利用體內共軛焦顯微鏡探討角膜內皮層移植術後之傷口癒合狀況

Abstract

【研究背景及目的】 傳統的全層角膜移植手術(penetrating keratoplasty)雖然在治療許多角膜病變上有很好的療效，但近幾年來對於治療內皮細胞失能的情形已漸漸被”戴氏膜撕除角膜內皮層移植手術” (DSAEK = Descemet's Stripping Automated Endothelial Keratoplasty)所取代。DSAEK手術的好處是開刀的傷口小、造成的散光少、術中不容易有猛爆性脈絡膜出血的合併症、術後的視力和傷口復原等等也有顯著的優勢。然而因為DSAEK手術目前仍相當新穎，有許多術中的條件設定仍未被充分了解；因此亟需客觀的研究來探討適當的手術方式。ㄧ般來說，DSAEK的手術過程中必須把受贈者本身角膜的戴氏膜(Descemet's membrane)和內皮細胞層(endothelial cell layer)移除，之後貼附上捐贈者之角膜內層瓣(posterior lamellar graft)。然而另一方理論認為在某些情況下角膜內皮細胞層和戴氏膜不需要移除(n-DSAEK, non-Descemet's Stripping Automated Endothelial Keratoplasty)，單純地把捐贈者之角膜內層瓣貼上即可，而壓縮在兩層之間的「戴氏膜/角膜內皮細胞層」不至於造成混濁甚至影響視力。到底何種理論正確，目前並無研究佐證。本研究乃針對以上臨床未解的問題，試圖用紐西蘭白兔的動物手術模式加以探討。 【研究方法與設計】 研究中將使用飛秒雷射切割製備角膜內層瓣，之後進行手術移植到兔眼上。被移植的兔眼又分為保留和去除「戴氏膜/角膜內皮細胞層」兩組。術後前三個月、每兩週一次利用「體內共軛焦顯微鏡」觀測夾在兩層之間的「戴氏膜/角膜內皮細胞層」隨時間所產生的變化，並測量其產生之角膜混濁現象。術後三個月取下兔眼，利用「前房光干渉斷層儀」(anterior segment OCT)輔助判斷角膜瓣的癒合和附著狀況，之後切片觀察顯微變化(H&E染色、免疫化學組織染色、穿透式電子顯微鏡)。 【結果】 在無戴氏膜撕除角膜內皮層移植手術(nDSAEK)中保留的受贈者內皮細胞層的細胞密度會隨著時間而逐漸下降。兩種手術方式所產生的交界面模糊程度(interface hazziness)都會隨著時間而下降，但”無戴氏膜撕除角膜內皮層移植手術”所產生的交界面模糊程度在所有的術後時間測量點均比”戴氏膜撕除角膜內皮層移植手術”來的大。 兩種手術方式術後的角膜厚度並沒有顯著的差異。利用H&E染色觀察”無戴氏膜撕除角膜內皮層移植手術”術後三個月之組織切片可以看見受贈者的戴氏膜層與細胞密度降低的內皮細胞層；利用穿透式電子顯微鏡則可以發現上述內皮細胞的型態因為擠壓而產生改變並且在內皮細胞內部可以觀察到和捐贈者基質層相同的纖維。免疫組織化學染色結果顯示這些受贈者內皮細胞原本的幫浦功能在術後三個月已經消失，但並沒有表現出上皮細胞的標誌，而部分受贈者內皮細胞表現出細胞凋亡現象。 【結論】 ”無戴氏膜撕除角膜內皮層移植手術”在術後結果與”戴氏膜撕除角膜內皮層移植手術”類似但有較明顯的交界面模糊程度。保留的受贈者內皮細胞會有型態上的改變並表現出吞噬能力，細胞密度可能因產生細胞凋亡而下降。

【Background and Study Purpose】 Although traditional penetrating keratoplasty (PK) was a widely accepted procedure, descemet's stripping automated endothelial keratoplasty (DSAEK) becomes popular and has replaced PK in the treatment of endothelial disorders. DSAEK is unique for the smaller wound size, lesser astigmatism, lesser chance of intraoperative expulsive hemorrhage, faster/better visual outcome and faster wound healing. However, there still exist many unknown factors for a better surgery. Most surgeons removed Descemet’s membrane and endothelial layer during DSAEK surgery follower by insertion of posterior corneal lamella. However, still some surgeons keep Descemet’s membrane and endothelial layer before the attachment of the posterior corneal lamella (n-DSAEK). They claim that the remained Descemet’s membrane/ endothelial layer between donor and recipient corneas will not inference corneal clarity and visual prognosis. The purpose of this project is to use New Zealand White Rabbit as the experimental model to find out the best surgical condition for DSAEK. 【Material and Method】 We will use femtosecond laser machine to create donor corneal posterior lamellas. The posterior corneal lamellas will be transplanted to rabbit corneas with or without stripping of Descemet’s membrane/corneal endothelial layer. After operation, the rabbit eyes will receive in vivo confocal microscopic examination every two weeks for totally 3 months. The Descemet’s membrane/corneal endothelial layer between donor and recipient corneas will be examined by morphology and Z-profile. At post-operative 3 months, the rabbit eyes will be obtained, and examined by anterior segment OCT to evaluate the attachment and healing of the transplanted grafts. In addition, the tissue section will be examined by H&E staining, immunohistochemical staining and transmission electron microscopy. 【Results】 The interface recipient endothelial cell density in nDSAEK gradually decreased in the follow-up period. The interface haziness of DSAEK and nDSAEK diminished gradually, and nDSAEK has greater interface opacity than DSAEK at all time points examined. There was no significant difference of total corneal thickness between DSAEK and nDSAEK at all time points. Tissue section using H&E staining showed preserved interface recipient Descemet’s membrane and decreased endothelial cell density three months after nDSAEK. Transmission electron microscopy revealed morphologically changed interface recipient endothelial cell and some collagen fibers similar as in graft stroma in recipient endothelial cell in nDSAEK three months after operation. The recipient endothelial cell didn’t express markers of pump function and epithelial cell by immunohistochemical staining. However some recipient endothelial cell went apoptotic pathway. 【Conclusion】 NDSAEK has similar surgical results as DSAEK in all parameters measured in this study. However, the interface haziness was greater than DSAEK, and recipient endothelial cell at the interface underwent morphological change and showed some phagocytic activity during the whole observational process. The endothelial cell density decreased due to cell apoptosis