| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 94 === 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.
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