通過多光子顯微鏡觀察角膜內皮癒合的動態

Abstract

角膜內皮是源自神經嵴的單層六邊形細胞，負責維持角膜的水分恆定。由於增殖能力有限，受損的成熟內皮細胞最終會導致角膜功能失常和視力喪失。然而，在生理平衡和受傷後修補期間角膜內皮的細胞動態仍不清楚。在這項研究中我們證實了N-鈣粘蛋白是角膜內皮細胞的特異性標誌物。我們透過多光子顯微鏡，發現細胞密度和細胞核體積明顯地從角膜中心向外圍減小。此外，連續3天追蹤，我們發現角膜內皮細胞都靜止於細胞週期G1並且沒有遷移。我們利用精密控制的雷射在活體小鼠的角膜內皮中製造50x50μm2的傷口。我們發現了傷口癒合過程可分為三個階段：潛伏期，遷移期和重塑期。潛伏期在6小時後結束，然後進入遷移階段。傷口在接近38小時左右關閉。有趣的是，我們也發現，在潛伏期和重塑期角膜有部分內皮細胞均會脫離彈力層(Descemet’s membrane)而進一步喪失。我們的研究不僅建立了一種新的角膜內皮損傷模型，而且還闡明了體內傷口癒合過程。此外，癒合過程中角膜內皮細胞脫離後彈力層造成進一步的細胞損失，使我們能夠在眼科治療上給予一個新見解。

The corneal endothelium is a monolayer of hexagonal cells derived from the neural crest and is responsible for maintaining the dehydration of the cornea. Due to the limited proliferative capacity, impaired mature endothelial cells ultimately results in corneal decompensation and loss of vision. However, the cell dynamics of corneal endothelium during homeostasis and regeneration remain unclear. In this study, we characterize that the N-cadherin is the specific marker of corneal endothelial cells. Using multi-photon live imaging, the cell density and nucleus volume in corneal endothelium significantly decreases from the center to periphery. In addition, the corneal endothelial cells are quiescent, arrested at G1 phase, without migration for consecutive 3 days. We use laser ablation with precise spatiotemporal control to create a 50x50μm2 wound in corneal endothelium in vivo. Here we show that the wound healing process can be divided into three phases: latent phase, migration phase, and remodeling phase. The latent phase initiates at 6 hours and followed by the migration phase from 24 hours. The wound approaches closed at 38 hours. Interestingly, our data suggest that corneal endothelial cells detach from the Descemet’s membrane in both the latent phase and remodeling phase. This study not only develops a novel corneal endothelial injury model but also elucidates the wound healing process in vivo. Additionally, the cell loss from Descemet’s membrane during regeneration enables us to validate the new insight into ophthalmic therapy.