大鼠三叉神經節及視丘神經元活性於下牙槽神經性疼痛發展與持續時期之變化

Abstract

神經性疼痛屬於慢性疼痛，許多人都深受其擾，加上神經性疼痛的致病機制仍不清楚，難以對症下藥而成為臨床上的重要議題。神經性疼痛的病徵包括自發性疼痛(spontaneous pain)、異覺痛(allodynia)與痛覺敏感(hyperalgesia)。下牙槽神經為下顎骨神經(三叉神經的第三分支)的分支。切斷下牙槽神經會造成該神經調控範圍外的大鼠鬍鬚區域(由三叉神經第二分支所調控, 簡稱V2)對輕觸覺刺激敏感(亦稱為跨範圍異覺痛, extraterritorial allodynia)，並持續一個月之久。該動物模式因類似神經性疼痛病患的病徵而成為三叉神經性疼痛的動物模式之一。研究發現，下牙槽神經切斷後，大鼠三叉神經的V2神經纖維與神經節(trigeminal ganglion)過度敏感，其傳入的三叉神經脊髓幹核之尾核(trigeminal subnucleus caudalis)中未受傷的V2神經元亦有過敏感化的現象。但直至目前為止，造成這些區域過敏感化的原因仍不清楚。根據文獻，一個可能的來源為受傷的下牙槽三叉神經節所產生的異位性放電(ectopic discharges)，另一個則可能來自視丘的過度興奮。因此，本研究將重心放在三叉神經節與視丘中接受臉部感覺訊息的腹後內側核區(ventroposterior medial nucleus, VPM)，並利用新研發之電極來長期記錄這兩個深腦核區的神經元活性，以比較在下牙槽神經切斷後，周邊神經與中樞神經的神經元活性變化。直至目前為止，這是第一篇觀察清醒動物的神經元活性於神經受損後變化的研究。 論文中的第一部分為電極研發。其中描述了類束狀微陣列電極的製程與埋設手術，以及功能性測試的結果。該電極重要的特色包括: (1) 以鎢微絲作為電線材料，突出導管尖端的鎢微絲用鑷子撥散呈扇形，就像微陣列電極的排列一樣，降低目標核區的傷害程度。鎢微絲的突出長度保持在1.5 mm，以維持硬度，有效突破大腦的硬腦膜與包覆三叉神經節的硬膜。(2) 使用不銹鋼針管，能輔助穿刺並利於接地，降低雜訊。(3) 參照金屬微絲位於記錄微絲附近，能有效去除局部雜訊。(4) 轉接頭垂直於導管，降低電極整體高度，以利特殊的動物行為測試，如老鼠鬍鬚區域的觸覺刺激。進行該項行為測試時，老鼠須鑽入前方有小洞的方盒中，並於洞口露出口鼻處以進行刺激。因此，必須將電極高度降低，才能讓老鼠順利進入小方盒中。透過連續的神經元活性紀錄，我們發現，類束狀微陣列多通道電極能長期並穩定記錄清醒動物的三叉神經節與視丘VPM的單一神經元活性長達80天。 論文中的第二部分描述三叉神經節與視丘VPM於下牙槽神經受損後的神經元活性長期變化時程，並將這些變化對應至大鼠鬍鬚區域痛覺行為(nocifensive behavior)的進程。利用一系列不同粗細的細絲(von Frey filament)於大鼠鬍鬚區域進行機械性刺激，測試產生縮頭反應的閾值克數。與前人研究相同，我們發現在下牙槽神經切斷後，大鼠的鬍鬚區域的閾值克數降低。與控制組比較後，此痛覺行為有三個階段的變化:早期發展階段、晚期維持階段與恢復階段。下牙槽神經受損後，我們發現 (1) 清醒時的自發性神經元活性上升由受傷的下牙槽神經先開始(第二個小時至第三十天)，而後是未受傷的V2三叉神經元(第六個小時至第三十天)，最後是視丘VPM V2神經元(第七至第三十天); (2)三叉神經節神經元不論是否來自受損的神經輸入，都有特殊的放電模式，如猛爆型放電(burst firing)、規則型放電(regular firing); (3) 在維持階段時，視丘VPM V2神經元的接受皮膚感覺區(receptive field)有擴大的現象，而且其感覺模式(modality)亦發生轉換 (modality shift)。這些VPM V2神經元原本只對輕觸覺刺激有反應，在神經受損後開始對疼痛刺激有反應; (4) 視丘VPM V2神經元在接受輕觸覺與痛覺刺激後，仍會繼續放電 (誘發後放電,evoked after-discharge); (5)在維持階段時，麻醉會引起三叉神經節 V2神經元的自發性神經元活性上升(與清醒時相比)。上述這些變化在晚期階段皆能發現到，並在最後恢復時期(第四十天至六十天)消失。從這些觀察，我們推測受傷的下牙槽神經所產生的異位性放電 (特別是猛爆型與規則型)可能會造成未受傷的三叉神經節V2 神經元敏感化，亦產生異位性放電與觸覺刺激誘發後放電，進而造成視丘V2的神經元活性過度興奮。視丘V2的神經元的感覺模式轉換與刺激誘發後放電等現象，顯示這些表型改變(phenotype switch)的神經元可能會於皮膚輕觸覺刺激後，因本身過度興奮而挾帶痛覺訊息傳至大腦皮質，引發觸覺疼痛。另外，晚期維持階段時，麻醉引起的三叉神經節V2神經元的自發性神經元活性上升，暗示當病理進程已發展完全後，很難透過麻醉止痛等藥物施予來緩解病徵。 綜合上述結果，我們推論在下牙槽神經受損後，跨範圍異覺痛的早期發展須藉由周邊神經敏感化促成，而在進入晚期時，則是中樞(視丘)與周邊神經敏感化共同作用而維持的。

Neuropathic pain is an important clinical issue, in that long lasting chronic pain arises spontaneously (spontaneous pain), after light touch (allodynia) or enhanced after noxious stimulation (hyperalgesia). It affects a large population of patients and is very difficult to treat. Inferior alveolar nerve (IAN) is a branch of the mandibular trigeminal nerve. Transection of IAN (IANx) is an animal model of trigeminal neuropathic pain and produces extraterritorial allodynia in the maxillary whisker pad area (a part of the maxillary division, V2) for one month. Neuronal hyperactivities in the uninjured V2 division of trigeminal subnucleus caudalis and their afferents have been found in IANx rats, whereas much less in known about how these uninjured V2 primary and secondary neurons became excited after IANx. One possible contributor is barrages of ectopic discharges from injured trigeminal ganglion (TG), and the other is central sensitization in the thalamus. Thus, we focused on TG and ventroposterior medial nucleus (VPM) neurons, and analyzed neuronal excitability using a newly developed long-term deep brain single-unit recording method to compare peripheral and central neuronal activity change in freely moving IANx rats. This is the first study to monitor neuronal activity changes in freely moving rats after nerve injury. The first part of this thesis is about electrode development. Fabrication and implantation of a bundled microarray electrode and results of functional test were described. The salient features of the design include: (1) short and separated tungsten microwires for stable chronic recording; (2) the use of a 30-guage stainless steel guide tube for facilitating penetration and aiming for deep targets as well as electrical grounding; (3) the inclusion of a reference of the same microwire material inside the bundle to enhance common mode rejection of far field noises; and (4) an adjustable connector. A 90 degree backward bending connector was used, so that implanted rats could perform the same hole-seeking behavior and their faces and whiskers could be stimulated in the behaving state. It was demonstrated that this multi-channel electrode caused minimal tissue damage at the recording site and was able to obtain good, stable single-unit recordings from the trigeminal ganglion and ventroposterior medial thalamus areas of freely moving rats for up to 80 days. In the second part of this thesis, time course of neuronal activity changes in TG and VPM was described and correlated to extraterritorial allodynia. Mechanical head withdrawal threshold showed three phases of allodynia by comparing to sham group: early development, late maintenance and recovery phases. After IANx, we found (1) sequential increase in of spontaneous activities from injured IAN ganglion neurons (2 hour-day 30) to uninjured V2 ganglion neurons (6 hour-day 30), and then to VPM V2 neurons (day 7-30); (2) bursty and regular firing patterns in IAN and V2 branches of TG neurons; (3) expanded receptive field and modality shift in VPM V2 neurons; (4) tactile- and pinch-evoked after-discharges in VPM V2 neurons; (5) anesthesia enhanced spontaneous activity in TG V2 neurons. All these changes can be found in the late phase and disappeared during recovery phase (day 40-60). From these observations, we suggested that ectopic barrages in injured IAN, fired as bursty or regular spiking patterns, may contribute to the developing sensitization of uninjured ganglion neurons and resulted in thalamic sensitization in V2 division after IANx. Modality shift and tactile- or pinch-evoked after-discharges in VPM V2 tactile neurons indicates that nociceptive information may be transmitted to the cortex via these phenotype switched VPM V2 neurons, even by light touch. Anesthesia enhanced spontaneous activity in TG V2 during late phase implied that it is difficult to relieve excitability via anesthetic or analgesic drug while the pathological process has been developed fully. We concluded that after IANx, peripheral sensitization in injured and uninjured peripheral nerves may be involved in the development of extraterritorial allodynia. During late phase of allodynia, central sensitization and peripheral sensitization may work together to maintain pathological process and are responsible for long-lasting nocifensive behavior.