A behavioral model of excitotoxicity: retinal degeneration, loss of vision, and subsequent recovery after intraocular NMDA administration in adult rats.

To establish a new behavioral animal model of excitotoxicity, we injected adult rats intraocularly with a single dose of 2, 20, or 100 nmol of N-methyl-D-aspartate (NMDA). We quantified visual impairment by plotting the size of the visual field in which the rats successfully oriented towards a small, moving target. In comparison to the saline-injected (contralateral) control side, the side injected with 2 nmol of NMDA was not significantly impaired. When injected with higher doses, the rats were nearly blind immediately after surgery, with only about 20% (20 nmol NMDA) or 10% (100 nmol NMDA) of residual vision. Within about 3 weeks, however, visual performance returned to near-normal levels. Simultaneous intraocular administration of a non-competitive NMDA-antagonist, MK-801 (1 nmol), resulted in complete behavioral protection. NMDA administration led to a dose-dependent loss of cells within the ganglion cell layer, as assessed in whole-mounted retinae which were retrogradely labelled with horseradish peroxidase (HRP). Whereas 2 nmol of NMDA led to the loss of about 30% of retinal ganglion cells (RGCs), at higher NMDA doses only 13% of the RGCs survived. After the injection of 20 nmol of NMDA, large-diameter RGCs (> 22 microns) survived the lesion to a greater extent than small diameter cells (8-21 microns); at 100 nmol cells of all diameters were equally affected. The number of Nissl-stained cells with small diameters (< 11 microns), presumed to be displaced amacrine cells, was also affected by NMDA, although to a lesser degree. Analysis of behavioral performance (vision score) and the number of cells in the retina revealed a correlation of r = 0.76 between visual performance and the number of HRP-filled RGCs immediately after surgery. Lower correlations were found between visual performance and cells stained with Nissl of diameters smaller than 11 microns (presumed RGCs without retinofugal connections; r = 0.55 and r = 0.58, respectively). Because of the spontaneous recovery of vision, all correlations declined to values near 0 after 3 weeks. Thus, despite a dramatic loss of RGCs following NMDA administration, visual deficits recover significantly in adult rats within 2-3 weeks.

A derivative of a rigid glutamate analog protects the retina from excitotoxicity.

In the retina, the activation of metabotropic glutamate receptors (mGluRs) reduces the toxic effect of N-methyl-D-aspartate (NMDA). We have induced NMDA-mediated excitotoxicity in the adult rat retina by a single intraocular injection of NMDA. The damage that resulted was estimated by assessing the NMDA-induced loss of retinal choline acetyltransferase (ChAT) activity. The new rigid glutamate analog, dimethyl ester of (+/-)-trans-azetidine-2,4-dicarboxylic acid (t-DMADA), with a putative mGluR-agonistic activity, protected the retina from NMDA-induced loss of ChAT activity. This study demonstrated that t-DMADA can be considered a prototype of new retino-protective agents.

Nerve growth factor promotes functional recovery of retinal ganglion cells after ischemia.

PURPOSE: To investigate the effect of a transient complete ischemia on the function of cat retina and to determine whether nerve growth factor (NGF), which was previously shown to enhance retinal ganglion cell (RGC) survival after optic nerve section in the adult rat, can promote recovery of retinal neurons after the ischemic insult. METHODS: Function of distal and proximal retina was assessed by recording the electroretinogram in response to both homogeneous flickering light (FERG) and contrast reversing gratings (PERG), respectively, 30 days after the induction of a 60-minute episode of ischemia. Visual evoked potentials in response to contrast reversing gratings were also recorded to evaluate visual acuity and contrast thresholds. Cell survival after ischemia was assessed in retinal whole-mounts stained with cresyl violet. Cats were intraocularly treated with NGF every other day, 3 times a week, for 30 days. Controls were treated with either phosphate buffered saline or cytochrome c. RESULTS: After ischemia, the FERG was not significantly affected. On the contrary, the PERG, visual acuity, and contrast thresholds were severely impaired. After NGF treatment, PERG response amplitudes were much less reduced compared to controls, and visual acuity and contrast thresholds were virtually normal. In addition, a larger number of presumed RGCs was present in the NGF-treated retinas compared to the cyt c-treated ones. CONCLUSIONS: The more proximally located retinal neurons, in particular RGCs, are highly vulnerable to ischemia. Intraocular NGF treatment was effective in enhancing the survival and functional recovery of these neurons. This suggests that NGF may represent a novel therapeutic agent for the treatment of ischemic ocular pathologies.

In rats, the metabotropic glutamate receptor-triggered hippocampal neuronal damage is strain-dependent.

The effect of intrahippocampal (i.h.) and intraocular (i.o.) administration of the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) was studied in different rat strains. A massive hippocampal damage was observed in CD/SD and Fischer 344 but not in SD/Rij and Brown Norway rats 7 days following the i.h. injection of 1S,3R-ACPD, while no retinal damage was observed following its i.o. administration. Moreover, 1S,3R-ACPD reduced the N-methyl-D-aspartate (NMDA) toxicity in the retina of both CD/SD and SD/Rij rats. Regardless of its toxic action on hippocampal neurons the i.h. injection of 1S,3R-ACPD caused an acute stimulation of motor activity in both CD/SD and SD/Rij rats. This effect was blocked by the intracerebroventricular (i.c.v.) administration of the putative mGluR antagonist L-2-amino-3-phosphono-propionic acid (L-AP3). It is suggested that the differential expression of mGluR subtypes might determine their role in brain pathology.

Activation of the glutamate metabotropic receptor protects retina against N-methyl-D-aspartate toxicity.

Intraocular pretreatment with the specific metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) in the adult rat reduced the excitotoxic effects induced in the retina by a single intraocular injection of N-methyl-D-aspartate (NMDA). Damage was estimated by assessing NMDA-induced loss of retinal choline acetyltransferase (ChAT) activity. The interaction between metabotropic and ionotropic glutamate receptors may, therefore, be considered an important target for in vivo pharmacological neuroprotection.

N-methyl-D-aspartate-induced neurotoxicity in the adult rat retina.

The present study provides evidence that the adult mammalian retina is highly sensitive to the excitotoxic action of NMDA. In particular, we have investigated the effects of a single intravitreal injection of different doses of N-methyl-D-aspartate (NMDA) (2-200 nmoles) on the adult rat retina. Morphological evaluation of transverse sections of retinae demonstrated a dose-dependent loss of cells in the ganglion cell layer (GCL) and a reduction in the thickness of the inner plexiform layer. No obvious alterations were noted in the more distal retinal layers. Quantitative analyses of Nissl-stained whole-mounted retinae revealed that administration of 20 nmoles of NMDA resulted in a 70% loss of cells with a soma diameter greater than 8 microns (presumed retinal ganglion cells); a 20% loss of cells with a soma diameter smaller than 8 microns (presumed displaced amacrine cells) was also observed. In addition, NMDA produced a dose-dependent decrease of retinal choline acetyltransferase (ChAT) activity, suggesting that NMDA affects cholinergic amacrine cells as well. MK-801, a non-competitive NMDA antagonist, completely prevented the NMDA-induced loss of cells in the GCL and blocked, in a dose-dependent manner, the NMDA-induced decrease of ChAT activity. The excitotoxic action of NMDA observed in these experiments is thus likely mediated through the NMDA receptor subtype. This "in vivo" model may be utilized to identify potential drugs that antagonize or limit the deleterious effects consequent to NMDA receptor overstimulation in the central nervous system.

Flash and pattern electroretinograms during and after acute intraocular pressure elevation in cats.

Retinal functionality during short-term intraocular pressure (IOP) elevation and simultaneous systemic blood pressure (BP) variations was evaluated in the cat by recording the electroretinogram in response to both homogenous flickering light (FERG) and contrast reversing gratings (PERG). The mean arterial blood pressure (BPm) was pharmacologically adjusted to different levels and a large range of IOP values was tested. Results indicate that both FERG and PERG responses are impaired when the eye perfusion pressure (PP = BPm - IOP) is reduced and they disappear at a critical PP value of about 20 mm Hg, irrespective of the absolute value of the IOP. In addition, when the critical perfusion pressure is maintained for periods longer than 5 min, the recovery of the PERG response, when present, is always delayed compared to the full recovery of the FERG response. These findings support the hypothesis that vascular factors, ie, the impairment of the retinal blood supply, may be responsible for the disappearance of the retinal electrical activity during short-term IOP elevation. Furthermore, the retinal ganglion cells, presumably the main source of the PERG response, appear less likely to recover from the acute ischemic episode.

Binocular suppression in cortical neurons.

Dichoptic presentation of patterns similar in shape but of very different contrast results in the perception of only the high contrast pattern (binocular suppression). When recording from binocular neurons of the cat visual cortex, we have found an effect which is strikingly similar to this perceptual phenomenon. If a high and a low contrast grating are presented simultaneously, one to each eye, the cell's response to the low contrast stimulus is suppressed.

Ganglioside treatment of genetic and alloxan-induced diabetic neuropathy.

Peripheral neuropathy is a common complication of diabetes. Using the mutant diabetic mouse C57BL/ks (db/db) and alloxan-treated rats, 30 days after intoxication, we investigated development and treatment with gangliosides of such a disease. The db/db mouse develops a neuropathy characterized by a loss in conduction velocity shown as early as 80-90 days after birth and maintained throughout life. At later stages (5-6 months of age) there is a drop in slow transport and myelin particle density. These changes are correlated by a lack of response to insulin treatment, which, prior to this stage, is capable of improving nerve conduction velocity (NCV). On the other hand gangliosides became effective, improving NCV, myelin particle density and sensory perception (auditory deficit) at 5 months of age in the db/db mouse. We presume that this differential neuronal response to insulin and gangliosides indicates a change of the neuropathy from a metabolic stage to neuronal. Alloxan induced diabetic neuropathy is treatable with gangliosides even 30 days after intoxication.

Muscle reinnervation--I. Restoration of transmitter release mechanisms.

Following sciatic nerve crush the restoration of neuromuscular transmission in the extensor digitorum longus muscle of rat proceeds in a well defined manner: (a) as soon as the nerve-muscle contact is reformed, a subthreshold end-plate potential is recorded; no 'non-transmitting stage' is observed; (b) 24 hours later muscle action potentials are induced by nerve stimulation; (c) miniature end-plate potentials are absent or very rare at the newly reinnervated end-plates; their frequency returns to normal in about 4 weeks; (d) the frequency is also very much reduced in 30 mM K+ and hypertonic solutions and recovers slowly, in 4 and 5 weeks, respectively, while black widow spider venom is from the beginning as powerful as in normal neuromuscular junctions; (e) at the early stages of reinnervation the Ca2+-dependent release mechanisms are much stronger than control cases, while the Ca2+-independent mechanisms are weaker and recover in 5 weeks. The gradual reassembly and restoration of neurotransmitter release mechanisms of the extensor digitorum longus nerve terminal indicate the complexity of pre-synaptic ending organization.

Brain cortex gangliosides and (Na+, K+)ATPase system of the stria vascularis in guinea pig.

This paper presents the results of an investigation into the possibility that gangliosides (brain cortex glycosphingolipids) are capable of being functionally incorporated into cell membranes and of interfering with enzymatic activity of the ATPase system on the stria vascularis and spiral ligament in the guinea pig. Labelled gangliosides (3H-GM1) were incorporated into the cell membranes of the tissues under examination. (Na+, K+)ATPase activity increased l0 minutes after intravenous injection of gangliosides and prevented the decrease in (Na+, K+)ATPase system produced by ethacrynic acid. The action of gangliosides on the (Na+, K+)ATPase system is discussed.