酸誘發ERK活化於皮質興奮性神經元與前端無顆粒狀島葉之興奮性突觸可塑性研究

Abstract

為了瞭解慢性肌肉疼痛的相關機制，本研究援引Sluka等人在2001年所發表的酸誘發慢性肌肉疼痛模型 (AIMP 模型) 作為研究工具，此模型可藉由在左腿腓腸肌重複注射酸性食鹽水而在雙側腳掌都誘發出長期的痛覺敏感現象。一般相信中樞敏感現象與酸誘發之慢性肌肉疼痛的機制相關，先前研究亦已指出脊髓、視丘與杏仁核等區域的角色，但大腦皮質所扮演的角色仍然不清楚，包括了到底是興奮性神經元或是抑制性神經元會在慢性肌肉疼痛的建立機制中被活化？本研究主要分為兩大部分：第一部分是型態實驗，著手於辯明在大腦皮質中在酸痛模型下活化的細胞種類；第二部分是電生理實驗，聚焦於前端無顆粒狀島葉(RAIC)的興奮性突觸可塑性之探討。 第一部分使用磷酸化細胞外信號調節激酶 (pERK) 來標定被活化的細胞，並結合雙重標定或三重標定之免疫染色方法確定其細胞種類。其中，NeuN 被用來標記神經細胞；Calmodulin kinase II （CaMKII) 被用來標定興奮性細胞；GAD-GFP 與 VGAT-tdtomato 兩品系之基因轉殖鼠被用來標定抑制性細胞。結果顯示，在重複注射第二針酸之後，pERK的活性在前扣帶皮質與島葉都有增加，且表現pERK的細胞幾乎全都是神經元而非神經膠細胞，且重要的是，pERK表現於興奮性神經元，而幾乎不表現於抑制性神經元。 第二部分使用全細胞鑲嵌技術紀錄前端無顆粒狀島葉的第五層錐狀細胞，並電刺激其第二/三層的位置來誘發出興奮性突觸後膜電位(eEPSPs)。藉由高頻電刺激的方式，可以誘導出長期增益現象(LTP)，此長期增益現象與高頻電刺激的重複次數相關，並與 NMDA 受器相關。除此之外，由於高頻電刺激的重複次數過少將不足以誘發長期增益現象，因此我們假定有誘發長期增益現象的閾值存在於前端無顆粒狀島葉。 結合以上結果，包括ERK在慢性疼痛模型當中活化於興奮性神經元，加上在正常小鼠中前端無顆粒狀島葉的興奮性神經元可以展現突觸可塑性，所以我們認為(前端無顆粒狀島葉的)興奮性神經元在慢性酸痛模型中扮演了重要角色，這也提供了關鍵點以供纖維肌痛症的相關研究。

In order to understand the mechanism of chronic muscle pain, the acid-induced muscle pain animal model (AIMP model) established by Sluka et al. (2001) was introduced. Bilateral widespread long-term hyperalgesia was reproduced by repeated acidic saline intramuscular injection into left gastrocnemius muscle. It was believed that central sensitization contributed the acid-induced muscle chronic pain and previous studies had discussed on the spinal cord or subcortical areas; however, the role of cortical areas was still unclear, including whether the nociceptive neurons was excitatory or inhibitory in the developing of chronic muscle pain. The study was interested in the cell-type of nociceptive neurons and synaptic plasticity within cortical areas, especially the rostral agranular insular cortex (RAIC). This study consisted of two main experiments, one was investigating the cell-type of nociceptive neurons in the cortical areas within AIMP model; the other was to examine whether a synaptic plasticity could be elicited in rostral granular insular cortex. First, the phosphorylated extracellular signal-related kinase (pERK), a biological activity and nociceptive marker, had been examined after the second acid injection. The pERK-IR cells were elevated in the bilateral anterior cingulate cortex (ACC) and insular cortex (IC), and combined with double labeled pERK-NeuN immunostaining, showing that the pERK-IR cells were almost all (at least 96%) neurons. Next, calmodulin kinase II (CaMKII), a general marker for pyramidal cells, were used for labeling excitatory principle neurons; in the other way, GAD-GFP mice and VGAT-tdtomato mice were also used for labeling GABAergic neurons. Triple-labeled of pERK&CaMKII&GAD (or VGAT) showed that around 40% pERK-IR cells were CaMKII-IR principle neurons, and rarely pERK-IR cells were GABAergic interneurons in the ACC and IC. Second, whole-cell patch clamp was done on the RAIC layer V pyramidal cell and evoking EPSPs by a local stimulation on RAIC layer II/III. Under high frequency stimulus with 100Hz in one second and repeated five or seven times (repeated times defined as train number ), a long-term potentiation of EPSP was elicited. The LTP on RAIC is train number-dependent and NMDA receptor-dependent. Furthermore, it was suggested that a threshold as train number of three within the RAIC for establishing LTP. In conclusion, the excitatory pyramidal cells played the critical roles in the ERK activation and the synaptic plasticity, which helped us much understand the mechanism of chronic muscle pain, and might provide a hint on the research of fibromyalgia.