Assessment of neuroprotective effects of glutamate modulation on glaucoma-related retinal ganglion cell apoptosis in vivo

Abstract

Purpose: To assess the neuroprotective effects of different glutamate modulation strategies, with a nonselective (MK801) and a selective (ifenprodil) NMDA receptor antagonist and a metabotropic glutamate receptor agonist (mGluR Group II, LY354740), in glaucoma-related in vivo rat models of retinal ganglion cell (RGC) apoptosis.

Methods: RGC apoptosis was induced in Dark Agouti (DA) rats by staurosporine (SSP) treatment. Single agents MK801, ifenprodil, or LY354740, or MK801 and LY354740 combined, were administrated intravitreally at different doses. Eyes were imaged in vivo using a recently established technique and the results confirmed histologically. The most effective combined therapy regimen of MK801 and LY354740 was then assessed in a chronic ocular hypertension (OHT) rat model with application at 0, 1, and 2 weeks after OHT surgery and the effects assessed as described before.

Results: All strategies of glutamate modulation reduced SSP-induced-RGC apoptosis compared with the control, in a dose-dependent manner: MK801 (R2= 0.8863), ifenprodil (R2= 0.4587), and LY354740 (R2= 0.9094), with EC50s of 0.074, 0.0138, and 19 nanomoles, respectively. The most effective combination dose of MK801 and LY354740 was 0.06 and 20 nanomoles (P < 0.05), respectively, and the optimal timing of the therapy was 0 weeks after OHT surgery (P < 0.05).

Conclusions: This novel SSP model was validated as a useful tool for screening neuroprotective strategies in vivo. Group II mGluR modulation may be a useful treatment for RGC death. Combination therapy optimized to limit neurotoxic effects of MK801 may be an effective neuroprotective approach in retinal degenerative disease. Furthermore, treatments that minimize secondary RGC degeneration may be most useful in glaucoma.

Figure 1

Visualization of RGC apoptosis using SLO imaging of in vivo glaucoma-related models. Staurosporine (SSP)-induced RGC apoptosis, appearing as white spots (A), was reduced by ifenprodil (0.6 nanomoles, B), MK801 (0.6 nanomoles, C), and LY354740 (20 nanomoles, D) at 2 hours after treatment. Similarly, RGC apoptosis at 3 weeks in a rat OHT model (E) was significantly reduced by treatment with a combined low dose of MK801/ LY354740 (0.06/20 nanomoles, F).

Figure 2

Histologic confirmation that apoptotic cells (A, labeled with Annexin V/Alexa-488, white) were localized to RGCs (B, retrograde labeled with DiAsp4, gray), as shown in the combined micrograph (C), in an SSP model.

Figure 3

Effect of single neuroprotective agents on RGC apoptosis in an SSP rat model. All treatments induced a dose-dependent reduction of RGC apoptosis (A, C MK801 and ifenprodil; B, D LY354740). The non-selective NMDA receptor antagonist MK801 was more effective than the selective antagonist ifenprodil. The EC₅₀s of MK801 (E) and LY354740 (F) were 0.074 and 19 nanomoles with R² regression of 0.8863 and 0.9094, respectively.

Figure 4

The effect of MK801 alone and with different dose combinations of LY354740 on RGC apoptosis in SSP-treated eyes. Results are shown as a percentage reduction of histologic RGC apoptosis compared with the vehicle-treated control. All treatments resulted in significant reduction of RGC apoptosis compared with the control (P < 0.05). The most effective combined regimen with low-dose MK801 was 0.06/20 (MK801/LY354740) nanomoles.

Figure 5

Effect of combined application of MK801/LY354740 (0.06/20 nanomoles) on RGC apoptosis at 3 weeks in an OHT model, when administrated at different times after surgery. All three treatment groups resulted in a reduction in RGC apoptosis. However, the most effective timing of the treatment application was at 0 weeks (at the time of OHT surgery), compared with 1 or 2 weeks (*P < 0.05).