新生兒時期發炎性疼痛對大鼠成年後 長期傷痛行為與脊髓背角神經元經突觸傳遞之影響

Long term effects of neonatal inflammatory pain on the nocifensive behaviors and trans-synaptical transmission in the adult rat spinal cord dorsal horn neurons

林至芃(Chih-Peng Lin)

指導教授 ： 孫維仁

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.00675

摘要

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。

關鍵字

新生兒 ； 發炎 ； 疼痛 ； 二氧化碳雷射 ； 疼痛閾值 ； 突觸 ； NK1受體 ； mitogen activated-protein kinase （MAPK） pathway ； extracellular signal-regulated kinase (ERK)

並列摘要

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”.

並列關鍵字

neonate ； inflammation ； pain ； CO2 pulse laser ； pain threshold ； synapse ； NK1 receptor ； mitogen activated-protein kinase (MAPK) pathway ； extracellular signal-regulated kinase (ERK)

參考文獻

Abbadie C, Brown JL, Mantyh PW, Basbaum AI. Spinal cord substance P receptor immunoreactivity increases in both inflammatory and nerve injury models of persistent pain. Neuroscience 1996;70(1):201-209.

Abbadie C, Trafton J, Liu H, Mantyh PW, Basbaum AI. Inflammation increases the distribution of dorsal horn neurons that internalize the neurokinin-1 receptor in response to noxious and non-noxious stimulation. Journal of Neuroscience 1997;17(20):8049-8060.

Adams JP, Sweatt JD. Molecular psychology: roles for the ERK MAP kinase cascade in memory. Annual Review of Pharmacology & Toxicology 2002;42:135-163.

Aley KO, Martin A, McMahon T, Mok J, Levine JD, Messing RO. Nociceptor sensitization by extracellular signal-regulated kinases. Journal of Neuroscience 2001;21(17):6933-6939.

Alvares D, Torsney C, Beland B, Reynolds M, Fitzgerald M. Modelling the prolonged effects of neonatal pain. Progress in Brain Research 2000;129:365-373.