Peptidergic innervation in the rat carotid body after 2, 4, and 8 weeks of hypocapnic hypoxic exposure

The distribution and abundance of neuropeptide-containing nerve fibers were examined in the carotid bodies of rats exposed to hypocapnic hypoxia (10% O2 in N2) for 2, 4, and 8 weeks. The carotid bodies after 2, 4, and 8 weeks of hypoxic exposure were enlarged by 1.2-1.5 times in the short axis, and 1.3-1.7 times in the long axis in comparison with the normoxic control ones. The enlarged carotid bodies contained a number of expanded blood vessels. Mean density per unit area (10(4) microm2) of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive fibers was transiently high in the carotid bodies after 4 weeks of hypoxic exposure, and decreased significantly to nearly or under 50% after 8 weeks of hypoxic exposure. Density of vasoactive intestinal polypeptide (VIP) immunoreactive fibers increased significantly in all periods of hypoxic exposure observed, and was especially high in the carotid bodies after 4 weeks of hypoxic exposure. Density of neuropeptide Y immunoreactive fibers was unchanged in the carotid bodies during hypoxic exposure. These characteristic changes in the density of SP, CGRP, and VIP fibers in the carotid bodies after 4 weeks of hypoxic exposure suggest that the role of these neuropeptide-containing fibers may be different in the carotid bodies after each of three periods of hypoxic exposure, and that the peptidergic innervation after 8 weeks of hypoxic exposure may show an acclimatizing state.     

Nicotinamide attenuates retinal ischemia and light insults to neurones

The aim of the present studies was to determine whether nicotinamide is effective in blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of light to transformed retinal ganglion cells (RGC-5 cells) in culture. Ischemia was delivered to the retina of one eye of rats by raising the intraocular pressure. Nicotinamide was administered intraperitoneally just before ischemia and into the vitreous immediately after the insult. Electroretinograms (ERGs) of both eyes were recorded before and 5 days after ischemia. Seven days after ischemia, retinas were analysed for the localization of various antigens. Retinal and optic nerve extracts were also prepared for analysis of specific proteins and mRNAs. Also, RGC-5 cells in culture were given a light insult (1000 lux, 48 and 96 h) and evidence for reduced viability and apoptosis determined by a variety of procedures. Nicotinamide was added to some cultures to see whether it reversed the negative effect of light. Ischemia/reperfusion to the retina affected the localization of Thy-1, neuronal nitric oxide synthase (NOS) and choline acetyltransferase (ChAT), the a- and b-wave amplitudes of the ERG, the content of various retinal and optic nerve proteins and mRNAs. Significantly, nicotinamide statistically blunted many of the effects induced by ischemia/reperfusion which included the activation of poly-ADP-ribose polymerase (PARP). Light-induced apoptosis of RGC-5 cells in culture was attenuated by nicotinamide and the PARP inhibitor NU1025. The presented data show that nicotinamide attenuates injury to the retina and RGC-5 cells in culture caused by ischemia/reperfusion and by light, respectively. Evidence is provided to suggest that nicotinamide acts as a PARP inhibitor and possibly an antioxidant.     

HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration

Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1alpha (HIF-1alpha) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.     

Morphological changes in the rat carotid body 1, 2, 4, and 8 weeks after the termination of chronically hypocapnic hypoxia

Morphological changes in the rat carotid bodies 1, 2, 4, and 8 weeks after the termination of chronically hypocapnic hypoxia (10% O2 for 8 weeks) were examined by means of morphometry and immunohistochemistry. The rat carotid bodies after 8 weeks of hypoxic exposure were enlarged several fold with vascular expansion. The carotid bodies 1 and 2 weeks after the termination of 8 weeks of hypoxic exposure were diminished in size, although their diameter remained larger than the normoxic controls. The expanded vasculature in chronically hypoxic carotid bodies returned to the normoxic control state. In the carotid bodies 1 week after the termination of chronic hypoxia, the density of NPY fibers was remarkably increased and that of VIP fibers was dramatically decreased in comparison with the density in chronically hypoxic carotid bodies. In the carotid bodies 2 and 4 weeks after the termination of hypoxia, the density of SP and CGRP fibers was gradually increased. In the carotid bodies 8 weeks after the termination of hypoxia, the appearance of the carotid body returned to a nearly normoxic state, and the density of SP, CGRP, VIP, and NPY fibers also recovered to that of normoxic controls. These results suggest that the morphological changes in the recovering carotid bodies start at a relatively early period after the termination of chronic hypoxia, and a part of these processes may be under the control of peptidergic innervation.     

Inhibition of Neutral Sphingomyelinase Activation and Ceramide Formation by Glutathione in Hypoxic PC12 Cell Death

Reduced glutathione (GSH) and N-acetylcysteine (NAC), but not other antioxidative or reducing agents, were found to inhibit cell death, both apoptosis and necrosis, induced by hypoxia in naive and nerve growth factor-differentiated PC12 cells. The level of intracellular total GSH decreased time-dependently during hypoxia, but exogenously added GSH prevented such a decrease in GSH. Pretreatment of cells with exogenous GSH or NAC resulted in inhibition of both neutral sphingomyelinase (SMase) activation and ceramide formation during hypoxia. In the in vitro assay system, neutral SMase activity was inhibited dose-dependently by GSH and NAC. Activation of caspase-3 induced by hypoxia was also inhibited by either GSH or NAC. NAC but not GSH inhibited caspase-3 activation induced by C2-ceramide. These results suggest that GSH protects cells from hypoxic injury by direct inhibition of neutral SMase activity and ceramide formation, resulting in inhibition of caspase-3 activation, and that NAC exerts an additional inhibitory effect(s) downstream of ceramide.     

Changes of superoxide dismutase, glutathione perioxidase and lipid peroxides in the brain of mice preconditioned by hypoxia.

We have developed an animal model of hypoxic preconditioning and assumed that oxygen radicals and their endogenous scavenging enzymes may play an important role in the preconditioning. To test this hypothesis, activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of lipid peroxides (LPO) were measured during the preconditioning. Compared with unpreconditioned control animals, in animals exposed to hypoxia only once, the activities of SOD and GSH-Px in whole brain were found to be significantly decreased while the LPO content in the hippocampus significantly increased. However, those in animals exposed to 4 runs of hypoxia tended to return to control levels and were lower than those in animals exposed to 1 and 2 runs of hypoxia. Our results indicate that the oxygen radicals and their specific scavenging enzymes seem to be involved in the development of tolerance to hypoxia.     

Normal and abnormal pathfinding of facial nerve fibers in the chick embryo.

Development of the facial nerve was studied in normal chicken embryos and after surgical disruption of ingrowing sensory facial nerve fibers at 38-72 h of incubation. Disruption of facial nerve fibers by otocyst removal often induced a rostral deviation of the facial nerve and ganglion to the level of the trigeminal ganglion. Cell bodies of the geniculate ganglion trailed their deviating neurites and occupied an abnormal rostral position adjacent to the trigeminal ganglion. Deviating facial nerve fibers were labeled with the carbocyanine fluorescent tracer DiI in fixed tissue. Labeled fibers penetrated the cranium adjacent to the trigeminal ganglion, but they did not follow the trigeminal nerve fibers into the brain stem. Rather, after entering the cranium, they projected caudally to their usual site of entrance and proceeded towards their normal targets. This rostral deviation of the facial nerve was observed only after surgery at 48-72 h of incubation, but not in cases with early otocyst removal (38-48 h). A rostral deviation of the facial nerve was seen in cases with partial otocyst removal when the vestibular nerve was absent. The facial nerve followed its normal course when the vestibular nerve persisted. We conclude that disruption of the developing facial pathway altered the routes of navigating axons, but did not prevent pathfinding and innervation of the normal targets. Pathfinding abilities may not be restricted to pioneering axons of the facial nerve; later-developing facial nerve fibers also appeared to have positional information. Our findings are consistent with the hypothesis that navigating axons may respond to multiple guidance cues during development. These cues appear to differ as a function of position of the navigating axon.     

Normal and abnormal pathfinding of facial nerve fibers in the chick embryo

Development of the facial nerve was studied in normal chicken embryos and after surgical disruption of ingrowing sensory facial nerve fibers at 38–72 h of incubation. Disruption of facial nerve fibers by otocyst removal often induced a rostral deviation of the facial nerve and ganglion to the level of the trigeminal ganglion. Cell bodies of the geniculate ganglion trailed their deviating neurites and occupied an abnormal rostral position adjacent to the trigeminal ganglion. Deviating facial nerve fibers were labeled with the carbocyanine fluorescent tracer Dil in fixed tissue. Labeled fibers penetrated the cranium adjacent to the trigeminal ganglion, but they did not follow the trigeminal nerve fibers into the brain stem. Rather, after entering the cranium, they projected caudally to their usual site of entrance and proceeded towards their normal targets. This rostral deviation of the facial nerve was observed only after surgery at 48–72 h of incubation, but not in cases with early otocyst removal (38–48 h). A rostral deviation of the facial nerve was seen in cases with partial otocyst removal when the vestibular nerve was absent. The facial nerve followed its normal course when the vestibular nerve persisted. We conclude that disruption of the devloping facial pathway altered the routes of navigating axons, but did not prevent pathfinding and innervation of the normal targets. Pathfinding abilities may not be restricted to pioneering axons of the facial nerve; later-developing facial nerve fibers also appeared to have positional information. Our findings are consistent with the hypothesis that navigating axons may respond to multiple guidance cues during development. These cues appear to differ as a function of position of the navigating axon. © 1992 John Wiley & Sons, Inc.     

GE-25-like immunoreactivity in the rat eye

This study aimed to investigate the presence and distribution of the chromogranin A-derived peptide GE-25 in the rat eye. The molecular form detected by the GE-25 antiserum was evaluated in the rat trigeminal ganglion, retina and remaining tissues of the rat eye by means of Western blots and the distribution pattern of GE-25-like immunoreactivity was studied in the rat eye and rat trigeminal ganglion by immunofluorescence. One single band of approximately 70kDa was stained in the trigeminal ganglion and retina which represents the uncleaved intact chromogranin A indicating that the proteolytic processing of chromogranin A to GE-25 is limited in these tissues. Sparse GE-25-like immunoreactive nerve fibers were visualized in the corneal stroma, at the limbus around blood vessels, in the sphincter and dilator muscle and stroma of the iris, in the stroma of the ciliary body and ciliary processes and in the stroma and around blood vessels in the choroid. This distribution pattern is characteristic for neuropeptides whereas the presence of immunoreactivity in the corneal endothelium and in Müller glia in the retina is atypical. GE-25-like immunoreactivity was found in small to medium-sized ganglion cells in the rat trigeminal ganglion clearly indicating that the nerve fibers in the rat eye are of sensory origin. The colocalization of GE-25-immunoreactivity with SP-immunoreactivity in the rat ciliary body is in agreement with the presumption of the sensory nature of the innervation of the anterior segment of the eye by GE-25.     

PE-11, a peptide derived from chromogranin B, in the rat eye

The aim of the study was to investigate the presence and distribution of PE-11, a peptide derived from chromogranin B, in the rat eye. For this purpose, newborn rats were injected with a single dosage of 50 mg/kg capsaicin subcutaneously under the neck fold and after three months, particular eye tissues were dissected and the concentration of PE-11-like immunoreactivity was determined by radioimmunoassay. Furthermore, PE-11-like immunoreactivities were characterized in an extract of the rat eye by reversed phase HPLC. Then, the distribution pattern of PE-11 was investigated in the rat eye and rat trigeminal ganglion by immunofluorescence. As a result, PE-11 was present in each tissue of the rat eye and capsaicin pretreatment led to a 88.05% (±7.07) and a 64.26% (±14.17) decrease of the levels of PE-11 in the cornea and choroid/sclera, respectively, and to a complete loss in the iris/ciliary body complex. Approximately 70% of immunoreactivities detected by the PE-11 antiserum have been found to represent authentic PE-11. Sparse nerve fibers were visualized in the corneal and uveal stroma, surrounding blood vessels at the limbus, ciliary body and choroid and in association with the dilator and sphincter muscle. Furthermore, immunoreactivity was present in the corneal endothelium. In the retina and optic nerve, glia was labeled. In the rat trigeminal ganglion, PE-11-immunoreactivity was visualized in small and medium sized ganglion cells with a diameter of up to 30 μm. In conclusion, there is unequivocal evidence that PE-11 is a constituent of capsaicin-sensitive sensory neurons innervating the rat eye and the distribution pattern is typically peptidergic in the peripheral innervation but in the retina completely atypical for neuropeptides and unique.     

The flavonoid baicalin counteracts ischemic and oxidative insults to retinal cells and lipid peroxidation to brain membranes

The purpose of the present study was to determine whether the flavonoid, baicalin is effective at blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of various insults to a transformed retinal ganglion cells (RGC-5 cells) in culture. Baicalin was administered intraperitoneally just before and after an ischemic insult to retina of one eye of a rat. Ischemia was delivered by raising the intraocular pressure above the systolic blood pressure for 50min. Seven days after ischemia, retinas were analysed for the localisation of various antigens. Retinal extracts were also analysed for various mRNAs. Moreover, the content of specific proteins was deduced in retinal and optic nerve extracts. Also, RGC-5 cells in culture were given one of three different insults, light (1000lx for 2 days), hydrogen peroxide (200microM H(2)O(2) for 24h) or serum deprivation (48h) where cell survival and reactive oxygen species (ROS) formation was assayed. Moreover, a lipid peroxidation assay was used to compare the antioxidant capacity of baicalin with the flavonoid, epigallocatechin gallate (EGCG). Ischemia/reperfusion to the retina affected the localisation of Thy-1 and choline acetyltransferase (ChAT) and the content of various proteins (optic nerve and retina) and mRNAs (retina). Importantly, baicalin statistically blunted most of the effects induced by ischemia/reperfusion. Only the increase in caspase-8 and caspase-3 mRNAs caused by ischemia/reperfusion were unaffected by baicalin treatment. Baicalin also attenuated significantly the negative insult of light, hydrogen peroxide and serum withdrawal to RGC-5 cells. In the lipid peroxidation studies, baicalin was also found to be equally effective as EGCG to act as an antioxidant. Significantly, the negative insult of serum withdrawal on RGC-5 cell survival was blunted by baicalin but not by EGCG revealing the different properties of the two flavonoids.     

Oxidative Stress in Rat Retina and Hippocampus after Chronic MDMA (‘ecstasy’) Administration

The effects of MDMA administration on oxidative stress markers in rat eye and hippocampus, and the neuroprotective effects of the antioxidant 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6) have been studied. MDMA effects on liver were used for comparison with those in eye and hippocampus and to test CR-6 protective effects. Another goal was to test for apoptosis in retinal cells, as it is known that happens in liver and brain. After 1 week of ecstasy administration, malondialdehyde (MDA) concentration increased, glutathione peroxidase (GPx) activity and glutathione (GSH) content decreased in liver, as previously described. MDA concentration increased and GPx activity decreased in hippocampus; whereas no change was observed in GSH concentration. MDMA decreased ocular GSH concentration and GPx activity; no change was observed in MDA concentration. The number of TUNEL-positive nuclei increased significantly in rat retinas after 1 week of MDMA administration. CR-6 normalized the modifications in liver, hippocampus and retina mentioned above.     

The effects of hypoxia and paraquat on the superoxide dismutase activity in different organs of carp, Cyprinus carpio L.

The effects of hypoxia and paraquat (PQ) were investigated on the superoxide dismutase (SOD) activity in carp gill, liver and brain. Increases in SOD activity occurred in these organs following exposure to both PQ and hypoxia. PQ administration under hypoxic conditions significantly increased the SOD activity in the gill as compared to the level in animals in an hypoxic state without PQ treatment. No such change was observed in the liver or brain.     

Effects of pyridoxine supplementation on blood glucocorticoids level, aspartate aminotransferase activity and glucose tolerance pattern under acute hypoxic stress

Studies were conducted on male adult rabbits to find out the changes in blood glucocorticoid levels along with the changes in aspartate aminotransferase activity in blood and the role of pyridoxine on the glucose tolerance pattern under hypoxic stress. Hypoxic stress was produced by exposing the animals to a simulated altitude of 7,000 m for 6 h. In the first set of experiments 10 rabbits were used. Blood haemoglobin level, plasma and erythrocyte glucocorticoid levels and erythrocyte GOT activity were measured just before and after the exposure to hypoxia. Erythrocyte GOT activity was measured both without and with 50 mg of pyridoxal phosphate addition to the incubation mixture. Glucocorticoid levels in plasma increased by 11% whereas in erythrocytes the increase was 55% after hypoxia. Percent stimulation of erythrocyte GOT activity with pyridoxal phosphate before exposure to hypoxia was 180% but increased to 321% after exposure. In the second set of experiments another 10 rabbits were used. First they were exposed to hypoxia without pyridoxine hydrochloride feeding and then after 7 days with 3 mg of pyridoxine hydrochloride feeding. For glucose tolerance tests the animals were fed with 1 g of glucose immediately after the hypoxic exposures. Plasma reduced glutathione (GSH), LDH and ICDH activities increased and GOT activity was depressed after hypoxic stress, but when the animals were fed pyridoxine hydrochloride prior to the exposure the enzyme activities remained unaltered after hypoxic stress. Pyridoxine hydrochloride did not alter the pattern of glucose tolerance after hypoxic stress.     

Silver and Gold Nanoparticles Exposure to In Vitro Cultured Retina – Studies on Nanoparticle Internalization,Apoptosis, Oxidative Stress,Glial- and Microglial Activity

The complex network of neuronal cells in the retina makes it a potential target of neuronal toxicity – a risk factor for visual loss. With growing use of nanoparticles (NPs) in commercial and medical applications, including ophthalmology, there is a need for reliable models for early prediction of NP toxicity in the eye and retina. Metal NPs, such as gold and silver, gain much of attention in the ophthalmology community due to their potential to cross the barriers of the eye. Here, NP uptake and signs of toxicity were investigated after exposure to 20 and 80 nm Ag- and AuNPs, using an in vitro tissue culture model of the mouse retina. The model offers long-term preservation of retinal cell types, numbers and morphology and is a controlled system for delivery of NPs, using serum-free defined culture medium. AgNO₃-treatment was used as control for toxicity caused by silver ions. These end-points were studied; gross morphological organization, glial activity, microglial activity, level of apoptosis and oxidative stress, which are all well described as signs of insult to neural tissue. TEM analysis demonstrated cellular- and nuclear uptake of all NP types in all neuronal layers of the retina. Htx-eosin staining showed morphological disruption of the normal complex layered retinal structure, vacuole formation and pyknotic cells after exposure to all Ag- and AuNPs. Significantly higher numbers of apoptotic cells as well as an increased number of oxidative stressed cells demonstrated NP-related neuronal toxicity. NPs also caused increased glial staining and microglial cell activation, typical hallmarks of neural tissue insult. This study demonstrates that low concentrations of 20 and 80 nm sized Ag- and AuNPs have adverse effects on the retina, using an organotypic retina culture model. Our results motivate careful assessment of candidate NP, metallic or-non-metallic, to be used in neural systems for therapeutic approaches.     

Enkephalin immunoreactivity in iris nerves: Distribution in normal and grafted irides,persistence and enhanced fluorescence after denervations

Summary The morphology and distribution of nerve fibers showing enkephalin-like immunoreactivity was studied in rat and mouse iris whole mounts. In adult rat, a relatively dense network of varicose fibers was seen throughout the iris. Individual, long, usually smooth fibers were observed running together with non-fluorescent fibers in bundles. Positive nerve fibers were also seen in the ciliary body and the choroid membrane. The fluorescence intensity was normally low. No enkephalin-positive fibers were detected in adult mouse iris.Extirpation or lesioning either one or all the three ganglia known to supply the rat iris with nerve fibers, the superior cervical, the ciliary and the trigeminal ganglia, caused no detectable decrease in amount of enkephalin-positive fibers. However, in irides grafted to the anterior eye chamber of adult recipients, no enkephalin-positive fibers could be observed 2–12 days postoperatively, strongly suggesting that degeneration of these fibers had occurred. When iris grafts were left longer in the eye, nerve fibers with enkephalin-like immunoreactivity reappeared. An increased fluorescence intensity was observed both in the ipsilateral and contralateral iris following extirpation or lesioning all three ganglia and in the ipsilateral iris after extirpation of the ciliary ganglion. Three days after a systemic injection of capsaicin, which causes a permanent disappearance of substance P fibers, the same phenomenon was often observed. This raises the possibility of an interaction between the enkephalin-positive and the substance P fiber systems in the iris.The present experiments thus demonstrate a rich network of enkephalin immunoreactive nerve fibers in the rat iris originating outside the iris but apparently not in the ciliary, trigeminal or superior cervical ganglion.     

Light exposure causes functional changes in the retina: increased photoreceptor cation channel permeability, photoreceptor apoptosis, and altered retinal metabolic function

Light exposure induces retinal photoreceptor degeneration and retinal remodeling in both the normal rat retina and in animal models of retinal degeneration. Although cation entry is one of the triggers leading to apoptosis, it is unclear if this event occurs in isolation, or whether a number of pathways lead to photoreceptor apoptosis following light exposure. Following light exposure, we investigated the characteristics of cation entry, apoptotic markers [using terminal deoxynucleotidyl transferase (EC 2.7.7.31) dUTP nick-end labeling (TUNEL) labeling] and metabolic properties of retina from Sprague-Dawley (SD) rats and a rat model of retinitis pigmentosa [proline-23-histidine (P23H) rat]. Assessment of cation channel permeability using agmatine (AGB) labeling showed that excessive cation gating accompanied the series of anomalies that occur prior to photoreceptor loss. Increased AGB labeling in photoreceptors was seen in parallel with the appearance of apoptotic photoreceptors detected by TUNEL labeling with only a smaller proportion of cells colocalizing both markers. However, SD and P23H retinal photoreceptors differed in the amounts and colocalization of AGB gating and TUNEL labeling as a function of light exposure. Finally, reduced retinal lactate dehydrogenase levels were found in SD and P23H rat retinas after a 24-h light exposure period. Short-term (2 h) exposure of the P23H rat retina caused an increase in lactate dehydrogenase activity suggesting increased metabolic demand. These results suggest that energy availability may be exacerbated during the early stages of light exposure in susceptible retinas. Also, the concomitant observation of increased ion gating and TUNEL labeling suggest the existence of at least two possible mechanisms in light-damaged retinas in both SD and the P23H rat retina.     

Glutathione system adaptation to acute stress in the heart of rats during different regimes of hypoxia training

The intensity of lipid peroxidation (LPO), reduced and oxidized glutathione (GSH and GSSG) contents, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase (G-6-PDH), and NADP-isocitrate dehydrogenase (NADP-IDH) activities were studied in the heart of male rats exposed to two modes of intermittent hypoxic training (IHT): I-breathing in normobaric chamber with 7% O2 gas mixture for 5 min with 15 min normoxic intervals 4 times daily during 3 weeks; II-breathing by 12% O2 gas mixture in the same manner). After adaptation to hypoxia, the rats were subjected to 6h-immobilization stress. It has been shown that stress action after IHT (regime I) caused the increase in LPO and the shift of GSH/GSSG to disulfides. A disbalance in antioxidative defense system was determined by the decrease in glutatione peroxidase, G-6-PDH activities, and GSH content. The support of glutathione reductase activity under stress in this group with simultaneous decrease of enzyme activity in the pentose phosphate pathway was realized through the participation of NADP-IDH. Hypoxic training in regime II induced LPO decrease in the heart tissue after stress. The increase in the heart GSH content, optimal balance of glutathione-related enzymes in this group evidences for the dependence of adaptation effects on the vigor of hypoxic exposition. Our results suggest the active participation of glutathione system in the formation of adaptation reactions under the extreme factor influences through the action on intracellular red/ox potential as well as effectiveness of antioxidant defense.     

Possible mechanism for the decrease of mitochondrial aspartate aminotransferase activity in ischemic and hypoxic rat retinas.

Glutamate is believed to be an excitatory amino acid neurotransmitter in the retina. Enzymes for glutamate metabolism, such as glutamate dehydrogenase, ornithine aminotransferase, glutaminase, and aspartate aminotransferase (AAT), exist mainly in the mitochondria. The abnormal increase of intracellular calcium ions in ischemic retinal cells may cause an influx of calcium ions into the mitochondria, subsequently affecting various mitochondrial enzyme activities through the activity of mitochondrial calpain. As AAT has the highest level of activity among enzymes involved in glutamate metabolism, we investigated the change of AAT activity in ischemic and hypoxic rat retinas and the protection against such activity by calpain inhibitors. We used normal RCS (rdy+/rdy+) rats. For the in vivo studies, we clamped the optic nerve of anesthetized rats to induce ischemia. In the in vitro studies, the eye cups were incubated with Locke's solution saturated with 95% N2/5% CO2. The activity of cytosolic AAT (cAAT) was about 20% of total activity, whereas mitochondrial AAT (mAAT) was about 75% in rat retina. Ninety minutes of ischemia or hypoxia caused a 20% decrease in mAAT activity, whereas cAAT activity remained unchanged. To examine the contribution of intracellular calcium ions to the degradation of mAAT, we used Ca2+-free Locke's solution containing 1 mM EGTA, ryanodine (Ca2+ channel blocker), and thapsigargin (Ca2+-ATPase inhibitor). In the present study, thapsigargin in Ca2+-free Locke's solution, but not ryanodine in this solution, was found to prevent AAT degradation. AAT degradation was also prevented by calpain inhibitors (Ca2+-dependent protease inhibitor) such as calpeptin at 1 nM, 10 nM, 0.1 microM, 1 microM and 10 microM, and by calpain inhibitor peptide, but not by other protease inhibitors (10 microM leupeptin, pepstatin, chymostatin). Additionally, we determined the subcellular localization of calpain activity and examined the change of calpain activity in ischemic rat retinas. Our results suggest that decreased activity of mAAT in ischemic and hypoxic rat retinas might be evoked by the degradation by calpain-catalyzed proteolysis in mitochondria.     

Expression of neurotransmitters and neurotrophins in neurogenic inflammation of the rat retina

Antidromic stimulation of the rat trigeminal ganglion triggers the release of substance P (SP) and calcitonin gene-related peptide (CGRP) from sensory nerve terminals of the capsaicin sensitive C-fibers. These pro-inflammatory neuropeptides produce a marked hyperemia in the anterior segment of the eye, accompanied by increased intraocular pressure, breakdown of the blood-aqueous barrier and myosis. To assess the effects of neurogenic inflammation on the retina, specifically on the immunostaining of neurotransmitters and neurotrophins, as well as on the expression of neurotrophin receptors in the retina. RT-PCR was also accomplished in control and stimulated animals to confirm the immunohistochemical results. In the electrically stimulated eyes, immunostaining for SP, CGRP, VIP and nNOS demonstrated a marked increase in the RPE/POS (Retinal Pigment Epithelium/Photoreceptor Outer Segments), in the inner and outer granular layers and in the ganglion cells in comparison to the control eyes. CGRP and SP were found increased in stimulated animals and this result has been confirmed by RT- PCR. Changes in neurotrophin immunostaining and in receptor expression were also observed after electric stimulation of trigeminal ganglia. Decrease of BDNF and NT4 in the outer and inner layers and in ganglion cells was particularly marked. In stimulated rat retinas immunostaining and RT-PCR showed a NGF expression increase. Neurotrophin receptors remained substantially unchanged. These studies demonstrated, for the first time, that antidromic stimulation of the trigeminal ganglion and subsequent neurogenic inflammation affect immunostaining of retinal cell neurotransmitter/neuropeptides and neurotrophins as well as the expression of neurotrophin receptors.