以大鼠周邊及中樞神經損傷模式探討調節細胞激素對於神經系統的保護機制

Elucidate the Neural Protective Mechanism of Regulation of Cytokines on the Peripheral Nerve Injury and Central Neuronal Damage Models in Rat

趙伯寬(Po-Kuan Chao)

指導教授 ： 呂國棟 ； 羅榮昇 ； 楊奕玲

國立臺灣師範大學/理學院/生命科學研究所/博士(2012年)

摘要

近年來的研究顯示，調節細胞激素有助於減少神經損傷後的神經發炎並且改善神經系統功能。在本研究當中，我們分別使用不同的大鼠模式來研究如何透過特定調節物質來改變周邊神經損傷及中樞神經傷害後細胞激素的表現。
首先，為了釐清組織病理評估結果與行為結果之間的關聯性，我們針對假手術對照組及長期壓迫神經損傷手術組大鼠的同側及對側脊髓神經背角及第五段腰椎的背根神經結，分別測量其c-Fos蛋白質表現以及小膠細胞活化作用。我們發現c-Fos蛋白質表現在長期壓迫神經損傷手術組大鼠的同側背根神經結並不會增加。此外，手術後72小時長期壓迫神經損傷引發c-Fos蛋白質表現增加只會出現在同側脊髓神經背角中。然而相對的，小膠細胞活化現象在脊髓神經背角兩側都非常顯著，同時也與機械性刺激引發痛覺過敏的行為結果一致。所以我們證實不僅有神經細胞、小膠細胞也在神經病變痛當中扮演角色，在中樞神經系統中，小膠細胞透過與神經細胞之間的交互作用對疼痛行為產生影響。
先前的研究中發現在神經系統中顆粒細胞刺激增生因子是一種重要的調節物質。所以本研究展示了與對照組相較的結果，在手術後的1到25天之間，投予顆粒細胞刺激增生因子有助於減輕長期壓迫神經損傷引發的熱痛覺過敏及機械性刺激過敏的現象。長期壓迫神經損傷手術手術後投予顆粒細胞刺激增生因子也減少動物熱痛覺過敏。推測若神經損傷後48小時內單次投與顆粒細胞刺激增生因子仍對於神經病變痛的治療具有效果。顆粒細胞刺激增生因子也驅動含鴉片類物質的聚多核細胞聚集進入受損的神經之中。長期壓迫神經損傷不僅導致背根神經結中的第六型細胞激素的mRNA及腫瘤壞死因子α亞型蛋白表現量增加，也使得脊髓中第一型細胞激素的蛋白表現量上升。這些細胞激素的產生都會被顆粒細胞刺激增生因子所抑制。我們更進一步發現長期壓迫神經損傷後，μ型鴉片受體出現在損傷的神經當中而且鴉片受體拮抗劑─naloxone methiodide減低了顆粒細胞刺激增生因子的抗痛效果，於是我們推論顆粒細胞刺激增生因子減輕熱痛覺過敏是透過鴉片類物質及其受體之間交互作用的結果。更進一步來看，投與顆粒細胞刺激增生因子可以抑制脊髓神經背角處因長期壓迫神經損傷所引發小膠細胞大量活化。這些結果推測在神經損傷初期單次全身性投予顆粒細胞刺激增生因子減輕神經病變痛的效果是透過活化聚多核細胞驅動的內生性鴉片類物質分泌，進而活化損傷神經當中的鴉片類物質受體，同時減少促發炎細胞激素，並減輕脊髓神經背角當中的小膠細胞活化現象。
而在大鼠神經損傷模式中，indomethacin曾被用來進行創傷的治療。基於西方墨漬法的分析結果, 我們發現Nogo-A蛋白表現在頭部創傷後八小時顯著增加。此外，頭部創傷引發受測動物的第一型細胞激素含量顯著提升，一般而言所有的頭部創傷相關的分子及細胞的後續反應都會被抗發炎藥物indomethacin所影響而顯著減少。更重要的是，頭部創傷刺激作用相關的Nogo-A及第一型細胞激素的含量明顯被具有專一性的反義寡核苷酸所抑制。我們的發現，出現Nogo-A表現受到抑制的初期反應是由indomethacin所賦予的，也減少第一型細胞激素的表現程度並且減輕頭部誘發的神經損傷。
總結以上，由於全身性投予顆粒細胞刺激增生因子或是indomethacin分別對於大鼠的周邊以及中樞神經損傷具有正面的影響，以顆粒細胞刺激增生因子或者是indomethacin來調節中樞神經系統中的促發炎細胞激素，很可能有成為治療中樞神經過敏化現象的嶄新療法。

關鍵字

顆粒細胞刺激增生因子 ； 腫瘤壞死因子 ； 小膠細胞 ； 神經損傷 ； 神經病變痛 ； 鴉片類物質 ； 細胞激素

並列摘要

Recent studies have shown that modulation of cytokine expression is helpful to reduce nerve inflammation and to improve dysfunction of nerve system. In this study, we use rat models to investigate the mediators for alterations of cytokines expression following peripheral nerve injury and central neural damage.
First, in order to clarify the correlation between histopathological assessment and behavior outcome, c-Fos protein expression and microglia activation were quantified in both of the ipsi- and contra-lateral spinal dorsal horn (DH) and/or Lumbar 5 (L5) dorsal root ganglia (DRG) in sham-operation and chronic constriction injury (CCI) rats. We found that c-Fos protein expression did not increase in the ipsilateral DRG of CCI rat. In addition, CCI evoked increase of c-Fos protein expression was only in the ipsilateral spinal DH after 72h. In contrast, however, microglia activation was notably induced at bilateral spinal DH and correlated with mechanical allodynia. Thus, neurons are not the only cells playing a role in neuropathic pain, we evidence that microglia are involved as well and there is a cross effect between neuron and microglia in the central nervous system (CNS) which associated to nociceptive behaviors.
The current studies show that granulocyte colony-stimulating factor (G-CSF) is an important mediator of nerve system. Secondary, this study shows the effectiveness of exogenous treatment for alleviating thermal hyperalgesia and mechanical allodynia in rats with CCI, during post-operative days 1–25, compared to that of vehicle treatment. G-CSF also increases the recruitment of opioid-containing PMN cells into the injured nerve. After CCI, single administration of G-CSF, relieved thermal hyperalgesia, indicated that its effect on neuropathic pain had a therapeutic window of 0–48 h after nerve injury. CCI led to increase in the levels of interleukin-6 (IL-6) mRNA and tumor necrosis factor-alpha (TNF-α) protein in the DRG and interleukin-1beta (IL-1β) protein in the spinal cord. These high levels of IL-6 mRNA, TNF-α and IL-1β were, respectively, suppressed by a single administration of G-CSF after CCI, respectively. Moreover, the μ-opioid receptor was observed in injured nerve and opioid receptor antagonist naloxone methiodide (NLXM) reversed G-CSF-induced antinociception after CCI, suggesting that G-CSF alleviates hyperalgesia via opioid/opioid receptor interactions. Furthermore, G-CSF administered after CCI suppressed the CCI-induced up-regulation of microglial activation in the ipsilateral spinal DH, which is essential for sensing neuropathic pain. These results suggest that an early single systemic injection of G-CSF alleviates neuropathic pain via activation of PMN cell-derived endogenous opioid secretion to activate opioid receptors in the injured nerve, down-regulate IL-6, IL-1β and TNF- pro-inflammatory cytokines, and attenuate microglial activation in the spinal DH.
In the neuronal damaged models, indomethacin was ever used to deliver a traumatic management to rats. Based on Western blot analyses, the expression of Nogo-A was found to be significantly up-regulated in the hippocampus beginning eight hours after traumatic brain injury (TBI). In addition, TBI caused an apparent elevation in IL-1β levels in the tested animals. All of the TBI-associated molecular and cellular consequences could be effectively reversed by treating the animals with the anti-inflammatory drug indomethacin. More importantly, the TBI-associated stimulation in the levels of both Nogo-A and IL-1β could be effectively inhibited by a specific Nogo-A antisense oligonucleotide. Our findings suggest that the suppression of Nogo-A expression appears to be an early response conferred by indomethacin, which then leads to decreases in the levels of IL-1β and TBI-induced neuron damage.
In conclusion, because systemic administration of G-CSF or indomethacin had positive effects in both models of peripheral nerve injury and central nervous system injury in rats, respectively, it is highly possible that treating with G-CSF or indomethacin to modulate pro-inflammatory cytokines of CNS is the new method to manage central sensitization.

並列關鍵字

granulocyte colony-stimulating factor ； tumor necrosis factor ； microglia ； nerve injury ； neuropathic pain ； opioid ； cytokine

參考文獻

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