Hypoxia activates calpains in the nerve fiber layer of monkey retinal explants

Masayuki Hirata, Thomas R. Shearer, Mitsuyoshi Azuma

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Purpose. 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.

Original languageEnglish (US)
Pages (from-to)6049-6057
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Issue number10
StatePublished - 2015


  • Calpain
  • Cell death
  • Monkey retinal explant culture
  • Nerve fiber layer
  • Proteolysis
  • Retinal ganglion cells
  • SNJ-1945

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience


Dive into the research topics of 'Hypoxia activates calpains in the nerve fiber layer of monkey retinal explants'. Together they form a unique fingerprint.

Cite this