Global and ocular hypothermic preconditioning protect the rat retina from ischemic damage

Abstract

Retinal ischemia could provoke blindness. At present, there is no effective treatment against retinal ischemic damage. Strong evidence supports that glutamate is implicated in retinal ischemic damage. We investigated whether a brief period of global or ocular hypothermia applied 24 h before ischemia (i.e. hypothermic preconditioning, HPC) protects the retina from ischemia/reperfusion damage, and the involvement of glutamate in the retinal protection induced by HPC. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. One day before ischemia, animals were submitted to global or ocular hypothermia (33°C and 32°C for 20 min, respectively) and fourteen days after ischemia, animals were subjected to electroretinography and histological analysis. Global or ocular HPC afforded significant functional (electroretinographic) protection in eyes exposed to ischemia/reperfusion injury. A marked alteration of the retinal structure and a decrease in retinal ganglion cell number were observed in ischemic retinas, whereas global or ocular HPC significantly preserved retinal structure and ganglion cell count. Three days after ischemia, a significant decrease in retinal glutamate uptake and glutamine synthetase activity was observed, whereas ocular HPC prevented the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate induced alterations in retinal function and histology which were significantly prevented by ocular HPC. These results support that global or ocular HPC significantly protected retinal function and histology from ischemia/reperfusion injury, probably through a glutamate-dependent mechanism.

Figure 1. Temporal course of body, ipsilateral, and contralateral retroocular temperature during unilateral ocular cooling.

A flow of ultrasound transmission gel cooled at 13°C was applied to one eye, which achieved a temperature of 32°C. Note that when one eye was perfused with the cooling gel, the contralateral eye, and the body temperature remained normothermic. Data are mean ± SE (n: 4 animals).

Figure 2. Effect of global HPC on retinal function.

Scotopic ERGs from rats subjected to 40-min ischemia without or with global hypothermia applied 24 h before ischemia, and registered 14 days after ischemia. Upper panel: average amplitudes of scotopic ERG a-wave, b-wave, and OPs. A significant decrease in the ERG a-wave, b-wave, and OP amplitude was observed in eyes exposed to 40-min ischemia without global HPC. In animals submitted to global HPC, a significant preservation of the ERG a-wave, b-wave, and OP amplitude was observed. Data are mean ± SE (n: 10 eyes/group). **P<0.01, by Tukey’s test. Lower panel: Representative scotopic ERG and OP traces from a non-ischemic eye without or with local HPC, and from an eye subjected to 40-min ischemia without or with global HPC applied 24 h before ischemia.

Figure 3. Effect of global HPC on retinal histology.

Upper panel: Representative photomicrographs showing histological appearance of non-ischemic retinas without (A) or with global HPC (B), ischemic retinas 14 days after 40 min of ischemia without global HPC (C), and ischemic retinas from animals submitted to global HPC, 24 h before ischemia (D). Severe retinal damage is shown in the retina from eyes submitted to ischemia without global HPC, whereas in animals submitted to global HPC, the retinal structure was notably preserved. Lower panel: Immunohistochemical detection of Brn3a(+) cells in the GCL from all the experimental groups. A decrease in GCL Brn3(+) cell number was observed in ischemic retinas without global HPC (G) as compared with non-ischemic eyes (E), whereas global HPC, which showed no effect in control eyes (F), partly preserved Brn3a(+) cell count in ischemic eyes (H). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer, OS, outer segment of photoreceptors.

Figure 4. Functional protection against I/R damage induced by ocular HPC

. Upper panel: Average amplitudes of scotopic ERG a-wave, b-wave and OPs. In eyes submitted to 40-min ischemia, local HPC significantly prevented the decrease in the amplitude of ERG b- (but not a-) wave and OP amplitude assessed 14 days after 40-min ischemia. Data are mean ± SE (n: 10 eyes/group). **P<0.01, by Tukey’s test. Lower panel: Representative scotopic ERG and OP traces from all the experimental groups.

Figure 5. Histological protection against I/R damage induced by local HPC.

Upper panel: Severe retinal damage is shown in the retina from eyes submitted to ischemia without local HPC (C) as compared with non-ischemic eyes (A), whereas local HPC applied 24 h before ischemia (D), notably preserved the retinal structure. Lower panel: A similar profile was observed for Brn3a(+) cell number in the GCL. Local HPC did not affect retinal histology and Brn3a(+) cell number in non-ischemic eyes (B and F, respectively). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer, OS, outer segment of photoreceptors.

Figure 6. Effect of ischemia and ocular HPC on retinal glutamate uptake and glutamine synthetase activity.

Three days after ischemia, a significant decrease of glutamate uptake (panel A) and glutamine synthetase activity (panel B) was observed in ischemic retinas, whereas local HPC, which showed no effect per se in non-ischemic eyes, significantly prevented the effect of ischemia on these parameters. Data are mean ± SEM (n = 10–12 animals per group). **P<0.01, by Tukey’s test.

Figure 7. Effect of an intravitreal injection of glutamate without or with local HPC on retinal function.

Eyes were intravitreally injected with vehicle or glutamate without or with local HPC applied 24 h before injections. Glutamate induced a significant decrease in the ERG b-wave and OP amplitude, whereas local HPC significantly prevented the effect of glutamate on these parameters. Upper panel: Average amplitudes of scotopic ERG a- and b-wave, and OP amplitudes. Shown are means ± SEM (n = 10 animals/group). *P<0.05, and **P<0.01, by Tukey’s test. Lower panel: Representative scotopic ERG and OP traces from eyes injected with glutamate without or with local HPC.

Figure 8. Effect of glutamate without or with local HPC on retinal histology.

Representative photomicrographs showing histological appearance of retinas obtained 7 days after injection of vehicle (A, D) or glutamate without (B, E) or with local HPC applied 24 h before glutamate injection (C, F). Glutamate induced alteration in the retinal structure (upper panel) and GCL Brn3a(+) cell number (lower panel), whereas local HPC prevented histological alterations from glutamate damage. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer, OS, outer segment of photoreceptors.