摘要:甘胺酸及伽傌-胺基丁酸為抑制性調控整合中的兩個重要成員,主要以突觸前或突觸後抑制作用參與訊息的調控。雖然有關甘胺酸及伽傌-胺基丁酸在楔狀神經核的研究報告已經相當詳實,但是相關的形態學研究大都以探討神經傳遞物質為主,缺乏有關於它們相對應受體的形態學資料。一般正常狀況下,楔狀神經核僅傳遞來自初級傳入神經有關於頸部、前肢及軀幹上部的觸覺、震動覺及本體覺等非傷害性訊息,爾後將訊息向上投射至丘腦腹基底核。根據我們先前的研究顯示,正中神經慢性纏繞損傷後,損傷同側上肢呈現神經病變疼痛行為,同時在同側的楔狀神經核有神經胜肽Y (NPY)及c-Fos免疫表現(Day et al., 2001; Tsai et al., 2008),而這些在正常未損傷側都不會表現。而早先的研究有學者將c-Fos 免疫表現作為神經元活性的指標,甚至於視為參與疼痛訊息傳遞的標誌(Hunt et al., 1987; Harris, 1998)。楔狀神經核在正中神經損傷後,有可能因抑制性調控整合降低而導致其神經元興奮性提高,進而誘導c-Fos 免疫表現的產生,以致於與神經病變疼痛傳遞有所關聯。另外,先前的研究發現坐骨神經慢性纏繞或截斷損傷後,脊髓背角淺層中伽傌-胺基丁酸性神經元有減少或死亡的情形,伴隨著實驗表現出痛覺過敏及觸覺痛等的神經病變疼痛行為(Ibuki et al., 1997; Eaton et al., 1998; Moore etal., 2002; Scholz et al., 2005);而神經損傷後也會造成脊髓內甘胺酸受體、伽傌-胺基丁酸A 受體和伽傌-胺基丁酸B 受體的數量減少(Bhisitkul et al., 1990; Castro-Lopes et al.,1995)。但是另有研究報告,卻指出周邊神經損傷並不會造成抑制性神經元數量的下降(Polgar et al., 2003; Polgar and Todd, 2008)。綜合以上所述,周邊神經損傷對抑制性神經元的影響尚有爭論,而目前所知的研究僅限於探討與脊髓相關的題材,但缺乏有關楔狀神經核的抑制性系統在正中神經損傷後所產生的改變及其對神經病變疼痛產生的可能角色。因此本研究計畫的主要目的在利用正中神經截斷與慢性纏繞傷害實驗模式、結合免疫細胞化學、西方墨點轉漬法、神經追蹤劑注射、胞外電生理技術、行為測試合併藥物使用及電子顯微鏡技術等研究法,預計分三年的研究實驗期來探討楔狀神經核及背根神經節的抑制性系統在神經受損後所產生的改變及其與神經病變疼痛產生的關係。茲將各年度的研究計畫主要目的及方法分別記載如下:《第一年研究計畫》:本年度計畫首先擬以免疫細胞染色法及電子顯微鏡技術的運用,來確認具Gly-R、GABAA-R 和GABAB-R 免疫反應神經元分別在正常的楔狀神經核和背根神經節內的數量及受體形成突觸的型態。再利用神經追蹤劑標誌或雙重免疫細胞染色確認楔狀神經核內具有Gly-R 或GABAA-R 和GABAB-R 免疫反應神經元為楔狀丘腦接轉神經元、甘胺酸及伽傌-胺基丁酸性神經元的數量及比例。另外,再配合包埋後免疫膠基金確定免疫標誌受體的突觸前神經終末的性質。最後使用西方墨點轉漬法分別地進一步確認Gly-R、GABAA-R 和GABAB-R 在楔狀神經核和背根神經節內的含量。《第二年研究計畫》:本年度計畫擬以正中神經截斷損傷模式配合抗甘胺酸、伽傌-胺基丁酸等免疫細胞化學,先探討在大白鼠正中神經截斷後二及四週,楔狀神經核內甘胺酸性和伽傌-胺基丁酸性神經元數量的改變。爾後配合抗Gly-R、GABAA-R 及GABAB-R等免疫細胞化學及神經追蹤劑標誌等研究方法,觀察大白鼠在正中神經截斷後二及四週,背根神經節和楔狀神經核內Gly-R、GABAA-R 和GABAB-R 的分布情形,以及在背根神經節內這些受體免疫表現為受傷神經元的數量及比例,至於在楔狀神經核它們為楔狀丘腦接轉神經元的數量及比例變化。最後以甘胺酸性或伽傌-胺基丁酸性受體的促效劑或拮抗劑在正中神經截斷後二或四週處理,配合電刺激及免疫細胞化學法,檢視楔狀神經核內NPY 及c-Fos 的免疫表現。《第三年研究計畫》:本年度計畫首先擬以免疫細胞染色法,探討正中神經纏繞損傷後二及四週,背根神經節和楔狀神經核內Gly-R、GABAA-R 和GABAB-R 的數量變化。神經纏繞損傷後二及四週,以相關受體的拮抗劑或促效劑處理,審視動物疼痛行為表現,楔狀神經核內NPY 及c-Fos 免疫表現。接著將擬以神經損傷手術前給予相關受體的促效劑或局部麻醉劑處理,在正中神經損傷後二及四週,再以胞外電生理技術記錄神經自發性動作尖棘;檢視動物行為表現,楔狀神經核內NPY 及c-Fos 免疫表現,甚至於背根神經節和楔狀神經核內Gly-R、GABAA-R 和GABAB-R 的數量變化。
Abstract: Glycine and -aminobutyric acid (GABA) are considered to be the two majorcomponents in the inhibitory modulation and integration, which are caused by pre- andpostsynaptic inhibition to regulate the information processing. The morphology and inhibitorysynaptic circuits of glycine and GABA have been well documented in cuneate nucleus (CN),but the morphology and topographic distributions of the receptors of glycine and GABAwithin this nucleus remain uncertain. Under normal conditions, the CN transmits theinformation related to innocuous tactile, vibratile and proprioceptive sensations from cervical,forelimb and upper thoracic areas through primary afferent fibers and relays the informationto the contralateral ventrobasal nucleus of thalamus. Our previous studies (Day et al., 2001;Tsai et al., 2008) had reveled that following median chronic constriction injury developed theneuropathic pain on the injured forepaw and upregulated the expressions of neuropeptide Y(NPY) and c-Fos in the ipsilateral CN, but all of the above were not detected in the controlintact group. The expression of Fos, which is the protein product of the immediate-earlyproto-oncogene c-fos, has been widely used to investigate populations of active neurons andhas been regarded as a neural marker of pain following noxious stimulation (Hunt et al., 1987;Harris, 1998). Median nerve injury might impair inhibitory modulation to result inhyperexcitability and Fos expression in cuneate neurons, which prossibly correlated thetransmission of neuropathic pain. In support of this, significant loss or death of GABAergicneurons in the superficial laminae of the dorsal horn following sciatic transection or chronicconstriction injury (CCI), paralleling with thermal hyperalgesia and allodynia (Ibuki et al.,1997; Eaton et al., 1998; Moore et al., 2002; Scholz et al., 2005). Moreover, reduction in thenumber of glycine (Gly-R), GABAA (GABAA –R) and GABAB (GABAB -R) receptor wasalso found in the spinal cord after peripheral nerve injury (Bhisitku et al., 1990; Castro-Lopeset al., 1995). In contrast to these findings, some studies reported that no significant loss inGABAergic neurons in the dorsal horn in the CCI model (Polgar et al., 2003; Polgar and Todd,2008). However, the effect of peripheral injury on the changes of inhibitory neurons isfocused on the spinal cord and is diverse. To our knowledge, the effect of median nerve injuryon the alterations of the inhibitory modulation in the CN associated with the contribution tothe neuropathic pain still needs to be investigated. In the present proposal, we will use mediannerve transection (MNT) and chronic constriction injury (CCI) models, immunocytochemistry,western blotting, neuronal tracer injection, extracellular electrophysiological recording,behavioral test, drug treatment combined with electron microscopic study, to explore thechanges in inhibitory modulation in the CN and dorsal root ganglion (DRG) and relationshipsbetween this change and the development of neuropathic pain during three years. The detailsof the experimental purpose and design will to be sequentially described in the following year,respectively:《The proposal of first year》:In this year proposal, we will first use immunolabeling alongwith electron microscopic methods to investigate the number and synaptic organization ofGly-R-, GABAA–R- and GABAB –R-immunoreactive neurons in the CN and DRG of thenormal rats. Using neuronal tracer labeling and double immunostaining the number andproportion of the Gly-R-, GABAA–R- and GABAB –R-immunoreactive neurons in the CNwere identified to be cuneothalamic projection neurons (CTNs), glycine- andGABA-immunoreative neurons, respectively. Then, along with post-embedding immunogoldlabeling the presynaptic terminals to the immunolabeling receptor elements werecharacterized in the CN. Finally, the expression level of Gly-R-, GABAA–R- and GABAB –Rin the CN and DRG were quantified by using western blot.《The proposal of second year》:We will utilize MNT model along with anti-glycine andGABA post-embedding immunocytochemistry to examine the expression pattern of glycineand GABA in the CN at 2 and 4 weeks after injury. Then, the combination of anti- Gly-R,GABAA–R and GABAB –R immunolabeling with neuronal tracer injection into the transectedmedian nerve or contralateral thalamus will use to identify the expression level andcharacteristic of the above-mentioned neuroelements in the DRG and CN at 2 and 4 weeksafter transection. Furthermore, we will use median nerve transection along with agonist(taurine, muscimol and baclofen) and antagonist (strychnine, bicuculline and phaclofen)treatment, electrical stimulation and immunocytochemical method to examine the expressionof NPY and c-Fos in the CN at 2 and 4 weeks after injury.《The proposal of third year》:We will subsequently utilize median nerve CCI modelcombined with immunocytochemical labeling to investigate the expression level of Gly-R,GABAA–R- and GABAB –R in the DRG and CN at 2 and 4 weeks after CCI. Furthermore, wewill employ median nerve CCI model along with agonist (taurine, muscimol and baclofen)and antagonist (strychnine, bicuculline and phaclofen) treatment, electrical stimulation andimmunocytochemical labeling to investigate the effect on the development of neuropathicbehaviors and expression of NPY and c-Fos in the CN at 2 and 4 weeks after CCI.Subsequently, we will use MNT or CCI model combined with preemptive agonist (taurine,muscimol and baclofen) and lidocaine treatment, extracellular electrophysiological recording,behavioral test, electrical stimulation and immunocytochemical labeling to examine the rateof spikes of the median nerve, magnitude of neuropathic pain, expression of NPY and c-Fosin the CN, immunoreactive level of Gly-R, GABAA–R- and GABAB –R in the DRG and CNat 2 and 4 weeks after injury.