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研究生：	邱馨誼 Chiu, Hsin-Yi
論文名稱：	粒線體移植在神經病變性疼痛大鼠之潛在治療效果 The Therapeutic Potential of Mitochondrial Transplantation in a Rat Model of Neuropathic Pain
指導教授：	李榮順 Lee, Jung-Shun
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：	2020
畢業學年度：	109
語文別：	英文
論文頁數：	51
中文關鍵詞：	細胞凋亡、粒線體、粒線體移植、神經發炎、神經病變性疼痛
外文關鍵詞：	Apoptosis, mitochondria, mitochondrial transplantation, neuroinflammation, neuropathic pain
相關次數：	點閱：133 下載：0
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神經病變性疼痛是由於體感覺系統受損所引發的一系列痛覺異常現象，常見症狀包含觸摸痛以及痛覺過敏。神經發炎、粒線體功能異常以及細胞死亡已經被證實和神經病變性疼痛的發生和持續具有高度相關性；其中又以粒線體功能異常最為關鍵，於是以改善粒線體功能異常為目標的療法開啟了治療神經病變性疼痛的新策略。粒線體移植是一個新穎的治療方法，在心臟缺血後再灌注以及脊髓損傷模式的實驗中都顯示其相當的組織功能復原效果。本篇研究旨在探討粒線體移植在大鼠神經病變性疼痛模式中的神經調節作用。吾人將粒線體移植入右側第五腰椎神經結紮之大鼠背根神經節內，結果顯示在粒線體移植七天後疼痛超敏反應得到顯著緩解。除此之外，粒線體移植也顯著降低在傷側脊髓背角的促發炎細胞因子以及細胞凋亡蛋白。免疫螢光染色結果顯示星狀膠細胞以及小膠細胞在傷側脊髓背角的活化情形隨粒線體移植得到降低。粒線體追蹤染色(MitoTracker deep red)顯示異源線粒體可以存活於第五腰椎背根神經節、第五腰椎脊髓以及坐骨神經。線粒體移植有追蹤潛在受傷組織之能力以調節細胞凋亡和發炎反應，因此透過本篇研究吾人認為粒線體移植具有神經保護作用。

Neuropathic pain is caused by damage to the somatosensory system and induces abnormal pain sensitivity, which is characterized by allodynia and hyperalgesia. Neuroinflammation, mitochondrial dysfunction, and cell death are hallmarks in the pathogenesis of neuropathic pain. Mitochondrial dysfunction, which is a consequence of neuroinflammation and results in further cell death, plays the most crucial role in neuropathic pain. Therefore, strategies that target mitochondrial dysfunction are emerging to treat neuropathic pain. A novel approach of mitochondrial transplantation has achieved therapeutic effects on myocardial ischemic-reperfusion and spinal cord injury models; however, little is known about its application to neuropathic pain. Therefore, this study aimed to investigate the neuromodulation effect of mitochondrial transplantation in a neuropathic pain model.
In this study, neuropathic pain was induced by right L5 spinal nerve ligation (SNL). Seven days after the mitochondrial transplantation into the right L5 dorsal root ganglion (DRG) of SNL rats, pain hypersensitivity was significantly palliated. Furthermore, TNF-α, IL-6, IL-1β, and caspase 3 expression reduced and the Bcl-2/Bax ratio significantly increased in the ipsilateral spinal cord of the mitochondrial transplantation group on post-SNL day (PSD) 7. Immunofluorescence showed plethora astrocytes and microglia activation in the ipsilateral spinal cord on PSD 7, which declined after mitochondrial transplantation. MitoTracker deep red staining showed that allogenic mitochondria were evident in the L5 DRG, L5 spinal cord, and sciatic nerve. Collectively, transplanted mitochondria migrate to potential injured sites to moderate neural apoptosis and inflammation, which is indicative of their neuroprotective effect.

中文摘要	I
Abstract	II
誌謝	IV
Abbreviations	V
Contents	VI
Chapter 1 Introduction	1
1.1    Pain	1
1.2    Neuropathic pain	1
1.3    Neuroinflammation and cell death	2
1.4    Mitochondrial dysfunction	3
1.5    Clinical symptoms and current treatment modalities	4
1.6    Mitochondrial transplantation	4
Chapter 2 Specific Aims	6
Chapter 3 Materials and Methods	7
3.1    Animals	7
3.2    Study design	7
3.3    Mitochondrial isolation	7
3.4    Mitochondrial labeling	8
3.5    Surgical procedure	8
3.6    DRG microinjection	9
3.7    Behavioral tests	9
3.7.1.	Mechanical test	10
3.7.2.	Thermal test	10
3.8.    Tissue preparation	10
3.9.    Immunoblotting	11
3.10.    Immunostaining	11
3.11.    Statistical analysis	12
Chapter 4 Results	13
4.1    SNL-induced neuropathic pain	13
4.2    Mitochondrial transplantation alleviated pain hypersensitivity	13
4.3    Mitochondrial transplantation attenuated apoptosis in the spinal cord	14
4.4	   Mitochondrial transplantation modulated inflammation in the spinal cord and DRGs	14
4.5    Mitochondrial transplantation inhibited glial activation in the spinal cord and DRG	14
4.6    Transplanted mitochondria signified the pain pathway	15
4.6.1	Distribution of transplanted mitochondria in the L5 DRG	15
4.6.2	Distribution of transplanted mitochondria in the L5 spinal cord	15
4.6.3	Distribution of transplanted mitochondria in the sciatic nerve	16
4.7    Colocalization of mitochondrial transplantation in the spinal cord and DRG		16
Chapter 5 Discussions	17
5.1    Neuromodulation effect of mitochondrial transplantation.	17
5.1.1.	Anti-inflammation of mitochondrial transplantation	17
5.1.2	Anti-apoptosis of mitochondrial transplantation	19
5.1.3	Functional maintenance of mitochondrial transplantation	19
5.2    Spatiotemporal distribution of transplanted mitochondria	20
5.3    Interactions between allogenic mitochondria and host cells	20
5.4    Limitations	21
Chapter 6 Conclusion	23
Chapter 7 References	24
Chapter 8 Tables	32
Chapter 9 Figures	33
Figure 1. L5 spinal nerve ligation (SNL) and Dorsal root ganglion (DRG) microinjection	33
Figure 2. Flow chart of the experiment	34
Figure 3. SNL-induced pain hypersensitivity	35
Figure 4. Pain hypersensitivity did not occur in the contralateral side after SNL	36
Figure 5. Transplanted mitochondria alleviated pain hypersensitivity	37
Figure 6. DRG microinjection did not affect the behaviors in the contralateral side	38
Figure 7. Mitochondrial transplantation reduced neural apoptosis in the spinal cord after SNL	39
Figure 8. Mitochondrial transplantation reduced neuroinflammation in the spinal cord after SNL	40
Figure 9. Mitochondrial transplantation did not reduce neuroinflammation in DRGs after SNL	41
Figure 10. Mitochondrial transplantation reduced astrocyte activation in the spinal cord dorsal horn	42
Figure 11. Mitochondrial transplantation reduced microglia activation in the spinal cord dorsal horn	43
Figure 12. Mitochondrial transplantation reduced astrocyte activation in the DRG	44
Figure 13. Mitochondrial transplantation reduced macrophage activation in the DRG		45
Figure 14. The spatiotemporal distribution of allogenic mitochondria in the DRG	46
Figure 15. The spatiotemporal distribution of allogenic mitochondria in the spinal cord	47
Figure 16. The distribution of allogenic mitochondria in the spinal cord	48
Figure 17. The spatiotemporal distribution of allogenic mitochondria in the sciatic nerve	49
Figure 18. Colocalization of allogenic mitochondria with neuron, astrocyte, and microglia	50
Figure 19. Colocalization of allogenic mitochondria with neuron and macrophage	51


                                    

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