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研究生： 蔡雲圃
Tsai, Yun-Pu
論文名稱： 定量分析高頻熱凝治療大鼠急性跟腱炎的行為以及痛覺表現
Quantitative analyses of gait behavior and nociception of radiofrequency treated acute Achilles tendinopathy in rat.
指導教授： 司君一
Sze, Chun-I
學位類別： 碩士
Master
系所名稱： 醫學院 - 細胞生物與解剖學研究所
Institute of Cell Biology and Anatomy
論文出版年： 2012
畢業學年度： 100
語文別： 中文
論文頁數： 54
中文關鍵詞： 高頻熱凝 、跟腱肌腱炎 、活動度 、步態 、疼痛
外文關鍵詞： Radiofrequency, RF, Achilles tendinopathy, ROM, Gait, Pain
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    • 過度使用或是肌腱本身負荷過大都會造成跟腱肌腱炎，通常發生在運動員身上。一般來講病患都接受保守治療，而高頻熱凝(RF)可以有效的讓病患減輕疼痛。目前來說，肌腱炎的病理機轉認為是一種失敗的癒合反應。本實驗使用Sprague-Dawley(SD)品系的老鼠，施打膠原溶解劑到跟腱部位來造成肌腱炎，並分析RF的效果，尤其是RF後的疼痛相關傳遞物質以及行為方面的改變。使用膠原酶類型I(0.3mg/20ul)或是磷酸鹽緩衝液(PBS)打入老鼠的跟腱後，分成實驗組以及sham對照組。SD老鼠切開跟腱部位的皮膚作為正常對照組。實驗的安排，檢測時間分別為膠原酶注射後的第1、3、5、7、9、11、13、15天，分別觀察病理組織以及步態的改變。在膠原酶注射後的第8天使用RF治療。使用免疫組織化學染色法(IHC)染P物質(Substance P,SP)、降鈣素基因相關肽(Calcitonin gene related peptide,CGRP)以及甘丙肽(Galanin)來評估疼痛的表現；使用免疫螢光染色法(IF)染β III tubulin來評估神經纖維的表現；使用特殊染色Masson trichrome來檢查膠原的變化。並分析步態行為的表現，參數包含時間(站立期、雙腳站立期、速率)、距離(步長、步寬、跨步、足底長度、2－4趾間距離、1－5趾間距離、阿基里斯功能指數)、活動度(接觸起始期、站立中期、擺盪前期、擺盪中期、內外八角度)。SP以及CGRP IHC染色法顯示在1、3、5、7天的時候，疼痛會隨時間而增加，但SP (n=5, p<0.001)以及CGRP(n=5, p<0.001)經過RF治療後會降低。Galanin IHC染色法顯示，經過RF治療後無明顯差異。對照正常組，免疫螢光染色法染β III tubulin時，神經纖維的密度及直徑在1、3、5、7天的時候，會隨時間而增加，而經過RF治療後只有直徑(n=5, p<0.001)會降低。經過化學傷害後，使用特殊染色Masson trichrome染色，會顯示張力有增加的現象。跟對照組及正常對照組比較後，可發現RF治療後的站立期(n=7, p<0.05)、步長(n=8, p<0.05)、2－4趾間距離(n=8, p<0.01)以及擺盪前期(n=8, p<0.05)均可獲得改善，這些改變可使老鼠的步態更趨近於正常。隨著時間的變化，疼痛的感覺也隨之變化，這會使得步態模式發生改變。RF可減輕疼痛、降低神經纖維直徑並且讓行走能力變好。所以總結就是，針對於老鼠的急性跟腱肌腱炎，本實驗可以提供詳細的RF效果分析，但對於應用在跟腱肌腱炎的病患需要再做進一步的評估分析。

    Achilles tendinopathy is caused by excessive use or loading of physical force of the tendon and it is commonly occurred among athletes. The patient is in general treated conservatively. Radiofrequency (RF) has been proven to be an effective method to relieve pain in those patients. Currently, the pathophysiology of tendinopathy is thought to be associated with a failed healing response. In this study, collagenolytic agent was injected to the Achilles tendon to generate tendinopathy in Sprague-Dawley (SD) rat. The effects of RF, specifically, on changes of pain-related neuropeptides release and gait behavior in acutely post RF treated SD rats were examined. Collagenase І (0.3mg/20ul) or Phosphate-Buffer Saline (PBS) was injected to the rat Achilles tendon as the experiment and sham control groups. SD rats underwent skin incision over the Achilles tendon were served as normal control. The experiment was following a time-course schedule to exam post collagenase I injection on 1,3,5,7,9,11,13,15 days to examine pathohistology and gait changes. RF treatment was on day 8 after collagenase injection. Substance P, Calcitonin gene related peptide(CGRP) and Galanin expressions were examined by immunohistochemical (IHC) staining to evaluate nociception. β III tubulin immunofluorescence (IF) staining was used to evaluated nerve fiber change. Masson trichrome staining of tendon was used to examine collagen change. The gait parameters recorded and analyzed in this study included gait pattern time (stance phase, double stance phase, walk speed), distance (step length, step width, stride length, print length, intermediary toe-spread), and range of motion (initial contact, mid‐stance, pre‐swing, mid‐swing, foot angle). Substance P and CGRP IHC staining showed that the nociception was increased on the days 1, 3, 5, 7, but Substance P (n=5, p<0.001) and CGRP (n=5, p<0.001) decrease after RF treatment. Galanin IHC staining showed that there was no significance change in rats underwent RF treatment. β III tubulin IF staining showed that the nerve fiber density and diameter increased on the days 1, 3, 5, 7 when compared to the normal controls. However, the nerve fibers diameter (n=5, p<0.001) were decreased after RF treatment. Masson trichrome staining showed that the tension increased after chemical injury. In post-RF treated groups, the stance phase (n=7, p<0.05), step length (n=8, p<0.05), intermediary toe-spread (n=8, p<0.01) and pre‐swing (n=8, p<0.05), were significantly different from shame and normal controls. These changes suggest that RF treatment improves gait behavior in acute tendinopathy, which allows rat ambulate toward to the normal gait pattern. Nociception is different in different time intervals, which may cause differences in gait pattern. RF treatment decreases nociception neuropeptides release, decrease nerve fiber diameter in injured Achilles tendon, improves pain and makes rat walk better. In conclusion, this study provides the first detailed analysis of RF effects on acute Achilles tendinopathy in rat. Whether these data would apply to the patients with pain in early stage of Achilles tendinopathy might require further evaluation.

    目錄 中文摘要...................................................I 英文摘要.................................................III 誌謝......................................................V 目錄.....................................................VI 圖目錄...................................................IX 第一章 緒論................................................1 1-1 肌腱炎 (Tendinopathy)的定義.............................2 1-2 跟腱的解剖與組織結構.....................................2 1-3 肌腱炎之失敗癒合理論.....................................3 1-4 跟腱肌腱炎(Achilles tendinopathy)......................4 1-5 高頻熱凝(Radiofrequency,RF)的效果.......................4 1-6 疼痛(Nociception)相關影響因子...........................4 1-7 動物模型(model)的挑選...................................5 1-8 肌腱炎的病理變化........................................5 1-9 研究動機與問題..........................................5 1-10 研究目的..............................................6 第二章 材料與方法...........................................7 2-1 實驗動物模式之建立.......................................8 2-2 形態病理切片組之建立.....................................8 2-3 行為組之建立...........................................9 2-4 Radiofrequency(RF)之手術操作...........................9 2-5 步態行為測試...........................................10 2-6 冷凍切片..............................................11 2-7 免疫組織化學染色(IHC staining)方法......................12 2-8 免疫螢光染色方法.......................................13 2-9 特殊染色Masson Trichrome Stains.......................13 2-10 量化與統計分析........................................14 第三章 結果...............................................15 3-1 P物質(Substance P)的表現..............................16 3-2降鈣素基因相關肽(CGRP)的表現..............................16 3-3 甘丙肽(Galanin)的表現..................................17 3-4 神經纖維的表現.........................................17 3-5 Masson Trichrome Stains的變化.........................18 3-6 步態行為測試...........................................26 3-6-1 步態行為分析總表......................................29 3-6-2站立期(stance phase).................................30 3-6-3雙腳站立期(Double Stance Phase).......................31 3-6-4步長(Step Length)....................................33 3-6-5 第2－4趾間距離(Intermediary toe-spread)..............34 3-6-6 第1－5趾間距離(Toe-spread)...........................36 3-6-7阿基里斯功能指數(Achilles Functional Index)............38 3-6-8 接觸起始期(Initial Contact)..........................39 3-6-9擺盪前期(Pre‐Swing)...................................41 3-6-10 擺盪中期(Mid‐Swing).................................43 3-6-11 腳踝活動度(Ankle ROM)...............................45 3-6-12 步態分析第15天平均值總表..............................46 第四章 討論與結論...........................................47 4-1 SP、CGRP、Galanin的IHC staining變化....................48 4-2 神經纖維的表現..........................................48 4-3 Masson Trichrome Stains的表現.........................49 4-4 步態行為...............................................49 4-5 未來深入研究探討方向.....................................51 4-6 結論..................................................51 參考文獻...................................................52 圖目錄 圖2-1 跟腱外觀，黑點為注射點...................................8 圖2-2 OWL universal RF system URF-3AP.....................10 圖2-3 步道系統同時擷取大鼠行走時側面及底部之影像資料..............11 圖3-1正常Substance P(SP)形態變化(200倍).....................19 圖3-2 注射膠原酶後Substance P(SP)的形態變化(200倍)............19 圖3-3 SP量化圖.............................................20 圖3-4正常Calcitonin gene related peptide(CGRP)形態變化(200倍).......................................................20 圖3-5 注射膠原酶後CGRP的形態變化(200倍).......................21 圖3-6 CGRP量化圖...........................................21 圖3-7正常Galanin形態變化(200倍).............................22 圖3-8注射膠原酶後Galanin的形態變化(200倍).....................22 圖3-9 Galanin量化圖........................................23 圖3-10正常β III Tubulin形態變化(200倍)......................23 圖3-11注射膠原酶後β III Tubulin的形態變化(200倍)..............24 圖3-12 β III Tubulin平均直徑量化圖..........................24 圖3-13 β III Tubulin總密度量化圖............................25 圖3-14 Masson Trichrome的形態變化(100倍)....................25 圖3-15 步態行為參數︰時間....................................27 圖3-16 步態行為參數︰距離-1..................................27 圖3-17 步態行為參數︰距離-2..................................28 圖3-18 步態行為參數︰距離-3..................................28 圖3-19 步態行為參數︰活動度..................................28 圖3-20步態行為分析總表......................................29 圖3-21站立期(stance phase)................................30 圖3-22雙腳站立期(Double Stance Phase)-1....................31 圖3-23雙腳站立期(Double Stance Phase)-1....................32 圖3-24步長(Step Length....................................33 圖3-25第2－4趾間距離(Intermediary toe-spread)-1............34 圖3-26第2－4趾間距離(Intermediary toe-spread)-2............35 圖3-27第1－5趾間距離(Toe-spread)-1.........................36 圖3-28第1－5趾間距離(Toe-spread)-2.........................37 圖3-29阿基里斯功能指數(Achilles Functional Index)...........38 圖3-30接觸起始期(Initial Contact)-1........................39 圖3-31接觸起始期(Initial Contact)-2........................40 圖3-32擺盪前期(Pre‐Swing)-1................................41 圖3-33擺盪前期(Pre‐Swing)-2................................42 圖3-34擺盪中期(Mid‐Swing)-1................................43 圖3-35擺盪中期(Mid‐Swing)-2................................44 圖3-36腳踝活動度(Ankle ROM)................................45 圖3-37 Normal/+ PBS /+ Collagenase Ι在第15天的平均值比較.....46 圖3-38 Normal/ Non-RF /RF在第15天的平均值比較................46

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