Blockade of both NMDA and non-NMDA receptors is required for optimal protection against ischemic neuronal degeneration in the in vivo adult mammalian retina.

Under ischemic conditions, the excitatory amino acids (EAA), glutamate and aspartate, accumulate in the extracellular compartment of brain and, by excessive stimulation of EAA receptors, trigger excitotoxic degeneration of CNS neurons. Since glutamate and aspartate exert excitotoxic activity through both of the generally recognized classes of EAA receptors [N-methyl-D-aspartate (NMDA) and non-NMDA], it follows that both receptor classes may play a role in ischemic neuronal degeneration. Although several laboratories have reported that NMDA receptor antagonists confer protection in vivo against ischemic neuronal degeneration, very little is known about the ability of non-NMDA antagonists to confer such protection, a major reason being that non-NMDA antagonists that penetrate blood-brain barriers have not been available. In the present study, we examined the ability of NMDA or non-NMDA antagonists, either individually or in combination, to prevent neuronal degeneration in vivo in the adult rat retina rendered ischemic by dye/photothrombotic occlusion of retinal blood vessels. In this model, delivery of drugs to the ischemic tissue is assured by intravitreal administration. Intravitreal administration of the NMDA antagonist, MK-801, reduced the severity of ischemic damage approximately 50% (a ceiling effect that could not be increased by administering higher doses). The predominantly non-NMDA antagonist, CNQX, when administered in the highest dose permitted by its solubility limitations, provided equivocal (statistically nonsignificant) protection, but the two drugs combined provided greater than 80% protection.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methyl-aspartate neurotoxicity.

The endogenous excitotoxin, glutamate (Glu), acting at the N-methyl-aspartate (NMA) subtype of Glu receptor, is thought to play a major role in hypoxic/ischemic neuronal degeneration. In the present study, the sensitivities of the developing rat CNS to hypoxic/ischemic neuronal degeneration and to the neurotoxic action of NMA were compared at various postnatal ages. In the hypoxic/ischemic experiments, ischemia was produced by unilateral common carotid artery ligation and hypoxia by subjecting the pups to a partial vacuum. Keeping the duration of the hypobaric episode constant at 75 min for all age groups, we observed that the vulnerability of the immature brain to hypobaric/ischemic damage increased during the early neonatal period (days 2-4), reached a peak at day 6 and then diminished progressively with increasing age. In the second part of the study, NMA was microinjected unilaterally into the head of the caudate nucleus at various postnatal ages (2-80 d). In the early neonatal period (days 2-6), injections of relatively small doses of NMA (6-15 nmol) produced a dose-dependent widespread excitotoxic reaction throughout the forebrain with peak sensitivity being observed on day 6. The cytotoxic reaction to NMA was identical in appearance and time course to that induced by hypobaric/ischemic methods. With increasing age, the excitotoxic response to a given dose of NMA decreased progressively and the lesions became more strictly confined to the injection site. Cell populations most sensitive to NMA toxicity in the 2-10 d period closely correlated with those most vulnerable to hypoxia/ischemia, and sensitivity to both types of injury reached a peak at 6 d. These findings reinforce other evidence linking an excitotoxic mechanism and the NMA subtype of Glu receptor to hypoxic/ischemic brain damage and suggest that there may be a period during development when NMA receptors are hypersensitive to excitotoxic stimulation, thus rendering the neurons possessing such receptors hypervulnerable to hypoxic/ischemic damage.

Photothrombosis-induced ischemic neuronal degeneration in the rat retina.

Here we describe a new model for inducing ischemic neuronal degeneration in the adult rat retina. Rose bengal dye was injected intravenously; then the retina was exposed to intense light which caused vascular photothrombosis resulting in acute degeneration of retinal neurons. By either light or electron microscopical criteria, the acute neurodegernative reaction was judged identical in pattern, cytopathological appearance, and time course to the excitotoxic type of reaction typically seen in the retina following exposure to exogenous glutamate. These results reinforce other accumulating evidence suggesting that ischemic CNS damage is mediated by glutamate or related excitotoxins. The advantages of this model of CNS ischemia include its noninvasiveness, ease of application, authentic simulation of vascular thrombosis, and accessibility for application of neuroprotective drugs.

Hypobaric-ischemic conditions produce glutamate-like cytopathology in infant rat brain.

We present a new animal model of perinatal hypoxic/ischemic brain damage and compare this type of brain damage with the excitotoxic type of damage previously described in the brains of infant rats and monkeys treated systemically with glutamate (Glu). Ten-d-old rats with unilateral occlusion of the common carotid artery were subjected to hypobaric conditions for 75 min and sacrificed 0-4 hr later for light and electron microscopic brain examination. The mortality rate was relatively low (12%), and brain damage was evident ipsilateral to the ligated carotid in 94% of surviving animals 4 hr after termination of the hypobaric event. Regions most frequently affected were the medial habenulum, dentate gyrus, caudate nucleus, frontoparietal neocortices, olfactory tubercle, and several thalamic nuclei. The acute cytopathological changes, primarily edematous degeneration of neuronal dendrites and cell bodies, evolved very rapidly, with some neurons manifesting end-stage necrosis at 0 hr (immediately after hypobaric exposure) and others developing such changes over a 1-4-hr period. We conclude that the neurodegenerative reaction induced in infant rat brain by hypoxia/ischemia is indistinguishable from the excitotoxic type of damage exogenous Glu is known to cause. Moreover, in a companion study (Olney et al., 1989) we show that MK-801, a powerful antagonist of the N-methyl-D-aspartate receptor complex (subtype of Glu receptor), protects against neuronal degeneration in this hypobaric/ischemic model. Our results reinforce other recent evidence suggesting that hypoxic/ischemic brain damage is mediated by endogenous Glu or related excitotoxins.

Hypothermia enhances protective effect of MK-801 against hypoxic/ischemic brain damage in infant rats.

Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor may play an important role in hypoxic/ischemic (H/I) brain damage. Accordingly, it has been shown that the NMDA antagonist, MK-801, partially protects the infant rat brain against H/I damage. Here we show that reducing the body temperature of the infant rat also confers partial protection against H/I brain damage and that mild hypothermia plus MK-801 treatment provides total protection against such damage. Relevance of these findings to the prevention of perinatal brain damage in humans is discussed.

MK-801 prevents hypobaric-ischemic neuronal degeneration in infant rat brain.

Recent evidence implicates the endogenous excitatory amino acids, glutamate (Glu) and aspartate, in hypoxic/ischemic neuronal degeneration. In a preceding article (Ikonomidou et al., 1989) we described a new model for studying hypoxic/ischemic neuronal degeneration in the infant rat brain that entails unilateral common carotid artery ligation followed by exposure to a partial vacuum for 75 min. Promising features of this model include a low mortality rate and high incidence of acute brain damage disseminated over numerous brain regions. In addition, there is a striking similarity between the type of cytopathology characterizing this model of hypoxic/ischemic neuronal degeneration and that which has been described in infant animals treated with Glu. MK-801 is a powerful antagonist of the N-methyl-D-aspartate (NMDA) receptor ionophore complex (a subtype of Glu receptor). In the present study, after unilateral carotid artery ligation was performed on 10-d-old rat pups, they were treated either with MK-801 (1 mg/kg i.p.) or saline 15 min before exposure to the hypobaric condition. MK-801 exerted a strong neuroprotective effect without serious side effects; the majority of saline control animals sustained severe brain damage, whereas the majority of MK-801-treated pups had no brain damage. These and other recent findings suggest that the NMDA receptor may play an important role in hypoxic/ischemic neuronal degeneration in the immature brain and provide hope that NMDA antagonists such as MK-801 may be effective in preventing such degeneration.

Comparison of adenosine uptake and endogenous adenosine-containing cells in mammalian retina.

Autoradiographic techniques were used to label [3H]-adenosine and [3H]-cyclohexyladenosine accumulating cells in rabbit, mouse, and ground squirrel retinas. Immunohistochemical methods revealed the distribution of cells that stained for endogenous adenosine. Comparisons of these two markers revealed for all three species that the distribution of specific subpopulations of retinal cells that store or accumulate the purine nucleoside, adenosine, is similar. For all three species, cells localized in the ganglion cell layer accumulated adenosine and exhibited adenosine-like immunoreactivity (ALIR). A smaller proportion of cells localized in the inner nuclear layer were labeled for ALIR, while a larger proportion of cells in this layer accumulated adenosine. Subtle differences between species are presented. However, the general similarities of the distribution of these two putative purinergic markers supports the evidence that a discrete adenosinergic neurotransmitter/modulatory system is present in the retina.

GABA-like immunoreactivity in the vertebrate retina: a species comparison.

Rabbit antisera directed against gamma-amino butyric acid (GABA) conjugated to bovine serum albumin was used to localize neurons containing GABA-like immunoreactivity in the retinas of nine species of animals: human, cat, rabbit, rat, chicken, turtle, frog, mudpuppy and goldfish. The retinas of all species contained GABA-like labeling in several populations of amacrine cells in the inner nuclear layer, cells in the ganglion-cell layer that may include displaced amacrine cells and in fibers in the inner plexiform layer and in the optic nerve fiber layer. Labeled horizontal cells were found in cat and in all non-mammalian retinas. Labeled interplexiform cells were found in rat, rabbit, cat and human retinas. Labeled bipolar cells were restricted to frog and mudpuppy retinas. The distribution of anti-GABA is usually similar to that of anti-glutamic acid decarboxylase and neuronal [3H]GABA uptake, indicating good correspondence between these 'GABAergic' markers. However, several significant differences among these markers are discussed.

Colocalization of GAD-like immunoreactivity and 3H-GABA uptake in amacrine cells of rabbit retina.

Rabbit retinas were double labeled to determine the degree of colocalization of glutamic-acid-decarboxylase-like immunoreactivity (GAD-like IR) and 3H-GABA uptake using light (LM) and electron microscopic (EM) autoradiography. Both GAD-like IR and 3H-GABA uptake were found in amacrine cell bodies in the inner nuclear layer (INL) as well as in cell bodies in the ganglion cell layer (GCL), and throughout the inner plexiform layer. GAD-like IR was found in 32% of the amacrine cells in the INL, 86% of which also showed 3H-GABA uptake; 3H-GABA uptake was observed in 38% of the amacrine cells. However, only 72% of these cells showed GAD-like IR. Labeled cells in the GCL were only 10-15% as common as similarly labeled cells in the INL. As in the INL, all GAD-positive cells in the GCL were double labeled, but only 53% of the cells taking up 3H-GABA were double labeled. We suggest that labeled cells in the GCL were ganglion cells rather than displaced amacrine cells. Cells, in both the INL and GCL, that showed 3H-GABA uptake but no GAD-like IR had a higher average grain density than double-labeled cells, indicating that uptake by these cells was specific. The relevance to GABAergic function of 3H-GABA uptake without an indication of GAD-like IR is yet to be determined. Statistical analysis at the EM level showed that one-third of the GAD-positive synaptic terminals of amacrine cells were double labeled after a 4-month exposure. Longer exposures at the EM level should reveal a higher percentage of GAD-positive terminals because at the LM level, one-half of the double-labeled cell bodies were "lightly" labeled with grains. The high degree of colocalization of GAD-like IR and 3H-GABA uptake suggests that both markers may be useful for labeling GABAergic neurons in the rabbit retina.

Aspartate aminotransferase-like immunoreactivity in the guinea pig and monkey retinas.

The excitatory amino acids, aspartate and glutamate, have been proposed as retinal neurotransmitters. Aspartate aminotransferase (AAT) is an enzyme which is involved in the routine metabolism of these amino acids and may be involved in the specific synthesis of glutamate and/or aspartate for use as a neurotransmitter. On the basis of the hypothesis that increased levels of aspartate aminotransferase may reflect a transmitter role for aspartate and/or glutamate, we have localized aspartate aminotransferase in the guinea pig and cynamolgus monkey retinas with light and electron microscopic immunohistochemical techniques. AAT-like immunoreactivity is localized to the cones of guinea pig retina and to monkey rods. Both species contain a subpopulation of immunoreactive amacrine cells as well as a subpopulation of immunoreactive cells in the ganglion cell layer. Immunostaining is seen in bipolar cells and terminals in the monkey but not in the guinea pig retina. We have performed quantitative analysis of the immunoreactive staining in the outer plexiform layer and described the synaptic organization of immunoreactive processes in the inner plexiform layer (IPL). Labeled amacrine processes in both species form synaptic contacts predominantly to and from bipolar terminals in the inner third of the IPL and to and from other amacrine and small unidentified processes in the outer portion of the IPL. The majority of labeled bipolar terminals in the monkey retina are seen in the inner third of the IPL where they synapse exclusively onto amacrine processes. Labeled bipolar terminals in the outer third of the IPL occasionally synapse onto ganglion processes.

Immunocytochemical localization of enkephalin-like immunoreactivity in the retina of the guinea pig.

The distribution of enkephalin-like immunoreactivity in the retina of the guinea pig was studied. Indirect immunofluorescence techniques were used on retinae with and without colchicine pretreatment. In retinae not receiving colchicine pretreatment, enkephalin-like immunoreactivity was seen in fibers in the inner plexiform layer, predominantly in laminae 1, 3, and 5. In colchicine-pretreated retinae, enkephalin immunofluorescent cell bodies were seen in the inner margin of the inner nuclear layer in addition to the immunoreactive fibers. These cells showed morphological characteristics of amacrine cells. No enkephalin-like immunofluorescence was seen in the optic nerve, ganglion cell layer, or outer nuclear or plexiform layers. These findings of enkephalin-like immunoreactive cells and fibers in a mammalian retina add to the findings in nonmammalian retinae and suggest that the enkephalins play a role in primary sensory systems of mammalian and nonmammalian species.