小鼠背根神經節於神經痛之分子表現：以坐骨神經損傷為模式

Molecular Expression in Dorsal Root Ganglia of Neuropathic Pain Mice Induced by Spared Nerve Injury

陳璟瑜(Ching-Yu Chen)

指導教授 ： 謝松蒼

國立臺灣大學/醫學院/腦與心智科學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.01212

摘要

神經痛為周邊及中樞神經系統因疾病或原發性損傷所造成，患者感受到程度不一的異常感覺，如觸摸痛、痛覺過敏、痛覺減退等。由於症狀在臨床的表現複雜且機制尚未全面了解，藥物治療效果不佳且有耐受性問題，對於疼痛機制的研究以及開發有潛力的藥物治療標的有其必要性。周邊神經損傷為造成神經痛的其中一種原因，而三磷酸腺苷 (ATP)及嘌呤受體 (Purinergic receptor)的活化則為痛覺活化的可能途徑。其中P2X3受體於先前研究發現主要表現在中小型感覺神經元，因此我們使用小鼠坐骨神經損傷(Spared nerve injury, SNI)為模型，藉此探討P2X3受體及相關分子在背跟神經節(Dorsal root ganglion, DRG)之表現。
手術後小鼠手術肢機械性刺激閾值 (mechanical threshold) 下降而敏感化 (mechanical allodynia)，相對於非手術肢及手術前測試結果都有顯著差異。背跟神經節L4免疫螢光染色結果顯示，P2X3受體主要表現於中小型神經元、以及少部分大型神經元; 而手術側與對照側之P2X3受體於整個神經元族群所佔比例並無顯著差異。利用雙重染色觀察P2X3型態上的分佈，主要和表現lectin IB4的non-peptidergic受體高度重疊，並且極少與表現calcitonin gene- related peptide (CGRP)的peptidergic 受體重疊，而IB4/P2X3神經元比例在手術側較對照側為低並達到統計上差異，因此表現P2X3的神經元族群於手術側與對照側可能有型態分佈上的改變。使用網路資料庫篩選參與P2X3啟動子結合調控的可能基因，其中activating transcription factor-3 (ATF3) 之染色結果於手術側顯著上升，然而ATF3剔除小鼠接受SNI後表現之神經痛行為測試結果與wild-type組並無顯著差異，ATF3與P2X3是否具有調控的相關性仍有待探討。
在SNI小鼠模型中，假手術組於機械性刺激閾值的絕對值與差值的統計結果呈現不一致的情形，代表需要更進一步探討小鼠的痛覺行為。此外，不同行為測量儀器、神經痛模型之間的行為與分子表現差異、免疫螢光染色中大型神經元在P2X3活化所扮演之角色、SNI小鼠模型中受損與未受損神經元之分子表現、以及P2X3神經元族群分佈型態上的特性仍有許多可以探究的問題。

關鍵字

坐骨神經損傷模型(SNI) ； 神經痛 ； P2X3受體 ； 激活轉錄因子-3(ATF3) ； 背跟神經節(DRG) ； 痛覺受體

並列摘要

Neuropathic pain refers to pains resulting from disease or damage of the peripheral or central nervous systems, and from dysfunction of the nervous system. Patients suffer from this chronic, intensively painful experience that is difficult to treat with conventional analgesic medications. Peripheral nerve injury is one of the etiologies causing neuropathic pain, and ATP seems to be strongly implicated in peripheral pain sensitization. The purinergic receptor, P2X3, a ligand-gated cation channel, is expressed in a subset of small-diameter, primary afferent neurons, and might be a potential therapeutic target to relieve pain. Previous studies showed controversial results in terms of P2X3 expression in several peripheral nerve injury models. Some claimed that a functional up-regulation of P2X3 in dorsal root ganglia, whereas some reported contradicted results. We hope to investigate the regulation of P2X3 gene expression.
Using spared nerve injury model (SNI) in mice, we found animals displayed mechanical allodynia in the spared sural territory one week after surgery and persisted at least two weeks. From the results of immunofluorescent staining to estimate the P2X3 (+) neuron ratio on lumbar (L4) dorsal root ganglia (DRG), P2X3 receptors were mainly expressed in small and medium sized neurons, while few percentages of large neurons were also discovered both in the contralateral and ipsilateral sides. The P2X3 immunoreactive neuron ratios between ipsilateral and contralateral DRG did not show significant difference, and the distribution were highly colocalized with the nonpeptidergic neurons that express IB4, but rarely co-expressed with CGRP immunoreactive neurons, which are peptidergic. The (IB4/P2X3) neuron ratios in ipsilateral DRG were statistically lower than the contralateral DRG, suggesting a morphological shift of P2X3 distribution could be happened. To screen the possible gene candidates regulating P2X3 expression on transcriptional level, we used bioimformatic web tools and further filtered by their functions. One possible molecule, activating transcription factor-3 (ATF3), was increased on the ipsilateral DRG significantly. Although the mechanical thresholds of ATF3-knockout mice after SNI were similar to those of the wild-type group, it is worth pursuing whether there exist relationship, probably with different neuropathic pain models.
In our study of SNI mouse model with mechanical allodynia, inconsistency of the sham group in difference of mechanical thresholds compared to absolute values, comparison of sensory abnormalities between dynamic plantar aesthesiometer and manual von Frey filaments, pain behaviors in different neuropathic pain models and species are possible issues to be investigated. From the results of immunofluorescent staining, quantities of large sized neuron in P2X3 (+) populations, degrees and location of nerve injury, and distribution of injured and intact neurons in DRG after SNI are also required to be further clarified.

並列關鍵字

Spared nerve injury(SNI) ； Neuropathic pain ； P2X3 ； Activating transcription factor-3(ATF3) ； Dorsal root ganglion(DRG) ； Nociceptor

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