Retinal ganglion cells are protected through hypothermia treatment in an organ culture model

Abstract

Purpose : Cobalt is important for the neuronal integrity but high quantities induce cytotoxic mechanisms. Treatment with Cobalt-chloride (CoCl₂) led to strong degeneration of porcine retina through hypoxic mechanisms. Neurons of the inner retina layers are particularly affected. In the presented project this model is used to study possible protective effects of hypothermia treatment.

Methods : Porcine retina explants were cultivated for four and eight days at 37°C. Four groups were compared: control, control+30°C, CoCl₂, and CoCl₂+30°C. Hypothermia (30°C) was applied in parallel to hypoxic damage using 300µM CoCl₂ for 48 h. Retinal explants were subject to immunohistochemistry, qRT-PCR, and Western blot analysis. The number of retinal ganglion cells (RGC), as well as microglia, bipolar, and amacrine cells was evaluated and compared between groups.

Results :
At four days of cultivation, in the control group 30.9±1.1 Brn-3a⁺ cells/mm were counted, exposure with CoCl₂ led to a significant loss of RGCs in the CoCl₂+37 °C group (23.7±0.9 Brn-3a⁺ cells/mm, p<0.001). The hypothermia treatment (CoCl₂+30°C) counteracted this effect, on average 27.4±1.5 Brn-3a⁺ cells were detected (p=0.19). Also apoptosis rate in the CoCl₂+30°C group (53.2±2.4% cleaved caspase3⁺) was lower than in the CoCl₂ +37°C group (65.6±3.2% cleaved caspase3⁺).
Furthermore, hypothermia treatment attenuated significantly gene expression of stress marker p21 (5-fold, after four and eight days, p<0.001) and HSP70 (23-fold after four days, 7-fold after eight days, p<0.001) compared to the CoCl₂ treated group at 37°C.
The microglia degenerated in the CoCl₂+37 °C group at both time points, (four days: 18.8±0.7 Iba1⁺cells/mm; eight days: 13.3±0.9 Iba1⁺cells/mm) compared to control (four days: 45.1±4.1 Iba1⁺cells/mm; eight days: 40.0±2.4 Iba1⁺cells/mm, p<0.05). Hypothermia treatment could stop this effect and rescued the number of microglia (four days: 37.1±1.7 Iba1⁺cells/mm, p<0.001; eight days: 19.5±0.8 Iba1⁺cells/mm, p=0.03).

Conclusions : The cultivation of porcine retinae with CoCl₂ lead to a hypoxia-like degeneration processes. The hypothermia treatment induced cell protection and saved especially retinal ganglion cells from apoptosis. Hence, this system may serve as a first analysis model for therapy screening for retinal diseases. This alternative model is suitable for drug and treatment screening and will therefore reduce the number of animal studies.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.