中樞神經系統由於外在的抑制環境和內在本身極低的生長能力而難以再生，目前研究上還有許多問題有待克服。一般神經細胞有較高的能量需求，須要粒線體提供能量以支持其進行細胞活動。而粒線體會藉由融合和分裂來維持自身的功能，此粒線體動態變化可能為神經再生所必須。在本研究中，我們利用十八天大的大鼠胚胎分離出海馬迴神經細胞，觀察粒腺體動態變化在神經再生過程中所扮演的角色。再生過程中粒線體型態依據其長度分為三種類型並進行統計，結果顯示受傷會造成粒腺體分裂，在受傷處附近的細胞其粒腺體長度較短，而在受傷細胞中加入粒腺體分裂的抑制劑並不影響神經再生。又由於粒線體功能的缺失跟很多神經退化性疾病有關，因此我們假設粒線體療法能促進神經的再生。實驗顯示外加入新鮮分離出的粒腺體能夠進入細胞當中，並且大幅地促進再生，甚至還能回復受傷海馬迴細胞的膜電位。綜合以上結果，本研究點出粒腺體在神經再生過程中的重要性，並對受傷的中樞神經系統提出一個新穎的治療方式。

Due to the inhibitory extrinsic microenvironment and the reduced intrinsic growth capacity of neurons, neuronal regeneration of central nervous system remains to be a challenge to overcome. Neurons are highly energy-demanding and require mitochondria to support cellular activities including regeneration. Mitochondria undergo fusion/fission cycles to ensure proper functions of cells. The plasticity of mitochondrial dynamics is thus required for neuronal regeneration. In this study, we examined the role of mitochondrial dynamics during neuronal regeneration by using embryonic day 18 rat hippocampal neurons as our model. The dynamics of mitochondrial morphogenesis during regeneration was analyzed and was defined into three categories- fission, intermediate, and fusion type according to their length. Quantitative analysis showed that injury-induced mitochondrial fission were more prominently near the injury sites. Treating neurons with mitochondrial fission inhibitor, Mdivi-1, had no much effect on neuronal regeneration. Since mitochondrial dysfunctions are highly associated with neurodegenerative diseases, we thus hypothesized that mitochondrial therapy may potentially promote neuronal regeneration. Freshly isolated mitochondria were added to the injured hippocampal neurons and were internalized into cells. Our results showed that addition of freshly isolated mitochondria significantly increased neurite re-growth. Moreover, treatment of mitochondria restored membrane potential of injured hippocampal neurons. Together, findings from this thesis highlight the important role of mitochondria during regeneration of hippocampal neurons and provide a novel therapeutic prospect to the injured central nervous system.

Table of contents
Abstract i
摘要 ii
致謝 iii
Table of contents vii
Abbreviations ix
Introduction 1
Traumatic brain injury 1
Regeneration of central nervous system 2
Mitochondria 3
Mitochondrial dynamics 4
Mitochondria in neurons 5
Mitochondrial therapy 8
Materials and methods 10
Reagents and antibodies 10
Experimental animals and primary culture of neurons 10
Experimental injury assay 11
Analysis of neurite re-growth 12
Transient transfection and live cell imaging 12
Mitochondrial isolation and internalization 12
Mitochondrial therapy 13
Immunofluorescence staining and confocal microscopy 13
Measurement of cell membrane potential 14
Statistical analysis 14
Results 16
Mitochondrial morphogenesis in response to injury 16
Effect on inhibiting mitochondrial fission by Mdivi-1 on neurite re-growth 16
Mitochondrial therapy promotes neuronal regeneration 17
Discussion 21
Reference 24
Figures 35
Figure 1. Mitochondrial morphogenesis and transport in hippocampal neurons after injury. 37
Figure 2. Mitochondria in hippocampal neurons became fragmented in response to injury. 39
Figure 3. Effect of inhibiting of mitochondrial fission by Mdivi-1 in injured hippocampal neurons. 42
Figure 4. Isolated mitochondria were co-stained with MitoTracker Red and anti-Tom20 antibody. 43
Figure 5. Exogenously added mitochondria were internalized into hippocampal neurons after 5 hrs. 44
Figure 6. Treatment of mitochondria promotes neurite re-growth of injured hippocampal neurons. 47
Figure 7. Treatment of mitochondria increases the numbers of regenerated neurites in injured hippocampal neurons. 48
Figure 8. Treatment of isolated mitochondria restores membrane potential of injured hippocampal neurons. 50
Figure 9. Working model 51

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