成體哺乳類視網膜節細胞的軸突在神經損傷後往往不易再生。其中，如神經活性等外在環境訊息被認為是促進神經再生關鍵。我們在先前幼年小鼠視網膜離體培養實驗中，意外發現因培養孔盤的下盤溫度上升而導致其蒸散作用增加，進而提高培養液的濃度，最終促進視網膜的神經纖維明顯增加。結果顯示，相較於1.5倍培養液濃度，當整體培養液濃度提升至1.25倍時，更能有效促進幼年小鼠視網膜節細胞的神經纖維生長。由此可知，增加滲透壓所造成的輕微逆境，可以有效的促進神經纖維再生。類似情形，以溫度作為操縱變因，在35 °C培養環境中，連續五天每日給予一小時38、40和42 °C的溫度刺激能顯著增加神經纖維的再生。由此得出，以短期升溫作為輕微逆境可有效促進神經纖維生長。在進一步實驗中，得出在此兩種輕微逆境中(包含長期提高滲透壓和短期升溫)，提高神經活性是作為促進小鼠離體培養之視網膜神經纖維再生的關鍵因素。重要的是，在開始給予升溫壓力的0至15分鐘內，部分視網膜節細胞的神經活性顯著上升，並且在連續五日離體培養中，每日以15分鐘42 °C的溫度壓力刺激即足以促進神經纖維的再生。此外，在短期42 °C溫度壓力下，小鼠視網膜組織中的熱休克蛋白27和70的表現量明顯增加。進而提出由輕微壓力所引起的熱休克蛋白的表現與輕微逆境促進的神經纖維再生之間存在著相當大的關聯性。這項研究以輕微逆境作為刺激，去探討其促進神經纖維再生的分子機制，並在未來有機會成為促進神經纖維生長的新興治療策略。

The axons of retinal ganglion cells (RGCs) in adult mammals fail to regenerate after injury. It has been suggested that some extrinsic factors, such as neural activity, may promote the regeneration process. During our previous investigation, it was serendipitously discovered that a slight increase in the concentration of the culture medium as a result of accidentally elevating the culture plate temperature from below and that the resulting increase in evaporation was able to significantly promote neurite outgrowth in retinal explants from P11 mice. In the present study, we found that RGC neurite outgrowth from P9-P11 mouse explants was significantly enhanced when the concentration of the culture medium was increased by 1.25 fold, but not when increased by 1.5 fold. This suggests that the mild stress caused by increased osmolarity promotes neurite outgrowth. Similarly, retinal explants from P9-P11 mice grew longer neurites when the overall temperature was increased from 35 °C to 38 °C, 40 °C or 42 °C for one hour each day, but not when they were kept at 40 °C or 42 °C constantly for five days. This supports the hypothesis that the mild stress induced by a short-term increase in temperature is able to enhance RGC neurite outgrowth. We further showed that there was increased neural activity during these two mild stresses, namely long-term increased osmolarity and short-term increased temperature, and these are the major driving force promoting neurite outgrowth by postnatal retinal explants. Importantly, the spiking rate of a subset of RGCs increased significantly during the first 15 min of temperature elevation. Subsequently, we observed using P9-P11 mouse explants that had only 15 min of heat stress at 42 °C each day for five days showed enhanced neurite outgrowth. In addition, it was found that short-term 42 °C heat stress increased the expression of heat shock proteins 27 and 70 (HSP 27 and HSP70) in postnatal retinas. This suggests that mild stress induced HSP expression is correlated with the observed neurite outgrowth enhancement by postnatal retinal explants. The present study thus provides insights into the cellular mechanism of retinal axon regeneration under mild stress and these findings could be potentially developed into a new therapeutic strategy to improve neural regeneration.

Contents
摘要 i
Abstract ii
致謝 iv
1. Introduction 1
2. Materials and Methods 4
2.1 Retinal explant preparation 4
2.2 Retinal explant culture 4
2.3 Immunohistochemistry 5
2.4 Neurite outgrowth quantification 6
2.5 Osmolarity measurement 6
2.6 Apoptotic cells assessment 7
2.7 Neural activity assessment 7
2.8 Heat shock protein expression quantification 8
2.9 Statistics 9
3. Results 10
3.1 Mild osmotic stress promotes neurite outgrowth of retinal explants in postnatal mice 10
3.2 Increased neural activity induced by mild osmotic stress facilitates neurite outgrowth of retinal explants 10
3.3 Mild heat stress enhances the neurite outgrowth of retinal explants 11
3.4 Increased neural activity induced by short-term mild heat stress promotes neurite outgrowth of retinal explants 12
3.5 Brief heat stress is sufficient to promote neurite outgrowth of retinal explants by increasing neural activity 13
3.6 Expression of two heat shock proteins is correlated with the short-term heat stress effect on neurite outgrowth 13
4. Discussion 15
4.1 Mild osmotic pressure elevation promotes neurite outgrowth of postnatal retinal explants 15
4.2 Mild heat stress contributes to neurite outgrowth without affecting the cell survival of postnatal retinal explants 16
4.3 Mild stress promotes neurite outgrowth by increasing neural activity of RGCs 17
4.4 Mild heat stress promotes neurite outgrowth by activating various heat shock proteins in RGCs 19
5. References 21
6. Figures 24

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