多光子螢光顯微鏡暨二倍頻顯微鏡於角膜屈光手術後傷口癒合之應用

The application of multiphoton fluorescence and second-harmonic generation microscopy in the evaluation of corneal wound healing after refractive corneal procedure

王宗仁(Tsung-Jen Wang)

指導教授 ： 胡芳蓉

共同指導教授 ： 董成淵(Chen-Yaun Dong)

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02227

摘要

角膜屈光手術是藉由改變角膜弧度來治療病人的屈光異常，手術後的傷口癒合是病人視力及角膜透明度恢復的關鍵，傷口癒合的過程涉及上皮癒合和實質層癒合等動態過程。傳統組織學檢查對於角膜傷口癒合雖然已有相當應用，但標本需固定後再檢查，這也使得對於觀察傷口癒合的動態變化，有研究上的限制。此外，屈光手術後度數的迴歸及疤痕的形成是和肌纖維母細胞(myofibroblast)的活化及膠原纖維(collagen fibers)的再生及排列有關，傳統檢查方式對肌纖維母細胞及膠原纖維需要經過免疫組織化學染色才能觀察，對標本的處理也無法做到非侵入性，因此無法做活體動態觀測。
多光子螢光顯微鏡是利用雙光子對組織做激發，可以偵測到生物組織中的自發螢光和二倍頻訊號，不需將組織標本做固定或標記即可觀測，同時可產生高品質及有深度的生物成像。本研究藉由傳導性角膜成型術(conductive keratoplasty)及雷射屈光角膜切除術(photorefractive keratectomy)二種角膜屈光手術，在兔子身上建立角膜傷口癒合的動物模式，這將涉及表皮細胞再生及實質層癒合的膠原蛋白重新排列。
在傳導性角膜成型術中，透過自發螢光我們能觀察到手術後表皮細胞的變化及恢復的過程，我們發現在手術後一週內，角膜上皮細胞層即已恢復，然而在傷口處，上皮細胞有補償性增生。透過二倍頻訊號我們能觀察到膠原纖維的再生過程及排列的變化，我們發現通入電流對角膜基質造成的熱破壞，直到八週後仍未完全復原，而熱破壞所造成的膠原纖維收縮，也可以從二倍頻影像中觀察到。在雷射屈光角膜切除術中，以多光子螢光顯微鏡及傳統組織學及免疫組織化學染色等檢查方式在術後不同階段做觀察，透過自發螢光我們能觀察到屈光手術後肌纖維母細胞活化的過程，更進一步地，我們以手動細胞計數計算傳統組織學檢查的黃金標準及多光子螢自發螢光影像中肌纖維母細胞的密度，並以Spearman’s correlation 檢驗，發現二者之間的關連性可達0.9 (p<0.05)，更加確立多光子螢光顯微鏡活體觀察角膜傷口癒合的基礎。在此同時，我們也發現Mitomycin-C能有效減少角膜細胞的數目並影響基質膠原纖維再生的速率。
我們企圖尋找替代的影像檢查儀器來研究角膜的傷口癒合，由我們體外觀察的研究結果顯示，在沒有切片及染色的情況下，多光子影像技術可以直接觀察到手術後傷口癒合的變化，包括肌纖維母細胞的活化及膠原纖維的再生及排列，藉由自發螢光訊號及二倍頻訊號，都可以清楚的觀察到，雖然離活體觀測還有許多問題需要克服，但是多光子影像技術似乎提供了一條可行的出路。

關鍵字

角膜屈光手術 ； 屈光性角膜切除術 ； 傳導性角膜成型術 ； 多光子螢光顯微鏡 ； 角膜傷口癒合

並列摘要

Corneal refractive procedures correct patients’ refractive errors by reshaping corneas and change corneal curvature. The corneal wound healing process after operation is the key point of the recovery of the visual acuity and the reestablishment of the corneal transparency. It is a dynamic process involving epithelial healing and stromal healing. Although conventional histological examination is useful in characterizing the process, the fixation procedure prevents the study of tissue dynamics during the repair process. In addition, the regression and scar formation after refractive corneal procedure are related to the reactivation of the myofibroblasts and the rearrangement of the collagen fibers. Traditionally, the observation of myoflibroblasts and the collagen fibers depends on immunohistochemical stain.
Recently, multiphoton microscopy, based on non-linear optical process, provides the feasibility of label-free pathological observation of corneas. The multiphoton excited autofluorescence from cytoplasma provides morphology of epithelial cells and keratocytes while second harmonic generation signals reveal collagen fibrils alignment in corneal stroma. There are a number of advantages associated with multiphoton microscopy. First, non-linear excitation of fluorescent species needs high photon density and only occurs at the objective focal point. Therefore, confocal-like image quality can be achieved without pinhole in multiphoton imaging. In addition, the typical excitation source in multiphoton microscopy is near infrared which scatters less in biological tissues. Therefore, multiphoton imaging has deeper penetration in biological specimens or turbid specimens. Due to the deeper penetration depth and optical section ability, three-dimensional reconstruction of biological specimens can also be achieved. Moreover, cytoplasmic autofluorescence and second harmonic generation from collagen which are the most abundant molecules in extracecullar matrices provide label-free and section-free observation of biological tissues. Using multiphoton microscopy in pathological imaging can avoid artificial effects in traditional histological process.
In this project, conductive keratoplasty (CK) and photorefractive keratectomy (PRK) were performed in the rabbit eyes to serve as the animal models for studying the epithelial and stromal healing after refractive surgery. Ex vivo observations of wound healing process with multiphoton microscopic examination and conventional histological examination were done in different stages after operation. In the CK experiment, the re-epithelialization is accomplished within 1 week after surgical procedure. However, compensatory epithelial hyperplasia is observed at wounding sites. Structural alternation of corneal stroma can be visualized without extra labeling by second harmonic generation (SHG) imaging. In SHG images, the absence of SHG at wounding site indicates current-induced collagen thermal damage has lasted for at least 8 weeks after CK procedure. The collagen contraction caused by thermal damage can be immediately observed right after the CK procedure.
In the PRK part, the re-epithelialization and keratocyte activation are observed by multiphoton excited autofluorescence (MAF) without labeling. Collagen regeneration in corneal stroma can be characterized by SHG imaging. To verify the correlation between activated keratocytes observed in MAF images and myofibroblasts observed in traditional immuohistochemical (IHC) imaging, keratocytes density are manually calculated in MAF and IHC. Positive correlation (0.9, p<0.05) is observed by using Spearman’s rank correlation analysis. The good correlation of activated keratocytes in MAF and myofibroblast in IHC suggest that MAF can be used to trace keratocyte activity during corneal wound healing process. Our results also suggest that, mytomicin-C can effectively reduce the popularity of keratocytes and the regeneration rate of collagenous stroma.
Our results show the proof of principle of using multiphoton microscopy, a novel and nonlinear optical process, in observing corneas after refractive surgery. Multiphoton microscopy provides a less invasive way in observing cellular activities and extracecullar matrices alternation during corneal wound healing process. Comparing to traditional histology methods, the minimal invasion of mulitphoton observation provides the feasibility of applying it in in vivo, clinical examination in the future.

並列關鍵字

Refractive corneal procedure ； Photorefractive keratectomy ； Conductive keratoplasty ； Multiphoton microscopy ； Corneal wound healing

參考文獻

4. Shih YF, Hsiao CH, Wen SH, et al. Prevalence of anisometropia in Taiwanese schoolchildren. J Formos Med Assoc 2005;104(6):412-7.

2. Song J, Lee YG, Houston J, et al. Neonatal corneal stromal development in the normal and lumican-deficient mouse. Invest Ophthalmol Vis Sci 2003;44(2):548-57.

5. Argento CJ, Cosentino MJ. Laser in situ keratomileusis for hyperopia. Journal of Cataract and Refractive Surgery 1998;24(8):1050-8.

6. Eggink CA, Bardak Y, Cuypers MHM, Deutman AF. Treatment of hyperopia with contact Ho : YAG laser thermal keratoplasty. Journal of Refractive Surgery 1999;15(1):16-22.

7. Koch DD, Abarca A, Villarreal R, et al. Hyperopia correction by noncontact holmium:YAG laser thermal keratoplasty clinical study with two-year follow-up. Ophthalmology 1996;103(5):731-40.