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

Masayuki Hirata, Thomas (Tom) Shearer, Mitsuyoshi Azuma

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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
Volume56
Issue number10
DOIs
StatePublished - 2015

Fingerprint

Calpain
Nerve Fibers
Haplorhini
In Situ Nick-End Labeling
Spectrin
Cell Death
Retinal Ganglion Cells
Retina
Synucleins
Ophthalmic Artery
Retinal Degeneration
Axonal Transport
Enzyme Activation
Hypoxia
Tubulin
Immunoblotting
Blood Vessels
Immunohistochemistry
Staining and Labeling
Membranes

Keywords

  • 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

Cite this

Hypoxia activates calpains in the nerve fiber layer of monkey retinal explants. / Hirata, Masayuki; Shearer, Thomas (Tom); Azuma, Mitsuyoshi.

In: Investigative Ophthalmology and Visual Science, Vol. 56, No. 10, 2015, p. 6049-6057.

Research output: Contribution to journalArticle

@article{db92047877654fdfa8b3b9fea5f636b4,
title = "Hypoxia activates calpains in the nerve fiber layer of monkey retinal explants",
abstract = "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.",
keywords = "Calpain, Cell death, Monkey retinal explant culture, Nerve fiber layer, Proteolysis, Retinal ganglion cells, SNJ-1945",
author = "Masayuki Hirata and Shearer, {Thomas (Tom)} and Mitsuyoshi Azuma",
year = "2015",
doi = "10.1167/iovs.15-17360",
language = "English (US)",
volume = "56",
pages = "6049--6057",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "10",

}

TY - JOUR

T1 - Hypoxia activates calpains in the nerve fiber layer of monkey retinal explants

AU - Hirata, Masayuki

AU - Shearer, Thomas (Tom)

AU - Azuma, Mitsuyoshi

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

KW - Calpain

KW - Cell death

KW - Monkey retinal explant culture

KW - Nerve fiber layer

KW - Proteolysis

KW - Retinal ganglion cells

KW - SNJ-1945

UR - http://www.scopus.com/inward/record.url?scp=84942154755&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84942154755&partnerID=8YFLogxK

U2 - 10.1167/iovs.15-17360

DO - 10.1167/iovs.15-17360

M3 - Article

VL - 56

SP - 6049

EP - 6057

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 10

ER -