利雪陽 王 云 孫雨晴 馬丹旭 吳安石 岳 云

(首都醫(yī)科大學(xué)附屬北京朝陽醫(yī)院麻醉科，北京 100020)

· 麻醉學(xué)與神經(jīng)科學(xué) ·

A型肉毒素復(fù)合低劑量加巴噴丁對切口痛大鼠脊髓背角神經(jīng)激肽-1受體內(nèi)化的影響

利雪陽 王 云*孫雨晴 馬丹旭 吳安石 岳 云

(首都醫(yī)科大學(xué)附屬北京朝陽醫(yī)院麻醉科，北京 100020)

目的 評價(jià)A型肉毒素復(fù)合低劑量加巴噴丁對切口痛大鼠行為學(xué)及脊髓背角神經(jīng)激肽-1(neurokinin-1， NK-1)受體內(nèi)化的影響。方法 雄性SD大鼠，體質(zhì)量280～300 g，6～8周齡。采用數(shù)字表法隨機(jī)分為5組(n=9)：對照組(Control組)、切口痛—鹽水組(Saline組)、切口痛—加巴噴丁組(GBP組)、切口痛—毒素組(BoNT/A組)、切口痛—毒素復(fù)合加巴噴丁組(G+B組)。BoNT/A組和G+B組于造模前1 d鞘內(nèi)注射BoNT/A 0.5U，GBP組和G+B組于造模前30 min鞘內(nèi)注射GBP 50 mg。切口痛模型制備后3 h，每組各隨機(jī)抽取6只進(jìn)行累計(jì)疼痛評分(cumulative pain scores， CPS)和機(jī)械縮足閾值(paw withdrawal threshold， PWT)的測量；每組各抽取3只，通過免疫熒光技術(shù)測定NK-1受體內(nèi)化情況。結(jié)果 與Control組比較，切口后3 h Saline組、GBP組、BoNT/A組和G+B組右后足CPS升高、PWT降低、脊髓背角NK-1受體內(nèi)化神經(jīng)元數(shù)目上調(diào)(P<0.05)；與Saline組比較，BoNT/A組和G+B組CPS降低、PWT升高、脊髓背角NK-1受體內(nèi)化神經(jīng)元數(shù)目下調(diào)(P<0.05)，GBP組差異無統(tǒng)計(jì)學(xué)意義(P>0.05)；與BoNT/A組比較，G+B組CPS降低、PWT升高、脊髓背角NK-1受體內(nèi)化神經(jīng)元數(shù)目下調(diào)(P<0.05)。結(jié)論 單獨(dú)應(yīng)用低劑量GBP對大鼠切口痛無效，BoNT/A復(fù)合低劑量GBP治療切口痛大鼠的術(shù)后疼痛有明顯的協(xié)同作用，療效優(yōu)于單獨(dú)應(yīng)用BoNT/A，其鎮(zhèn)痛機(jī)制可能與抑制切口痛大鼠脊髓背角NK-1受體內(nèi)化有關(guān)。

A型肉毒桿菌毒素；加巴噴?。惶弁?；神經(jīng)激肽1受體；脊髓

術(shù)后疼痛是一種常見的急性疼痛，嚴(yán)重影響病人術(shù)后生理功能的恢復(fù)和生活質(zhì)量的改善。由于術(shù)后鎮(zhèn)痛的不完善，約有25%～55%的術(shù)后急性疼痛可轉(zhuǎn)化為慢性疼痛，遷延數(shù)月或數(shù)年，給家庭和社會帶來沉重負(fù)擔(dān)[1]。A型肉毒素(botulinum toxin A，BoNT/A)是肉毒桿菌在繁殖中分泌的一種有毒性的蛋白質(zhì)，可阻斷神經(jīng)遞質(zhì)在突觸前膜的釋放，可減輕神經(jīng)病理性痛等慢性疼痛[2]及切口痛模型大鼠的術(shù)后疼痛[3]。大劑量加巴噴丁(gabapentin，GBP)可以有效緩解神經(jīng)病理性痛，但GBP作為一種抗癲癇藥，大劑量使用可引起嗜睡等不良反應(yīng)[4]。而BoNT/A復(fù)合低劑量GBP對切口痛影響尚不清楚。P物質(zhì)作為一種與疼痛傳遞相關(guān)的神經(jīng)遞質(zhì)或調(diào)質(zhì)，在術(shù)后疼痛和術(shù)后急性痛向慢性痛轉(zhuǎn)化過程中起非常重要的作用[5]。脊髓背角神經(jīng)激肽-1(neurokinin-1， NK-1)受體是G蛋白偶聯(lián)受體，其內(nèi)化是反映初級感覺神經(jīng)元末梢釋放P物質(zhì)的重要指標(biāo)[6]。本研究擬評價(jià)A型肉毒素復(fù)合低劑量加巴噴丁對切口痛大鼠行為學(xué)及NK-1受體內(nèi)化的影響。

1 材料與方法

1.1 動物與藥品

SPF級雄性SD大鼠，體質(zhì)量280～300 g，6～8周齡，由北京維通利華實(shí)驗(yàn)動物技術(shù)有限公司提供，實(shí)驗(yàn)動物許可證號：SCXK(京)2012-0001。飼養(yǎng)于晝夜周期12 h的恒溫房間內(nèi)，自由進(jìn)食、飲水。

A型肉毒素(批號：C3786 C3，Allergan公司，美國)，小鼠抗神經(jīng)元核抗原(NeuN)抗體(批號：ab104224，Abcam公司，美國)，兔抗神經(jīng)激肽-1(NK-1)受體抗體(批號：NB300-101，Novus公司，美國)，TRITC標(biāo)記的抗兔IgG抗體(批號：111-025-003，Jackson公司，美國)，F(xiàn)ITC標(biāo)記的抗小鼠IgG抗體(批號：115-095-003，Jackson公司，美國)。

1.2 實(shí)驗(yàn)方法

1)鞘內(nèi)置管的實(shí)施參照文獻(xiàn)[7]：腹腔注射10%(質(zhì)量分?jǐn)?shù))水合氯醛300 mg/kg麻醉，碘伏消毒并正中剪開腰椎表面皮膚，剪斷L4、L5棘間韌帶，鈍性分離，暴露椎間隙，向上提起L4棘突，7號針頭刺破硬膜囊，沿針孔置入無菌PE10導(dǎo)管3 cm，置入蛛網(wǎng)膜下腔后可見甩尾反應(yīng)或腦脊液流出。PE10導(dǎo)管的另一端經(jīng)皮下隧道從大鼠頸部背側(cè)引出并固定，0.9%(質(zhì)量分?jǐn)?shù))氯化鈉注射液10μL沖洗管腔，熱熔封口?？p合腰部創(chuàng)面。術(shù)后每只大鼠單籠飼養(yǎng)，第6天時(shí)鞘內(nèi)注射2%(質(zhì)量分?jǐn)?shù))利多卡因10 μL，若注射后30 s內(nèi)出現(xiàn)雙后肢癱瘓說明鞘內(nèi)置管成功。鞘內(nèi)置管后7 d沒有神經(jīng)功能損傷的大鼠用于實(shí)驗(yàn)。

2)切口痛模型的制備：參照Brennan等[8]介紹的方法制備大鼠切口痛模型。吸入異氟醚麻醉下，消毒右后肢足底后，在大鼠右后足掌中間距足跟部0.5 cm處沿腳趾方向做一縱形切口，切開皮膚和筋膜，長度約1 cm，用小彎鑷提起跖肌，縱行切開，保持肌肉起止部位完整，輕壓止血后縫合皮膚，局部涂抹氧氟沙星軟膏預(yù)防感染。

3)分組與給藥：采用數(shù)字表法，隨機(jī)將其分為5組(n=9)：對照組(Control組)、切口痛-鹽水組(Saline組)、切口痛-加巴噴丁組(GBP組)、切口痛-毒素組(BoNT/A組)、切口痛-毒素復(fù)合加巴噴丁組(G+B組)。Saline組于切口手術(shù)前1 d鞘內(nèi)注射0.9%(質(zhì)量分?jǐn)?shù))氯化鈉注射液10 μL，切口手術(shù)前30 min 鞘內(nèi)注射0.9%(質(zhì)量分?jǐn)?shù))氯化鈉注射液10 μL。BoNT/A組于切口手術(shù)前1 d鞘內(nèi)注射BoNT/A 0.5 U(0.5 U/10 μL)，切口手術(shù)前30 min 鞘內(nèi)注射0.9%(質(zhì)量分?jǐn)?shù))氯化鈉注射液10 μL。GBP組于切口手術(shù)前1 d鞘內(nèi)注射0.9%(質(zhì)量分?jǐn)?shù))氯化鈉注射液10 μL，切口手術(shù)前30 min鞘內(nèi)注射GBP 50 mg (50 mg/10 μL)。G+B組于切口手術(shù)前1 d鞘內(nèi)注射BoNT/A 0.5 U(0.5 U/10 μL)，切口手術(shù)前30 min 鞘內(nèi)注射GBP 50 mg(50 mg/10 μL)。

4)行為學(xué)測試：每組取6只大鼠，分別于術(shù)后3 h時(shí)測定右后足累計(jì)疼痛評分(cumulative pain scores，CPS)和機(jī)械縮足反應(yīng)閾值(paw withdrawal threshold，PWT)。CPS的評分標(biāo)準(zhǔn)：右后足完全負(fù)重為0分；右后足僅僅接觸地面而沒有變白或者扭曲為1分；右后足抬離地面為2分。每5 min評價(jià)1次，每次觀察1 min，持續(xù)觀察1 h，1 h內(nèi)評分之和作為CPS。參照文獻(xiàn)[9]介紹的方法測定PWT，將大鼠置于金屬網(wǎng)上適應(yīng)環(huán)境30 min，用一系列標(biāo)準(zhǔn)化的von Frey纖維絲(North Coast Medical公司，美國)采用序貫法刺激右后足掌根部縫合線外側(cè)皮膚，使其彎曲成S型，持續(xù) 6～8 s，出現(xiàn)快速的縮足反應(yīng)或擺腿運(yùn)動為陽性。初始刺激力度為2.041 g，當(dāng)出現(xiàn)陰性反應(yīng)時(shí)，則使用高一級的刺激力度，當(dāng)出現(xiàn)陽性反應(yīng)時(shí)，則使用低一級的刺激力度，從第1次出現(xiàn)陽性反應(yīng)和陰性反應(yīng)騎跨開始，測定5次，相鄰刺激間隔5 min，計(jì)算50%機(jī)械縮足反應(yīng)刺激力度即為PWT。為防止刺激力度過大損傷大鼠，最大刺激力度設(shè)定為15.136 g。

5)脊髓背角NK-1受體內(nèi)化的測定：術(shù)后3 h時(shí)每組取3只大鼠，腹腔注射10%(質(zhì)量分?jǐn)?shù))水合氯醛300 mg/kg麻醉，迅速開胸暴露心臟，經(jīng)升主動脈插管，剪開右心耳。先以400 mL 0.9%(質(zhì)量分?jǐn)?shù))氯化鈉注射液沖凈血液，隨即灌注4%(質(zhì)量分?jǐn)?shù))多聚甲醛400 mL。掀開椎板，取L4、L5脊髓節(jié)段置于4%(質(zhì)量分?jǐn)?shù))多聚甲醛4 ℃后固定4～6 h，蔗糖脫水沉底后冰凍包埋。于-20 ℃連續(xù)冠狀面切片，厚度12 μm，貼片，42 ℃烤片30 min。切片復(fù)水后用0.4%(體積分?jǐn)?shù))Triton X-100室溫通透20 min， PBS漂洗5 min×3次，用10%(體積分?jǐn)?shù))山羊血清封閉1 h；PBS漂洗后加入兔抗NK-1受體抗體(稀釋度1∶50)和小鼠抗NeuN抗體(稀釋度1∶1 000)，4 ℃孵育過夜；PBS漂洗5 min×3次，加入TRITC標(biāo)記的抗兔IgG(稀釋度1∶100)和FITC標(biāo)記的抗小鼠IgG(稀釋度1∶100)，室溫避光孵育1 h，封片。采用BX-51熒光顯微鏡(Olympus公司，日本)觀察并拍片，計(jì)數(shù)NK-1受體陽性神經(jīng)元，以此反映NK-1受體的內(nèi)化水平。

1.3 統(tǒng)計(jì)學(xué)方法

2 結(jié)果

2.1 各組大鼠累計(jì)疼痛評分和機(jī)械縮足閾值的比較

行為學(xué)實(shí)驗(yàn)結(jié)果顯示， Control組右后足CPS為(1.05±0.43)分，與Control組相比，切口后3 h Saline組升高至(21.83±0.70)分、GBP組升高至(19.83±0.98)分、BoNT/A組升高至(15.17±1.14)分、G+B組升高至(10.83±0.60)分(P<0.001，n=6)；與Saline組比較，BoNT/A組和G+B組CPS顯著降低 (P<0.001，n=6)，GBP組差異無統(tǒng)計(jì)學(xué)意義(P>0.05，n=6)；與BoNT/A組比較，G+B組CPS降低(P<0.01，n=6)。結(jié)果詳見圖1A。

Control組右后足PWT為(15.14±0)g，與Control組相比，切口后3 h Saline組降低至(2.69±0.49)g、GBP組降低至(3.54±0.78)g、BoNT/A組降低至(7.54±1.05)g(P<0.001，n=6)，G+B組降低至(11.35±1.38)g(P<0.05，n=6)；與Saline組比較，BoNT/A組和G+B組PWT均顯著升高(P<0.01，n=6)，GBP組差異無統(tǒng)計(jì)學(xué)意義(P>0.05，n=6)；與BoNT/A組比較，G+B組PWT升高(P<0.05，n=6)。結(jié)果詳見圖1B。

2.2 各組大鼠脊髓背角NK-1受體內(nèi)化神經(jīng)元數(shù)目比較

Control組脊髓背角NK-1受體內(nèi)化神經(jīng)元數(shù)目(2.47±0.46)個(gè)，切口后3 h 平均每個(gè)視野，Saline組上調(diào)至(9.53±1.27)個(gè)(P<0.001，n=15)、GBP組上調(diào)至(9.60±1.11)個(gè)(P<0.001，n=15)，而BoNT/A組(5.8±0.86)個(gè)和G+B組(2.13±0.49)個(gè)較Control組差異無統(tǒng)計(jì)學(xué)意義(P>0.05，n=15)；與Saline組比較，BoNT/A組和G+B組脊髓背角NK-1受體內(nèi)化神經(jīng)元數(shù)目下調(diào)(P<0.05，n=15)，GBP組差異無統(tǒng)計(jì)學(xué)意義(P>0.05，n=15)；與BoNT/A組比較，G+B組脊髓背角NK-1受體內(nèi)化神經(jīng)元數(shù)目下調(diào)(P<0.05，n=15)。結(jié)果詳見圖2。

圖1 大鼠術(shù)后3 h累計(jì)疼痛評分(A)和機(jī)械縮足閾值(B)比較Fig.1 Cumulative pain score (A) and paw withdrawal threshold (B) of rats 3 h after operation

**P<0.01，***P<0.001；###P<0.001；△P<0.05，△△P<0.01；n=6;GBP:gabapentin; BoNT/A:botulinum toxin A; G+B: gabapentin+botulinum toxin A.

圖2 各組大鼠術(shù)后3h脊髓背角NK-1受體/NeuN免疫熒光雙標(biāo)結(jié)果Fig.2 DoubleimmunofluorescencestainingofspinalcorddorsalhornofratswithNK-1receptor(red)andNeuN(green)*P<0.05;###P<0.001;△P<0.05;n=15;GBP:gabapentin;BoNT/A:botulinumtoxinA;G+B:gabapentin+botulinumtoxinA;NK-1r:neurokinin-1receptor.

3 討論

Brennan等[8]建立的大鼠后足掌切口痛模型是經(jīng)典的術(shù)后痛模型。CPS評分主要反映非誘發(fā)性疼痛反應(yīng)，通常與術(shù)后靜息痛有關(guān)。PWT測試中采用von Frey纖維絲刺激切口，可對術(shù)后機(jī)械性痛覺過敏進(jìn)行量化。前期研究[8-10]結(jié)果表明，切口痛大鼠術(shù)后3 h時(shí)痛覺過敏最明顯。因此本研究選擇切口痛術(shù)后3 h作為觀察時(shí)點(diǎn)。本研究結(jié)果表明，切口痛組痛行為學(xué)變化同文獻(xiàn)[10]的研究結(jié)果一致，提示切口痛模型制備成功。

參照文獻(xiàn)[11-12]并結(jié)合預(yù)實(shí)驗(yàn)結(jié)果，本研究選擇鞘內(nèi)注射BoNT/A的劑量為0.5 U，于術(shù)前24 h時(shí)給藥；鞘內(nèi)注射GBP的劑量為50 mg，于術(shù)前30 min時(shí)給藥。傷害性刺激可誘發(fā)脊髓背角NK-1受體出現(xiàn)明顯的內(nèi)化而進(jìn)入神經(jīng)元胞質(zhì)。本研究采用免疫熒光雙標(biāo)脊髓背角神經(jīng)元和NK-1受體的方法，檢測NK-1受體在脊髓背角神經(jīng)元的內(nèi)化水平，保證了收集數(shù)據(jù)的客觀性。

P物質(zhì)作為一種與疼痛傳遞相關(guān)的神經(jīng)遞質(zhì)或調(diào)質(zhì)，在術(shù)后疼痛和術(shù)后急性疼痛向慢性疼痛轉(zhuǎn)化過程中起非常重要的作用[13]。NK-1受體是G蛋白偶聯(lián)受體，其內(nèi)化是反映初級感覺神經(jīng)元末梢釋放P物質(zhì)的重要指標(biāo)，傷害性刺激可誘發(fā)脊髓背角NK-1受體出現(xiàn)明顯的內(nèi)化而進(jìn)入神經(jīng)元胞質(zhì)[6,14]。本研究結(jié)果表明，BoNT/A復(fù)合低劑量GBP可有效緩解大鼠切口痛，其效應(yīng)優(yōu)于單獨(dú)應(yīng)用BoNT/A，但單獨(dú)應(yīng)用低劑量GBP不能有效緩解切口痛。同時(shí)發(fā)現(xiàn)切口痛引起脊髓背角NK-1受體內(nèi)化神經(jīng)元表達(dá)上調(diào)，而BoNT/A或BoNT/A復(fù)合GBP逆轉(zhuǎn)NK-1受體內(nèi)化神經(jīng)元表達(dá)上調(diào)。因此，聯(lián)合用藥可以產(chǎn)生更佳的療效、降低彼此用量、減少不良反應(yīng)，提示BoNT/A復(fù)合GBP可能從不同通路不同層次協(xié)同下調(diào)脊髓背角神經(jīng)元NK-1受體內(nèi)化，進(jìn)而減少P物質(zhì)釋放。

本研究結(jié)果提示，單獨(dú)應(yīng)用低劑量GBP對大鼠切口痛無效，BoNT/A復(fù)合低劑量GBP治療切口痛大鼠的術(shù)后疼痛有明顯的協(xié)同作用，治療效果優(yōu)于單獨(dú)應(yīng)用BoNT/A，其鎮(zhèn)痛機(jī)制可能與抑制切口痛大鼠脊髓背角NK-1受體內(nèi)化有關(guān)。

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編輯 陳瑞芳

Effects of botulinum toxin A combined with low dose gabapentin on the NK-1 receptor internalization at spinal cord dorsal horn in rats with incisional pain

Li Xueyang, Wang Yun*, Sun Yuqing, Ma Danxu, Wu Anshi, Yue Yun

(DepartmentofAnesthesiology,BeijingChaoyangHospital,CapitalMedicalUniversity,Beijing100020,China)

Objective To evaluate the effects of botulinum toxin A combined with low dose gabapentin on the neurokinin-1 (NK-1) receptor internalization at spinal cord dorsal horn in rats with incisional pain. Methods Male Sprague-Dawley rats, weighing 280-300 g, aged 6-8 weeks, were used in the study. Rats were randomly selected and divided into 5 groups (n=9 each) using a random number table: control group (Control group), incisional pain group (Saline group), gabapentin group (GBP group), botulinum toxin A group (BoNT/A group), botulinum toxin A combined with gabapentin group (G+B group). At 24 h before operation, botulinum toxin A 0.5U (in 10 mL of normal saline) was injected intrathecally in BoNT/A group and G+B group. At 30 min before operation, gabapentin 50 mg was injected intrathecally in GBP group and G+B group. At 3 h after operation, 6 rats in each group were selected to measure the cumulative pain scores (CPS) and mechanical paw withdrawal threshold (PWT) in the right hindpaw; besides, 3 rats in each group were selected and sacrificed, and the lumbar segment (L4,5) of the spinal cord was removed for determination of the expression of NK-1 receptors in the spinal dorsal horn by immunofluorescence. Results Compared with Control group, the CPS was significantly increased, the PWT was significantly decreased, and the expression of NK-1 receptors in the spinal dorsal horn was significantly up-regulated in Saline group, GBP group, BoNT/A group and G+B group at 3 h after operation(P<0.05). Compared with Saline group, the CPS was significantly decreased, the PWT was significantly increased, and the expression of NK-1 receptors in the spinal dorsal horn was significantly down-regulated in BoNT/A group and G+B group at 3 h after operation (P<0.05), and no significant change was found in group GBP (P>0.05). Compared with BoNT/A group, the CPS was significantly decreased, the PWT was significantly increased, and the expression of NK-1 receptors in the spinal dorsal horn was significantly down-regulated in G+B group at 3 h after operation (P<0.05). Conclusion Low doses of gabapentin alone may have no effect on postoperative pain, but when it is coadministrated with BoNT/A, it can greatly enhance the analgesic effect of BoNT/A. The analgesic mechanism may be due to inhibition of the internalization of NK-1 receptor at spinal cord horn in a rat model of incisional pain.

botulinum toxin type A；gabapentin； pain； neurokinin-1 receptors；spinal cord

國家自然科學(xué)基金(81571065,81428008，81400909)。This study was supported by National Natural Science Foundation of China(81571065,81428008，81400909).

時(shí)間：2017-06-09 17∶50 網(wǎng)絡(luò)出版地址：http://kns.cnki.net/kcms/detail/11.3662.r.20170609.1750.056.html

10.3969/j.issn.1006-7795.2017.03.001]

R971

2017-03-20)

*Corresponding author， E-mail：wangyun129@ccmu.edu.cn