The aim of this study was to determine whether agmatine, a channel permeable probe, can identify photoreceptor dysfunction in the Royal College of Surgeons (RCS) retina at an earlier stage to that shown by apoptosis or anatomical markers, and also characterize the neurochemical development of the inner retina in the normal and degenerating rat. We used isolated retinas at different ages incubated in physiological media containing agmatine. Subsequently, postembedding immunocytochemistry was used to determine the number of labelled photoreceptors and the labelling pattern within postreceptoral neurons. Agmatine labelling patterns revealed a sequential development of retinal neurons beginning at postnatal day (PND) 11/12 with most horizontal cells, a few ganglion and amacrine cells, showing a strong signal. The neurochemical development progressed rapidly, and reflects to a large part the known distribution of glutamate receptors, with inner nuclear labelling being evident by PND14, continuing with the same pattern of labelling in adulthood for the control retina. The RCS retina showed markedly reduced agmatine labelling in the inner retina at PND20. A rapid increase in photoreceptor AGB labelling was evident during the degeneration phase. Multiple samples at PND14 and PND16 confirmed a significant increase of labelled photoreceptors in the RCS retina.

The current study aims to assess the vulnerability of photoreceptors in rat retina to variations in tissue oxygen levels. Young adult Sprague-Dawley rats were exposed to air with the concentration of oxygen set at 10% (hypoxia), 21% (room air, normoxia), and four levels of hyperoxia (45%, 65%, 70%, and 75%), for up to 3 weeks. Their retinas were then examined for cell death, using the TUNEL technique. Hypoxia (10% oxygen) for 2 weeks caused a limited but significant rise in the frequency of TUNEL+ (dying) cells in the retina, the great majority (>90%) being located in the outer nuclear layer (ONL). Hyperoxia also induced an increase in the frequency of TUNEL+ cells, again predominantly in the ONL. The increase rose with duration of exposure, up to 2 weeks. At 2 weeks exposure, the increase was limited yet significant at 45% oxygen, and maximal at 65%. Where the frequencies of TUNEL+ cells were high, it was evident that photoreceptor death was maximal in the midperipheral retina. The adult retina is vulnerable to maintained shifts in oxygen availability to the retina, both below and above normal. The vulnerability is specific to photoreceptors; other retinal neurons appeared resistant to the exposures tested. Shifts in retinal oxygen levels caused by variations in ambient light, by the persistence of light through the normally dark (night) half of the day-night cycle, or by depletion of the photoreceptor population, may contribute to photoreceptor death in the normal retina.

No abstract

We assessed the effect of the in vivo application of monocarboxylate transport inhibitors on retinal function and amino acid immunocytochemistry. We wanted to determine the impact that altered aerobic metabolite availability has on retinal function and the characteristics of amino acid shunting into metabolic pools. Electroretinograms were collected from anaesthetized rats at various times after intravitreal injection of the monocarboxylate transport inhibitors alpha-cyano-4-hydroxycinnamate (4-CIN; 2 micro L, 0.1-10 mm) or p-(dipropylsulphamoyl)benzoic acid (probenecid; 1-10 mm). Changes in retinal function were compared with quantitative amino acid immunocytochemical changes in retinas harvested 20 and 40 min after either 4-CIN or vehicle treatment. The injection of 4-CIN resulted in a dose-dependent reduction of the ON-bipolar cell P2 wave amplitude (20-80%) and delay in its implicit time. The phototransduction sensitivity was mildly reduced whereas the ON-bipolar cell P2 sensitivity was unaffected. Probenecid induced functional changes similar to those observed with 4-CIN. We also mapped the amino acid alterations within specific cell classes induced by 4-CIN application. All neurones displayed a reduced glutamate content averaging 48%; reduced GABA (31%) and glycine (28%) were found within amacrine cells and glutamine was reduced in all cell classes except photoreceptor and Müller cells. All cell classes in the retina demonstrated increases in aspartate (57%), whereas leucine (24%) and ornithine (21%) were only significantly increased in photoreceptor and bipolar cells. The reduction in glutamate immunolabelling in specific retinal cell classes was mirrored by an increase in aspartate levels at these locations. In addition, attenuated glutamine immunolabelling also closely matched the spatial pattern observed for glutamate. Our immunocytochemical analysis provides evidence that monocarboxylate transport inhibition induces a shift in the equilibrium of glutamate transamination reactions involving aspartate throughout the retina whereas photoreceptor and bipolar cells also use glutamate transamination reactions involving ornithine and leucine. The distribution pattern of glutamine secondary to monocarboxylate inhibition suggests that this amino acid is a major precursor for glutamate throughout the retina.