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研究生：	李佳龍 Li, Chia-Lung
論文名稱：	以老鼠模型探討高能量雷射於阿基里斯腱癒合之治療 High intensity laser therapy for Achilles tendon healing in a rat model
指導教授：	蔡一如 Tsai, Yi-Ju
學位類別：	碩士 Master
系所名稱：	醫學院 - 物理治療學系 Department of Physical Therapy
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	51
中文關鍵詞：	高能雷射治療、雷射治療、阿基里斯腱斷裂、阿基里斯腱病變、肌腱病變、急性肌腱傷害
外文關鍵詞：	High intensity laser therapy, HILT, Laser therapy, Achilles tendon rupture, Achilles tendinopathy, tendinosis, Acute tendon injury
相關次數：	點閱：260 下載：1
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研究背景與目的:阿基里斯腱損傷是一種常見的運動傷害。反覆的肌腱損傷而無法有適當癒合是導致慢性肌腱病變最常見的原因之一。肌腱本身的低細胞與低血管的環境導致其受傷後癒合緩慢。低能量雷射是一種非侵入性的裝置，被廣泛使用在物理治療上來促進組織再生，肌腱的癒合過程可以被其『光生物調節作用』來促進。高能雷射裝置是近期進入骨科領域被用來治療肌肉骨骼相關疾病。雖然高能雷射治療越來越被廣泛使用在治療肌腱傷害，目前並無基礎的動物實驗來認證其功效及探討其可能促進肌腱癒合的治療機轉。本篇研究的目的就是使用老鼠的肌腱部分傷害的模型，來探討高能雷射促進肌腱癒合的效用及可能機轉。

研究材料與方法:60隻雄性大鼠被隨機分配實驗組及控制組，每組各30隻老鼠，其中每組使用3個不同時間(7天，14天及28天)再各細分成3個小組，每小組10隻老鼠。所有的老鼠其右側阿基里斯腱皆接受手術引發肌腱部分損傷。接著實驗組的老鼠其右側的阿基里斯腱接受每周3次為期2周的高能雷射治療。實驗組中的7天小組因為在手術後第7天犧牲，因此只接受3次高能雷射照射。所以的老鼠皆是照其小組的術後天數被犧牲，每小組老鼠的右腿阿基里斯腱10隻中的一半檢體(5隻)送交即時逆轉錄聚合酶鏈反應檢查，另一半(5隻)送交組織切片分析。本實驗中的肌腱部分損傷是在皮膚切開分離出阿基里斯腱後使用14號針頭穿刺阿基里斯腱的方式進行。高能的銣雅鉻脈衝雷射可以產生瞬間的高能量，其探頭產生直徑約5mm的治療光點在離皮膚約10公分的距離給予能量治療，本實驗控制在360mJ/cm2的強度每秒15發的頻率，給予每次總能量4.2焦耳治療。每周3次共治療2周。和疼痛、發炎及膠原蛋白合成相關的信使核糖核酸(mRNA)將會被評估。組織切片分析將會使用modified Movin score system半定量的方法由不知道實驗設計的相同觀察者來評估。組織分析的細項分數會被加總起來，所有的資料將會以平均值加減標準差來表示。統計上會使用unpaired t Test來做組間之比較，P值定在小於0.05有顯著差異。

研究結果:與疼痛相關物質substance P其信使核糖核酸(mRNA)在第7天的實驗組明顯低於控制組，而第一型膠原蛋白的信使核糖核酸(mRNA)在第14天實驗組明顯高於控制組並達統計意義，第三型膠原蛋白在14天時有類似表現但是未達統計意義。而在第14天時，和發炎相關因子(IL-6, TNF-α, and COX-2)的信使核糖核酸(mRNA)在實驗組明顯低於控制組並達到統計意義。而組織切片分析在第28天實驗組總分明顯低於控制組並達統計意義，代表實驗組肌腱癒合優於控制組。

討論與結論:肌腱癒合會被一些促進及危害的不同因子所調控，不當的調控將會導致細胞外基質的代謝與平衡。這些被提出可能在肌腱病變的扮演一定角色。過多的發炎因子有可能引發破壞性及毀滅性的過程。低能量雷射被報告在肌腱癒合上可減少發炎反應，在本實驗也應證了高能雷射也可減少發炎。此外本實驗也發現高能雷射治療可以減少與疼痛相關的物質substance P的產生，此和臨床上高能雷射可以止痛互相呼應。在組織學上可以看到高能雷射治療於28天時可觀察到較佳的癒合情況，其中膠原蛋白的外觀與排列方向性都是較好的。膠原蛋白的穩定與機械性刺激在肌腱癒合上扮演關鍵角色。高能雷射可以促進第一型膠原蛋白合成及排列較佳，更多的研究應該投入來了解高能雷射於肌腱癒合過程中其細胞內的機轉。在老鼠的實驗中，使用高能雷射給予4.2焦耳的能量於受傷的阿基里斯肌腱可以藉由減少疼痛、調控發炎及增加膠原蛋白合成來促進其癒合。

臨床運用:本動物實驗提出高能雷射治療對於急性肌腱損傷可能有正面的效果。而在臨床上，高能雷射治療在促進急性肌腱損的癒合及減少肌腱病變也許有一定的角色。

Background and Purpose: Achilles tendon injury has presented a high incidence in sport-related injury. Repeat tendon injury without adequate healing is through to be the common cause of tendinosis. The hypocellular and hypovascular environment of tendon itself delay the healing process. Low level laser therapy (LLLT), a non-invasive modality, has been widely used in the physical therapy for tissue regeneration. The tendon healing process is stimulated by the photobiomodulation(PBM) effect. The high intensity laser therapy (HILT) has been recently introduced into the orthopedic field for the musculoskeletal disease. Despite of its increasing clinical practice in tendon injury, there is no basic animal study to validate its effects and investigate the possible mechanism in promoting tendon healing. The objective of this study is to evaluate the effects and possible mechanism of HILT on the healing process of Achilles tendon in rats after partial tenotomy.

Materials and Methods: Sixty male Sprague-Dawley rats received right Achilles partial tenotomy were divided into control and experimental groups (n=30). Rats in both groups were then randomly assigned to one of 3 time-points subgroups (n=10) : day 7, day 14, and day 28. The right Achilles tendons of experimental group had received the high intensity laser therapy 3 times a week for 2 weeks. The subgroup day 7 in experimental group had only irradiated for 3 times and then euthanized on day 7. All of the rats in both control and experimental group were euthanized on the post-surgery days indicated in the subgroup names: days 7, 14, and 28. After sacrifice, the right Achilles tendons were harvested and prepared for further studies. Half tendons of each subgroup (N=5) was submitted for histological analysis. The other half (N=5) was sent for quantitative real-time reverse transcription-PCR studies. The right Achilles partial tenotomy was made by the G14 needle puncture after skin incision and tendon exposure. The high intensity laser irradiation was delivered with a high peak power, pulsed Nd:YAG laser apparatus (HILTERAPIA®, SH1), using a optic ﬁber in a non-focused way, producing a circular spot about 5mm in diameter at a distance of 10 cm from the skin. The total energy of delivery was 4.2J, by controlling the exposure time under the frequency of 15 Hz and ﬂuency rate (360mJ/cm2).The right Achilles tendons in experimental group were irradiated 3 times a week in the first and second week after partial tenotomy. Levels of mRNAs associated with pain, inﬂammation and collagen synthesis were evaluated. The histologic assessment of Achilles tissue samples was based on the modifed semiquantitative method of Movin (method by Bonar). The grade of the tendon healing was assessed by the same observers unaware of the protocol in the study. Sum of the histologic scores was calculated. Data were expressed as mean ± SEM. Statistical significance between two groups was assessed using unpaired t test. P values less than 0.05 were considered significant.

Results: The mRNA level of Substance P on Day 7 was much lower in the experimental group (P < 0.05). The mRNA level of collagen I in the experimental group was significant higher than the control group on D14. (P<0.05) Similarly, the mRNA level of collagen III in the experimental group on D14 was seen higher than control group but not reaching the statistical difference. (P>0.05) The mRNA level of IL-6, TNF-, and COX-2 in the experimental group was significant lower than the control group on D14. (P<0.05) The sum of histologic grading score in tendon healing on D28 was much lower (P<0.05) in experimental group, which meant better quality of tendon healing.

Discussion: Tendon healing is regulated by some promoting and some compromising factors. Improper regulation of these factors may lead to imbalance of extracellular matrix metabolism. It was proposed to play a role in the development of tendinopathy. Excessive inflammatory cytokines may promote catabolic and destructive processes. LLLT was reported to reduce the inflammation response after tendon injury. In this study, HILT was able to reduce the inflammation. Besides, the mRNA level of substance P was much lower in experimental group on D7. This finding was comparable to previous clinical study of HILT for pain relief. By the histologic grading of tendon healing, it showed better quality of tendon healing in experimental group on Day 28. The shape and orientation of collagen fibers were better after HILT. Hemostasis of collagen and mechanical stimulation may play a role on tendon healing. There were the positive effects of HILT in increasing type I collagen synthesis and realignment. More researches are needed to determine the exact intracellular mechanism in tendon healing after high intensity laser irradiation. Application of HILT under the dosage of 4.2J in injured rat Achilles tendon may promote the tendon healing by decreasing the pain, modulating the inflammation and improving the collagen synthesis and realignment.

Clinical Relevance :
This animal study proposed the possible positive effect of HILT in acute tendon injury. In the clinical practice, there may be a role of HILT in acute tendon injury to promote the healing process and reduce the development of tendinopathy.

中文摘要(Chinese Abstract)	I
英文摘要(English Abstract)	III
致謝 (Acknowledgement)	V
目錄(Contents)	VI
表目錄(List of Tables)	VIII
圖目錄(List of Figures)	IX

Chapter I Introduction	1
    1.1  Backgroundand Purpose 	1
    1.2  Hypotheses	3

Chapter II Literature Review	4
    2.1 Introduction of Tendon	4
       2.1.1 Tendon structure	4
    2.1.2 Epidemiology and Etiology of Tendon Injury	5
    2.1.3 The nature of tendon healing	6
    2.2 Introduction of laser therapy	7
       2.2.1 Photobiomodulation (PBM) 	8
       2.2.2 Low level laser therapy in tendinopathy and  
             tendon tear	10
       2.2.3 High intensity laser therapy	10

Chapter III Materials and Methods	12
    3.1 Ethics statement	12
    3.2 Animal model	12
    3.3 Surgical protocol	13
    3.4 Instrumentation	14
    3.5 Laser exposure and protocol	14
    3.6 Outcome measures	15
       3.6.1 Quantitative real-time reverse transcription-PCR (quantitative RT-PCR) 	15
       3.6.2 Histologic evaluation	16
    3.7 Statistical Analysis	17
Chapter IV Result	18
    4.1 Quantitative real-time reverse transcription-PCR (quantitative RT-PCR) 	18
       4.1.1 The neurotransmitter, substance P	18
       4.1.2 The inflammatory cytokines	19
       4.1.3 The status of proliferation	19
    4.2 The histologic analysis	19
Chapter V Discussion	20
Chapter VI Conclusion	25
Reference	26
Table 1 :  Primers in RT-PCR	30
Table 2 :  Modifed semiquantitative method of Movin score	30
Table 3 :  Result of RT-PCR	31
Figure 1. Three phase of tendon healing 	32
Figure 2. Therapy windows	32
Figure 3. BPM : Scheme of mitochondrial retrograde signaling pathways	33
Figure 4. Grouping of rats 	33
Figure 5. needle puncture induced partial tenotomy 	34
Figure 6. Instrumentation	34
Figure 7.Gross view of tendon healing in HILT on D28 .	35
Figure 8. Quantitative RT-PCR : Substance P 	36 
Figure 9. quantitative RT-PCR : IL-1 beta 	37 
Figure 10. quantitative RT-PCR : IL-6	38 
Figure 11. quantitative RT-PCR : TNF-alpha 	39 
Figure 12. quantitative RT-PCR : COX-2	40
Figure 13. quantitative RT-PCR : tenomodulin 	41
Figure 14. quantitative RT-PCR :Collagen type I 	42 
Figure 15. quantitative RT-PCR :Collagen type III	43
Figure 16. quantitative RT-PCR :Ration of collagen type III /I 	44
Figure 17. quantitative RT-PCR : histology score 	45 
Figure 18. slide, D7, HILT	46
Figure 19. slide, D7, control	47
Figure 20. slide, D14, HILT	48
Figure 21. slide, D14, control	49
Figure 22. slide, D28, HILT	50
Figure 23. slide, D28, control	51 


                                    

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