title: The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage.
abstract: OBJECTIVE
  In this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway.
  
  
  METHODS
  Oxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH, 30mM, 2μl). Control eyes were injected with 2μl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100mg/kg, intraperitoneal administration) in these eyes.
  
  
  RESULTS
  Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7days after treatment (control: 3806.7±575.2RGCs/mm(2), AAPH: 3156.1±371.2RGCs/mm(2), P<0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0±226.9RGCs/mm(2), AAPH+SNJ-1945: 3717.1±614.6RGCs/mm(2), P<0.01).
  
  
  CONCLUSIONS
  AAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Yokoyama
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Kazuichi
authors.initials: K
authors.lastname: Maruyama
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Omodaka
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yasuda
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Himori
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Morin
authors.initials: M
authors.lastname: Ryu
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Koji M
authors.initials: KM
authors.lastname: Nishiguchi
authors.affiliation: Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan; Department of Retinal Disease Control, Tohoku University Graduate School of Medicine, Miyagi, Japan; Department of Advanced Ophthalmic Medicine, Tohoku University Graduate School of Medicine, Miyagi, Japan. Electronic address: ntoru@oph.med.tohoku.ac.jp.
authors.forename: Toru
authors.initials: T
authors.lastname: Nakazawa
mesh_terms: D000578:Amidines / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002154:Calpain / Q000502:physiology*; D002219:Carbamates / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D006023:Glycoproteins / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000378:metabolism; D023421:Models, Animal; D018384:Oxidative Stress / Q000502:physiology*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism*; D013049:Spectrin
chemical_list: D000578:Amidines; D002154:Calpain; D002219:Carbamates; D002352:Carrier Proteins; D006023:Glycoproteins; D008840:Microfilament Proteins; D013049:Spectrin; C515164:((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester; C039304:calpain inhibitors; C046728:2,2'-azobis(2-amidinopropane)
keywords: AAPH; Apoptosis; Calpain; Oxidative stress; Retinal ganglion cell; SNJ-1945