Metallothionein, an endogenous antioxidant, protects against retinal neuron damage in mice.
OBJECTIVE
To clarify the functional role of metallothionein (MT) in retinal damage in mice deficient in both MT-I and -II (MT-I/-II-deficient mice [C57BL/6J background]) and wild-type (C57BL/6J) mice and MT induction (zinc sulfate [ZnSO4] and 1alpha, 25-dihydroxyvitamin D3 [Vit. D3]).


METHODS
Retinal, cell damage was induced by intravitreous injection of N-methyl-D-aspartate (NMDA; 40 nmol/eye). Retinal MT-I, -II, and -III mRNA expression was monitored by real-time reverse-transcription-PCR of total retinal RNA from eyes injected or not injected with NMDA. In wild-type mice, MT-I and -II immunohistochemistry was performed (with antibody that recognizes both proteins) 12 and 24 hours after intravitreous NMDA injection. To examine the involvement of induced retinal MT, ZnSO4 (10 nmol/eye) or Vit. D3 (0.2 or 2 ng/eye) was intravitreously injected 24 hours before NMDA injection in wild-type or MT-I/-II-deficient mice, and ganglion cell layer (GCL) cell loss and inner plexiform layer (IPL) thinning were evaluated 7 days after the NMDA injection. The protective effect of Vit. D3 was assessed against the RGC-5 cell death induced by oxidative stress (using buthionine sulfoximine [BSO] to deplete glutathione in combination with glutamate to inhibit cystine uptake).


RESULTS
In wild-type mice, MT-II mRNA expression was time-dependently elevated by NMDA (5.9 and 7.4 times versus the nontreated control at 4 and 12 hours, respectively, after injection), with the normal level being regained within 24 hours. In contrast, MT-I and -III showed persistent decreases (to <50% control) from 4 to 24 hours. In wild-type mice, MT-like immunoreactivity was increased in the inner retina (GCL and IPL) 12 and 24 hours after NMDA injection. At 7 days after NMDA injection in MT-I/-II-deficient mice (versus wild-type mice), GCL cell loss was increased, but IPL thickness was not different. Pretreatment with ZnSO4 or Vit. D3 increased inner retinal MT-like immunoreactivity 24 hours after NMDA injection and significantly attenuated NMDA-induced GCL cell loss in wild-type mice, but ZnSO4 pretreatment did not protect against such cell loss in MT-I/-II-deficient mice. In vitro, Vit. D3 pretreatment (100 nM) reduced BSO+glutamate-induced RGC-5 cell death.


CONCLUSIONS
These findings suggest that MT, especially MT-II, protects against retinal neuron damage, by acting as an endogenous antioxidant.
Suemori
Shinsuke
S
Department of Biofunctional Molecules, Gifu Pharmaceutical University, Gifu, Japan.
Shimazawa
Masamitsu
M
Kawase
Kazuhide
K
Satoh
Masahiko
M
Nagase
Hisamitsu
H
Yamamoto
Tetsuya
T
Hara
Hideaki
H
D000818:Animals; D000975:Antioxidants / Q000502:physiology*; D019328:Buthionine Sulfoximine / Q000633:toxicity; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D002762:Cholecalciferol / Q000494:pharmacology; D018698:Glutamic Acid / Q000633:toxicity; D007124:Immunoenzyme Techniques; D007267:Injections; D008297:Male; D008668:Metallothionein / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016202:N-Methylaspartate / Q000633:toxicity; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000378:metabolism; D012164:Retinal Diseases / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D014822:Vitreous Body; D019287:Zinc Sulfate / Q000494:pharmacology
D000975:Antioxidants; D012333:RNA, Messenger; D002762:Cholecalciferol; D018698:Glutamic Acid; D019328:Buthionine Sulfoximine; D016202:N-Methylaspartate; D019287:Zinc Sulfate; D008668:Metallothionein