title: Hypoxia Activates Calpains in the Nerve Fiber Layer of Monkey Retinal Explants.
abstract: OBJECTIVE
  The vascular ischemic hypothesis attributes nerve damage in the retina to decreased blood flow in the ophthalmic artery, reduced oxygenation, and impaired axonal transport. Activation of calpain enzymes contributes to retinal cell death during hypoxia. However, we still do not know in which specific retinal layers calpains are activated. Thus, the purpose of the present study was to investigate where and when calpains are activated in an improved culture model of hypoxic monkey retina.
  
  
  METHODS
  Monkey retinal explants were cultured on microporous membranes with the retinal ganglion cell (RGC) side facing up. Explants were incubated under hypoxic conditions, with or without additional reoxygenation. When it was used, the calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting assays for α-spectrin, calpains 1 and 2, calpastatin, β-III tubulin, and γ-synuclein were performed with specific antibodies. Cell death was assessed by TUNEL staining.
  
  
  RESULTS
  Under normoxic conditions, TUNEL-positive cells were minimal in our improved culture conditions. As early as 8 hours after hypoxia, the 150-kDa calpain-specific α-spectrin breakdown product appeared in the nerve fiber layer (NFL), where calpains 1 and 2 were localized. TUNEL-positive RGCs then increased at later time periods. The calpain inhibitor SNJ-1945 ameliorated changes induced by hypoxia or hypoxia/reoxygenation.
  
  
  CONCLUSIONS
  During hypoxia/reoxygenation in an improved, relevant monkey model, calpains were first activated in the NFL, followed by death of the parent RGCs. This observation suggest that calpain-induced degeneration of retinal nerve fibers may be an underlying mechanism for RGC death in hypoxic retinal neuropathies.
authors.affiliation: Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Portland, Oregon, United States.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Hirata
authors.affiliation: Department of Integrative Biosciences, Oregon Health and Science University, Portland, Oregon, United States.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Shearer
authors.affiliation: Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Portland, Oregon, United States 2Department of Integrative Biosciences, Oregon Health and Science University, Portland, Oregon, United States.
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Azuma
mesh_terms: D000818:Animals; D002154:Calpain / Q000378:metabolism*; D016923:Cell Death; D002478:Cells, Cultured; D004195:Disease Models, Animal; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology; D015151:Immunoblotting; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008253:Macaca mulatta; D009412:Nerve Fibers / Q000378:metabolism* / Q000473:pathology; D012164:Retinal Diseases / Q000378:metabolism* / Q000473:pathology; D012165:Retinal Ganglion Cells / Q000378:metabolism* / Q000473:pathology
chemical_list: D002154:Calpain