and amplitude in pyramidal neurons. In a parallel neuronal viability study using TO-PRO-3 iodide staining, 20 min OGD induced irreversible CA1 pyramidal neuronal death which was significantly reduced by 100 microM RVT. No similar effects were found with PQQ treatment, an antioxidant also showing potent neuroprotection in the rat rMCAO ischemia model. This suggests that antioxidant action per se, is unlikely accounting for the observed early effects of RVT. RVT also markedly reduced the frequency and amplitude of AMPA mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons, which is also an early consequence of OGD. RVT effects on neuronal excitability were inhibited by the large-conductance potassium channel (BK channel) inhibitor paxilline. Together, these studies demonstrate that RVT attenuates OGD-induced neuronal impairment occurring early in the simulated ischemia slice model by enhancing the activation of BK channel and reducing the OGD-enhanced AMPA/NMDA receptor mediated neuronal EPSCs.

Topics: Action Potentials; Animals; Antioxidants; Carbocyanines; Cell Death; Hippocampus; Hypoxia-Ischemia, Brain; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Nerve Degeneration; Organ Culture Techniques; Potassium Channel Blockers; Pyramidal Cells; Rats; Resveratrol; Stilbenes; Time Factors

Glaucoma is a major cause of vision impairment, which arises from the sustained and progressive apoptosis of retinal ganglion cells (RGC), with ocular hypertension being a major risk or co-morbidity factor. Because RGC death often continues after normalization of ocular hypertension, growth factor-mediated protection of compromised neurons may be useful. However, the therapeutic use of nerve growth factor (NGF) has not proven effective at delaying RGC death in glaucoma. We postulated that one cause for the failure of NGF may be related to its binding to two receptors, TrkA and p75. These receptors have distinct cellular distribution in the retina and in neurons they induce complex and sometimes opposing activities. Here, we show in an in vivo therapeutic model of glaucoma that a selective agonist of the pro-survival TrkA receptor was effective at preventing RGC death. RGC loss was fully prevented by combining the selective agonist of TrkA with intraocular pressure-lowering drugs. In contrast, neither NGF nor an antagonist of the pro-apoptotic p75 receptor protected RGCs. These results further a neurotrophic rationale for glaucoma.

Topics: Animals; Carbocyanines; Cell Death; Cell Survival; Female; Glaucoma; Intraocular Pressure; Nerve Degeneration; Nerve Growth Factor; Neuroprotective Agents; Peptides; Rats; Rats, Wistar; Receptor, Nerve Growth Factor; Receptor, trkA; Retinal Degeneration; Retinal Ganglion Cells; Stilbamidines

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

Human amniotic epithelial cells (HAEC) possess certain properties similar to that of neural and glial cells. In the present work, the potential of HAEC as stem cells for spinal cord injury repair was tested. HAEC obtained from human placenta were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyllindocarbocyanine perchlorate (Dil) in the culture medium. These labeled cells were transplanted into the transection cavities in the spinal cord of bonnet monkeys. Results were analyzed after 15 and 60 days of post-transplantation. HAEC cells survived in the monkey spinal cord for up to the maximum period of observation in the present study, i.e. 60 days. HAEC graft was penetrated by the host axons. There was no glial scar at the transection lesion site. Some of the host spinal neurons and axons were labeled with Dil (used to label HAEC) whereas in lesion control group, there was no such host-neuron labeling. This may be either due to the prevention of death in the axotomized neuron's ensuing lesion or due to the neurotrophic effect exhibited by the transplanted HAEC. Further studies would be required to verify these speculations. Therefore from this pilot study it appears that HAEC survive in the transplanted environment, support the growth of host axons through them, prevent the formation of glial scar at the cut ends and may prevent death in axotomized cells or attract the growth of new collateral sprouting. The abovementioned properties, i.e. serving as a suitable milieu for the host axons to grow, preventing glial scar at the lesion site and rescuing axotomized neurons from death were previously reported in the case of neural transplantation studies. Thus it is speculated that HAEC may be having certain properties equal to the beneficial effects of neural tissue in repairing spinal cord injury. Apart from this speculation, there are two more reasons for why HAEC transplantation studies are warranted to understand the long-term effects of such transplantations. First, there was no evidence of immunological rejection probably due to the non-antigenic nature of the HAEC. Second, unlike neural tissue, procurement of HAEC does not involve many legal or ethical problems.

Topics: Amnion; Animals; Carbocyanines; Cell Communication; Cell Differentiation; Cells, Cultured; Epithelial Cells; Female; Gliosis; Graft Survival; Growth Cones; Humans; Macaca radiata; Nerve Degeneration; Nerve Regeneration; Neuroglia; Neurons; Pregnancy; Spinal Cord Injuries; Stem Cell Transplantation; Transplantation, Heterologous; Treatment Outcome

Axonal injury to CNS neurons results in apoptotic cell death. The processes by which axotomy signals apoptosis are diverse, and may include deprivation of target-derived factors, induction of injury factors, bursts of reactive oxygen species (ROS), and other mechanisms. Our previous studies demonstrated that death of a dissociated retinal ganglion cell, an identified CNS neuron, is ROS-dependent. To better define the mechanisms by which ROS induce retinal ganglion cell death after axotomy, we studied their effects in dissociated neonatal rat retinal cultures. Postnatal day 2-4 Long-Evans rat retinal ganglion cells were retrogradely labeled with the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). Postnatal day 7-9 retinas were dissociated and cultured in the presence of specific ROS generating systems, scavengers, or redox modulators. Retinal ganglion cells were identified by DiI positivity and viability determined by metabolism of calcein-acetoxymethyl ester. We found that ROS scavengers protected against retinal ganglion cell death after acute dissociation, and the effects of ROS appeared to be due to shifts in the redox potential, as retinal ganglion cell survival was critically dependent on redox state, with greatest survival under mildly reducing conditions. Culture of retinal ganglion cell with the non-thiol-containing reducing agent tris(carboxyethyl)phosphine resulted in long-term survival equivalent to or better than with neurotrophic factors. Our data suggest that axotomy-associated neuronal death induced by acute dissociation may be partly dependent on ROS production, acting to shift the redox state and oxidize one or more key thiols. Understanding the mechanisms by which ROS signal neuronal death could result in strategies for increasing their long-term survival after axonal injury.

Topics: Animals; Animals, Newborn; Apoptosis; Carbocyanines; Cell Survival; Cells, Cultured; Dithionitrobenzoic Acid; Dithiothreitol; Enzyme Inhibitors; Fluorescent Dyes; Indicators and Reagents; Nerve Degeneration; Nerve Growth Factors; Phosphines; Rats; Rats, Long-Evans; Reactive Oxygen Species; Retinal Ganglion Cells; Sulfhydryl Compounds; Sulfhydryl Reagents

A technique has been developed for cutting single nerve fibers in mammalian spinal cord. In the presence of diaminobenzidine (DAB), a laser microbeam was applied to carbocyanine (Dil) stained sensory fibers in cultured spinal cords of the newly born opossum Monodelphis domestica. Digital images of fluorescent fibers were acquired with an intensified video CCD-camera coupled to an image processor. Laser illumination of two spots on a fiber in the presence of 3 mg/ml DAB cut it, so that following DAB wash out, Dil fluorescence did not return after the intermediate segment was bleached. In contrast, when a similar procedure was carried out without DAB, fluorescence of the bleached segment was recovered within minutes in darkness, by dye diffusion from adjacent regions of the uncut fiber. After exposure to DAB, through-conduction of compound action potentials continued in undamaged fibers. The DAB reaction product remained as a dark precipitate, helping to localize the lesion sites. By illuminating a continuous series of spots it was possible to cut whole nerve roots. Fluorescent fibers extended across the cut segment 24 h later. With minor modifications, the procedure described here allows a precise lesioning of single fibers within an intact nervous system.

Topics: 3,3'-Diaminobenzidine; Action Potentials; Animals; Animals, Newborn; Axons; Axotomy; Carbocyanines; Female; Fluorescent Dyes; Ganglia, Spinal; Lasers; Nerve Degeneration; Nerve Regeneration; Opossums; Organ Culture Techniques; Photic Stimulation; Photochemistry; Spinal Cord; Spinal Nerve Roots

The inability of axons to grow across damaged central nervous system tissue is a well-known consequence of injury to the brain and spinal cord of adult mammals. Our previous studies showed that predegenerated peripheral nerve grafts facilitate neurite outgrowth from the injured hippocampus and that this effect was particularly distinct when 7-, 28-, and 35-day-predegenerated nerve grafts were used. The purpose of the present study was to use the above method to induce and support the regrowth of injured nerve fibers as well as the survival of retinal ganglion cells (RGCs). Adult Sprague-Dawley rats were assigned to three groups. In the experimental groups transected optic nerve was grafted with peripheral nerve (predegenerated for 7 days (PD) or nonpredegenerated). In the control group, the optic nerve was totally transected. RGCs and growing fibers labeled with fluorescent tracers were examined. They were counted and the results were subjected to statistical analysis. Retinal ganglion cells survived in the groups treated with predegenerated as well as nonpredegenerated grafts; however, the number of surviving retinal ganglion cells was significantly higher in the first one. In both groups the regrowth of the transected optic nerve was observed but the distance covered by regenerating fibers was longer in the PD group. No fibers inside grafts and no labeled cells in retinas were present in the control animals. On the basis of the obtained results we can state that the predegeneration of grafts enhance their neurotrophic influence upon the injured retinal ganglion cells.

Topics: Animals; Axotomy; Carbocyanines; Cell Count; Cell Division; Cell Survival; Fluorescent Dyes; Male; Nerve Degeneration; Nerve Regeneration; Optic Nerve; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; Rhodamines

Retinal cell death induced by over-stimulation of glutamate receptors is related to the programmed cell death or apoptosis. However, little is known about the intracellular events that lead to this cell death process in the retina. In this study, we asked if caspase-3 family cysteine proteases regulate cell death in an explant culture of adult rat retina after exposure to excessive glutamate. Cells with DNA fragmentation were first detected in the ganglion cell layer 3 h after a brief exposure to 20 mM glutamate; whilst those in the inner nuclear layer were first observed 6 h after the glutamate lesion. Caspase-3-like activity, as indicated by immunostaining of the fractin antibody that recognizes actin fragments generated by caspase-3 family proteases, was seen 40 min after glutamate treatment. Staining was first detected in the ganglion cell layer and then in the inner nuclear layer, preceding the appearance of cells with DNA fragmentation in these layers. Colocalization study showed that all cells with DNA breaks were fractin positive, indicating that caspase-3 family activity was involved in the glutamate-induced cell death in the adult rat retina. Furthermore, DEVD-CHO, a tetrapeptide inhibitor for caspase-3 family members, reduced dramatically the fractin staining and significantly alleviated glutamate-induced cell death and DNA fragmentation in the ganglion cell layer and inner nuclear layer. Inhibitor for caspase-1-like activity, YVAD-CHO, neither reduced the fractin staining nor showed comparable neuroprotective effects to the retina. We conclude that glutamate-induced apoptotic cell death in adult rat retina is mediated by a specific activation of cysteine proteases related to the caspase-3 family, and an intervention to the caspase-3 proteases provides effective protection to retinal neurons against glutamate excitotoxicity.

Topics: Animals; Apoptosis; Carbocyanines; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; DNA Fragmentation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Fluorescent Dyes; Glutamic Acid; In Situ Nick-End Labeling; Nerve Degeneration; Oligopeptides; Rats; Rats, Sprague-Dawley; Retina; Retinal Ganglion Cells

Cholinergic neurons of the basal forebrain form one of the neuron populations that are susceptible to excitotoxic injury. Whereas neuropharmacological studies have aimed at rescuing cholinergic neurons from acute excitotoxic attacks, the short-term temporal profile of excitotoxic damage to cholinergic nerve cells remains largely elusive. The effects of N-methyl-D-aspartate (NMDA) infusion on cytochemical markers of cholinergic neurons in rat magnocellular nucleus basalis were therefore determined 4, 24 and 48 h post-lesion. Additionally, the influence of excitotoxic damage on the efficacy of in vivo labelling of cholinergic neurons with carbocyanine 3-192IgG was investigated. Carbocyanine 3-192IgG was unilaterally injected in the lateral ventricle. Twenty-four hours later, NMDA (60 nM/microl) was infused in the right magnocellular nucleus basalis, while control lesions were performed contralaterally. Triple immunofluorescence labelling for carbocyanine 3-192IgG, NMDA receptor 2A and B subunits and choline-acetyltransferase (ChAT) was employed to determine temporal changes in NMDA receptor immunoreactivity on cholinergic neurons. The extent of neuronal degeneration was studied by staining with Fluoro-Jade. Moreover, changes in the numbers of ChAT or p75 low-affinity neurotrophin receptor immunoreactive neurons, and the degree of their co-labelling with carbocyanine 3-192IgG were determined in basal forebrain nuclei. The effects of NMDA-induced lesions on cortical projections of cholinergic nucleus basalis neurons were studied by acetylcholinesterase (AChE) histochemistry. Characteristic signs of cellular damage, as indicated by decreased immunoreactivity for NMDA receptors, ChAT and p75 low-affinity neurotrophin receptors, were already detected at the shortest post-lesion interval investigated. Fluoro-Jade at 4 h post-lesion only labelled the core of the excitotoxic lesion. Longer survival led to enhanced Fluoro-Jade staining, and to the decline of ChAT immunoreactivity reaching a maximum 24 h post-surgery. Significant loss of p75 low-affinity neurotrophin receptor immunoreactivity and of cortical AChE-positive projections only became apparent 48 h post-lesion. Carbocyanine 3-192IgG labelling in the ipsilateral basal forebrain exceeded that of the contralateral hemisphere at all time points investigated and progressively declined in the damaged magnocellular nucleus basalis up to 48 h after NMDA infusion. The present study indicates that excitotoxic lesion

Topics: Acetylcholine; Animals; Basal Nucleus of Meynert; Carbocyanines; Choline O-Acetyltransferase; Excitatory Amino Acid Agonists; Fluoresceins; Fluorescent Dyes; Immunoglobulin G; Immunohistochemistry; Injections, Intraventricular; Male; Microscopy, Confocal; N-Methylaspartate; Nerve Degeneration; Neural Pathways; Neuroglia; Neurons; Neurotoxins; Organic Chemicals; Rats; Rats, Wistar; Receptor, Nerve Growth Factor; Receptors, N-Methyl-D-Aspartate; Sensitivity and Specificity

Both antennal receptor cell axons and uniglomerular projection neurons of the antennal lobe were specifically labeled, and their synaptic relationship was studied at the fine structural level. The labelings were applied in different combinations: i) Experimentally induced anterograde degeneration of sensory-afferent axons was combined with injection of horseradish peroxidase into uniglomerular projection neurons. ii) Lucifer Yellow was injected into uniglomerular projection neurons, and receptor cell axons were anterogradely labeled with the lipophilic dye DiI. The fluorescent dyes were transformed by immuno- or photochemical treatment into electron-dense markers. In both types of preparations, a considerable number of monosynaptic output synapses from antennal receptor neurons onto processes of uniglomerular projection neurons were identified within the glomeruli of the lobe. In most cases, the receptor axon was connected in a dyadic fashion firstly to a process of a projection neuron and secondly to a nonlabeled process. The results clearly demonstrate a direct connection between receptor cells and output neurons of the cockroach antennal lobe which exists in parallel to the already proposed and demonstrated polysynaptic connection via inhibitory local interneurons.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Male; Microscopy, Electron; Nerve Degeneration; Neural Pathways; Neurons; Olfactory Receptor Neurons; Periplaneta; Sense Organs; Signal Transduction; Synapses

The present work was undertaken to assess the fate of ganglion cell debris in the axotomized retina of adult rats and employed a new technique to label phagocytosing microglia via the internalized material. In the main experiment, transection axotomy was performed on the intraorbital segment of the optic nerve, and a fast-transported, vital fluorescent styryl dye (4Di-10ASP) was deposited at the ocular stump of the nerve in order to pre-label retrogradely the ganglion cells destined to die because of the axotomy. Optic nerve transection resulted in progressive degradation of ganglion cell axons, perikarya, and dendrites within the retina and in release of fluorescent material, which was then incorporated into cells identified as microglia. No other retinal cells stained, although astrocytes and Müller's cells also responded to neuron degeneration by accumulating glial fibrillary acidic protein. Incorporation of labelled material into microglia topo-chronologically paralleled the ganglion cell degeneration starting within the optic fibre layer (OFL) and proceeding towards the ganglion cell layer (GCL) and the inner plexiform layer (IPL) of the affected retina. Long-term labelling of microglia monitored up to 3 months after optic nerve transection indicated that labelled microglial cells persisted within the retina. Microglia displayed a strong territorial arrangement within the GCL and IPL, and staggered, bilaminated distribution in both layers. These studies directly prove that microglia in the retina can be transcellularly labelled during traumatic degeneration of ganglion cells. The findings suggest that microglial cells play an important role in axotomy-induced wound healing and removal of cell debris.

Topics: Animals; Carbocyanines; Cell Count; Dendrites; Female; Fluorescent Dyes; Nerve Degeneration; Optic Nerve; Phagocytosis; Rats; Rats, Inbred Strains; Retinal Ganglion Cells; Staining and Labeling; Thiamine Pyrophosphatase; Time Factors

The present work employed a new technique for labelling phagocytizing microglia in the axotomized retinal of adult rats. Transection axotomy was performed within the intraorbital segment of the optic nerve, and the fast-transported, vital fluorescent carbocyanine dyes DiI and 4Di-10ASP were deposited at the ocular stump of the nerve in order to retrogradely prelabel the ganglion cells which were destined to die. Optic nerve transection resulted in progressive degradation of ganglion cell axons, perikarya and dendrites within the retina and in release of fluorescent material which was then incorporated into cells identified as microglia but not into other cells of the retina. Incorporation of labelled material into microglia occurred only when the ganglion cells degenerated and not when the non-lesioned ganglion cells were labelled from the superior colliculus. Double-staining of microglia with both dyes helped to compare the pattern of labelling for each dye. After progression of ganglion cell degeneration, microglia displayed a staggered, bilaminated distribution within the ganglion cell layer and within the inner plexiform layer. Fluorescent microglia were not found within the deeper layers of the retina indicating that transneuronal degeneration and subsequent labelling of microglial cells do not occur. The results show that one major function of microglia within the ganglion cell and inner plexiform layers of the lesioned retina is to remove debris produced after degradation of neurons.

Topics: Animals; Carbocyanines; Denervation; Female; Microscopy, Fluorescence; Nerve Degeneration; Neuroglia; Optic Nerve; Rats; Rats, Inbred Strains; Retina; Retinal Ganglion Cells