Basic research to achieve reconstitution of optic tract and the retinotopic projection

实现视束重建和视网膜专题投影的基础研究

• 批准号: 13671839
• 负责人: KOSAKA Jun
• 金额: $ 2.56万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671839/
• 关键词: retinotopy; DNA array; in situ hybridization; retina; optic nerve; BH3 only protein; 末梢神経移植; Hrk; BimEL; 視神経切断; 網膜神経細胞; DNAアレイ; ラット; 網膜視覚中枢投射; RT-PCR

The purpose of the present study is to develop new strategy for reconstitution of visual pathway, from optic nerve regrowth to formation of retinotopic map. Peripheral nerve grafting is usually carried out against, the transected stump of optic nerve to stimulate axonal regeneration of retinal ganglion cells, however, the retinotopic map is not reconstituted in mammals. In the present study, I tried to graft two pieces of peripheral nerve neuraxis into the rat eye ball. One grafting was put into the nasal sclera, and another to the temporal sclera. This "double grafting" was not succeeded because of severe tissue damage, however, some important information were piled up. Especially, peripheral nerve grafting should be penetrated at the anterior portions of the Ora serrata to avoid severe intraocular hemorrhage.From the results of DNA array and cDNA differential screening between the normal retina and the retina 1 day after axotomy in the rat, I focused BH3 only proteins and new genes. … More One of BH3-only proteins, Hrk mRNA was not detectable by RT-PCR in the normal rat retina, however, Hrk expression was induced after optic nerve transection. In situ hybridization revealed that Hrk mRNA was localized in retinal ganglion cells after axotomy. Realtime RT-PCR revealed 3 times larger expression of ARG357 mRNA in axotomized rat retina than in the intact. BimEL expression was also enhanced after axotomy. Quantitative in situ hybridization using TSA systems and quantification of fluorescent strength with the confocal microscope clearly demonstrated that stronger signals for BimEL and ARG 357 were localized in the axotomized retinal ganglion cells than the intact cells. This strategy which I developed in this study is so useful to carry out molecular approach against small amount of isolated mRNAs, like as the nasal retina when analysis of reconstitution of retinotopic map.The inductions of Hrk, BimEL and ARG357 have broken through on molecular analysis of retrograde degeneration of retinal ganglion cells after axotomy. These findings have brought a valuable improvement of strategy to promote functional recovery of visual system from severe damages. Less

本研究的目的是从视神经再生到视网膜定位图的形成，为视路重建提供新的策略。周围神经移植通常是在视神经切断的残端上进行的，以刺激视网膜神经节细胞的轴突再生，然而，在哺乳动物中，视网膜定位图不能重建。本研究尝试将两段周围神经轴突移植到大鼠眼球内。将一个移植物置于鼻侧巩膜，另一个置于颞侧巩膜。由于严重的组织损伤，这种“双移植”没有成功，但却积累了一些重要的信息。特别是，周围神经移植应穿透锯齿缘的前部，以避免严重的眼内出血。从DNA阵列和cDNA差异筛选的结果，在正常的视网膜和大鼠轴突切断后1天的视网膜，我集中BH 3只有蛋白质和新的基因。 ...更多信息 Hrk是一种BH 3特有的蛋白质，在正常大鼠视网膜中RT-PCR检测不到Hrk mRNA的表达，但在视神经切断后，Hrk表达被诱导。原位杂交显示Hrk mRNA定位于轴突切断后的视网膜神经节细胞。实时RT-PCR显示，ARG 357 mRNA在轴突切断的大鼠视网膜中的表达是完整视网膜中的3倍。轴突切断后BimEL表达也增强。使用TSA系统的定量原位杂交和用共聚焦显微镜定量荧光强度清楚地表明，BimEL和ARG 357的更强的信号被定位在轴突切断的视网膜神经节细胞比完整的细胞。本研究中开发的这一策略对于针对少量分离的mRNA（如鼻侧视网膜）进行视网膜定位图重建分析时非常有用。Hrk、BimEL和ARG 357的诱导在轴突切断后视网膜神经节细胞退行性变性的分子分析方面取得了突破。这些发现为促进严重视觉系统损伤后功能恢复的策略带来了有价值的改进。少

项目成果

Kimura T, Kosaka J, Nomura T, Yamada T, Miki Y, Takagi K, Kogami T, Sasaki J.: "Quantification of in situ hybridization signals in rat testes"Journal of Histochemistry and Cytochemistry. 52(印刷中). 1-8 (2004)

Kimura T、Kosaka J、Nomura T、Yamada T、Miki Y、Takagi K、Kogami T、Sasaki J.：“大鼠测试中原位杂交信号的量化”《组织化学和细胞化学杂志》52（出版中）。 8 (2004)

Kosaka J, Wakabayashi T, Sasaki J.: "Histochemical Evidence for the Differential Involvement of α and β Isoenzymes of Protein Kinase C in ON- and OFF-Pathways in the Rat Retina"Frontiers in Life Science. (印刷中). (2004)

Kosaka J、Wakabayashi T、Sasaki J.：“蛋白激酶 C α 和 β 同工酶在大鼠视网膜 ON 和 OFF 通路中差异参与的组织化学证据”生命科学前沿（2004 年出版）。 ）

Wakabayashi T, Kosaka J, Hommura S: "Up regulation of Hrk, a regulator of cell death, in retinal ganglion cells of axotomized rat retina"Neuroscience Letters. 318. 77-80 (2002)

Wakabayashi T、Kosaka J、Hommura S：“在轴突大鼠视网膜的视网膜神经节细胞中，细胞死亡调节剂 Hrk 的上调”《神经科学快报》。

Kosaka J, Wakabayashi T, Sasaki J: "Histochemical Evidence for the Differential Involvement of Alpha and Beta Isoenzymes of Protein Kinase C in ON- and OFF-Pathways in the Rat Retina"Advancement of Life Science (edt.By Setsu K.and Roan C.-H.) AMVO Pub.Com

Kosaka J、Wakabayashi T、Sasaki J：“蛋白激酶 C 的 Alpha 和 Beta 同工酶在大鼠视网膜 ON 和 OFF 通路中差异参与的组织化学证据”生命科学进展（由 Setsu K. 和 Roan 编辑）

Wakabayashi T, Kosaka J, Honmura S.: "Up regulation of Hrk, a regulator of cell death, in retinal ganglion cells of axotomized rat retina"Neuroscience Letters. 318(2). 77-81 (2002)

Wakabayashi T、Kosaka J、Honmura S.：“在轴突大鼠视网膜的视网膜神经节细胞中，Hrk（细胞死亡调节剂）的上调”《神经科学快报》。