Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Portland, Oregon, United States.
Department of Integrative Biomedical & Diagnostic Sciences, Oregon Health & Science University, Portland, Oregon, United States.
Invest Ophthalmol Vis Sci. 2020 Nov 2;61(13):13. doi: 10.1167/iovs.61.13.13.
Activation of proteolytic enzymes, calpains and caspases, have been observed in many models of retinal disease. We previously demonstrated calpain activation in monkey retinal explants cultured under hypoxia. However, cellular responses are often species-specific. The purpose of the present study was to determine whether calpains or caspase-3 was involved in retinal ganglion cell (RGC) damage caused by hypoxia/reoxygenation in human retinal explants. The explant model was improved by use of an oxygen-controlled chamber.
Human and monkey retinal explants were cultured under hypoxic conditions in an oxygen-controlled chamber and then reoxygenated. Calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting were performed for calpains 1 and 2, calpastatin, α-spectrin, calpain-specific α-spectrin breakdown product at 150 kDa (SBDP150), caspase-3, and apoptosis-inducing factor (AIF). Propidium iodide (PI) staining measured membrane disruption, and TUNEL staining detected DNA fragmentation.
Activation of calpains in nerve fibers and increases of PI-positive RGCs were observed in retinal explants incubated for 16-hour hypoxia/8-hour reoxygenation. Except for autolysis of calpain 2, SNJ-1945 ameliorated these changes. In longer incubations under 24-hour hypoxia/16-hour reoxygenation, TUNEL-positive cells appeared, although activated caspase-3 and truncated AIF were not observed. DNA fragmentation was inhibited by SNJ-1945.
An improved human retinal explant model showed that calpains, not caspase-3, were involved in cell damage induced by hypoxia/reoxygenation. This finding could be relevant for patient treatment with a calpain inhibitor if calpain activation is documented in human retinal ischemic diseases.
在许多视网膜疾病模型中，已观察到蛋白水解酶、钙蛋白酶和半胱天冬酶的激活。我们之前证明了在缺氧条件下培养的猴视网膜外植体中钙蛋白酶的激活。然而，细胞反应通常具有物种特异性。本研究的目的是确定钙蛋白酶或半胱天冬酶-3 是否参与了人类视网膜外植体中缺氧/复氧引起的视网膜神经节细胞（RGC）损伤。通过使用含氧可控室，改进了外植体模型。
人类和猴视网膜外植体在含氧可控室中于缺氧条件下培养，然后再复氧。在整个培养过程中维持钙蛋白酶抑制剂 SNJ-1945。用免疫组织化学和免疫印迹法检测钙蛋白酶 1 和 2、钙蛋白酶抑制剂、α- spectrin、150 kDa 的钙蛋白酶特异性α- spectrin 断裂产物（SBDP150）、半胱天冬酶-3 和凋亡诱导因子（AIF）。碘化丙啶（PI）染色测量膜破坏，TUNEL 染色检测 DNA 片段化。
在孵育 16 小时缺氧/8 小时复氧的视网膜外植体中，观察到神经纤维中钙蛋白酶的激活和 PI 阳性 RGC 的增加。除了钙蛋白酶 2 的自溶外，SNJ-1945 改善了这些变化。在 24 小时缺氧/16 小时复氧的较长孵育中，尽管未观察到激活的半胱天冬酶-3 和截断的 AIF，但出现了 TUNEL 阳性细胞。SNJ-1945 抑制了 DNA 片段化。
改进的人类视网膜外植体模型表明，钙蛋白酶（而不是半胱天冬酶-3）参与了缺氧/复氧诱导的细胞损伤。如果在人类视网膜缺血性疾病中证实钙蛋白酶激活，这一发现可能与使用钙蛋白酶抑制剂治疗患者有关。
