神經痛與神經受損於缺血性神經病變之研究

神經缺血目前已知是引起末梢神經病變的相當重要的一項原因且越來越受重視。當負責供應末梢神經血液的神經膜血管出現問題、且導致神經得不到足夠的血液供應而缺血，進一步神經因缺血傷害而產生病變，即所謂的缺血性神經病變(ischemic neuropathy)。已有許多臨床研究指出缺血傷害於末梢神經的型態及功能受損與末梢血管病變的關連性，而臨床上諸如周邊動脈血管阻塞疾病、血管炎、嚴重長時間休克、以及糖尿病的病人也有研究指出存在末梢神經功能受損症狀，且其神經切片顯現缺血性神經病變的病理變化。缺血性神經病變臨床上的神經病變類型可為表現為單一神經病變，或多發性不對稱性神經病變，但以多發性神經病變居多，且其發生過程多為急性或亞急性發作。臨床症狀常見有肢體感覺異常，無力甚至伴隨疼痛。我們注意到在這些缺血性神經病變的病人的症狀中，難耐的疼痛不適感是常見的抱怨與就醫的理由。考慮同以缺血為主要疾病本質的疾病，如周邊動脈血管阻塞疾病，之前也有臨床研究提及於周邊動脈血管阻塞疾病中，除了血管缺血的症狀外，約有43%-58.8%的病患也伴有肢體疼痛不適感之症狀。臨床上另一類可能導致神經缺血的疾病就是血管炎，其特點在於它侵犯血管而造成發炎性反應及壞死現象，進而導致血管阻塞及所支配器官的缺血性變化。若血管炎發生在供應神經血液的神經膜血管，導致神經因此得不到足夠的血液供應而缺血，病人就可能產生缺血性神經病變的臨床神經症狀。缺血性神經病變臨床症狀常見有肢體感覺異常，無力甚至伴隨疼痛。由其症狀來分析，動作無力意指末梢神經中的運動神經(屬於粗髓鞘神經，large-myelinated nerves)出問題，而感覺異常暗示感覺神經(屬於粗、細或無髓鞘神經，large-myelinated, small-myelinated or unmyelinated nerves)受到影響。這些缺血性神經病變的病人症狀提示我們神經缺血對末梢神經的影響可能包含了粗髓鞘神經、細髓鞘神經甚至無髓鞘神經的病變。過去對於小纖維神經是否比大纖維神於缺血傷害時更容易受到影響未有深入探討，也未將病理結果與臨床上的疼痛症狀做聯結與分析。前述提到細或無髓鞘神經病變時特別容易出現疼痛感，而病患常抱怨的難耐疼痛感這個現象，令我們開始去懷疑神經缺血與疼痛感之關連性、且這疼痛是否是因為傳遞疼痛感的痛覺神經本身受損所產生的呢？因此，由血管炎此疾病本質上可能令神經產生缺血的特質配合上病人臨床出現的疼痛及其他神經相關的症狀，啟發了我們想以血管炎合併末梢神經病變的病人進一步探究血管炎與缺血性神經病變及神經痛之產生的關聯性。目前對缺血與神經痛是否產生及背後相關機制尚不清楚；特別是兩個議題至今顯少有人深入探討，那就是神經缺血的程度與神經痛產生之關連性，及皮膚內小纖維感覺神經對缺血的耐受性。一些臨床上或實驗室的電生理研究關於神經類型對缺血傷害耐受性之探討，發現結果差異很大；從以感覺神經影響為主，到以運動神經影響為主的報告都有。神經受損後，可能在周邊或中樞神經的神經纖維與細胞本體出現型態或功能上的改變，這些包含有位於初級傳入神經元(primary afferent neuron)鈉離子通道的變化、與強化痛覺有關的神經傳導物質(例如substance P, N-methyl-D-aspartate (NMDA)與galanin等)及其受器(receptor)的合成增加，以及減弱痛覺有關的神經傳導物質及其受器的合成(例如opioid receptor等)受到抑制，而相似的變化也可見於脊髓背角及背根神經結內的感覺神經元細胞中，且被認為與神經對疼痛刺激過度敏感，痛覺反應異常強烈的現象有關。愈來愈多的研究指出，神經受損後不單是神經纖維與細胞出現型態或功能上的改變，連過去被認為只是負責提供營養及修復功能的神經膠細胞，如星狀細胞(astrocytes)與微膠細胞(microglia)，巨嗜細胞與嗜中性球等免疫發炎細胞以及一些細胞激素(例如IL-1, IL-6及TNF等))也被指出可能與神經痛的產生及持續有關。有研究指出，神經於缺血傷害後，細胞膜電位逐漸下降，進而導致神經興奮度短暫增加接著出現電流傳導障礙。至於神經電生理上出現神經興奮度短暫增加這變化，對臨床上血管炎或糖尿病合併神經病變病人初期出現的痛麻感與皮膚對感覺刺激過度敏銳這現象可能有很重要的意義。神經於受損後與痛覺傳遞有關的感覺神經元細胞出現神經長期過度興奮性，並進一步強化往中樞神經系統的痛覺傳遞迴路，則可能與神經受損後疼痛長期存在未消失有關。在缺血後因血液再灌注對血管內皮細胞的氧化性傷害伴隨神經內膜水腫、脂肪過氧化物的產生，以及神經纖維的退化也被注意到。神經傳導檢查是臨床上最常用來評估神經功能的檢查。然而神經傳導檢查僅能評估粗髓鞘神經的功能，無法檢測細或無髓鞘神經(簡稱小纖維神經)之功能是否有問題。感覺定量測試(quantitative sensory test)雖包含檢測小纖維神經的功能是否正常，但其受限於受試者的配合度，因此感覺定量測試並非全然客觀的檢查。神經切片屬於從病理觀點客觀評估小纖維神經病變的工具。然而神經切片會造成切除部位神經的不可逆傷害，病人的施行意願多半不高，也因此常使小纖維神經病變的診斷及病因探究出現困難。近年來已有多個包含我們研究室在內的研究單位建立出另一種客觀評估小纖維神經病變的檢查方式，那就是皮膚切片。隨著免疫組織化學法(immunohistochemistry)的發展與應用，學者們可利用各種神經軸突標記，清楚的標示出表皮內神經(epidermal nerves)，也就是俗稱的游離神經末梢(free nerve endings)的存在，其中最敏感的標記為protein gene product 9.5 (簡稱 PGP 9.5)。PGP 9.5可以標示出所有種類的神經纖維，尤其是清楚標示出其它神經軸突標記不易染出的無髓鞘神經(unmyelinated nerves)，而這類神經正是我們感興趣、與疼痛相關的神經。利用免疫細胞化學染色的方法研究皮膚上的這些小纖維感覺神經並加以定性及定量，便得以客觀檢視表皮內的小纖維感覺神經是否有退化與數量減少。這對小纖維神經病變之診斷無疑提供了具體又客觀的證據。除了前述可供檢視皮膚內神經狀態外，皮膚切片也可檢視存在於表皮層中的小血管是否有血管病變或其他異常之訊息，這對臨床上懷疑血管炎的病人等於提供了除了進行內臟器官切片以外另一個評估血管狀態的途徑。具備表皮內神經部分去神經化但又不至於嚴重到完全去神經化的局部神經傷害模式對建立一個神經痛模式是其中一項重要的考量。當神經受損後，感覺神經位於皮膚的末梢端與位於坐骨神經的近端是否有不同的變化，以及可否呼應神經痛之行為表現一直是討論的重點。一個具神經痛行為表現且可提供感覺神經皮膚末梢端客觀評量並且加以定量的動物模式對此問題的深入探討也就愈顯重要。以目前大部份的缺血動物模式來看，要符合可模擬局部缺血性神經傷害且具有高重現性又接近實際病生理狀況這些要求，現有的缺血動物模式都令人不甚滿意。常見的缺血動物模式主要是利用綁住血管、栓塞微小血管、或對血管進行壓迫阻斷血流方式來製造缺血效應，這些方式包括:綁住或切斷脊髓或坐骨神經，以雷射照射或以光化學反應搭配光感物質使用使血管產生栓塞，將主要供應後肢的血管進行栓塞，以止血帶捆綁營造肢體缺血，或是利用人造微栓子進行阻斷血流等等。這當中有些模式的方式存在著一些問題，例如因綑綁作用所帶來對坐骨神經的直接機械性壓迫效應，或雷射直接照射效應對神經的傷害，都可能干擾判讀缺血傷害對神經的影響。若要避開這些干擾, 將動脈綁住使其供應的神經產生缺血似乎是較單純且接近病生理的狀態。然而過去以將動脈綁住方式的動物模式大多是在體型較大的動物身上以方便進行，僅有少數報告曾於小鼠身上進行綁住動脈手術阻斷血液製造神經缺血狀態，且這些模式報告不是將重點放在探討關於神經痛的議題。考量到目前已有許多模擬人類疾病的基因轉殖鼠可供使用，若能直接於小鼠上建立一套手術方式容易進行且具備神經痛產生之缺血性神經病變之動物模式，將對未來探討缺血與神經痛之相互關係與病生理機制有莫大助益。我們的研究計畫目的是要探討︰1. 缺血傷害對末梢神經的影響及神經類別對缺血之耐受性有無差異 2. 神經痛之產生與缺血傷害之關聯性。我們將計畫分成兩個階段︰第一階段進行臨床病人的觀察與研究：就臨床上以缺血為主要疾病特質來考慮, 我們選擇血管炎同時合併末梢神經症狀的病人為研究對象，藉由觀察其臨床表現及實驗室檢查結果，分析探討兩個主要議題︰(1) 血管炎病人神經痛之類型及其皮膚內神經的病理變化及特徵 (2) 血管炎病人皮膚內血管發炎的狀況。第二階段設計研發一個模擬臨床神經缺血合併有神經痛表現的動物模式:基於將來方便適用於基因轉殖鼠之考量，我們欲在小鼠身上建立一個操作簡單且結果重現性高的缺血性神經病變之動物模式，並利用此模式來探究神經缺血是否會引起神經痛。利用簡單易行的股動脈綁住手術搭配血液再灌注，然後從疼痛行為觀察及測試，電生理評估, 病理學分析等方式多面向探討缺血傷害對末梢神經的影響，及不同類別之神經纖維對缺血耐受度之差異。並利用此缺血性神經病變之動物模式測試神經痛藥物對神經痛行為的改善效果。我們的假說為缺血的程度會影響神經痛的嚴重度及神經痛的類型，此與掌管痛覺的神經受損有關；經由檢查皮膚內的感覺神經末梢狀態評估小纖維感覺神經對此缺血傷害之耐受度與神經痛行為之關聯性，進一步測試神經痛藥物對神經痛之改善效果。第一階段進行臨床觀察與研究血管炎併末梢神經病變與皮膚小纖維神經退化。研究所選取的病患需同時符合下述兩項條件：(1)臨床表現為急性或亞急性多發性神經病變(2)具有符合血管炎認定的神經切片病理檢查結果。所有收案的病人都經過詳細的神經學評估及一系列的實驗室檢查以排除代謝性或感染性疾病相關的末梢神經病變，並於病患單側小腿外側位置臨床上無明顯血管炎病灶處進行皮膚切片，然後以免疫組織化學分析皮膚中的浸潤細胞類型，包括標記巨嗜細胞的CD68，標記T細胞的CD3，以及標記B細胞的CD20。表皮內神經支配狀態的定量分析則是以計算表皮層中PGP 9.5陽性反應的神經的數量算出表皮神經密度(epidermal nerve density)。結果顯示，所有病人都有急性或亞急性表現的肢體無力及感覺異常。大多數的病人都有疼痛的抱怨(六個病人當中有五個)。有三位病人的脊髓液蛋白質呈現異常增高。神經傳導檢查結果為軸突型多發性神經病變。經過免疫相關方式治療後(類固醇, 血漿置換或cyclophosphamide)，所有病人在肢體無力都有顯著進步，且神經痛也都有減輕。血管炎的病理檢查結果方面，在腓腸神經及皮膚的真皮層血管可見到T細胞及巨嗜細胞標記為主的發炎細胞圍繞血管浸潤的現象。這些血管炎病人的表皮內神經數量明顯的減少，甚至有些病人的皮膚表皮層為完全的去神經變化(completely denervated)。所有血管炎病人的表皮神經密度與正常人相比都有顯著的下降(0-3.18纖維/公釐)。此外，真皮層內的神經亦呈現神經退化的型態表現；有些病人的皮膚切片中甚至可見環繞汗腺的神經也有神經退化現象。第二階段進行研發一個模擬臨床神經缺血合併有神經痛表現的動物模式，即小鼠缺血-再灌流之動物模式，將單側股動脈綁住搭配血液再灌流，然後以雷射都卜勒血流儀評估供應脛神經之神經表層血管之血流狀態。行為測試包括了機械刺激引起之感覺異常冷刺激引起之感覺異常之定量分析。神經的電生理評估則是進行坐骨神經的運動功能檢查。腳底足墊(後腳的第一對足墊)的免疫組織化學染色是以 PGP 9.5標示出有髓鞘與無髓鞘神經，並且進行表皮神經支配的定量分析算出表皮神經密度。藥物試驗則分別於以腹腔注射生理食鹽水或溶解於無菌水的gabapentin。結果顯示，在股動脈綁住後，手術側的神經外膜的血管血流(血液流速:術後流速/術前基礎值)在術後10分鐘內明顯降到術前的40%以下(流速比值flow ratio 37.5% ± 9.2%, p = 0.002)且神經外膜的血管血流減少狀況在動脈綁住的過程中持續存在，直到鬆開綁線才恢復；鬆開綁線後1小時內，原本減少的血流即回升到綁線前的流速。缺血傷害導致小鼠產生明顯的神經痛相關之行為。這些行為改變及異常的走路型態在術後4到14天之間最明顯，然後約於術後21天開始減輕。股動脈綁住經3、4、5或6個小時不等的缺血時間後在術後1週可產生機械刺激引起之感覺異常。在缺血5小時的這組，於術後第4到第56天可見手術側的機械刺激縮腳閥值相較於對照側有顯著降低的現象(POD 4：0.04 ± 0.06 vs. 0.1 ± 0.03 g，p < 0.001；POD 56：0.01 ± 0.008 vs. 0.06 ± 0.03 g，p = 0.017)。小鼠經股動脈綁住缺血5小時後也產生丙酮刺激引起之感覺異常。在術後第1到第56天相較於對照側來看，手術側對冷刺激()的反應相較於對照側來看顯著更為強烈(POD 1: 2.81 ± 0.97 vs. 1.33 ± 0.47，p < 0.001; POD 56: 2.57 ± 0.53 vs. 1.71 ± 0.7, p = 0.003)。在術後第7天，缺血效應導致手術側的複合肌肉動作電位的振幅較對照側明顯下降且存在劑量效應(p = 0.009 on ANOVA and post-hoc analysis)。在缺血5小時的這組，手術側其複合肌肉動作電位振幅下降的現象持續到術後第56天且未見改善(3.38 ± 1.83 mV on POD 7 vs. 4.00 ± 1.68 mV on POD 56, p = 0.559)。缺血傷害於術後第7天可見相較於對照側來說，手術側的表皮神經密度顯著減少。真皮層內的神經也可見明顯的退化。此表皮去神經化之效應與缺血時間長短也有關係，缺血時間較長的相較於缺血時間較短的組別表皮神經密度明顯更低 (p = 0.027 on ANOVA and post-hoc analysis)。此外，比較缺血5小時的這組術後第7天的神經密度8.34 ± 1.62 fibers/mm及術後第56天的神經密度7.89 ± 1.44 fibers/mm 兩者並無顯著差異，這代表了皮膚去神經化的現象直至術後第56天仍未見改善。若比較術後第56天複合肌肉動作電位的振幅與表皮神經密度兩者在手術側/對照側之比值，發現表皮神經密度之比值(27.5% ± 6.1%)，顯著低於複合肌肉動作電位振幅之比值(50.5% ± 24.0%) (p = 0.026)，此說明了缺血對小纖維神經的傷害比對大纖維神經來得嚴重。機械刺激引起之感覺異常及丙酮刺激引起之冷感覺異常在給予gabapentin後(劑量各為50 與 100 mg/kg)30分鐘到3小時之間明顯的被改善，效果最顯著是在給藥後1小時且有劑量效應。從第一階段關於血管炎合併末梢神經病變的臨床研究中，我們得到了兩個重要的結果以評估及了解末梢神經病變與血管炎的關係：1.神經痛是這些病人常見的感覺症狀；除了過去認為易受缺血傷害影響的大纖維神經外，小纖維神經於這些血管炎的病人也同樣出現受損現象。2.即使在臨床上皮膚外觀無明血管炎病灶處仍可存在明顯的血管發炎病理變化。我們研究的這些血管炎病人的表皮神經密度與正常人相比都有顯著的下降，這證據客觀說明了於血管炎併神經變的病人皮膚內的感覺神經末梢存在著退化現象，而這點正呼應了臨床上這些病人的神經痛及感覺不適之症狀。我們的研究結果清楚的證實在血管炎存在下皮膚內小纖維感覺神經末梢退化受損的現象。研究的第二階段進行了模擬缺血性神經病變的動物實驗。在此我們提出了一個新的小鼠模式：以股動脈綁住及再灌流產生缺血性神經病變。由於股動脈綁住處直接產生的血管物理性傷害距離坐骨神經還有一段距離，因此這模式避開了可能對坐骨神經直接產生物理壓性傷害的疑慮。這套缺血及再灌流系統重現了兩件臨床上重要的現象：神經痛與神經退化。其中神經痛的部分又包含了於術後7天出現且持續到術後56天的自發性神經痛相關行為變化及經刺激誘發之神經痛相關行為(包含機械刺激引起之感覺異常及丙酮引起冷刺激之感覺異常)，且這些神經痛相關行為可經由給予抗神經痛藥物獲得改善。本研究之主要貢獻在於：我們發展了一套簡單且結果重現性高的新的缺血性神經病變小鼠模式以模擬臨床上的缺血性神經病變與神經痛。此局部缺血性神經傷害系統包含了：1.在病理及電生理上提供關於大纖維神經及小纖維神經受損神經退化的證據 2.神經痛的產生，此包含了自發性神經痛相關行為、以及可被抗神經痛藥物減輕的機械與冷刺激誘發之感覺異常。此動物模式可供我們經由多面向探究缺血性神經傷害之程度與神經痛之產生彼此間的關聯性，而評估的方式可由行為觀察、電生理檢測、以及病理學研究多種客觀方式進行。由於我們的模式是建立在小鼠，具有可利用基因轉殖鼠進一步探究缺血性神經病變神經受損與神經痛之產生彼此間的關聯性及致病機轉，並以之測試欲研究的分子在其中扮演的角色及相關的藥物研發之優勢。我們期待新建立的缺血性神經病變小鼠模式能為缺血性神經病變及相關疼痛的病生理研究帶來曙光。

關鍵字

缺血性神經病變；缺血及再灌流；神經痛；小纖維神經病變；皮膚神經退化

並列摘要

Introduction Ischemia has been reported to be important in the pathogenesis of human neuropathies caused by acute peripheral arterial occlusion and in neuropathies caused by vasculitis. It has also been implicated in metabolic disorders, e.g. diabetes mellitus and in disseminated neuropathy due to prolonged coma (critical illness polyneuropathy). The main clinical presentation of ischemic neuropathy is a mononeuropathy simplex or multiplex with asymmetric pain, sensory symptoms and motor deficits. Several clinical studies have confirmed the association between morphological and functional impairments of peripheral nerves in peripheral vascular diseases. Among the neurological deficits in these patients with peripheral vascular insufficiency or vasculopathy, neuropathic pain of variable incidence (43%-58.8%) is frequently mentioned, depending on the severity of the diseases. Skin denervation in vasculitic neuropathy has rarely been documented despite frequent manifestations of small-fiber neuropathy including reduced sensitivity and neuropathic pain. Recently, skin biopsy has been established as a new approach to diagnose small fiber sensory neuropathy. Small-diameter sensory nerves innervating the skin are responsible for conveying noxious and thermal stimuli, and damage to these nerves is presumably related to neuropathic pain behaviors. However, the mechanisms of ischemia-induced neuropathic pain are largely not clear at present. Specifically, two issues are seldom mentioned: the relationship between the extent of ischemia and the occurrence of neuropathic pain, and the susceptibility of small-diameter sensory nerves in the skin to ischemia. Neurophysiological studies of the preferential involvement of nerve types in ischemia show wide discrepancies in several clinical investigations, ranging from predominantly sensory neuropathy to mainly involvement of motor nerves. After ischemia, there is a progressive reduction in membrane potential resulting in a transient increase in excitability followed by a failure of impulse conduction. This increased excitability may be important for the clinical symptoms of hyperesthesia, pain and numbness in patients presenting with vasculitis or diabetic neuropathy in early stages. Nerve injury elicits regenerative growth, which occurs in most peripheral neurons, and long-term hyperexcitability that appears selectively in nociceptive sensory neurons; the long-term hyperexcitability enhances inputs to nociceptive circuits in the CNS and can contribute to chronic pain after nerve injury. How information from the site of a distal lesion is communicated to the cell nucleus is not completely understood, but positive injury signals, i.e. axonal proteins that are activated at the site of the injury and retrogradely transported to the cell soma, are thought to have an import role. Several studies indicate that protein kinase G activated in axons is a limited signal that is responsive for the induction of long-term hyperexcitability. The pathological changes of ischemic neuropathy are well described as axonal degeneration and regeneration, endoneurial edema, abnormally thin in myelinated fibers and endothelial swelling of endoneurial microvessels. The energy requirements is lower in peripheral nerves, which indicate that peripheral nerve has a greater metabolic safety factor than the central nervous system and there is a much wider window of time for therapeutic intervention in peripheral nerve than in the brain. Although restoration of blood flow to the ischemic nerve is essential to prevent irreversible damage, reperfusion can result in oxidative injury to endothelial cells with ensuing endoneurial edema, lipid peroxidation and fiber degeneration. There are various studies of neuroprotective approaches using the aforementioned models including hypothermia, hyperbaric oxygenation, and the antioxidant α-lipoic acid. However, further studies are needed to investigate the mechanisms of the treatments. Neuropathic pain results in a series of complex but coordinated behaviors mediated by damage to large myelinated, small myelinated, and unmyelinated nerve fibers. Several groups including ours have demonstrated the rich innervation of the skin by immunohistochemistry with various neuronal markers, particularly, protein gene product (PGP) 9.5. PGP 9.5 is an ubiquitin carboxyhydrolase and probably functions as an immediate early gene for processing sensory information in the neurons. Our previous study on chronic constriction injury and laser-induced focal neuropathy indicate that PGP 9.5(+) nerve terminals in the skin were moderately depleted compared with those in completely denervated skin. These findings suggest that partial injury is another important principle for creating experimental models of neuropathic pain. Apparently, large-diameter and small-diameter nerve fibers are differentially vulnerable in models of neuropathic pain. Whether terminals of sensory nerves in the skin retain the same patterns as they have at the sciatic nerve level is an open issue. Thus, it was intriguing to investigate whether there is a relationship between nerve injury to fibers of different categories and the magnitude of neuropathic pain. A critical issue in ischemia-induced neuropathic pain studies is to develop an experimental system with quantifiable degree of injury. Most of the currently available ischemic animal models tend to be less than satisfactory in their ability to simulate focal ischemic nerve damage in a reproducible and physiologically relevant manner. Models based on ligation, microembolisation or compression have been developed by Adams (1943) and Roberts (1948) and improved by Korthals and Wisniewski (1975). Nukada and Dyck (1984) obtained dose-related effects using polystyrene microspheres. Sladky (1991) and coworkers produced chronic regional nerve ischemia in rats by creating proximal arteriovenous shunts. For the ischemic models induced by arterial ligation, most studies were performed in the rats or rabbits; seldom experiments were performed in mice and did not specify whether neuropathic pain developed. There are many mice with many genetic models mimicking human disease and an ischemic neuropathy model in mice will shed light on the pathophysiology of ischemia-induced neuropathic pain. The purposes of this study were (1) to investigate the pathologic features of cutaneous nerves in ischemic neuropathy, (2) to generate a mouse system of ischemic neuropathy with neuropathic pain behaviors and quantifiable degree of nerve injury by ligating the femoral artery with reperfusion, (3) to investigate the influence of ischemia on nerve fibers of different categories and the magnitude of neuropathic pain behaviors using multidisciplinary approaches, (4) to evaluate the susceptibility of small-diameter sensory nerves in the skin to ischemic injury by examining the skin innervation, and (5) to evaluate effects of ant anti-nociception treatment on allodynia in ischemic nerve injury. We hypothesize that the degree of ischemia has influence on categories and the magnitude of neuropathic pain. Material and Method I. Skin denervation in vasculitis In the study of skin denervation in vasculitis, clinical data of patients were retrieved from the database of hospitalized patients at National Taiwan University Hospital, Taipei, from January 1, 2000, to December 31, 2003. All patients fulfilled 2 criteria: (1) clinical presentation of a mononeuropathy multiplex of acute or subacute onset and (2) pathological evidence of vasculitis on nerve biopsy specimens following the consensus criteria. All underwent detailed neurologic examinations and laboratory investigations to exclude metabolic and infectious diseases that affect the peripheral nerves, including plasma glucose level, and functional tests of the liver and kidneys. Specific autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, were excluded by the following relevant laboratory tests: antinuclear antibody, antibody against double-stranded DNA, anti–Sjögren syndrome A antigen, anti-–Sjögren syndrome B antigen, anti–Smith antigen, anti–scleroderma antigen (SCL-70), anti-RNP, rheumatoid factor, C3 and C4 complement levels, cryoglobulin, hepatitis profiles (hepatitis B surface antigen, anti–hepatitis B surface antibodies, and anti–hepatitis C virus antibodies), syphilis (VDRL/rapid plasma reagent test), anti–human immunodeficiency virus antibodies, lead levels in serum, immunoelectrophoresis of proteins in the serum and cerebrospinal fluid, and tumor markers (α-fetoprotein, carcinoembryonic antigen, CA125, and CA19-9). Patients had 3-mm punch biopsy specimens taken from the distal part of the leg (without active vasculitic lesions) and a sural nerve biopsy specimen was taken in addition to detailed neurologic examinations, laboratory investigations, and nerve conduction studies. Results of nerve conduction studies, epidermal nerve fiber density studies, and immunohistochemistry were analized. II. Patterns of nerve injury and neuropathic pain in ischemic neuropathy after ligation-reperfusion of femoral artery in mice Effect of femoral arterial ligation on epineurial arteries and blood flow In the study of patterns of nerve injury and neuropathic pain in ischemic neuropathy after ligation-reperfusion of femoral artery in mice, the femoral artery on one side was tied in the proximal portion with a 7-0 prolene suture using a slipknot technique for rapid release, and reperfusion was achieved by the release of the ligature on adult male 8-week-old ICR mice. Epineurial blood flow of the tibial nerve was measured with a laser Doppler flow meter before and immediately after arterial ligation, and immediately after reperfusion. Behavioral testing assessed after arterial ischemia including mechanical allodynia using a set of calibrated von Frey filaments, and cold allodynia using acetone test with modifications. Electrophysiological studies for evaluation about the motor function of the sciatic nerve after ischemia was performed by measuring the compound muscle action potential (CMAP). Immunohistochemistry of the footpads with an antibody to protein gene product (PGP) 9.5 and quantitation of epidermal innervation expressed as the epidermal nerve density (END) were performed. Pharmacological studies using gabapentin was performed for assessment about the neuropathic pain behaviors. Experimental designs and statistical analyses In the first phase of the experiment, we investigated the duration of the ischemic effect of inducing neuropathic pain. The criterion of successful induction was the presence of sustained mechanical allodynia in the interval between postoperative day (POD) 7 and 14. We studied the effects of different ischemic durations on the development of neuropathic pain behaviors. Mice were randomly divided into four groups and respectively received 3, 4, 5, and 6 h of ischemia, followed by reperfusion. Neuropathic pain was determined by the presence of sustained mechanical allodynia. There were six mice at each time point for each laboratory procedure. Behavioral and laboratory data are presented as the mean ± SEM at different time points after ischemia. For the statistical analysis of values obtained from nerve-conduction studies and ENDs over the experimental period, analysis of variance (ANOVA) and a post-hoc analysis were used. Differences in the CMAP amplitude and END between the control and operated sides were tested using a paired Student's t-test. Any difference with p < 0.05 was considered statistically significant. Results I. Skin denervation in vasculitis Clinical presentations and laboratory examinations All patients had motor and sensory impairments with acute or subacute onset. Motor and sensory symptoms usually began focally or asymmetrically in one leg or thigh and then progressed to affect the contralateral side and the upper limbs. Three patients had simultaneous symptoms in the upper and lower limbs as the initial manifestation. Pain was noted in most patients (5 of 6). Elevated protein levels in the cerebrospinal fluid were noted in 3 patients. Nerve conduction studies showed a pattern of axonal neuropathy or mononeuropathy multiplex. After immunotherapy with corticosteroids, plasma exchange, or cyclophosphamide, there were significant improvements in muscle strength (at least 1 grade in the weakest limb according to the grading of the Medical Research Council) and reduction of neuropathic pain in all patients. Pathological demonstration of vasculitis Inflammatory cells surrounding vessels were demonstrated in the sural nerves and dermal vessels of the skin, and cells were immunoreactive for markers of macrophages (CD68) and T cells (CD3), but not for B cells (CD20). In patients with vasculitis, the quantity of epidermal nerves was markedly reduced. The epidermis of some patients had even become completely denervated. We quantified epidermal innervation by measuring the epidermal nerve density. All patients had significantly reduced epidermal nerve fiber density (0-3.18 fibers per millimeter) compared with the aforementioned normative values from our laboratory. In addition, dermal nerves showed a pattern of degeneration; sweat glands became denervated in some patients. II. Patterns of nerve injury and neuropathic pain in ischemic neuropathy after ligation-reperfusion of femoral artery in mice Effect of femoral arterial ligation on epineurial arteries and blood flow After femoral arterial ligation, the blood flow on the operated side (flow velocity of post-operated/baseline level) was significantly reduced to < 40% of the baseline value (flow ratio 37.5% ± 9.2%, p = 0.002) within 10 min. The decrease in epineurial blood flow persisted through the entire period of arterial ligation. After reperfusion, the blood flow returned to the pre-ligation level within 1 h after releasing the ligature. Behavioral observations after arterial ligation with subsequent reperfusion Ischemic injury resulted in distinct neuropathic behaviors in the mice, showing significant changes in behaviors, gait, and stance, showing guarding of the affected paw from touching the floor which was most pronounced during PODs 4~14, and these gradually decreased after POD 21. In the first week after the operation, mice showed spontaneous pain behaviors, for example, elevating the hind paw with shaking movements, particularly when walking on uneven surfaces. When walking, the mice always elevated the hind paw of the affected side to avoid touching the floor with the footpads. Effects of the ischemic duration on neuropathic pain behaviors: mechanical allodynia and acetone-induced cold allodynia Femoral arterial ligation could induce mechanical allodynia during the first postoperative week after various ischemic duration of 3, 4, 5, or 6 h. In the 6-h ischemic group, there was a transient increase in the mechanical threshold on the operated side compared to that on the contralateral side from PODs 1 to 2 (POD 1: 0.31 ± 0.14 vs. 0.13 ± 0.06 g, p = 0.039; POD 2: 0.29 ± 0.18 vs. 0.14 ± 0.02 g, p = 0.037, Fig. 2). In the 5-h ischemic group, the withdrawal thresholds to mechanical stimuli on the operated sides were significantly reduced compared to those on the control sides from PODs 4 to 56 (POD 4: 0.04 ± 0.06 vs. 0.1 ± 0.03 g, p < 0.001; POD 56: 0.01 ± 0.008 vs. 0.06 ± 0.03 g, p = 0.017, Fig. 3). In preliminary studies, a shorter ischemic duration (3 h) produced less-sustained mechanical allodynia lasting for < 2 weeks, compared to 4 weeks with 4-h ischemic injury, and > 2 months with 5- and 6-h ischemia. The presence of longer initial hypoesthesia in the 6-h ischemic group made it less suitable for assessing acute neuropathic pain behaviors after ischemic injury, compared to the 5-h ischemic group. Therefore we determined that femoral arterial ligation for 5 h was an optimal ischemic duration and used this setting to induce neuropathic pain in animals in subsequent studies. Mice with ischemic neuropathy after 5-h ligation of femoral artery also showed significant acetone-induced allodynia. The response to cold stimuli (application of acetone to the plantar skin) on the operated side became accentuated compared with that on the control side from PODs 1 to 56 (POD 1: 2.81 ± 0.97 vs. 1.33 ± 0.47, p < 0.001; POD 56: 2.57 ± 0.53 vs. 1.71 ± 0.7, p = 0.003). Changes in nerve conduction studies after femoral arterial ligation with reperfusion On POD 7, ischemia for 3, 4, 5, or 6 h resulted in significant reductions in CMAP amplitudes on the operated side compared to that on the control side, providing evidence of large-fiber deficits. There was a dose effect of ischemic duration, i.e., the longer the ischemic duration, the smaller the CMAP amplitude was (p = 0.009 on ANOVA and post-hoc analysis). In the 5-h ischemic group, the reduction in CMAP amplitudes on the operated side persisted through POD 56 without improvement (3.38 ± 1.83 mV on POD 7 vs. 4.00 ± 1.68 mV on POD 56, p = 0.559). Skin innervation in the vicinity of the sciatic nerve after femoral-arterial ligation On POD 7, the abundance of epidermal nerves was significantly reduced as validated by lower ENDs on the operated side than that on the control side, indicating the effect of ischemia on small-diameter sensory nerve terminals in the skin. The effect of skin denervation was related to the ischemic duration, and a longer ischemic duration caused much-lower ENDs compared to those with a shorter ischemic duration (p = 0.027 on ANOVA and post-hoc analysis), for example, 6.67 ± 1.65 fibers/mm for the 6-h ischemic group vs. 13.52 ± 3.55 fibers/mm for the 3-h ischemic group, p = 0.032). In the 5-h ischemic group, the skin remained denervated on POD 56 with no sign of recovery in skin innervation (8.34 ± 1.62 fibers/mm on POD 7 vs. 7.89 ± 1.44 fibers/mm on POD 56, p = 0.84). Vulnerability of large and small fibers to ischemic injury The aforementioned results indicated there was damage to both large- and small-diameter nerve fibers after ischemic injury as quantified by reductions in CMAP amplitudes and ENDs, respectively. The damage to small fibers was more robust than that to large fibers as determined by comparing the ratios (operated/control sides) betweens ENDs and CMAP amplitudes on POD 56 (27.5% ± 6.1% vs. 50.5% ± 24.0%, respectively, p = 0.026). Effects of an intraperitoneal injection of gabapentin on allodynia with ischemic nerve injury To understand whether the above behaviors could be alleviated by pharmacological treatment, we administrated gabapentin (25, 50, and 100 mg/kg) intraperitoneally on POD 7. Mechanical allodynia was significantly reduced by the use of gabapentin (50 and 100 mg/kg) between 0.5 and 3 h after the intraperitoneal injection, with a maximal effect at 1 h. Gabapentin (50 and 100 mg/kg, ip) also significantly reduced cold allodynia at 0.5~3 h with a maximal effect at 1 h. Gabapentin alleviated mechanical allodynia in a dose-dependent manner. Gabapentin at 25 mg/kg did not significantly affect the neuropathic pain behaviors. Discussion The main contribution of this study included revealing small-diameter sensory nerves are affected in vasculitis in addition to the well-known effect of vasculitis on large-diameter nerves; the denervation of cutaneous small-fiber may correlate to the neuropathic pain. Significant inflammatory vasculopathy is present in the skin despite the absence of clinically active vasculitic lesions. We generate a new mouse system of ischemic neuropathy after ligation-reperfusion of the femoral artery. Direct vascular injury of the femoral artery was remote from the sciatic nerve and thus eliminated any concerns about direct mechanical compressive injury of the sciatic nerve. This system of ischemia-reperfusion injury replicated two major clinical manifestations: neuropathic pain and nerve degeneration. For neuropathic pain, these included spontaneous pain behaviors and evoked pain behaviors (mechanical allodynia and cold allodynia) which developed within 1 week after nerve injury and persisted through POD 56 of the experimental period. The neuropathic pain can be alleviated by gabapentin. The reduction of ENDs in the current model provides an index to assess the ischemic effect on small-diameter sensory nerve terminals in the skin, and this model replicates what was observed in human vasculitic neuropathy. This system produced by a simple procedure provides an opportunity to investigate mechanisms and further treatments of ischemic neuropathy on genetically engineered mice.

並列關鍵字

ischemic neuropathy ； neuropathic pain ； ischemia-reperfusion ； skin denervation ； skin biopsy

參考文獻

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國際替代計量

神經痛與神經受損於缺血性神經病變之研究

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