福尔马林致炎大鼠鞘内注射布托啡诺后脊髓背角NMDA受体的表达

研究背景与目的: 酒石酸布托啡诺是一种阿片类镇痛剂,对阿片受体具有激动/拮抗双重作用。其在临床和动物实验中的应用已有报道,但还未见鞘内应用于福尔吗林炎性痛大鼠模型的报道.另外在疼痛机制形成过程中,NMDA受体激活起着非常重要的作用。故我们假设鞘内注射酒石酸布托啡诺应用于福尔马林炎性痛大鼠时能产生明显的镇痛效果,其产生镇痛的机制可能是通过抑制NMDA受体表达来实现. 方法: 在大鼠的左后足掌面皮下注射5%福尔马林50μl致痛前30min,健康雄性SD大鼠接受生理盐水,低剂量布托啡诺（12.5μg）,高剂量布托啡诺（25μg）鞘内注射;对照组在福尔马林注射前不注射任何试剂,为了更进一步的研究,我们在设计中加入鞘内注射非选择性NMDA受体拮抗剂氯胺酮,也分别在福尔马林致痛前30min,鞘内注射低剂量（50μg）,高剂量（100μg）或低剂量（50μg）混合低剂量布托啡诺进行注射。与疼痛相关的行为学测定通过计算大鼠后爪理毛行为（后爪的离地、舔足/咬足）总计反应时间来确定,记录福尔马林诱导出注射后爪的双相理毛行为时间(在福尔马林注射后第1时相, 0-5 min; 第 2时相, 10-60 min ).所有大鼠均在注射后2h处死,用免疫组化法测定大鼠L5节段脊髓背角NMDA受体的表达。 结果：鞘内单独用布托啡诺当剂量增加到25μg时,以及鞘内低剂量布托啡诺和氯胺酮联合使用时都引起第1和2时相的后爪理毛行为的总计反应时间明显减少;其余的组别对福尔马林1和2时相都没有明显影响。鞘内单独用高剂量布托啡诺以及低剂量布托啡诺和氯胺酮联合处理都可以显著降低福尔马林致痛大鼠L5脊髓背角NMDA受体表达。 结论：本研究结果揭示鞘内用布托啡诺能够对福尔马林诱导的疼痛产生明显的镇痛作用,并具有剂量依赖性,其镇痛机制可能是通过抑制NMDA受体激活产生;不过具体NMDA受体抑制机制是否与鞘内布托啡诺激活к受体或μ受体有关还需要更进一步的研究来确定。     

多巴胺D1和谷氨酸NMDA受体参与调控异动症大鼠纹状体△FosB蛋白的表达

目的研究多巴胺D1和D2受体以及谷氨酸NMDA受体对△FosB蛋白表达水平的影响,由此探讨它们在左旋多巴诱导的异动症(Levodopa-induced Dyskinesia,LID)发病机制中的作用。方法对单侧黑质纹状体6-羟多巴胺(6-OHDA)损毁致帕金森病(PD)大鼠给予左旋多巴治疗28d制作LID模型,将大鼠分为7组:正常对照组、PD组、LID组、SCH23390治疗组、MK-801治疗组、raclopride治疗组和非LID组,分别观察各组行为学改变并进行异常不自主运动(abnormal involuntary movement,AI M)评分,用免疫组化和免疫印迹方法测定各组△FosB蛋白表达水平。结果多巴胺D1受体阻断剂SCH23390和谷氨酸NMDA受体阻断剂MK-801明显减轻LID大鼠行为学异常,而多巴胺D2受体阻断剂raclopride对异常不自主运动无明显影响;LID大鼠损毁侧纹状体△FosB蛋白表达较PD大鼠和非LID大鼠明显增加;与LID大鼠相比,MK-801和SCH23390均使△FosB蛋白表达显著下降,而raclopride没有这种效应;各组大鼠健侧纹状体△FosB蛋白表达水平没有显著差异。结论多巴胺D1受体和谷氨酸NMDA受体均通过参与调控纹状体△FosB蛋白的表达而影响大鼠LID的发生。     

NMDA受体拮抗剂剂量与脑缺血再灌注大鼠海马内源性神经干细胞的增殖

背景：脑缺血再灌注后,过度释放的兴奋性氨基酸可通过NMDA受体激活内源性神经干细胞,促使其增殖、分化,修复神经细胞,但同时也导致细胞内钙离子超载,引起神经细胞的损伤。通过控制NMDA受体活化的程度,刺激内源性神经干细胞激活的同时把损伤减到最小。 目的：观察NMDA受体拮抗剂MK-801质量浓度对脑缺血再灌注大鼠海马内源性神经干细胞增殖的影响。 方法：健康雄性SD大鼠54只随机数字表法分为对照组(不进行任何处理)、手术组(缺血模型制作)及不同剂量MK-801组。采用大鼠四条血管阻断方法(Pulsinelli-4VO法)制备大鼠全脑缺血再灌注模型。不同浓度MK-801组在模型制作前30 min按照0.2,0.4,0.6,0.8,1.0 ,1.2 mg/kg侧脑室注射MK-801。Western-blot、RT-PCR技术检测各组nestin蛋白及mRNA水平及表达。 结果与结论：MK-801剂量在0.8 mg/kg以下时,nestin mRNA及蛋白的表达差异无显著性意义(P > 0.05),呈现高表达;当剂量为0.8 mg/kg时,可明显抑制内源性神经干细胞增殖;在0.8mg/kg以上,对神经干细胞的抑制作用随着剂量的升高呈递增趋势;在1.2 mg/kg时,大鼠有躁动、共济失调等严重不良反应。nestin mRNA表达结果与蛋白表达趋势相吻合。说明MK-801的剂量与脑梗死后神经功能的修复有一定的关系,实验中0.6 mg/kg是一个比较合适的实验应用剂量,在此剂量下既可使MK-801减少兴奋性氨基酸对神经元的毒性作用,保护神经细胞,又使内源性神经干细胞的增殖不受较大影响。     

NMDA受体拮抗剂MK-801对新生大鼠室管膜下区胶质纤维酸性蛋白表达的影响

目的研究N-甲基-D-天冬氨酸(NMDA)受体拮抗剂MK-801对新生7 d大鼠室管膜下区(SVZ)胶质纤维酸性蛋白(GFAP)表达的影响。方法将40只新生SD大鼠分成对照组和MK-801组,各组按出生后(P)时间点再随机分成4个亚组:P7 d、P14 d、P21 d、P28 d组。新生大鼠均于生后第3天给药,MK-801组腹腔注射MK-801 10 mg.kg-1;对照组腹腔注射同量生理盐水。通过免疫组化学方法观察大鼠SVZ区GFAP阳性细胞数。结果①对照组GFAP阳性细胞数于P14 d开始增加,至P21 d达最大值;但P28 d时阳性细胞迅速下降;②MK-801组GFAP阳性细胞数与对照组相比,P7 d和P28 d无明显差异,P14 d(65.40±6.11)和P21 d(239.60±12.92)细胞数明显减少;而对照组P14 d(79.20±5.26)、P21 d(265.20±7.40)GFAP阳性细胞数明显增多,差异有显著统计学意义(P<0.01)。结论 NMDA受体拮抗剂MK-801对正常新生大鼠SVZ区GFAP的表达有抑制作用,能够抑制SVZ区神经干细胞的增殖和分化。     

MK-801对左旋多巴诱导异动症大鼠模型基底节区神经元活性的影响

目的:研究NMDA(N-methyl-D-aspartate)受体拮抗剂MK-801对左旋多巴诱导的帕金森病大鼠异常不自主运动行为学及其基底节区Fos表达的影响,探讨谷氨酸对长期左旋多巴治疗后帕金森病大鼠基底节输出通路活性改变的影响。方法:帕金森病大鼠给予左旋多巴治疗28d,第29d左旋多巴治疗前15min腹腔注射MK-801一次。观察其行为学变化,并用免疫组织化学方法观察尾壳核和苍白球Fos表达情况。结果:长期间断左旋多巴治疗后,帕金森病大鼠出现刻板运动和进行性增加的对侧旋转等行为学改变。提前用MK-801抑制了其刻板动作,而增加了对侧旋转行为。与左旋多巴治疗组比较,MK-80l治疗组损毁侧尾壳核区Fos表达明显增多而苍白球区Fos表达明显减少。结论:慢性间断性左旋多巴治疗诱导帕金森病大鼠异常不自主运动和旋转期缩短是帕金森病患者左旋多巴诱导异动症和疗效减退的啮齿类动物模型,谷氨酸在其发生机制中发挥重要作用,NMDA受体拮抗剂可能通过逆转直接通路的活动而抑制异动症和疗效减退的发生。     

N-Methyl-D-Aspartate受体拮抗剂对大鼠海马神经干细胞内源性激活的实验研究

目的研究N-Methyl-D-Aspartate(NMDA)受体拮抗剂MK-801对大鼠神经干细胞(NSC)内源性激活的作用.方法将不同年龄阶段(10 d、3月、10月)的SD大鼠分为实验组和对照组,实验组腹腔注射MK-801,对照组腹腔注射生理盐水,用免疫组织化学方法测定两组大鼠海马齿状回颗粒层(SGZ)的Brdu阳性细胞、Nestin阳性细胞表达数.结果2组的10 d幼鼠,Brdu阳性细胞、Nestin阳性细胞增殖均不明显,2组无显著差异(P＞0.05),而在3月成年鼠实验组Brdu阳性细胞7 d达高峰,Nestin阳性细胞11 d达高峰,较对照组增殖较明显(P＜0.05);实验组10月老龄鼠Brdu阳性细胞、Nestin阳性细胞表达更明显,并可持续到18 d,2组比较统计学有显著意义(P＜0.01).结论NMDA受体拮抗剂MK-801可明显促进3月成鼠、10月老年鼠脑中NSC的增殖、分化.     

左旋多巴诱导异动症大鼠模型纹状体棘状神经元电活动的研究

目的研究左旋多巴诱导异动症(levodopa induced-dyskinesias LID)大鼠模型纹状体棘状神经元的自发性电活动变化。方法帕金森病(Parkinson disease PD)大鼠模型应用左旋多巴(L-dopa)治疗28d诱发LID大鼠模型,29d L-dopa治疗前15min腹腔注射N-methyl-D-aspartic acid(NMDA)受体拮抗剂地佐环平(MK-801)1次。采用微电极细胞外记录技术检测大鼠模型纹状体棘状神经元的电生理活动。结果LID大鼠纹状体神经元的自发性电活动较对照组(P<0.01)及PD组(P<0.05)明显增多,MK-801治疗后显著减少(P<0.01)。结论纹状体棘状神经元的自发性电活动改变是LID的重要发病机制之一,NMDA受体拮抗剂可能通过逆转纹状体棘状神经元的电活动抑制LID的发生。     

地卓西平马来酸盐对雄性大鼠自发活动和前脉冲抑制及再认记忆的影响

目的 观察急性腹腔注射N-甲基-D-天冬氨酸(NMDA)受体拮抗剂地卓西平马来酸盐(MK-801)刘大鼠自发活动、感觉运动门控和物体再认记忆的影响,探讨MK-801模拟精神分裂症不同内表现型的适宜剂量.方法 成年雄性SD大鼠共104只,按照体质量采用分层随机化方法进行分组.(1)取SD大鼠48只,分为MK-801小剂量组、MK-801中剂量组、MK-801高剂量组和对照组,每组12只,观察不同剂量MK-801 (0.1、0.2、0.4 mg/kg)对大鼠自发活动的影响;(2)取SD大鼠32只,分为MK-801小剂量组、MK-801中剂量组、MK-801高剂量组和对照组,每组8只,观察不同剂量MK-801(0.1、0.2、0.4 mg/kg)对大鼠前脉冲抑制(PPI)的影响;(3)取SD大鼠24只,分为MK-801小剂量组和对照组,每组12只,观察小剂量MK-801 (0.1 mg/kg)对大鼠物体辨别测试的影响.结果 (1)中高剂量MK-801 (0.2,0.4 mg/kg)呈剂量依赖性诱导大鼠自发活动的增加以及PPI的损害(P＜0.05或P＜0.01),小剂量(0.1 mg/kg)组在自发活动和PPI上与对照组差异无统计学意义(P＞0.05).(2)小剂量MK-801组在物体辨别测试中对新物体的偏爱指数显著低于对照组[(57.79±10.66)％比(73.34±18.52)％,P＜0.05].结论 中高剂量MK-801可引起自发活动和感觉运动门控功能异常,而小剂量在排除运动系统异常的前提下可特异性地破坏大鼠的再认记忆,提示MK-801模拟精神分裂症的不同内表现型时应根据研究目的选择适宜剂量.     

代谢型谷氨酸受体1亚型选择性拮抗剂LY367385对抗大鼠缺血性脑水肿

目的研究代谢型谷氨酸受体1亚型（mGluR1）选择性拮抗剂LY367385对大鼠缺血性脑水肿的影响.方法Wistar雄性大鼠（280～320g）线栓法复制大脑中动脉闭塞（MCAO）脑缺血模型.动物随机分为生理盐水（NS）对照组、LY367385给药组及MK-801给药组,于MCAO后1min,侧脑室内注射NS或LY367385（500nmol）5μl,或腹腔注射MK-801（1mg/kg）.各组动物分别于MCAO 6、24h进行神经病学评分、脑含水量测定及脑梗死面积测定.结果LY367385明显改善大鼠脑缺血引起的神经症状,而MK-801在MCAO 6h增加神经病学评分.LY367385降低大鼠MCAO引起的脑含水量增加,MK-801无明显作用.LY367385及MK-801均降低脑梗死面积百分率,且LY367385作用优于MK-801.结论LY367385能对抗大鼠脑缺血性脑水肿,作用明显优于MK-801.     

地卓西平马来酸盐对抑郁大鼠行为学和前额叶BDNF表达的影响

目的观察地卓西平马来酸盐(MK-801)预处理对利血平诱导的抑郁模型大鼠抑郁行为的改善作用及大脑前额叶脑源性神经营养因子(BDNF)表达的影响。方法采用随机数字表法将32只成年雄性SD大鼠分为4组:对照组、利血平模型组、MK-801+利血平组和MK-801组,每组8只。MK-801+利血平组和MK-801组预先给予腹腔注射MK-801(0.3 mg/kg),对照组和利血平模型组腹腔注射相应体积的生理盐水。30 min后,利血平模型组和MK-801+利血平组腹腔注射利血平(4 mg/kg),对照组和MK-801组腹腔注射相同体积的乙酸溶剂。注射利血平48 h后利用强迫游泳实验观察大鼠的抑郁样行为表现,并在行为实验完成后处死大鼠,以酶联免疫吸附实验(ELISA)检测大脑前额叶BDNF的表达水平。结果在强迫游泳实验中,利血平模型组强迫游泳不动时间[(49.38±7.85)s]长于对照组[(15.59±5.43)s],差异有统计学意义(t=11.91,P0.01);MK-801+利血平组强迫游泳不动时间[(12.32±4.25)s]短于利血平模型组,差异有统计学意义(t=13.06,P0.05)。ELISA结果显示,利血平模型组前额叶BDNF表达水平[(10.09±0.88)ng/mL]低于对照组[(13.29±1.10)ng/mL],差异有统计学意义(t=6.44,P0.01);MK-801+利血平组大鼠前额叶的BDNF表达水平[(12.56±1.83)ng/mL]高于利血平模型组,差异有统计学意义(t=3.44,P0.05)。结论 MK-801可改善大鼠的抑郁样行为,其机制可能与调节脑内BDNF的表达有关。     

Alterations in spinal cord gene expression after hindpaw formalin injection

Heme oxygenase type 2 (HO-2) is an enzyme that uses heme as a substrate to produce iron, biliverdin, and carbon monoxide (CO). This enzyme participates in regulation of nociceptive signal transmission in spinal cord tissue. We set out to identify genes undergoing alterations in expression in a model of inflammatory pain and to determine whether HO-2 participates in that regulation. After the hindpaw injection of formalin in mice, we measured changes in expression of immediate early genes including c-fos, c-jun, jun B, nerve growth factor induced genes (NGFI-A and NGFI-B) and activity-related cytoskeletal protein (ARC) using real-time PCR. The mRNA corresponding to these genes increased in abundance in the first hour after formalin injection and then slowly declined. Changes in the abundance of prodynorphin, extracellular signal related kinases (ERK1 and ERK2) and N-methyl-D-aspartate (NMDA) receptor R1 subunit mRNA generally peaked between 8 and 12 hr after formalin injection. In HO-2 null mutant mice, the enhancement of expression was less for all genes studied. We went on to quantify gene expression in superficial dorsal horn tissue using laser capture microdissection followed by RNA amplification and real-time PCR. The results confirmed that the changes in gene expression were occurring in regions of the spinal cord involved in nociceptive processing. We conclude that the hindpaw injection of formalin leads to enhanced early and late expression of many genes in spinal cord dorsal horn tissue, and that this enhancement of expression relies to a degree on the presence of HO-2.     

Knock down of spinal NMDA receptors reduces NMDA and formalin evoked behaviors in rat

Chronic pain remains a major health problem afflicting an estimated 70% of patients with advanced cancer and inflammatory disorders, and up to 94% of patients with spinal cord injuries. Although progress has been made in the pharmacotherapy of chronic pain management, such as usage of adjuvant drugs and more effective methods of drug delivery, the mainstay of clinical pain management still depends on opiates. NMDA receptor activation, at the level of the spinal cord has been shown to play an important role in the facilitation of nociception (pain) in several animal models. Unfortunately, potent NMDA receptor antagonists, such as MK-801 and APV, have toxic properties and low safety margins that preclude their clinical use. We present evidence which indicates that the use of antisense oligonucleotides targeted to the NMDA-R1 receptor subunit (AS-NMDA-R1), but not sense, abolishes NMDA and formalin induced behaviors. Moreover, we demonstrate that spinal administration of AS-NMDA-R1 results in the abolition of staining for immunoreactive NMDA-R1 in the spinal cord. These data provide novel evidence supporting the feasibility of the use of gene therapy approaches in the management of neuropathic pain.     

Intrathecal injection of GluR6 antisense oligodeoxynucleotides alleviates acute inflammatory pain of rectum in rats

Objective

To investigate whether the kainate (KA) receptor subunit GluR6 is involved in the acute inflammatory pain.

Methods

Formalin was injected into the mucosa of rectum in Sprague-Dawley rats to induce visceral pain. The antisense oligodeoxynucleotides (ODNs) of GluR6 were injected once per day for 3 d before formalin injection, after which GluR6 protein level was examined by immunoblotting method. The change of visceral pain was also investigated.

Results

The expression of GluR6 in the spinal cord of rats increased after the formalin injection. Moreover, pre-treatment of GluR6 antisense ODNs could suppress GluR6 expression in the spinal cord of rats and decrease the scores of visceral pain at 45 min following formalin injection.

Conclusion

Kainate receptor subunit GluR6 plays an important role in the visceral pain induced by injection of formalin into the wall of rectum. GluR6 may serve as a potential target for the treatment of acute inflammatory visceral pain.     

Reduced GABA neurotransmission underlies hyperalgesia induced by repeated forced swimming stress

We determined if cutaneous hyperalgesia and pain-induced c-Fos overexpression in the spinal cord produced by repeated forced swimming (FS) stress in the rat were related to changes in GABA neurotransmission by studying spinal release of GABA and the effect of positive modulation of GABA-A receptors with diazepam. Male rats were daily submitted to 10-20 min of either forced swimming or sham swimming (SS) for 3 consecutive days. Two days later, spinal GABA release was estimated by in vivo microdialysis. In other set of rats, either diazepam (2 mg/kg, i.p.) or saline was administered 1h before either SS or FS and inflammatory nociception was assessed with the formalin test; it was followed by removal of lumbar spinal cords for c-Fos immunocytochemistry. Basal and pain-evoked release of GABA in the spinal cord was lower in FS rats than in SS rats. In contrast, pain scores during formalin test late phase and pain-induced c-Fos expression in laminae I-VI of ipsilateral dorsal horn were significantly higher in FS rats than in SS rats. In FS rats, diazepam did not have effect on GABA release but reduced pain scores and overexpression of c-Fos whereas flumazenil (0.1 mg/kg, i.p.), an antagonist of the benzodiazepine binding site, reversed these effects. When diazepam was given only 1h before the formalin test, it slightly but significantly reduced pain scores during late phase in FS rats but not in SS rats. In conclusion, stress-induced reduction in GABA-A receptor activation is involved in the development of FS stress-induced hyperalgesia.     

New role for spinal Stargazin in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated pain sensitization after inflammation

Considerable evidence has demonstrated that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blockade has an antinociceptive effect on inflammatory pain. Stargazin (STG) is the first transmembrane protein known to associate with AMPA receptors and regulate their synaptic targeting. However, it is not known whether STG is involved in inflammatory pain processing by regulating AMPA receptor function. In the present study, we investigated the effect of knockdown of spinal STG on AMPA receptor-mediated pain sensitization after inflammation. Antisense technology was employed to knock down STG expression in the spinal cord. We show that STG was expressed and interacted with AMPA receptor subunit GluR2 in the spinal cord. Intrathecally injected STG antisense oligodeoxyribonucleotide (ODN) specifically decreased STG expression in the lumbar spinal cord and dose dependently inhibited formalin-induced inflammatory pain in the second phase. More important was our finding for the first time that this specific STG antisense ODN diminished AMPA (0.1 mug)-enhanced formalin pain and lost its effect if pretreated with AMPA receptor antagonist CNQX. Our results demonstrate a new role for STG in central sensitization of inflammatory pain by interacting with AMPA receptors in the spinal cord.     

Spinal inhibition of p38 MAP kinase reduces inflammatory and neuropathic pain in male but not female mice: Sex-dependent microglial signaling in the spinal cord

Previous studies have shown that activation of p38 mitogen-activating kinase (MAPK) in spinal microglia participates in the generation of inflammatory and neuropathic pain in various rodent models. However, these studies focused on male mice to avoid confounding effects of the estrous cycle of females. Recent studies have shown that some spinal pro-inflammatory signaling such as Toll-like receptor 4-mediated signaling contributes to pain hypersensitivity only in male mice. In this study we investigated the distinct role of spinal p38 in inflammatory and neuropathic pain using a highly selective p38 inhibitor skepinone. Intrathecal injection of skepinone prevented formalin induced inflammatory pain in male but not female mice. Furthermore, intrathecal skepinone reduced chronic constriction injury (CCI) induced neuropathic pain (mechanical allodynia) in male mice on CCI-day 7 but not CCI-day 21. This male-dependent inhibition of neuropathic pain also occurred in rats following intrathecal skepinone. Nerve injury induced spinal p38 activation (phosphorylation) in CX3CR1-GFP⁺ microglia on CCI-day 7, and this activation was more prominent in male mice. In contrast, CCI induced comparable microgliosis and expression of the microglial markers CX3CR1 and IBA-1 in both sexes. Notably, intraperitoneal or local perineural administration of skepinone inhibited CCI-induced mechanical allodynia in both sexes of mice. Finally, skepinone only reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in lamina IIo neurons of spinal cord slices of males 7 days post CCI. Therefore, the sex-specific p38 activation and signaling is confined to the spinal cord in inflammatory and neuropathic pain conditions.     

Blocking NMDA receptors in the hippocampal dentate gyrus with AP5 produces analgesia in the formalin pain test.

The hippocampus is an integral component of the "limbic" system and, as such, may contribute to the negative affect and avoidance motivation experienced during pain. A substantial body of evidence indicates that the hippocampus processes pain-related information, that some hippocampal neurons respond exclusively to painful stimulation, and that long-term anatomical changes occur in dentate gyrus neurons, following noxious physical stimulation. NMDA receptor antagonist drugs administered to the hippocampus interfere with long-term potentiation, learning, and memory; these same drugs, when applied to the spinal cord, prevent the long-term neurophysiological changes caused by noxious physical stimulation. This experiment tested whether blocking NMDA receptors in the hippocampal formation reduces nociceptive behaviors in an animal model of persistent human pain. The competitive NMDA receptor antagonist AP5 was injected into the dentate gyrus of alert, unrestrained rats either 5 min before or 15 min following the administration of a subcutaneous injection of formalin irritant. Pain behaviors in both acute and tonic phases of the formalin test were significantly reduced by AP5 treatments. These results support the hypothesis that the hippocampal formation is involved in pain-related neural processing and that NMDA receptor-sensitive mechanisms in the hippocampus are involved in pain perception and/or the expression of pain-related behaviors.     

Intrathecal MK-801 inhibits formalin-induced activation of spinal p38-MAPK in rats

BACKGROUND: p38 mitogen-activated protein kinase (MAPK) plays an instrumental role in signal transduction from the cell surface to the nucleus, while subcutaneous injection of formalin can induce increased activation of spinal p38 MAPK. However, the mechanisms underlying the formalin-induced activation of spinal p38 MAPK in rats are unclear. OBJECTIVE: To observe the effects of N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801 on the formalin-induced activation of spinal p38 MAPK in rats. DESIGN, TIME AND SETTING: This randomized grouping, controlled animal experiment was performed at the Department of Physiology and Neurobiology, Shanxi Medical University between May and November 2007. MATERIALS: Forty eight healthy, adult Wistar rats were randomly divided into two groups: formalin + normal saline (n = 12) and formalin + MK-801 (n = 36). The formalin + MK-801 group was further divided into three subgroups according to the dosage of MK-801 (10, 50, and 100 nmol/L, 12 rats for each subgroup) METHODS: Following anesthesia, polyethylene tubing filled with sterile normal saline was implanted into the subarachnoid cavity. On postoperative days 5-8, rats received a 15 minute perfusion of normal saline or MK-801 (10, 50, and 100 nmol/L) in the formalin + normal saline and formalin + MK-801 groups, respectively, followed by formalin injection for the induction of nociceptive behavior. MAIN OUTCOME MEASURES: Detection of total p38 MAPK and of phosphorylated p38 MAPK by western Blot analysis; observation of nociceptive behaviors in the 1 hour after formalin injection. RESULTS: Western Blot analysis revealed that injection of formalin had no effect on total p38 MAPK expression but resulted in increased phosphorylation of p38 MAPK in the spinal cord. This increase was apparent after 5 minutes, peaked at 20 minutes, and thereafter descended and reached control levels after 45 minutes. Pretreatment with MK-801 (10, 50, 100 nmol/L) resulted in a dose-dependent reduction of p38 MAPK phosphorylation in the spinal cord, 20 minutes after formalin injection. Injection of 50 and 100 nmol/L MK-801 produced a suppression of the first phase of nociceptive behaviors, and all three doses of MK-801 resulted in dose-dependent inhibition of the second phase of nociceptive behaviors. CONCLUSION: The NMDA receptor participates in formalin-induced activation of p38 MAPK in the rat spinal cord.     

Maturation of NK1 receptor involvement in the nociceptive response to formalin

Administration of NK1 antagonists in adult animals attenuates the nociceptive response in the formalin test, indicating that the neurokinins and the NK1 receptor play a role in mediating this pain response. The number and distribution of NK1 receptors change dramatically during development, and the age at which they become involved in pain processing is not known. We examined the role of NK1 receptors in the formalin model in rats ranging in age between 3- and 21-days old. An NK1 antagonist, CP99,994, and its less active enantiomer CP100,263 were administered to the spinal cord (intrathecal), systemically (subcutaneous), or locally (intraplantar). Intrathecal administration of CP99,994, but not CP100,263, attenuated pain behaviors in the second phase of the formalin response in 14-day and 21-day old rats, but did not alter the pain response in 3-day or 10-day old rats. CP99,994 also reduced the expression of the c-fos protein in the superficial dorsal horn of 21-day old rats. Systemic and intraplantar injection of either CP99,994 or CP100,263 reduced the pain response to formalin in 3-day and 21-day old rats, suggesting a non-NK1 mediated mechanism of action. These results indicate that, within the spinal cord, NK1 receptors start to play a role in the pain response to formalin between 10 and 21 days. Moreover, analgesia induced by systemic or local injection of NK1 antagonists involves mechanisms other than, or in addition to, the NK1 receptor.     

Formalin- or adjuvant-induced peripheral inflammation increases neurokinin-1 receptor gene expression in the mouse

Substance P (SP) has been widely studied as a mediator of nociception. The release of SP from primary afferent neurons is increased during nociception, and SP activates neurokinin-1 (NK-1) receptors in the spinal cord and periphery. Nociception-evoked alterations in NK-1 receptor gene expression have been studied in rat models of persistent pain but have not been characterized in any murine models of peripheral inflammation. This study assessed behavioral responses and NK-1 receptor mRNA gene expression in mice receiving formalin or Freund's complete adjuvant (CFA) as an inflammatory stimulus. Mechanical withdrawal thresholds were measured before injection of formalin or CFA and hind paw licking/biting timed during the late-phase of the formalin response. Two and 24 hours after formalin or CFA injection, mechanical withdrawal thresholds were measured and the mice euthanized. Solution hybridization-nuclease protection assays were used to quantify NK-1 receptor mRNA levels. Results demonstrated that inflamed hind paws were edematous, and the withdrawal thresholds of the inflamed hind paws were significantly lower after formalin or CFA injection. Neurokinin-1 receptor mRNA levels in the ipsilateral dorsal spinal cords of mice were higher at 24 h after formalin injection or 4 days after CFA injection. These results confirm that mice are hyperalgesic at late time points after formalin or adjuvant injection when NK-1 receptor gene expression is elevated in the dorsal spinal cord. This supports the hypothesis that increased NK-1 receptor gene expression contributes to the development and maintenance of a hyperalgesic state.