1999 Fiscal Year Final Research Report Summary
The elucidation of molecular mechanism in delayed neruonal death and its control in rat retina
| Project/Area Number |
10307042
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Ophthalmology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
HONDA Yoshihito Kyoto University, Graduate School of Medicine, Professor, 医学研究科, 教授 (90026930)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Masayo Kyoto University, Graduate School of Medicine, Assistant Professor, 医学研究科, 助手 (80252443)
TAKAGI Hitoshi Kyoto University, Graduate School of Medicine, Lecturer, 医学研究科, 講師 (70283596)
KASHII Satoshi Kyoto University, Graduate School of Medicine, Associate Professor, 医学研究科, 助教授 (50194717)
MANDAI Michiko Kyoto University, Graduate School of Medicine, Asistant Professor, 医学研究科, 助手 (80263086)
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| Project Period (FY) |
1998 – 1999
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| Keywords | delayed neruonal death / Glutamate / N-methyl-D-aspartate(NMDA) / Nitric oxide / Bax / Bcl-2 / DNA fragmentation / Apoptosis |
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| Research Abstract |
We evaluated the involvement of apoptosis and contributions of Bcl-2 family genes in delayed neruonal death after retinal ischemia in Sprague-Dawley rat. Retinal ischemia was induced by elevating IOP to 130 mmHg for 45 min. Histological specimens were obtained at various time after reperfusion and examined by TUNEL staining. A significant number of TUNEL positive cells were observed in the ganglion cell layer (GCL) and inner nuclear layer (INL) starting at 6 to 48 hr after transient ischemia and reached a maximum 24 hr after ischemia. DNA was extracted from the-ischemic retina at 24 and 48 hr after reperfusion and the contralateral non-ischemic retina and electrophoresed. DNA laddering was observed on agarose gel electrophoresis in retinas 24 and 48 hr after ischemia but not in normal retina. RT-PCR analysis was carried out with the retina at various time after transient ischemia using specific primers for Bcl-2 and Bax. It demonstrated that Bax gene expression gradually increased as e
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arly as 6 hr, reached a peak at 24 hr, then decreased to near the baseline level at 168 hr, while Bcl-2 gene expression did not show any obvious change at any time point after transient ischemia. Immunohistochemical study using a specific antibody for Bax was performed using retina 24 hr after transient ischemia and fingings were compared with those of the contralateral non-ischemic retina. The number of TUNEL-positive cells after transient ischemia were measured using retinas pretreated by intra-vitreous injection of a Bax antisense oligodeoxynucleotide (ODN) or a randomly sequenced ODN. intense Bax protein immunoreactivity was detected in the GCL and INL of the post-ischemic retina 24 hr after reperfusion, while little immunoreactivity was present in the non-ichemic retina. The inhibition of Bax protein expression by antisense ODNs reduced the number of TUNEL-positive cells. We conclude some of the neuronal death caused by transient retinal ischemia involves an active cell death process of apoptosis induced by the upregulaion of Bax.
Furthermore, we examine histological changes after transient retinal ischemia in transgenic mice in which neurons overexpress Bcl-2, in comparison with those of wild-type littermates. Histological sections were obtained 7 days after ischemic insult. Morphometric analysis were performed to quantify the ischemic injury in transgenic mice overexpressing Bcl-2 (NSE73a/ab) vs wild type littermates (C57BL/6). The number of cells in the GCL and the thickness of the inner plexiform layer (IPL), INL, and outer nuclear layer (ONL) were quantified. For each animal, these parameters in the post-ischemic eye were normalized to those in the intact contralateral eye and shown as a percentage. The GCL cell number was approximately 60% greater in transgenic mice than that in wild-type littermates. The IPL thickness was 33.7±1.8 μm in wild-type and 43.8±6.7 μm in transgenic mice. In wild-type mice, ischemia reduced the GCL cell number and IPL thickness to 64.6±7.5, and 42.3±0.6% of the control, respectively, whereas ischemia reduced the GCL cell number and IPL thickness to 81.9±5.6, and 82.4±19.6 in transgenic mice. These results suggest that retinal neurons overexpressing Bcl-2 become torelant to ischemic injury. Less
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Research Products
(14 results)
