iNOS-inhibitor treatment leads to cell rescue in a porcine retina organ culture model

Abstract

Purpose : Nitrite oxide (NO) plays an important role in the pathogenesis of various retinal diseases, especially when hypoxic processes are involved. This degeneration process can be simulated by incubating porcine retinal explants with cobalt-chloride (CoCl₂). Based on this model, therapeutic approaches can be tested. Hence, treatment with the iNOS-inhibitor 1400W as a possible protectant was evaluated.

Methods : Explants of porcine retinae were cultivated for 4 and 8 days. Degeneration through 300µM CoCl₂ (for 48h) and treatment with the iNOS-inhibitor 1400W (for 72h) were applied simultaneously from day one. Three groups were compared: control, CoCl₂, and CoCl₂+iNOS-inhibitor. At days 4 and 8, retinal ganglion cells (RGCs), bipolar and amacrine cells were analyzed via immunohistology and qRT-PCR. Furthermore, the influence on the glia cells and different markers (HIF1α, HSP70, iNOS) involved in hypoxic/stress processes were evaluated.

Results : Treatment with CoCl₂ resulted in a significant loss of RGCs already after 4 days (Co: 40.0±1.0 RBPMS⁺cells/mm; CoCl₂: 25.7±2.5 RBPMS⁺cells/mm; p=0.0002), which was counteracted by the iNOS-inhibitor (33.9±2.2 RBPMS⁺cells/mm; p=0.021). After 8 days the CoCl₂ group displayed a significant loss in amacrine cells (Co: 17.7±3.5 calretinin⁺cells/mm; CoCl₂: 3.4±0.5 calretinin⁺cells/mm; p=0.0003) and downregulation of PVALB mRNA expression (2.8-fold; p<0.05). A drastic reduction in bipolar cells was observed after 8 days (Co: 59.2±2.4 PCKα⁺cells/mm; CoCl₂: 17.1±4.7 PCKα⁺cells/mm p=0.0001), which was prevented by 1400W (45.4±7.1 PCKα⁺cells/mm p=0.001). A significant decrease of Iba⁺ microglia (p=0.0001) and the corresponding mRNA marker was found in the CoCl₂ group after 4 and 8 days, which could not be prevented by the iNOS-inhibitor. CoCl₂ induced a significant increase in the mRNA expression of HSP70 (5-fold; p=0.008), HIF1α and iNOS (4.5-fold; p-p=0.045). Again, this effect was counteracted by the iNOS-inhibitor.

Conclusions : CoCl₂ induces strong degeneration in porcine retinae by mimicking hypoxia, damaging RGCs, amacrine, bipolar and microglia cells. Treatment with the iNOS-inhibitor counteracted these effects to some extent and clearly prevented the loss of retinal ganglion and bipolar cells. Hence, the iNOS-inhibitor 1400W seems to be a very promising treatment for retinal diseases.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.