CXCR4對大鼠背根神經(jīng)節(jié)中交感芽生形成的干預(yù)作用

楊悅橙 馬冰潔 馬 柯 △

(1復(fù)旦大學(xué)附屬腫瘤醫(yī)院麻醉科 上海 200032; 2上海交通大學(xué)醫(yī)學(xué)院附屬新華醫(yī)院疼痛科 上海 200092)

?

CXCR4對大鼠背根神經(jīng)節(jié)中交感芽生形成的干預(yù)作用

楊悅橙1馬冰潔2馬 柯2 △

(1復(fù)旦大學(xué)附屬腫瘤醫(yī)院麻醉科 上海 200032;2上海交通大學(xué)醫(yī)學(xué)院附屬新華醫(yī)院疼痛科 上海 200092)

目的 研究CXCR4在大鼠脊神經(jīng)結(jié)扎(spinal nerve ligation,SNL)后對背根神經(jīng)節(jié)(dorsal root ganglion,DRG)交感芽生形成的干預(yù)作用。方法 將大鼠隨機分為AMD3100(CXCR4特異抑制劑)實驗組(n=16)、AMD3100對照組(n=8)、對照組(n=16)和空白對照組(n=16),對AMD3100實驗組和對照組的大鼠行SNL術(shù),術(shù)后7天內(nèi)對AMD3100實驗組大鼠每天給予AMD3100,術(shù)后3、5、7、10、14天(d3、d5、d7、d14)檢測機械痛閾(mechanical withdrawal threshold,MWT),分別用實時PCR和Western blot檢測神經(jīng)生長因子(nerve growth factor,NGF)的RNA和蛋白質(zhì)水平,d7和d14行免疫熒光檢測DRG中交感芽生。結(jié)果 與對照組相比, AMD3100實驗組大鼠在d5、d7和d14的MWT均顯著緩解,抑制CXCR4后,AMD3100實驗組大鼠DRG中交感芽生在d7較對照組顯著減少,但在d14減少不明顯,且NGF的RNA和蛋白質(zhì)水平均較對照組顯著降低。結(jié)論 抑制CXCR4能改善大鼠SNL術(shù)后d5～d14的疼痛行為,并能減少d7的藍狀結(jié)構(gòu)數(shù)量。

交感芽生; 趨化素; 背根神經(jīng)節(jié); 大鼠

針對交感神經(jīng)的治療能在復(fù)雜區(qū)域疼痛綜合征中使部分患者獲益[1-2],但在帶狀皰疹后遺神經(jīng)痛中療效一般[3]?；A(chǔ)研究已在神經(jīng)損傷、關(guān)節(jié)炎癥動物模型中發(fā)現(xiàn)交感芽生現(xiàn)象,即在背根神經(jīng)節(jié)(dorsal root ganglion,DRG)中交感纖維繞著神經(jīng)元生長形成藍狀結(jié)構(gòu)。被交感神經(jīng)包繞著的神經(jīng)元更易產(chǎn)生自發(fā)電活動[4],這也可能是交感神經(jīng)相關(guān)治療的機制。

研究發(fā)現(xiàn)神經(jīng)生長因子(nerve growth factor,NGF)在交感芽生中起著重要的作用,大鼠鞘內(nèi)注射NGF可引起交感芽生[5],且抑制NGF受體P75會導(dǎo)致交感芽生的形態(tài)紊亂[6]。近年來,趨化因子在神經(jīng)病理疼痛中的作用日益受到關(guān)注[7],在神經(jīng)損傷模型中發(fā)現(xiàn)趨化因子CXCL12及其受體CXCR4的表達上調(diào)[8],大鼠鞘內(nèi)注射CXCL12可誘發(fā)疼痛至少2 h,且疼痛可被CXCR4的特異性抑制劑AMD3100所逆轉(zhuǎn)。在胚胎發(fā)育階段,CXCR4的表達可決定神經(jīng)嵴細胞向DRG或交感神經(jīng)節(jié)的分化。關(guān)于CXCR4與交感芽生的研究尚不多見。本研究旨在探索CXCR4對神經(jīng)損傷后交感芽生形成的作用及其與NGF的關(guān)系。

材 料 和 方 法

動物模型與分組 SD大鼠56只,體質(zhì)量200～250 g,飼養(yǎng)于新華醫(yī)院SPF動物房,室溫23 ℃,濕度50%～60%,每日光照12 h,自由獲取水和飼料,實驗前大鼠適應(yīng)性喂養(yǎng)3天,所有動物實驗均符合動物疼痛研究倫理學(xué)的要求。大鼠隨機分為4組:空白對照組(n=16)、AMD3100實驗組(n=16)、AMD3100對照組(n=8)和對照組(n=16)。AMD3100實驗組和對照組大鼠腹腔注射10%水合氯醛(0.3 mL/100 g)麻醉后,同時行鞘內(nèi)置管術(shù)和脊神經(jīng)結(jié)扎(spinal nerve ligation,SNL),術(shù)后即刻AMD3100實驗組給予AMD3100(0.5 μg/μL,10 μL),對照組予以生理鹽水10 μL,兩組大鼠術(shù)后第2天行利多卡因試驗,置管成功大鼠繼續(xù)實驗。AMD3100實驗組、AMD3100對照組和對照組大鼠在術(shù)后第3、5、7、10、14天(d3、d5、d7、d14)檢測機械痛閾(mechanical withdrawal threshold,MWT),并在d3對AMD3100實驗組和對照組大鼠取材行RT-PCR檢測NGF,d7取材行Western blot檢測NGF,d7和d14取材行免疫熒光觀察交感芽生。

MWT檢測 MWT由一位不了解分組情況且不參與數(shù)據(jù)處理的研究人員來實施。在基礎(chǔ)狀態(tài)和d3、d5、d7、d14檢測MWT,檢測時間限于上午9點至下午4點。檢測前大鼠置于獨立、避光暗盒至少15 min,MWT通過Von-frey纖維刺激大鼠術(shù)側(cè)足底,刺激5次中3次能引起大鼠明顯抬腿的最低纖維即為大鼠MWT,檢測最高值限定為26 g。

Western blot檢測 大鼠麻醉后置干冰上,快速取同側(cè)L5 DRG, -80 ℃凍存。取材完畢后,加裂解液提上清,加入上樣緩沖液,100 ℃加熱3 min變性。應(yīng)用十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳轉(zhuǎn)至PVDF膜,5%脫脂奶粉室溫封閉1 h,一抗于4 ℃封閉過夜(NGF,1∶2 000,美國CST公司;GAPDH,1∶1 000,碧云天生物技術(shù)研究所),對應(yīng)二抗室溫孵育1 h。增強化學(xué)發(fā)光法顯影,化學(xué)發(fā)光成像系統(tǒng)記錄影像,目標(biāo)蛋白質(zhì)與GAPDH標(biāo)準(zhǔn)化灰度值表示該蛋白質(zhì)相對表達量,重復(fù)3次。

免疫熒光大鼠腹腔麻醉,4%多聚甲醛PBS灌注后取同側(cè)L5 DRG,固定后蔗糖脫水。OTC包埋后于恒冷切片機中切片,切片厚度為15 μm,切片時每隔2張取1張。檢測時,樣本復(fù)溫后置于5%驢血清室溫封閉1 h,TH抗體(1∶1 000,美國Pel-freeze公司)4 ℃孵育48 h,沖洗后熒光二抗37 ℃孵育1 h。采用萊卡DMI-3000B型倒置熒光顯微鏡觀察樣本,TH陽性纖維包繞神經(jīng)元50%的周長定義為TH陽性細胞[9]。在20倍物鏡下隨機選擇6個視野,分別記錄每個視野下TH陽性細胞數(shù)量。

RT-PCR檢測 大鼠腹腔麻醉,無菌下快速取同側(cè)L5 DRG置于Trizol中,-80 ℃凍存。NGF引物序列:5′-CCTTCAACAGGACTCACA-GG-3′ (正向),5′-TCTCCAACCCACACACTGAC-3′ (反向)。GAPDH引物序列:5′-GACATGCCG-CCTGGAGAAAC-3′ (正向),5′-AGCCCAGGAT-GCCCTTTAGT-3′ (反向)。應(yīng)用PrimeScript RT Master MIX試劑盒(日本TaKaRa公司)逆轉(zhuǎn)錄為cDNA,采用SYBR Premix Ex TaqTM試劑盒(日本TaKaRa公司)擴增DNA,NGF相較于GAPDH的表達采用2△△CT法,同時檢測熔解曲線確定擴增的特異性。

結(jié) 果

大鼠MWT改變AMD3100對照組和空白對照組大鼠在各個時間點的MWT均無顯著差異。AMD3100實驗組和對照組大鼠在d3的MWT均顯著降低,兩組之間差異無統(tǒng)計學(xué)意義。實驗組大鼠MWT在d5較對照組顯著緩解(t=2.8,P<0.01),并維持到d14(t=3.5,P<0.01)。然而,兩組大鼠MWT之差約為3g(圖1)。

交感芽生與藍狀結(jié)構(gòu) 在空白對照組中無TH陽性纖維。SNL術(shù)后,大鼠DRG中交感芽生在d7顯著增多,在d14有所減少但依然存在。在AMD3100實驗組中,抑制CXCR4后大鼠DRG交感芽生在d7較對照組減少,且兩組TH陽性細胞數(shù)的差異在d7有統(tǒng)計學(xué)意義(t=3.38,P<0.01),而在d14無統(tǒng)計學(xué)意義(P>0.05,圖2)。

NGF的RNA和蛋白質(zhì)水平 建立模型后d7,DRG中NGF的RNA和蛋白質(zhì)水平均較空白對照組顯著升高(P<0.01),且AMD3100實驗組大鼠DRG中NGF表達均較對照組顯著降低(P<0.01,圖3、4)。

討 論

本研究發(fā)現(xiàn)CXCR4在SNL大鼠的MWT和交感芽生中都起著重要的作用。雖然有研究發(fā)現(xiàn)鞘內(nèi)抑制CXCR4可以緩解疼痛達2～4h,但AMD3100抑制CXCR4的效果無法持續(xù)24 h[10-11],但每日鞘內(nèi)給予ADM3100仍然是一種長期抑制CXCR4的干預(yù)手段[12]。在本研究中,大鼠在脊神經(jīng)結(jié)扎后出現(xiàn)疼痛行為和交感芽生,且CXCR4表達上調(diào),提示模型建立成功且穩(wěn)定。因疼痛模型的穩(wěn)定與良好的對照,我們認為AMD3100實驗組和對照組的差異是由鞘內(nèi)注射AMD3100引起的。研究結(jié)果中行為學(xué)的顯著性差異提示每日鞘內(nèi)抑制CXCR4可以降低大鼠痛閾,且在停止給予AMD3100后,鎮(zhèn)痛效果仍能維持7天。

There was no significant difference in mechanical withdrawal threshold between AMD3100 control group and naive group in all time points. There was no significant difference in mechanical withdrawal threshold between AMD3100 group and control group at the baseline and d3. The MWT in AMD3100 treatment group was significantly improved compared with those in control group at d5 (P<0.01), d7 (P<0.001), d10 (P<0.05) and d14 (P<0.01).

圖1 SNL術(shù)后各組大鼠在不同時間點MWT的變化

Fig 1 MWT after SNL of rats in different groups at different time points

電生理研究認為藍狀結(jié)構(gòu)是連接疼痛和交感系統(tǒng)的關(guān)鍵[4, 13],但其在疼痛行為中確切的功能至今仍是未知的。在許多研究中,干預(yù)措施包括注射曲安奈德和敲除IL-6都能減少藍狀結(jié)構(gòu),并能緩解疼痛[7, 14]。然而,至今尚無確切的研究證明其痛閾緩解是因為藍狀結(jié)構(gòu)減少。在Kim等[15]的研究中,疼痛大鼠對酚妥拉明的敏感性與藍狀結(jié)構(gòu)的密度無明顯相關(guān)性,在Pertin等[9]的研究中,早期的交感神經(jīng)毀損也不能緩解大鼠建立疼痛模型后的MWT,上述研究提示交感芽生在疼痛中并不起決定性的作用。在本研究中,術(shù)后14天藍狀結(jié)構(gòu)的數(shù)量和交感芽生的密度顯著少于術(shù)后7天,與文獻[16]中術(shù)后28天藍狀結(jié)構(gòu)少于術(shù)后7天的結(jié)果相符。研究發(fā)現(xiàn)早期的化學(xué)交感毀損不能改善保留坐骨神經(jīng)結(jié)扎的大鼠術(shù)后28天的疼痛行為[9]。在本研究中,與術(shù)后7天相比,術(shù)后14天的疼痛行為并沒有因為藍狀結(jié)構(gòu)的減少而改善,這提示了交感芽生和藍狀結(jié)構(gòu)在SNL大鼠術(shù)后14天的疼痛行為中并未起到?jīng)Q定性作用。

Immunofluorescence of TH in DRG in blank control group atd7 (A), AMD 3100 control group at d7 (B), control group at d7 (C), AMD3100 treatment group at d7 (d), control group at d14 (E), and AMD3100 group at d14 (F). G: The feature of the neuron wrapped by TH positive fibers at d14. The arrows in these figures indicated sympathetic sprouting, and the arrow heads indicated the basket-like structures. H: A significant difference in the numbers of neurons wrapped by TH positive fibers between two groups at d7 (t=3.38,P<0.01). A-G: Calibration bar=100 μm.

圖2 SNL術(shù)后大鼠DRG中的藍狀結(jié)構(gòu)與交感芽生

Fig 2 Sympathetic sprouting and basket-like structures in DRG of rats after SNL

The mRNA expression of NGF in the control group significantly increased compared to the blank control group (P<0.01). There was a significant difference in the mRNA expression of NGF between AMD3100 treatment group and control group (P<0.01).

圖3 SNL術(shù)后d3大鼠DGR中NGF的mRNA水平

Fig 3 mRNA levels of NGF in DRG of rats at d3 after SNL

The grayscale of NGF band was defined as ratio of the grayscale of NGF and GAPDH, and the blot in the blank control group was regarded as a standardized control. The protein expression of NGF significantly increased atd7 after SNL (P<0.01). There was a significant difference in the protein expression of NGF between the AMD3100 treatment group and control group at d7 (P<0.01).

圖4 SNL術(shù)后d7大鼠DGR中NGF的蛋白質(zhì)水平

Fig 4 Protein levels of NGF in DRG of rats at d7 after SNL

免疫細胞在神經(jīng)病理痛的發(fā)生和維持中起著重要的作用[7],巨噬細胞和T細胞均與交感芽生緊密相關(guān),且缺乏IL-6的小鼠表達交感芽生減少,這提示可能是巨噬細胞釋放的炎性因子參與交感芽生的發(fā)生和維持。研究提示全身或局部注射皮質(zhì)醇藥物能減少交感芽生,這可能是由于抑制了DRG中的炎性反應(yīng)。在本研究中,我們發(fā)現(xiàn)持續(xù)抑制CXCR4在術(shù)后7天能顯著減少藍狀結(jié)構(gòu)數(shù)量,這提示CXCL12/CXCR4參與了交感芽生的過程。然而,此現(xiàn)象在術(shù)后14天并不明顯,這可能是由于術(shù)后14天藍狀結(jié)構(gòu)數(shù)量較少。因此我們認為藍狀結(jié)構(gòu)的減少并不是改善疼痛行為最關(guān)鍵的原因,CXCL12/CXCR4可能通過獨立于藍狀結(jié)構(gòu)的其他機制改善大鼠疼痛。如在骨癌痛模型中,CXCL12/CXCR4可以通過興奮神經(jīng)元,激活星型膠質(zhì)細胞和小膠質(zhì)細胞參與疼痛的發(fā)生和維持[2]。

本研究中減少藍狀結(jié)構(gòu)的機制可能包括神經(jīng)細胞自發(fā)性放電和NGF的表達。研究顯示,通過抑制神經(jīng)元自發(fā)性放電可以減少藍狀結(jié)構(gòu)[17],而趨化因子可以直接興奮DRG神經(jīng)元[18],抑制CXCR4可能通過抑制神經(jīng)元電活動從而減少藍狀結(jié)構(gòu)的形成,神經(jīng)損傷后CXCL12的上調(diào)能激活鈉離子通道NAV1.8[19],而藍狀結(jié)構(gòu)的形成與NAV1.6相關(guān)[20],CXCL12/CXCR4是否通過鈉離子通道參與藍狀結(jié)構(gòu)的形成仍有待研究。另一方面,研究發(fā)現(xiàn)外源性NGF能顯著增加藍狀結(jié)構(gòu)的數(shù)量[5],本研究中抑制CXCR4后NGF的表達在RNA和蛋白質(zhì)水平均下調(diào),提示CXCR4使藍狀結(jié)構(gòu)減少可能和NGF有關(guān)。已知DRG中衛(wèi)星膠質(zhì)細胞可以分泌NGF[21],且衛(wèi)星膠質(zhì)細胞和NGF的受體P75也有緊密的關(guān)系[6]。

本研究存在一定的局限性,尚不能確認大鼠疼痛行為改善是通過持續(xù)抑制CXCR4而減少藍狀結(jié)構(gòu)的機制,也無法明確CXCR4調(diào)節(jié)NGF的機制,在疼痛模型中,NGF、藍狀結(jié)構(gòu)、衛(wèi)星膠質(zhì)細胞的機制有待于進一步研究。綜上所述,抑制CXCR4能改善大鼠SNL術(shù)后d5到d14的疼痛行為,并且能減少d7的藍狀結(jié)構(gòu)數(shù)量,其機制可能與抑制NGF表達有關(guān)。

[1] ROCHA RDE O,TEIXEIRA MJ,YENA LT,etal. Thoracic sympathetic block for the treatment of complex regional pain syndrome type I: A double-blind randomized controlled study [J].Pain, 2014,155(11): 2274-2281.

[2] O′CONNELL NE,WAND BM,GIBSON W,etal. Local anaesthetic sympathetic blockade for complex regional pain syndrome [J].CochraneDatabaseSystRev, 2016,7:7CD004598.

[3] DWORKIN RH,O′CONNOR AB,KENT J,etal. Interventional management of neuropathic pain: NeuPSIG recommendations [J].Pain, 2013,154(11):2249-2261.

[4] XIE W,STRONG JA,MAO J,etal. Highly localized interactions between sensory neurons and sprouting sympathetic fibers observed in a transgenic tyrosine hydroxylase reporter mouse [J].MolPain, 2011,7: 53.

[5] JONES MG,MUNSON JB,THOMPON SW. A role for nerve growth factor in sympathetic sprouting in rat dorsal root ganglia [J].Pain, 1999,79(1):21-29.

[6] WALSH GS,KROL KM,KAWAJA MD. Absence of the p75 neurotrophin receptor alters the pattern of sympathosensory sprouting in the trigeminal ganglia of mice overexpressing nerve growth factor [J].JNeurosci, 1999,19(1):258-273.

[7] SCHOLZ J,WOOLF CJ. The neuropathic pain triad: neurons,immune cells and glia [J].NatNeurosci, 2007,10(11):1361-1368.

[8] DUBOVY P,KLUSAKOVA I,SVIZENSKA I,etal. Spatio-temporal changes of SDF1 and its CXCR4 receptor in the dorsal root ganglia following unilateral sciatic nerve injury as a model of neuropathic pain [J].HistochemCellBiol, 2010,133(3):323-337.

[9] PERTIN M,ALLCHORNE AJ,BEGGAH AT,etal. Delayed sympathetic dependence in the spared nerve injury (SNI) model of neuropathic pain [J].MolPain, 2007,3: 21.

[10] MENICHELLA DM,ABDELHAK B,REN D,etal. CXCR4 chemokine receptor signaling mediates pain in diabetic neuropathy [J].MolPain, 2014,10: 42.

[11] WILSON NM,JUNG H,RIPSCH MS,etal. CXCR4 signaling mediates morphine-induced tactile hyperalgesia [J].BrainBehavImmun, 2011,25(3):565-573.

[12] SHEN W,HU XM,LIU YN,etal. CXCL12 in astrocytes contributes to bone cancer pain through CXCR4-mediated neuronal sensitization and glial activation in rat spinal cord [J].JNeuroinflammation, 2014,11: 75.

[13] MCLACHLAN EM,JANIG W,DEVOR M,etal. Peripheral nerve injury triggers noradrenergic sprouting within dorsal root ganglia [J].Nature, 1993,363(6429):543-546.

[14] LI JY,XIE W,STRONG JA,etal. Mechanical hypersensitivity,sympathetic sprouting,and glial activation are attenuated by local injection of corticosteroid near the lumbar ganglion in a rat model of neuropathic pain [J].RegAnesthPainMed, 2011,36(1):56-62.

[15] KIM HJ,NA HS,SUNG B,etal. Is sympathetic sprouting in the dorsal root ganglia responsible for the production of neuropathic pain in a rat model? [J].NeurosciLett, 1999,269(2):103-106.

[16] LEE BH,YOON YW,CHUNG K,etal. Comparison of sympathetic sprouting in sensory ganglia in three animal models of neuropathic pain [J].ExpBrainRes, 1998,120(4):432-438.

[17] ZHANG JM,LI H,MUNIR MA. Decreasing sympathetic sprouting in pathologic sensory ganglia: a new mechanism for treating neuropathic pain using lidocaine [J].Pain, 2004,109(1-2):143-149.

[18] DAWES JM,MCMAHON SB.Chemokines as peripheral pain mediators [J].NeurosciLett, 2013,557 Pt A:1-8.

[19] YANG F,SUN W,YANNG Y,etal. SDF1-CXCR4 signaling contributes to persistent pain and hypersensitivity via regulating excitability of primary nociceptive neurons: involvement of ERK-dependent Nav1.8 up-regulation [J].JNeuroinflammation, 2015,12: 219.

[20] XIE W,STRONG JA,ZHANG JM. Local knockdown of the NaV1.6 sodium channel reduces pain behaviors,sensory neuron excitability,and sympathetic sprouting in rat models of neuropathic pain [J].Neuroscience, 2015,291: 317-330.

[21] CHENG CF,CHENG JK,CHEN CY,etal. Mirror-image pain is mediated by nerve growth factor produced from tumor necrosis factor alpha-activated satellite glia after peripheral nerve injury [J].Pain, 2014,155(5):906-920.

Effect of CXCR4 on sympathetic sprouting in dorsal root ganglion of rats

YANG Yue-cheng1, MA Bing-jie2, MA ke2 △

(1DepartmentofAnesthesiology,FudanUniversityShanghaiCancerCenter,Shanghai200032,China;2DepartmentofPainManagement,XinhuaHospital,SchoolofMedicine,ShanghaiJiaoTongUniversity,Shanghai200092,China)

Objective To explore the role of CXCR4 in sympathetic sprouting in dorsal root ganglion (DRG) after spinal nerve ligation (SNL) in rats. Methods Rats were randomly divided into AMD3100 treatment group (n=16), control group (n=16),AMD3100 control group (n=8) and naive group (n=16). SNL was performed in the rats in AMD3100 treatment group and control group. AMD3100, the inhibitor of CXCR4, was injected intrathecally into the rats of AMD3100 treatment group 1-7 days after SNL. Mechanical withdrawal threshold (MWT) was detected at d3, d5, d7, d10 and d14. Real-time PCR and Western blot were applied to detect the levels of RNA and protein of nerve growth factor (NGF), respectively. Sympathetic sprouting in DRG was detected by immunofluorescence at d7 and d14. Results The MWT of rats in AMD3100 treatment group was significantly improved compared with which in control group from d5 to d14. After the inhibition of CXCR4, sympathetic sprouting in DRG was dramatically decreased compared with which in control group at d7, but not at d14. In addition, The RNA and protein levels of rats in AMD3100 treatment group was significantly decreased compared with which of control group. Conclusions The inhibition of CXCR4 could not only improved pain behavior from d5 to d14, but also decreased basket-like structures at d7 after SNL.

sympathetic sprouting; chemokines; dorsal root ganglion; rat

國家自然科學(xué)基金面上項目(81371246)

R441.1

A

10.3969/j.issn.1672-8467.2016.06.005

2016-03-07;編輯:段佳)

△Corresponding author E-mail:marke72@163.com

*This work was supported by the General Program of National Natural Science Foundation of China (81371246).