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Masayuki Hirata, Emi Nakajima, Thomas R Shearer, Mitsuyoshi Azuma; Hypoxia activates calpain in the nerve fiber layer of monkey retinal explants. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1744. doi: https://doi.org/.
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The vascular ischemic theory of retinal nerve damage assumes that decreased blood flow in the ophthalmic artery reduces oxygenation and thereby damages neurons. Optic nerve damage from hypoxia in glaucoma includes ischemia, impaired axonal transport, and free radical formation. Hypoxia also leads to retinal cell death by activating calpain enzyme. Data from our cultured monkey retinas show that: 1) Hypoxia induces retinal cell death. 2) An active form of calpain is observed in retina during hypoxia. 3) Calpain substrates are proteolyzed. 4) All such changes are ameliorated by calpain inhibitor SNJ1945. However, we still do not know in which specific retinal layer(s) calpains are activated. The purposes of the present experiment were to: 1) determine where calpain is activated in retina, and 2) improve the methodology for culture of monkey retina.
Equal-sized pieces of retinas were dissected from enucleated monkey eyes and cultured with the RGC side facing up on micro porous membranes in 6-well culture plates. After 3 hrs of normoxia, the retinal explants were incubated under hypoxia for 16 hrs in medium with 0.5 mM glucose, and then reoxygenated for 8 hrs in medium with 5.5 mM glucose. When used, calpain inhibitor SNJ-1945 was added 1 hr before hypoxia treatment. Formalin-fixed paraffin embedded sections were subjected to TUNEL staining and immunohistochemistory for α-spectrin.
Under improved culture conditions, TUNEL positive cells were minimal under normoxia and indicated that proper culture time was somewhat less than 2 days. During hypoxia/reoxygenation, the 150 kDa calpain-specific, α-spectrin breakdown product first accumulated in the nerve fiber layer, and SNJ-1945 inhibited it’s formation. TUNEL staining then increased in the ganglion cell layer.
During hypoxia/reoxygenation, calpain is activated in the nerve fiber layer, the first point of injury. This is followed by death of the parent retinal ganglion cells. These observations suggest that calpain may be involved in the degeneration of retinal nerve fibers during hypoxia in glaucoma. Dr. Shearer receives a research contract and consulting fees from, and Drs. Hirata, Nakajima and Azuma are employees of, Senju Pharmaceutical Co. Ltd.
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