眼角膜手術之臨床與基礎相關研究

Clinical and Basic Research of Corneal Surgeries

陳偉勵(Wei-Li Chen)

指導教授 ： 胡芳蓉

共同指導教授 ： 尹相姝

國立臺灣大學/醫學院/臨床醫學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00616

摘要

本論文的宗旨為眼角膜手術之臨床與基礎相關研究。由於眼角膜手術涵蓋範圍非常廣泛，因此將重點放在兩大最常見的手術-"準分子雷射近視手術(在論文中側重LASIK)"和"角膜移植手術"方面。在第一部分，準分子雷射近視手術方面，論文將著重在睫狀體在LASIK術後的變化；由於經眼坦部玻璃體切除術也可能經由類似機轉導致睫狀體的變化，因此論文附帶研究經眼坦部玻璃體切除術，睫狀體相關構造的變化。這兩個有關睫狀體變化的研究，乃是用超高頻生物顯微鏡(UBM)來進行測試。至於第二部分，有關角膜移植手術方面，則以臨床和基礎研究的角度來探討角膜移植手術和角膜內皮細胞的相關性，並且對於角膜內皮細胞對類固醇的藥理反應，和促進細胞分裂的可能方法加以探討。
論文中的第一部分為睫狀體的相關研究。此一部分研究又再分為兩個子題，從臨床和基礎研究的角度，探討睫狀體在LASIK 和經眼坦部玻璃體切除後的變化。第一個關於睫狀體在準分子雷射近視手術(LASIK)後變化的研究，目的是評估兔子的眼睛在歷經LASIK 的角膜瓣切割過程中鞏膜吸力環的吸引過後，中心前房深度、睫狀體/隅角的截面積，以及眼壓的變化過程。研究的實驗方法是將三十隻紐西蘭白兔分派在以下的實驗組當中:(第一組)，並不加上鞏膜吸力環的吸引；(第二組)，鞏膜環吸引兩分鐘；(第三組)，鞏膜環吸引一分鐘；(第四組)，鞏膜環吸引二十秒鐘；(第五組)，鞏膜環吸引十秒鐘。術前和術後十分鐘，一小時，兩小時，一天，兩天，一週和兩週之後，以超高頻生物顯微鏡用以測量眼前房中心深度和睫截狀體/隅角的截面積等等因子。這部分的研究發現，在第二組到第五組的眼睛當中，術後十分鐘到一天，睫狀體有水腫的現象；而且其嚴重程度與鞏膜吸力環吸引的時間成正比。所有超高頻生物顯微鏡偵測出來的變化，在術後兩天之後都無法再偵測出來。至於前房中心深度或是眼壓方面，在整個觀察期間都不曾發現變化。本部分研究的結論是，兔子眼睛在歷經LASIK手術的角膜瓣切割過程中鞏膜吸力環的吸引過後，會有暫時性的睫狀體和隅角變化；而這些變化的嚴重程度與鞏膜吸力環吸引的時間成正比。睫狀體的水腫變化與隅角的狹窄有關，但是對於角膜和水晶體之間的距離並沒有影響。
由於動物實驗中發現睫狀體會在LASIK 術後產生變化，論文中進一步探討第二個子題，也就是在臨床上是否另一個器械在睫狀體附近進出的手術-"經眼坦部玻璃體切除術"也會引發睫狀體附近結構的變化。這一部分研究的目的是探討"經眼坦部玻璃體切除術"後，睫狀體剝離的機率，持續時間，危險因子和臨床後果的分析。此研究為人體試驗，研究的方式是分析103個病人的109隻眼睛，而這些眼睛是因為各種原因接受、經眼坦部玻璃體切除術"。研究中利用超高頻生物顯微鏡在術前、以及術後一天、三天和七天來檢測這些變化。之後，每週檢查一次一直到術後兩個月。之後分析病患的基本資料，術前，術中和術後因子對術後是否產生睫狀體剝離的影響。這個子題的研究發現，術後有46隻眼睛(42%)罹患睫狀體剝離，並且46隻眼睛中有40隻眼睛持續了3週。睫狀體剝離最常出現在罹患"增殖性糖尿病網膜病變"proliferative diabetic retinopathy (PDR) (64%)，以及網膜血管阻塞的病患 (or retinal vascular obstructive diseases (RVO) (47%)身上。"廣泛性的網膜雷射光凝固法"以及"冷凍治療"都會增加術後睫狀體剝離之機率；然而液體/氣體置換術則會減少術後睫狀體剝離之機率，因為在所有的病患當中，都沒有發現有人產生術後睫狀體剝離的現象。從這部分的研究可以發現，睫狀體剝離在"經眼坦部玻璃體切除術"病患身上是相當常見的術後後遺症。"增殖性糖尿病網膜病變"，"網膜血管阻塞病變"，和一些手術術式，例如"廣泛性的網膜雷術凝固法"和"冷凍治療"都是導致此後遺症發生的危險因子。
總結論文第一部分有關睫狀體的研究，不管在動物實驗或臨床研究方面，研究中都發現在常見的眼科手術過後，睫狀體都可能產生變化。雖然這些變化看似都是暫時性，並且沒有嚴重的後果，不過相關的問題仍應特別注意；而這也可用來解釋某些術後病患近距離視力滿意度不高的部分原因。
本論文第二部分為角膜移植和角膜內皮細胞的相關研究。研究分為四個子題，從臨床和基礎研究的角度，分別來研究角膜內皮細胞病變在角膜移植手術所扮演的角色，角膜內皮細胞對類固醇的藥物學反應，以及研究促進角膜內皮細胞分裂的機轉的可能發展。本論文的研究目的，乃在整合臨床與基礎研究，從巨觀和微觀的角度來研究角膜內皮細胞。
　　角膜移植和角膜內皮細胞的相關研究中的第一子題，是探討有關國內角膜移植手術的現況，並以之與國外的報告相比。雖然角膜移植手術在台灣已經引進數十年，然而有關接受此手術的病患病因的本土分析，資料還是非常欠缺。由於台大醫學院附設醫院為北台灣重要的教學後送醫院，本院的分析資料應可代表台灣的狀況。研究中分析探討過去十二年來(1987年到1999年)，在本院接受角膜移植手術的病患病因探討；並就不同的時間分期，來探討是否隨著時間的演變，醫療水準的變更，和各國國情的不同，這些接受手術的病患病有改變的趨勢。這個研究的研究方法乃為病歷的回溯分析。論文中將所有從1987年到1999年這十二年當中，在台大醫院接受角膜移植手術的病患作詳盡的病歷回溯分析。在盡可能的範圍之內，臨床上的病因將與病理學上的診斷作適當的評估。而研究的結果發現，這十二年內總共有770個角膜移植瓣是在本院所執行的，而最常見的術前診斷，按多寡順序排列分別為角膜?疤(corneal scar) (27.9%)，再次角膜移植手術 (regraft) (21.0%)，急性壞死性及潰瘍性角膜炎 (acute necrotic/ulcerative keratitis )(17.9%)，偽/無水晶體性水泡性角膜病變 (pseudophakic or aphakic bullous keratopathy) (17.6%), 傅氏角膜失養症(Fuchs' dystrophy) (4.5%)，以及角圓錐角膜 (keratoconus) (2.5%)。 這段時間隨著時間的推進，可發現”再次角膜移植手術”和”急性壞死性及潰瘍性角膜炎”的病患有逐年增加的趨勢；角膜瘢疤有逐年減少的趨勢；而”偽/無水晶體性水泡性角膜病變”則有先減少後增加的趨勢。在”再次角膜移植”的病患當中發現最常見的病因是”急性壞死性及潰瘍性角膜炎”。從這個研究當中我們可以下此結論，角膜?疤，再次角膜移植手術，急性壞死性及潰瘍性角膜炎，偽/無水晶體性水泡性角膜病變，傅氏角膜失養症以及角圓錐角膜都是常見接受角膜移植的病因，”偽/無水晶體性水泡性角膜病變”會隨著時間而有有先減少後增加的趨勢，背後的原因應是人工水晶體的變化趨勢，以及白內障手術的方法演進。與其他國家的大部分研究相比，此論文發現”急性壞死性及潰瘍性角膜炎”在本研究中所佔的比例特別的高，而圓錐角膜的比例特別的低。由於大部分的”再次角膜移植”病患和”偽/無水晶體性水泡性角膜病變”都是因為角膜內皮細胞受損而需要接受手術，本研究中發現角膜內皮細胞病變在接受角膜移植手術的病患當中，佔了相當大的比例。
　　角膜移植和角膜內皮細胞的相關研究中的第二子題，是探討有關再國內接受治療性角膜移植手術以治療感染性角膜炎的現況。而進行此一研究的背景，乃因在第一子題的研究當中，我們發現了國內因為”急性壞死性及潰瘍性角膜炎”而接受角膜移植手術的病患，在所有移植角膜瓣中所佔的比例，比起其他國家的大部分研究都來的高。是什麼原因造成此一現象，以及不同的感染性角膜炎是否有不一樣的表現，都是本部分的研究重點。因此，此一子題研究的目的乃在分析探討用"治療性角膜移植手術"以治療"感染性角膜炎"的成效。研究的設計方式為介入性病例分析。而在研究的方法方面，是採取回溯性的病歷分析法，分析從1987到2001年這十四年當中，在本院接受"治療性角膜移植手術"以治療"感染性角膜炎"的案例。病患被分為以下數類 (1)細菌性角膜炎; (2)黴菌性角膜炎;以及(3)阿米巴性角膜炎。研究中著重在分析以下數個因子(1)術後一個月和一年後的角膜瓣清晰度，(2)感染治癒率，以及(3)解剖學上的成功率。這個子題的研究發現，總共有108個"治療性角膜移植"的病患符合分析條件。在細菌性角膜炎方面，治療性角膜移植治癒了37/41的病患；在黴菌性角膜炎方面，治療性角膜移植治癒了36/52的病患；在阿米巴性角膜炎方面，治療性角膜移植治癒了13/15的病患。在術後一年後仍能保持角膜瓣清澈的比例，細菌性角膜炎佔了22/32，黴菌性角膜炎占了 20/39，阿米巴性角膜炎佔了 11/14。在所有的各類角膜炎當中，若是移植角膜瓣的大小等於或小於8.5毫米，術後一年角膜瓣保持清澈的比率會比大於8.5毫米要來的高。而所有五個在手術當時同時罹患續發性眼內炎的病患，即使經過積極的內科或外科治療，最後都因為治療無效而必須接受眼球挖除術。本部分研究的結論是，”治療性角膜移植”在處理藥物治療無效的”感染性角膜炎”病患身上是非常有價值的。在所有三種診斷當中，都發現越小的角膜瓣有較大的術後成功率。無論是在角膜瓣的清澈率，解剖成功率或是感染清除率上，黴菌性角膜炎都是所有病患當中預後最糟糕的一群。本研究中也發現，在角膜瓣失敗的病患當中，相當高比例的人是因為角膜內皮細胞功能受損所造成。因此若能想辦法維持或修復角膜內皮細胞的受損功能，將可以大大提高術後的成功率，減少需要再次手術的機率。角膜內皮細胞在維繫角膜功能的重要性在此可見一斑。
　　角膜內皮細胞的相關研究中的第三子題，是探討否有任何藥物可能促進角膜內皮細胞的排水功能，而研究目標是在最常用在角膜內皮細胞的藥物--類固醇身上。這部分研究的目的，是評估是否在體外的情況下，皮質類固醇dexamethasone (DEX) 會影響牛類角膜內皮細胞的分裂，自殺行為或是Na+-K+-ATPase 的活性。研究的方法是將牛類角膜內皮細胞培養在 10-10 到 10-3M的皮質類固醇當中。皮質類固醇對細胞分裂的影響，乃由 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS)分析法分析。細胞自殺行為和壞死乃由annexin V和propidium iodide螢光染色，之後用流式細胞儀來分析。皮質類固醇對Na+-K+-ATPase 活性的影響則用非同位素方法來定量。本研究的結果發現，皮質類固醇的濃度在10-10 到10-5 M之間，並不會對細胞分裂造成影響；在濃度在10-4 和10-3 M時，DEX顯著地減少細胞分裂，並且增加細胞自殺和壞死的比例。皮質類固醇也在10-8 到10-6 M時顯著地增加了 Na+-K+-ATPase的活性 ，而在濃度為10-6 M時影響達到最大 (P < 0.01)。而這個效應可以被RU38486，一個拮抗皮質類固醇作用的分子所抑制。而本研究的結論是，牛類角膜內皮細胞可以表現皮質類固醇的受體之mRNA 和蛋白。皮質類固醇在高濃度的情況下會減少細胞的分裂，並增加細胞自殺行為和壞死。它同時在某些特定濃度下增加了 Na+-K+-ATPase的活性。
　　角膜內皮細胞的相關研究中的第四子題，是探討SOV對於角膜內皮細胞分裂的影響。先前有許多研究中顯示，接觸抑制(Contact inhibition)是讓角膜內皮細胞維持在不分裂狀態的主要原因之一。 Protein tyrosine phosphatases (PTPs)已知在許多重要的細胞功能，例如調節細胞與細胞間的接觸，分化和生長有極大的關係。 本研究旨在探討protein tyrosine phosphatases(PTP)是否牽涉在角膜內皮細胞的接觸抑制(Contact inhibition)所調控的細胞分裂機轉之上，並且分析到底有哪些相關的PTPs與這個機制有關。 本研究的方法，是在培養到生長匯合(confluent)的大鼠角膜內皮細胞，或在ex vivo培養的角膜組織的角膜內皮細胞上，加上 phosphatase 的廣泛抑制劑- sodium orthovanadate (SOV)。研究中利用細胞免疫化學染色來觀測加上SOV之後對細胞與細胞間的接觸的影響， ZO-1是研究的指標蛋白。另外細胞分裂周期的進展乃是用Ki67的染色來探知。老鼠角膜的冷凍切片或是培養的角膜內皮細胞，都用來測試是否表現重要的PTPs: 例如PTP-mu，PTP-LAR，PTP1B，SHP-1，SHP-2，和PTEN。另外，西方點墨法和RT-PCR也用來輔助證明細胞免疫化學染色的結果。研究的結果發現，ZO-1 的染色顯示SOV引發了時間性的"細胞與細胞間接觸"的變化。Ki67 的染色暗示了 SOV 促進有限的細胞分裂週期進展。PTP-mu、PTP1B、SHP-1、SHP-2 和 PTEN(但不包含PTP-LAR)，在角膜上或培養的大鼠角膜內皮細胞上都有表現。在細胞匯合與否的情況下，細胞內PTP-mu 和PTP1B的表現位置並不相同；然而 SHP-1, SHP-2, 和PTEN則無不同。西方點墨法則顯示了PTP1B、SHP-1、SHP-2、和PTEN的表現。 RT-PCR則確認了 PTP-mu mRNA的表現。
Phosphatases已知在細胞的嵌合和分裂上都扮演重要角色。PTP-mu、PTP1B、 SHP-1、SHP-2和PTEN 都表現在大鼠的角膜內皮細胞上，並且牽涉在調節"細胞與細胞間接觸"和正常情況下不分裂的生理現象上。實驗中雖然嚐試用Phosphotase的廣泛抑制劑來促進細胞分裂，但若要達到臨床上可應用的程度，仍需進一步的研究。

關鍵字

角膜移植 ； 皮質類固醇 ； 角膜內皮細胞 ； 睫狀體 ； 細胞分裂

並列摘要

For treating various corneal diseases, corneal surgery is one of the majoy treatment strategies. Corneal itself is a non-vascular, transparent, and elastic tissue. It need to be maintained in 78 % water saturations. Any insults damage the transparency of corneal tissue will lead to blurred vision, possible eye pain (due to corneal epithelial edema) and halo visions (due to increased corneal hydrations). Since corneal tissue is quite different from most tissues in human bodies, the post-operative concerns are also different from other tissues. The main purpose of this study is to do clinical and basic research about corneal surgeries. Since there are a lot of corneal associated surgeries, this study select the commonest two surgeries as the targets--- excimer laser refractive surgery (LASIK)，and corneal transplantation。In the first part of this study, I focus on excimer laser refractive surgery, especially on the ciliary body changes after LASIK. Since ciliary body may also change after pars plana vitrectomy, the study also focus on the change of ciliary body associated tissues after pars plana vitrectomy。For these two parts of ciliary body associated studies, ultrasound biomicroscopy was used as the tool for evaluating ciliay body associated changes。For the second main part of this study---Research on corneal transplantation, clinical and basic researches were performed to retrospectively study the outcome of corneal transplantations performed at National Taiwan University Hospital during recent 10+ years. For basic research, the effect of dexamethasone on corneal endothelial cells, and the possible pharmacologic strategy to promote corneal endothelial proliferation were done.

Project I.

This part of the study try to evaluate the possible ciliay body changes after lamellar refractive surgery, and the result has been accepted and published in IOVS.

Chen WL, Shih YF, Liao SL, Hu FR, Hung P. Ultrasound biomicroscopic findings in rabbit eyes undergoing scleral suction during lamellar refractive surgery. Invest Ophthalmol Vis Sci. 2002;43:3665-72.
The main purpose of this study is to evaluate changes of the central anterior chamber depth, cilio-angular cross-sectional surface area, and intraocular pressure in rabbit eyes undergoing application of the scleral suction ring during lamellar refractive surgery. First of all, this study defined some parameters (central anterior chamber depth (CACD); Ciliary body cross-sectional surface area (CBCSA); ASA-2000;ASA-1000 (ASA-1000) and existence of ciliary detachment of not ) to evaluate the ciliary body associated tissues in rabbit eyes. n this project, thirty eyes of 30 rabbits were used. The eyes were assigned to one of the following five surgical groups: group 1, no application of the suction ring; group 2, suction for 2 minutes; group 3, suction for 1 minute; group 4, suction for 20 seconds; and group 5, suction for 10 seconds. Ultrasound biomicroscopy (UBM) was performed to determine tomographic features, including central anterior chamber depth, cross-sectional surface area of the ciliary body, and chamber angle structure before and 10 minutes, 1 hour, 2 hours, 1 day, 2 days, 1 week, and 2 weeks after surgery. Intraocular pressure was also measured at each of these time points. The study result was discribed as followed: swelling of the ciliary body occurred in groups 2 to 5 of eyes from 10 minutes up to 1 day after the operation, and its severity was positively related to the duration of suction. Shallowness of the chamber angle was positively related to swelling. All UBM-detectable changes became insignificant compared with baseline values at 2 days after the operation. No significant change was found in the central anterior chamber depth and intraocular pressure during the 2-week postoperative observation period. From this part of the study, we can concluded that ransient change in the ciliary body and the chamber angle occurred frequently after application of the scleral suction ring during lamellar refractive surgery in rabbit eyes. Its severity was positively related to the duration of suction. Swelling of the ciliary body corresponded with the shallowness of the chamber angle without alteration of the corneal-lenticular distance and intraocular pressure. However, if we want to apply this study result to human eyes, we have to be very careful. Human eyes are different from rabbit eyes since the latter have thinned scleral tissues, thicker lens, thinned iris tissue and more crowded anterior segments compared to human eye. All theses factors may make the interpretation of this study result very difficult. Primate eyes may be a good study candidate. However, the difficult to get such study animals make this quite impossible. It is necessary to do more associated researches to prove that the findings in this research can be applied to human eyes.

Project II.

This part of the study try to evaluate the possible ciliay body changes pars plana vitrectomy, and the result has been accepted and published in Retina

Chen WL, Yang CM, Chen YF, Yang CH, Shau WY, Huang JS, Ho TC, Chen MS, Hung PT. Ciliary detachment after pars plana vitrectomy: an ultrasound biomicroscopic study. Retina. 2002;22:53-8.
Since the previous study has demonstrated the change of ciliary body associated tissues after lamellar refractive surgery, he main purpose of this study is to determine the incidence, duration, risk factors, and clinical outcomes for ciliary detachment after pars plana vitrectomy (PPV).Since the instrument of pars plana vitrectomy will theoretical damage ciliry body during surgery, its quite reasonable to suspect that ciliry body is proned to change and injuried during pars plana vitrectomy. In this study, a total of 109 eyes of 103 patients who underwent PPV for various disease entities at National Taiwan University Hospital by several different doctors were included. Ultrasound biomicroscopy (UBM) was applied to determine the tomographic features of the ciliary body before and 1, 3, and 7 days after the surgery. All eyes were then examined once weekly for 2 months. Demographic, preoperative, intraoperative, and postoperative parameters were evaluated to assess their predictive value in the formation of postvitrectomy ciliary detachment. The result of this project were described as followed: Ciliary detachment was observed in 46 eyes (42%) after pars plana vitrectomy surgeries, and may persist for less than 3 weeks in 40 of 46 eyes. It most frequently occurred in eyes of patients with proliferative diabetic retinopathy (PDR) (64%) or retinal vascular obstructive diseases (RVO) (47%). Extensive retinal photocoagulation and retinal cryopexy positively predisposed to its formation whereas fluid-gas exchange had a protective effect. No clinical complications were observed in eyes with postoperative ciliary detachment. From this study, we can conclude that ciliary detachment occurred frequently after PPV. A diagnosis of PDR or RVO and surgical procedures with extensive retinal photocoagulation and retinal cryopexy may have a higher incidence of its occurrence. According to these results. We found that it's better to evaluate the ciliary body conditions routinely after pars plana vitretomy surgeries. Although all cases in this study have only temporary ciliary detachments, and without permanent side effects, we will need to be cautious. Quite a lot of patients undergoing pars plana vitrectomy need to receive another pars plana vitrectomy surgery due to different reasons (e.g. vitreous or retinal hemorrhage, retinal detachment), if the surgeons did not notice the existence of ciliary/choroidal detachment, the mis-inserted infusion probe placed in suparchoroidal/suprociliay space will lead to severe ciliary/choroidal detachment, or even retinal detachment, voitreous/subretinal/suprochoroidal hemorrhage after the infusion fluid begin to flow into eyeballs. Careful observation of the existence of ciliary/choroidal detachment after pars plana vitrectectomy is this very important. In addition, possible side effects due to ciliary body changes (e.g. glaucoma, hypotomy, angle closure) also need to be observe in order to get to better surgical outcomes.

Project III.

This part of the study tries to evaluate the Changing indications for penetrating keratoplasty in Taiwan from 1987 to 1999, and the results have been published in Cornea.
Chen WL, Hu FR, Wang IJ. Changing indications for penetrating keratoplasty in Taiwan from 1987 to 1999. Cornea. 2001;20:141-4

Although corneal transplantation has been performed in Taiwan and worldwide for many years, there is only limited study focusing on the trand of etiology change during these few years. Since National Taiwan University Hospital is an important tertiary referral center in North Taiwan, it represents the corneal transplantation market in Taiwan. The main purpose of this part is to determine the leading indications for penetrating keratoplasty and to identify the changing trends in these indications during the past 12 years, namely, from 1987 to 1999. To achieve this goal, a retrospective study was performed (a chart review of the hospital records of all patients undergoing penetrating keratoplasty at the National Taiwan University Hospital during a 12-year period (1987-1999) was preformed). When possible, the clinical indication was corroborated by the pathologic report. A total of 770 corneal transplants were performed during the observative period. The leading indications for penetrating keratoplasty, in order of decreasing frequency, were corneal scars (27.9%), regraft (21.0%), acute necrotizing and ulcerative keratitis (17.9%), pseudophakic or aphakic bullous keratopathy (17.6%), Fuchs' dystrophy (4.5%), and keratoconus (2.5%). A trend of increasing frequency of regraft and acute necrotizing and ulcerative keratitis, a decreasing frequency of corneal scar, and an initially decreasing then increasing frequency of pseudophakic and aphakic bullous keratopathy were found during the 12-year study period. Acute necrotizing and ulcerative keratitis was found to be the most frequent indication for regraft. From this study, we can conclude that corneal scars, regraft, and acute necrotizing and ulcerative keratitis were the leading indications for penetrating keratoplasty. A changing incidence of pseudophakic and aphakic bullous keratopathy noted during the study period was related to the type of intraocular lens implanted and the method of cataract surgery performed. This study found a comparatively high frequency of acute necrotizing and ulcerative keratitis and an extremely low frequency of keratoconus compared with previous reports. The reason for the difference from other studies can be explained by following reasons: (1) Since most of the corneal buttons from this study are imported from the United States instead of domestically gained, it is possible that surgeons prone to keep the corneal buttons for more severed corneal diseases. (2) The different climates/humidity of different countries will make the tolerance of contact lens wearing different. Taiwan in wet and warm, patients may feel more comfortable to wear contacts lens compared to those countries which are dray and cold. Thus the treatment strategy of keratoconus may be different in these countries. (3) Since National Taiwan Univerisity is an important tertially referral medical center in Northern Taiwan, those cases with infectious corneal ulcers are usually difficult to treat, and make medial treatment quite difficult. In such cases, corneal transplantations is the only way to cure these diseases. All these factors can explain why our result is different from some important studies performed in other countries.

Project IV.

This part of study tries to evaluate outcomes of therapeutic penetrating keratoplasty for microbial keratitis in Taiwan from 1987 to 2001. This part of the study has been publied in Am J Ophthalmol.

Chen WL, Wu CY, Hu FR, Wang IJ. Therapeutic penetrating keratoplasty for microbial keratitis in Taiwan from 1987 to 2001. Am J Ophthalmol. 2004;137:736-43.

According to previous study, we know that therapeutic penetrating keratoplasty for microbial keratitis is a common etiology for corneal transplantation. However, it is quite possible that the surgical outcomes of corneal transplantations for microbial keratitis is different from other corneal transplatational surgeries, since uncontrolled corneal infection and severe corneal inflammation both lead to possible poor surgical outcomes. The main purpose of this study is to determine the surgical outcomes of therapeutic penetrating keratoplasty (PKP) for treating various infectious keratitis, and to evaluate its role in the management of microbial keratitis. The study was an interventional case series. In this study, retrospective chart review of 151 patients undergoing therapeutic PKP for culture-proven microbial keratitis at the National Taiwan University Hospital during a 14 year-period (1987-2001) was done. The surgeries were performed by several different surgeons. Patients were divided into three categories: (1) bacterial keratitis;(2) fungal keratitis; and(3) acanthamoebic keratitis. Each of the following criteria was evaluated: (1) graft clarity 1 month and 1 year postoperatively; (2) cure of the disease (control of the infections); and (3) anatomical success rate (no need for enucleation/evisceration). The result demonstrated that a total of 108 therapeutic PKP met the criteria, and were included in this study. Therapeutic PKP was found to eradicated the infection in 37/41 of patients with bacterial keratitis, 36/52 of patients with fungal keratitis, and 13/15 of patients with acanthamoebic keratitis. 22/32 of grafts of bacterial keratitis, 20/39 of fungal keratitis, and 11/14 of acanthamoebic keratitis remained clear at 1 year postoperatively. A higher percentage of graft clarity at 1 year postoperatively was achieved in all three categories when grafts were 8.5 mm or less compared with larger grafts. All five patients with secondary endophthalmitis observed at the time of therapeutic PKP experienced a progression of infection and had to be enucleated despite of aggressive surgical treatment. According to these results, we can concluded that therapeutic PKP is valuable in the management of various microbial keratitis that is unresponsive to medical therapy. Different microorganisms may have different surgical outcomes. A higher percentage of clear graft was found when grafts were smaller in all three categories, meaning that pre-operative conditions may lead to poorer surgical outcomes.. Surgical results are worse for patients with fungal keratitis, regardless of graft clarity, anatomical success, or infection eradication rate. Since the postoperative result is quite good, surgeons can be more aggressive to use corneal transplantation to treat those with severe microbial corneal infections unresposive to medical treatment, or those cases with impending or real corneal perforations. In addition, we found that corneal endothelial decompenstaion is a major factor leading to corneal graft failure. The methods to cure the corneal endothelial dysfunctions are thus clinically important, and need to be performed.
Project V.

This part of the study tries to evaluate the direct effect of dexamethasone on corneal endothelial cells.

Wei-Li Chen, Chung-Tien Lin, Chung-Chen Yao, Yu-Hua Huang, Yu-Bin Chou, Hsiang-Shu Yin, Fung-Rong Hu. In Vitro Effects of Dexamethasone on Cellular Proliferation, Apoptosis and Na+-K+-ATPase activity of Bovine Corneal Endothelial Cells

The main purpose of this study is to assess the in vitro effects of dexamethasone (DEX) on proliferation, apoptosis and Na+-K+-ATPase activity of bovine corneal endothelial cells. There are various types of corneal endothelial diseases, such as graft rejection, corneal endothelitis, trauma, etc. In all these diseases, corticosteroid is widely used to suppress inflammation and to cure corneal endothelial dysfuction. However, there are limited studies undertaken to evaluate the direct effect of dexamethasone on corneal endothelial cells. To evaluate the possible pharmacological effect of dexamethasone on these cells, bovine corneal endothelial cells were cultured with DEX from 10-10 to 10-3M for different durations (2,4 and 6 days). Effect of DEX on proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Apoptosis and necrosis were detected by staining with fluorescein-conjugated annexin V and propidium iodide, followed by flow cytometry. The effect of DEX on Na+-K+-ATPase activity was evaluated using non-isotopic methods. We found that DEX did not affect cellular proliferation or induce apoptosis/necrosis from 10-10 to 10-5 M on day 2, day 4 or day 6. At 10-4 and 10-3 M, DEX significantly decreased proliferation and increased apoptosis and/or necrosis on day 6. DEX significantly increased the Na+-K+-ATPase activity from 10-8 to 10-6 M at day 6 instead of day 2 or day 4, with the maximal effect at 10-6 M (P < 0.01), which was inhibited by RU38486, an antiglucocorticoid molecule. We can conclude from this project that bovine corneal endothelial cells express GR mRNA and protein. DEX decreases cell proliferation and induces cellular apoptosis and/or necrosis at high concentrations. DEX also increases the Na+-K+-ATPase activity at certain concentrations. According to this result, it may be beneficial to use DEX to promote corneal endothelial functions. However, we need to pay attention to its well-known side effects of steroid usage in ocular diseases. DEX induces cataract or glaucoma when used intensively. It delays the corneal epithelial migration and wound healing, and also decreases the ocular surface immunity and leading to unwanted corneal infections. Our study result also show that DEX can trigger cornel endothelial apoptosis/necrosis, or decrease cellular proliferation at higher concentrations. Thus we have to be very cautious when we use dexamethasone to cure corneal endothelial diseases.

Project VI.

This part of the study tries to evaluate the effect of SOV-a generalized PTP inhibitors on corneal endothelial cells. This part of the study has been published in Experimental Eye Research.

Paper IV. Chen WL, Harris D, Joyce NC. Effects of SOV-Induced Phosphatase Inhibition and Expression of Protein Tyrosine Phosphatases in Rat Corneal Endothelial cells. Experimenal Eye Research. (accepted)
Human corneal endothelial cells are non-proliferative in vivo, and they will migrate and enlarge, instead of proliferate, after injury. When the corneal endothelial cells decreases to a certain level, corneal endothelial decompensation will occur. In such conditions, corneal transplantation is the only way to cure this disease. Triggering the corneal endothelial cells to regain proliferative ability is thus very important clinically. Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Some studies have demonstrated that when confluented-cultured corneal endothelial cells were treated with EDTA, the cells entered proliferation when bovine serum was added. In ex vivo cultured human cornea, corneal endothelial cells will enter limited cell proliferatve cycle when scrape wounds were made, which means that breakthrough of corneal endothelial cell-cell junctions will trigger the non-proliferative cells to proliferate. According to this phenomenon, it may be clinically useful to disrupt the cell-cell junctions of corneal endothelial cells, make the cells to proliferate, and thus use non-surgical methods to cure corneal endothelial diseases. Protein tyrosine phosphatases (PTPs) are well known to play a role in regulating the integrity of cell-cell contacts, differentiation, and growth in many different cell types. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells, and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of the contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV) for various times under different concentrations. Immunocytochemistry (ICC) was used to evaluate the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contact in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B was different in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2 and PTEN. RT-PCR confirmed the expression of PTP-mu mRNA. Phosphatases are involved in the regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in the regulation of contact inhibition in these normally non-proliferating cells. The result of this study demonstrated that, as predicted in the previous experiments, disruption of rat corneal endothelial cells by SOV can trigger the cells to enter limited corneal endothelial proliferations. Although which PTP/PTPs play a role in this mechanism was not explored, at least this finding points out one possible treatment method to cure corneal endothelial cells. However, in vivo condition is quite different from in vitro or ex vivo condition. In addition, human corneal endothelial cells are quite different from other species, e.g. rat corneal endothelial cells. Although the results presented here are quite exciting, we still have to be very careful to apply this result to human cells.

並列關鍵字

corneal transplantation ； dexamethasone ； corneal endothelium ； ciliary body ； proliferation

參考文獻

Tayler DM, Atlas BF, Romanchuk KG, et al. Pseudophakic bullous keratopathy.

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Almawi WY, Melemedjian OK. Molecular mechanisms of glucocorticoid antiproliferative effects: antagonism of transcription factor activity by glucocorticoid receptor. J Leukoc Biol, (2002),71:9-15.

Al-hazzaa SA, Tabbara KF. Bacterial keratitis after penetrating keratoplasty. Ophthalmology (1988) 95:1504-1508.