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Long term effects of neonatal inflammatory pain on the nocifensive behaviors and trans-synaptical transmission in the adult rat spinal cord dorsal horn neurons
No full text醫療科技的進步，大幅提升早產兒與重症嬰幼兒的存活率。但此時期之疼痛刺激若未得到適當治療，會引發長期生理與行為上的異常反應。動物模式中更證實，新生大白鼠接受發炎性之持續疼痛刺激後，周邊及脊髓感覺神經系統的發育會產生永久性的變化。包括解剖構造上，初級疼痛神經元在脊髓後角的異樣分佈和分枝增生；電生理上，脊髓背角神經元對疼痛及非疼痛刺激產生過度興奮；疼痛刺激的生化證據c-Fos蛋白在脊髓背角的表現增高等。
實驗材料與方法：將Complete Freund，s Adjuvant （CFA）注射於出生第一天（P1）及第21天（P21）大白鼠之左側後腳掌皮下，引發持續的發炎性疼痛為期五到七天。對照組（N）則不給予致發炎物質。然後於成鼠期（出生後8週），對這些大白鼠進行疼痛行為學上的觀測，利用二氧化碳脈衝雷射熱能作為短暫性疼痛刺激源，以固定輸出功率5 watt，以5ms為單位，調整脈衝輸出時間，刺激兩側後腳掌皮膚，測定疼痛閾值。並以超閾值刺激，進行傷痛行為的觀測。
對於初級感覺神經元經突觸傳遞疼痛訊息至脊髓內二級神經元的訊息強度之活體內觀察以疼痛的神經傳導物質substance P與其受體NK1Receptor的作用為指標。以歷經新生鼠時期疼痛的成年大鼠（P1）雙側後腳掌皮下注射5%福馬林引發週邊疼痛，8分鐘後進行動物犧牲、灌流固定後取得腰椎第四節第五節之脊髓（L4-L5），冷凍切片後對脊髓切片進行NK1受體的免疫螢光染色，比較兩側螢光免疫反應相對強度，並進行分析。
對於脊髓背角二級感覺神經元被疼痛刺激所活化的程度，則以mitogen activated-protein kinase（MAPK）pathway中extracellular signal-regulated kinase（ERK）被磷酸化的程度作為指標，成年大鼠雙側後腳掌皮下注射5%福馬林後5分鐘犧牲，以4℃冰生理食鹽水及固定液進行灌流，冷凍切片後進行pERK之免疫組織染色，並定量脊髓切片中被活化的神經元個數，進行比較分析。
接受CFA注射側，與未注射CFA側，以Paired-T test 比較；三組不同處理的老鼠的反應以ANOVA進行統計分析。統計分析軟體採用STATA 8.0。數據結果以（Mean ± S.E.M）表示，P值小於0.05視為統計上有差異。
結果：共分為三個部分，總結如下：
（1）二氧化碳脈衝雷射熱刺激的疼痛閾值與傷痛行為測定結果顯示：歷經新生兒時期發炎性疼痛的P1組其”未”接受CFA注射之右後腳（P1_nonCFA）疼痛閾值較接受發炎疼痛的左腳（P1_CFA）為高（71.66±1.47 vs 65.00±1.86 p=0.004）；同時也較P21組兩側及N組為高（ANOVA，F=5.38，p=0.0030）。P1組CFA注射腳（P1-left_CFA）的疼痛閾值與P21組（無論接受CFA注射與否）及N組，皆無統計上差異。P21組左右兩側無組內差異。P21組與N組之間亦無差異。以超閾值之二氧化碳脈衝雷射熱刺激（5 watts，200 ms）引發之傷痛行為包含抬腳與舔腳，其結果皆顯示歷經新生兒疼痛的P1組CFA注射腳（P1_CFA）表現明顯較強烈的傷痛行為，強於P1_ nonCFA、P21組及N組（ANOVA，F=10.57，p=0.0001）。
（2）P1組於成年期再次接受疼痛刺激，支配後腳掌感覺傳入訊息的腰椎脊髓第四節第五節（L4－L5）其脊髓背角經突觸訊息傳遞強度的指標NK1 Receptor免疫螢光反應，在CFA注射側（P1_CFA side）明顯強於對側（P1_nonCFA side）。
（3）在支配後腳掌感覺傳入訊息的腰椎脊髓第四節第五節（L4－L5）其脊髓背角二級感覺神經元，接受週邊疼痛刺激後短期內以ERK的磷酸化作為其活化程度的指標。pERK（＋）神經元數量在L4與L5皆顯示：歷經新生兒疼痛的P1 CFA注射側（P1_CFA）明顯多於P1未接受CFA注射的對側（P1_nonCFA）又多於P21組與N組。（ANOVA，L4：F=30.6，p<0.0001；L5：F=49.36，p<0.0001）。
結論：新生鼠時期歷經發炎性疼痛的成鼠，在行為上表現較高的疼痛閾值及較強烈之傷痛行為。同時在功能上，新生鼠時期歷經疼痛側，其脊髓背角經突觸訊息傳遞較未曾有疼痛暴露的另一側，明顯有被強化的情形。當成鼠接受相同強度外界刺激時，曾歷經新生兒疼痛側脊髓背角中二級神經元活化程度增加。而類似的變化並不會出現於青少年鼠時期歷經疼痛經驗的成鼠，這暗示了新生兒時期為神經發育關鍵期，此期間內的疼痛經驗會造成永久性的影響。Advances in medical technology have significantly improved survival among medically compromised premature babies. However, poorly managed pain during this period will result in long-term physiologic and behavioral consequences. Animal studies reveal that neonatal peripheral inflammatory pain results in long-standing changes on the development of nociceptive neuronal circuitry. Morphologically, fine primary afferent fibers termination area in spinal cord dorsal horn expands longitudinally and sprouts into deeper lamina. Electrophysiologic study shows increase in background activity and responses to noxious and non-noxious stimuli. The distribution and extent of “Fos like immunoreactivity” (a marker of post-synaptic activation) in spinal cord dorsal horn is also increased.
Materials and methods: On postnatal day 1 (P1) and day 21 (P21), rat pups were intraplantarlly injected with complete Freund’s adjuvant (CFA). Inflammatory pain was thus induced on the left hind paw and lasted for 5-7 days. Control group (N) rats wer left untreated. All animals were allowed to mature into adulthood without further manipulation. Further assessments were done at 8 weeks of age.
We determined the pain thresholds and nocifensive behaviors among all 3 different treatment groups (P1, P21 and N) bilaterally by CO2 pulse laser stimulation. Pain thresholds were determined by serially increases of the laser pulse duration (5 ms increaments) with fixed CO2 pulse laser output energy to 5 watts until leg withdrawal were elicited. Nocifensive behaviors were induced by supra-threshold laser stimulation (5watts, 200ms). Leg-lifting and licking times were recorded.
For accessing the trans-synapticall transmission, interactions of nociceptive neuromodulator- substance P and it receptor- NK1 receptor were analysed. P1 rats were intraplantarlly injected bilaterally with 5% fomalin. Rats were sacrificed 8 minutes after the intense pain rechallenge. After transcardially perfusion and fixation, L4-L5 spinal cord were retrieved, cryosectioned and processed for substance P receptor - NK1 Receptor (NK1R) immunofluorescent staining. Relative immunofluorescent density was measured.
For analysis of dorsal horn secondary sensory neuron activation, all 3 treatment groups (P1, P21 and N) were intraplantarlly injected bilaterally with 5% fomalin to induce intense pain rechallenge. Five minutes later, rats were sacrificed and transcardially perfused and fixed by 4℃ fixatives. L4-L5 spinal cord were retrieved, cryosectioned and processed for phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry staining by ABC method. Numbers of pERK (+) neurons were counted and compared.
All data were presented as mean±SEM, Paired T test was performed for side-by-side comparison. ANOVA test was performed for comparison among the 3 treatment groups. P<0.05 was considered statistically significant. All analysis was assisted by statistic software STATA 8.0.
Results:
(1) Pain thresholds assessed by CO2 pulse laser stimulation showed that thresholds were significantly higher in P1_nonCFA side than in P1_CFA side (71.66±1.47 vs 65.00±1.86 p=0.004). P1_nonCFA side had greater pain threshold than P21 and N groups as well. (ANOVA，F=5.38，p=0.0030). The difference of pain thresholds among P1_CFA side, P21 and N groups were not significant. Nocifensive behaviors induced by supra-threshold pain stimulation was significant greater in P1_CFA than in P1_non CFA, P21 and N groups. (ANOVA, F=10.57, p=0.0001)
(2)In both L4 and L5, after 8 minutes of intense pain rechallenge, the NK1R relative immunofluorescent densities were significantly greater in P1_CFA side than in P1_nonCFA side.
(3)In both L4 and L5, after intense pain rechallenge, the number of pERK (+) neurons of superficial dorsal horn was significantly greater in P1_CFA side than in P1_nonCFA side and than in P21 and N groups. (P1_CFA > P1_nonCFA > P21=N).
Conclusion: Adult rat experienced neonatal inflammatory insult has higher pain threshold and stronger nocifensive behaviors. When suffering from intense peripheral pain rechallenge, it has stronger dorsal horn trans-synaptical transmission at the neonatal insult site. It has more superficial dorsal horn neurons activated by peripheral noxious stimulation, especially at neonatal insult site. Similar responses cannot be elicited in adult rat experienced juvenile inflammatory pain. These long term changes of nociceptive signal transduction happen only when the neonatal insults occurred during “window of vulnerability”.一 中文摘要 4
二 英文摘要 6
三 研究背景與目的 8
四 研究方法 15
五 結果 19
六 討論 22
七 臨床意義與未來展望 29
八 結論 31
九 參考文獻 32
十 圖表 4
Translational Research on Opioid Analgesic Tolerance: from Bedside back to Bench
No full text鴉片類藥物是臨床處理中重度疼痛，最有效也最被廣泛使用的藥物。然而長期使用鴉片類藥物止痛卻也會伴隨產生藥物的耐受性。脊椎管內鴉片類藥物投與將藥物直接送至中樞神經產生藥效，是非常有效的止痛方式但卻也更容易產生藥物耐受性。傳統神經科學以神經元為中心的思維下，鴉片類藥物耐受性的致病機轉著重於Opioid receptor的internalization、NMDA receptor的upregulation或glutamate transporter 的down regulation。然而這些現象都只能部分解釋耐受性的成因。最近在齧齒類實驗動物上的研究結果顯示神經膠細胞的活化及發炎性物質的過度表現、也就是所謂的神經炎性反應在神經病變疼痛與類鴉片耐受性的致病機轉上有重要的角色。然而相關的人體研究證據則相當稀少。 在本研究的臨床部分，我們首先嘗試建立國內最完整的，有關體外可程控式植入型脊椎內給藥系統的照護流程。包括合適病患的選取、脊椎內嗎啡藥物輸注測試、手術方式的改進、長期追蹤與品質提升計畫。我們紀錄並分析了流程建立初期的病患，其脊椎管內嗎啡的劑量的改變、治療相關併發症的發生率與後續處理及病患日常生活功能的改善程度。藉由給予足夠劑量的脊椎管內嗎啡，病患的疼痛控制與生活品質皆能大幅改善。然而在這當中我們也發現所有接受脊椎內嗎啡輸注療法的病患，其嗎啡的劑量皆快速的增加，遠遠超過病情的演進。 以臨床的照護觀察到的現象為起點，在研究倫理委員會核准後，我們進行了一系列的轉譯醫學研究。我們首先分析了已產生類鴉片藥物耐受性病患的腦脊髓液中發炎相關因子的濃度。包括TNF-alpha、 CXCL1、CXCL10、CCL2、CX3CL1及CXCL12並與未暴露類鴉片藥物的對照組受試者比較。研究結果發現，已經對類鴉片止痛藥產生耐受性的病患群，其腦脊髓液中的CXCL1及CXCL12濃度明顯高於未暴露類鴉片藥物的對照組。進一步我們更發現CXCL1的濃度與病患所接受的類鴉片止痛藥物劑量成高度正相關。 接著我們建立轉譯動物實驗模式，藉由實驗鼠的閃尾反應，評估嗎啡的止痛效果及相關發炎因子對類鴉片耐受性產生的影響。在實驗大鼠投予嗎啡誘發藥物耐受性後，大鼠脊髓組織之CXCL1 及CXCL12 mRNA皆顯著增加。雖然單獨給予椎管內CXCL1或CXCL12並不會改變老鼠的基礎閃尾行為，然而椎管內給予CXCL1或CXCL12卻會大幅降低腹腔內給予嗎啡所造成的急性止痛效果。接著我們參照臨床長期椎管內類鴉片輸注用於頑固疼痛的處置，建立植入皮下微幫浦進行長期椎管內嗎啡輸注並誘發大鼠產生藥物耐受性的實驗模式。我們發現耐受性的發生會因同時給予嗎啡與CXCL1或CXCL12 而加速。反之 若被嗎啡輸注時一併給予CXCL1或CXCL12的中和抗體則會延緩耐受性的發生。針對CXCL1 訊息傳遞給予其受體CXCR2的拮抗劑antileukinate hexapeptide，或針對CXCL12 訊息傳遞給予其受體CXCR4的拮抗劑 AMD3100，則可更有效的延緩嗎啡耐受性的發生。 綜合以上的實驗結果，我們藉由臨床到實驗動物的轉譯醫學研究模式驗證了趨化激素CXCL1與CXCL12可能參與鴉片類止痛藥耐受性的形成。阻斷 CXCL1/CXCR2 與 CXCL12/CXCR4 的訊息傳遞路徑則可以延緩藥物耐受性的產生並降低其嚴重度。因此針對CXCL1/CXCR2 與 CXCL12/CXCR4 的訊息傳遞路徑進行介入將是治療類鴉片止痛藥耐受性的新藥研發之潛力標的。Opioid analgesics remain the most effective and widely used analgesics for the management of moderate to severe pain. However, the efficacy of long-term opioid analgesics is progressively attenuated by tolerance, preventing adequate pain relief under stable opioid dosages for chronic pain patients. Although intrathecal opioid delivery provides very effective analgesia by acting directly on central nervous system, opioid analgesic tolerance is also accelerated. Classical neuron-centered concepts such as internalization of opioid receptors, up-regulation of N-methyl-D-aspartate receptor function, or down-regulation of glutamate transporter activity can only partially explain the phenomenon of tolerance. Recent evidence showing glial activation and upregulated inflammatory mediators in the rodent central nervous system has confirmed the pivotal role of neuroinflammation in neuropathic pain or opioid tolerance, or both. However, human evidence is still sparse. In clinical part of this study, we developed comprehensive management protocol for totally implantable programmable intrathecal drug delivery system from patient selection, intraspinal morphine trial, surgical procedure to follow up program. Intrathecal morphine dosage adjustment, treatment related complications and patient functional outcomes are recorded regularly and analyzed. By delivering liberal dose of intrathecal morphine, pain severity decreased significantly. Due to much better pain control and improved quality of life, functional performance status also improved. Intrathecal morphine delivery by using totally implantable programmable device is an effective alternative method to relieve refractory cancer pain. Based on our clinical practice, we further conducted subsequent translational research by investigating the intraspinal cytokine and chemokine profiles of opioid-tolerant cancer patients after research ethic committee approval. Cerebrospinal fluid (CSF) samples from opioid-tolerant cancer patients and opioid-naive subjects were compared. The CSF levels of tumor necrosis factor-alpha, CXCL1, CXCL10, CCL2, CX3CL1 and CXCL12 were assayed. CXCL1 and CXCL12 levels in CSF were significantly upregulated in the opioid-tolerant group. Further analysis revealed that CXCL1 level was strongly positively correlated with opioid dosage. The rat tail flick test was utilized to assess the effects of intrathecal CXCL1 or CXCL12 on morphine-induced acute antinociception and analgesic tolerance. After induction of tolerance by intrathecal morphine infusion, the spinal cord CXCL1 and CXCL12 messenger RNA were significantly upregulated. Although CXCL1 or CXCL12 infusion alone did not affect baseline tail flick latency, the analgesic tolerance was accelerated by intrathecal infusion of CXCL1 or CXCL12 in daily intraperitoneal morphine injection of paradigm. After establishing tolerance by intrathecal continuous infusion of morphine, its development was accelerated by co-administration of CXCL1 or CXCL12. On the contrary, tolerance was attenuated by co-administration of CXCL1 or CXCL12 neutralizing antibody or corresponding receptor antagonists. CXCL1 and CXCL12 were upregulated in both opioid-tolerant patients and rodents. The onset and extent of opioid tolerance was affected by antagonizing intrathecal CXCL1/CXCR2 and CXCL12/CXCR4 signaling. Therefore, the CXCL1/CXCR2 and CXCL12/CXCR4 signal pathways may be novel drug targets for the treatment of opioid tolerance
Prolonged retraction on the normal common carotid artery induced lethal stroke after cervical spine surgery.
No full textPPARγinhibits osteogenesis via the down-regulation of the expression of COX-2 and iNOS in rats
No full text
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