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Jose Hurst, Sandra Kuehn, Farina Rensinghoff, Teresa Tsai, Yathavan Satgunarajah, Karl Ulrich Bartz-Schmidt, Burkhard Dick, Stephanie C Joachim, Sven Schnichels; Degenerative effects of cobalt-chloride treatment on neurons and microglia in a porcine retina organ culture model. Invest. Ophthalmol. Vis. Sci. 2017;58(8):284.
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© ARVO (1962-2015); The Authors (2016-present)
To understand the pathological processes of retinal diseases, experimental models are necessary. High quantities of cobalt chloride (CoCl2) induce cytotoxic mechanisms via hypoxia mimicry. We aimed to establish an alternative porcine retina degeneration model for hypoxia via CoCl2 in order to reduce the number of animal experiments.
Organotypic cultures of porcine retina obtained from eyes from the local abattoir were cultivated and treated with different concentrations of CoCl2 (0, 100, 300 and 500 µM) for 48 h from day (d) 1 onwards. At day 4 and 8, retinas were processed for histological, Western blot and qRT-PCR analysis. RGCs (Brn-3a) microglia (Iba1), and macroglia response (GFAP, vimentin) as well as cellular stress response (Caspase3, p21, hypoxia induced factor-1a (HIF1α) and heat shock protein 70 (HSP70)) was evaluated.
After 4 and 8 days, CoCl2 induced a strong degeneration of the porcine retina. Compared to control a loss of RGCs, was observed after 4 days (Co: 20.1±2.2; 300 µM: 12.0±1.2; 500 µM: 12.3±1.3 Brn-3a+ cells/mm) as well after 8 days (Co: 11.6±0.8; 300 µM: 6.4±0.8; 500 µM CoCl2 3.9±0.5 Brn-3a+ cells/mm). An induction of HIF-1a mRNA (4d: 500 µM 4.8-fold; 8 d: 300 µM-2.3-fold; 500 µM: 2.6-fold) and HSP70 mRNA (4d: 300µM: 12-fold, 500µM: 344-fold; 8 d: 300µM: 16-fold, 500µM: 319-fold) expression were noted at both points in time. Also, the caspase 3 protein was activated and the p21 mRNA expression induced (4 d: 300µM: 2.7 fold; 500µM: 5.5-fold; 8d 300µM: 17.9-fold; 500µM: 24.3-fold). However, the effect of CoCl2 was not restricted to neurons; the macroglia response was reduced in comparison to the control group at day 4. Also, microglia population revealed a reduced cell amount and activity after CoCl2 treatment. After 4 days, the treatment with 500 µM CoCl2 reduced the amount of Iba1 cells significantly (227.8±18.0 cells/mm2) compared to the control (288.2±11.5 cells/mm2). At day 8, all concentrations had an effect on the Iba1 microglia in comparison to control. High concentrations of CoCl2 seemed also to be toxic for these cells.
In comparison to retinal hypoxia animal models, similar degenerative mechanisms were observed, while the glia response was unusual. Therefore, an effective and reproducible hypoxia-mimicking model for retinal degeneration was established, which is easy to handle and ready for drug studies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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