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Upregulation of doublecortin in the rat retina after ischemia-reperfusion injury

Upregulation of doublecortin in the rat retina after ischemia-reperfusion injury
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대학원 의학과
이화여자대학교 대학원
Horizontal cells are interneurons of the retina contributing to the processing of light stimuli in the outer plexiform layer. It has been known that the horizontal cells of the rat retina are resistant to degenerative processes induced by ischemia-reperfusion injury. Ca^2+ overload by ischemic insult is a common step in various neurodegenerative processes. One of the intracellular calcium regulating systems is calcium binding protein which act to buffer the bulk of Ca^2+. One of these proteins, calbindin D28k (calbindin), an intracellular protein of the EF-hand family, has been shown to have a neuroprotective role to various stimuli including ischemia. In the rat retina, calbindin labeling is most prominent in the horizontal cells. Doublecortin(DCX) is 40 kDa microtubule-associated phospho-protein. DCX is also known to have regions that are homologous to the putative Ca^2+ calmodulin-dependent protein kinase, which may be involved in neuronal migration through Ca^2+ -dependent signaling pathways. To identify the possibile roles of calbindin and DCX in the ischemic retina, immunocytochemistry and Western blot analysis were conducted by using an anti-DCX antibody in the retina ischemia-reperfusion injury. Additionally, DCX-expression cells were characterized by double immunocytochemistry using an antibody for anti-calbindin. The results are follwing. 1. Transient ischemic conditions induced by the elevation the intraocular pressure(IOP) for 60 minutes resulted that the thickness of the entire retina declined gradually and was reduced to about 30% after 4 weeks. The thickness of inner nuclear layer (INL) was changed early between 1 and 3 days after insults but decreased by less than 40% after 4 weeks. The thickness of the outer nuclear layer (ONL), in contrast, was nearly unchanged during the first week, but then decreased by more than 75% during the next 2 weeks and finally consisted of only 1 or 2 rows of cells. 2. Western blot analysis demonstrated a single band of 28 kDa molecule, that of calbindin during all the postlesional periods, the intensity of which was not distinguishably different from that of the normal retina to 4 weeks following ischemia-reperfusion in accordance with the immunocytochemical observations. Strong calbindin immunoreactivity was not distinguishably different from that of the normal retina during 4 weeks after reperfusion. 3. In immunoblot analysis, antibodies to DCX recognized a single band with an apparent molecular mass of 40 kDa, the intensity of which increased up to 4 weeks following ischemia-reperfusion in accordance with the immunocytochemical observations. Densitometric analysis revealed that DCX protein levels were upregulated to about 370% of control by 28 days after reperfusion. In normal rat retina immunostained for DCX, labeling was very weak and labeled profiles were horizontally oriented. 4 weeks after reperfusion, strong DCX immunoreactivity appeared in the somata located in the outer part of the INL. After reperfusion, DCX-labeled processes are descended into the IPL and ramified mainly in the IPL. 4. Double-labeling using antiserum against DCX and antiserum against calbindin, a specific marker for the horizontal cells showed coexpression of DCX and calbindin in the cells located in the outer part of the INL at 1 week, 4 weeks. These results demonstrate clearly that DCX labeled cells that appeared in the outer part of the INL from retina ischemia-reperfusion injury are horizontal cells. Consequently, these results suggest that DCX expressed in horizontal cell in ischemia-reperfusion injury may play an important role in the protection of horizontal cells in ischemia-reperfusion injury. However, more detailed studies are clearly needed to clarify the possible mechanisms responsible for the expression of DCX in the retina after ischemia-reperfusion injury.;Doublecortin (DCX)은 발생중인 중추 신경계의 여러 부분과 뇌에서 허혈-재관류 손상받은 신경의 이동, 신경돌기의 성장과 분화에 필요한 40 kDa의 크기를 가진 미세소관 연관 인산단백질(microtubule- associated phosphoprotein)이다. 본 연구는 흰쥐 망막에서 허혈-재관류 손상후의 DCX의 발현을 알아보고자 시행되었다. 90-120 mmHg까지 안압을 올려 60분간 유지함으로써 흰쥐의 망막 조직에 일시적인 허혈을 유도하였으며 재관류후 1일, 3일, 7일, 14일, 28일에 흰쥐를 희생시키고 안구를 적출한 후 망막조직을 절취 하였다. 망막조직은 DCX와 calbindin D_28k (calbindin)의 항혈청으로 면역세포화학염색과 western blot 분석을 시행하였다. 허혈성 손상을 유도한 망막은 허혈 손상후 재관류의 기간이 늘어날수록 그 두께가 감소하였으며 4주째에는 정상 망막 두께의 30%정도로 감소하였다. 하지만 내과립층에 존재하는 수평세포는 대조군과 허혈 손상군에서 모두 비슷한 배열 및 세포밀도를 보였으며 허혈 손상후 재관류의 기간이 늘어남에 따라서도 차이를 보이지 않았다. 수평세포내의 DCX의 발현은 재관류후 1일부터 증가하기 시작하여 28일까지 대조군의 370%수준까지 증가하였고 calbindin의 발현은 재관류후 실험기간 동안 정상에 비하여 의미있는 변화를 보이지 않았다. DCX로 표지된 수평세포로부터 나온 돌기는 재관류 7일후에 속그물층까지 뻗어내려갔다. DCX과 수평세포의 표지인자인 calbindin의 항혈청으로 이중 염색하였을 때 재관류후 1주 및 4주의 조직에서 내과립층의 바깥쪽에 존재하는 세포층에 DCX과 calbindin이 함께 발현됨을 확인하였으며 이를 통해 DCX이 발현되어 나타난 세포가 수평세포임을 확인하였다.
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