胶质细胞和促炎性细胞因子参与鞘内注射血小板活化因子诱发的触诱发痛和热痛敏

目的:探讨脊髓胶质细胞、促炎性细胞因子TNF-α和IL-1β以及NF-κB通路在鞘内注射血小板活化因子(PAF)诱发大鼠痛敏中的作用。方法:鞘内置管成功的雄性Sprague-Dawley大鼠64只随机分为6组:人工脑脊液(artificial cerebral spinal fluid,ACSF),对照组(n=16),鞘内注射ACSF 10μl;PAF组(n=16),鞘内注射PAF 10μg,溶解于10μl人工脑脊液;二甲基亚砜(DMSO)对照组(n=8),腹腔注射0.1%DMSO生理盐水2 ml;SC-514(10 mg/kg)组Ⅰ,SC-514(50 mg/kg)组Ⅱ和SC-514(100 mg/kg)组Ⅲ(n=16)。SC-514溶解于2 ml 0.1%DMSO生理盐水。DMSO组和SC-514组在鞘内注射PAF前2 h分别腹腔注射给药。鞘内给药后测机械缩爪阈值和热缩爪潜伏期,5 h后免疫组织化学染色检测腰段脊髓GFAP和OX-42的表达,ELISA检测脊髓TNF-α和IL-1β表达。结果:鞘内注射PAF激活大鼠脊髓星形胶质细胞和小胶质细胞,GFAP和OX-42标记免疫阳性反应增强,脊髓TNF-α和IL-1β表达增强;IKKβ抑制剂SC-514剂量依赖性减轻PAF诱发的触觉痛敏和热痛敏,并抑制TNF-α和IL-1β的表达增强。结论:鞘内注射PAF诱发大鼠触诱发痛和热痛敏,脊髓胶质细胞和NF-κB通路的激活以及促炎性细胞因子TNF-α和IL-1β表达增强可能参与其机制。     

炎性痛小鼠脑内3种环氧合酶亚型的变化及不同选择性环氧合酶抑制剂的镇痛效应比较

目的:比较炎性疼痛小鼠脑内环氧合酶1、环氧合酶2及环氧合酶3的表达变化,以及选择性环氧合酶抑制剂单独或联合应用后的镇痛效应。方法:实验于2004-03-01/12-20在第四军医大学药理教研室完成。138只小鼠足底注射甲醛溶液诱导炎性痛。①用54只小鼠放射免疫分析及54只小鼠反转录聚合酶链反应评估脑环氧合酶1、环氧合酶2及环氧合酶3在甲醛溶液注射前、注射后1,12h,1,3,7,14,30,60d的变化(升高表示疼痛增强)。②30只小鼠随机分成5组,每组6只。对照组灌胃生理盐水0.3mL;SC-560组灌胃SC-560(环氧合酶1选择性抑制剂)300μg/kg;NS-398组灌胃NS-398(环氧合酶2选择性抑制剂)150μg/kg;吲哚美辛组灌胃吲哚美辛(低选择性环氧合酶抑制剂)300μg/kg;以上各组灌胃1次/d,连续2个月。NS-398+SC-560组前1个月灌胃NS-398300μg/kg,后1个月灌胃SC-560150μg/kg,1次/d。应用行为学测定各组动物在甲醛溶液注射前、注射后1,12h,1,3,7,14,30,60d对热痛刺激的反应时间(延长表示热痛敏感性下降)。结果:①甲醛溶液注射前、后环氧合酶的表达:108只小鼠环氧合酶2在注射后12h,3d升高显著(P<0.05),环氧合酶1在注射后14d,30d,60d升高显著(P<0.05)。在整个观察时限内环氧合酶3的表达无明显变化。②对热痛刺激的反应时间:注射甲醛溶液的30只小鼠均出现对热痛刺激反应时间的明显缩短。与对照组相比,NS-398+SC-560组反应时间明显较长。NS-398组动物在注射后1,12h,1,3d对热痛刺激反应时间明显延长。SC-560组动物在整个观察过程中均未出现热痛刺激反应时间的改变。吲哚美辛组对热痛刺激反应时间明显短于NS-398+SC-560组而长于SC-560组。结论:与单纯使用环氧合酶1或环氧合酶2抑制剂相比,早期应用环氧合酶2抑制剂结合后期应用环氧合酶1抑制剂对炎性痛有更好的镇痛效应。     

炎性痛小鼠脑内3种环氧合酶亚型的变化及不同选择性环氧合酶抑制剂的镇痛效应比较

目的：比较炎性疼痛小鼠脑内环氧合酶1、环氧合酶2及环氧合酶3的表达变化，以及选择性环氧合酶抑制剂单独或联合应用后的镇痛效应。方法：实验于2004—03—01／12—20在第四军医大学药理教研室完成。138只小鼠足底注射甲醛溶液诱导炎性痛。①用54只小鼠放射免疫分析及54只小鼠反转录聚合酶链反应评估脑环氧合酶1、环氧合酶2及环氧合酶3在甲醛溶液注射前、注射后1，12h，1，3，7，14，30，60d的变化(升高表示疼痛增强)。②30只小鼠随机分成5组，每组6只。对照组灌胃生理盐水0．3mL；SC-560组灌胃SC-560(环氧合酶1选择性抑制剂)300μg／kg；NS-398’组灌胃NS-398(环氧合酶2选择性抑制剂)150μg／kg；吲哚美辛组灌胃吲哚美辛(低选择性环氧合酶抑制剂)300μg／kg；以上各组灌胃1次／d，连续2个月。NS-398+SC-560组前1个月灌胃NS-398300μg／kg，后1个月灌胃SC-560150μg／kg，1次／d。应用行为学测定各组动物在甲醛溶液注射前、注射后1，12h，1，3，7，14，30，60d对热痛刺激的反应时间(延长表示热痛敏感性下降)。结果：①甲醛溶液注射前、后环氧合酶的表达：108只小鼠环氧合酶2在注射后12h，3d升高显著(P&;lt;0．05)，环氧合酶1在注射后14d，30d，60d升高显著(P&;lt;0．05)。在整个观察时限内环氧合酶3的表达无明显变化。②对热痛刺激的反应时间：注射甲醛溶液的30只小鼠均出现对热痛刺激反应时间的明显缩短。与对照组相比，NS-398+SC-560组反应时间明显较长。NS-398组动物在注射后1，12h，1，3d对热痛刺激反应时问明显延长。SC-560组动物在整个观察过程中均未出现热痛刺激反应时间的改变。吲哚美辛组对热痛刺激反应时间明显短于NS-398+SC-560组而长于SC-560组。结论：与单纯使用环氧合酶1或环氧合酶2抑制剂相比，早期应用环氧合酶2抑制剂结合后期应用环氧合酶1抑制剂对炎性痛有更好的镇痛效应。     

鞘内注射银杏内酯B对神经病理痛大鼠痛阈及脊髓c-fos表达的影响

目的:观察鞘内注射银杏内酯B(BN52021)对SNI神经病理痛大鼠痛阈及脊髓c-fos表达的影响,探讨脊髓血小板活化因子及其受体参与痛觉信号调节的可能机制.方法:鞘内置管后的SD大鼠24只随机等分为4组:假手术组(sham组),SNI组,SNI+DMSO对照组和SNI+BN52021组,建立SNI疼痛模型,手术后1,3,5,7,10和14d鞘内给药并测痛阈,第14d取大鼠腰段脊髓,冰冻切片,免疫组织化学染色检测Fos免疫阳性细胞.结果:SNI神经损伤大鼠机械缩爪阈明显降低(P<0.05),同侧脊髓背角浅层内Fos阳性神经元明显增多(P<0.05);鞘内应用银杏内酯B明显减少脊髓背角神经元c-fos的表达,同时伴有大鼠机械异常痛敏的减轻,各组大鼠辐射热缩爪潜伏期无明显差异.结论:鞘内注射银杏内酯B可减轻SNI大鼠机械异常痛敏,抑制神经损伤后脊髓背角c-fos的表达.     

鞘内注射氯胺酮对镜像痛大鼠热痛敏的影响及可能机制

目的：探讨氯胺酮鞘内注射对镜像痛大鼠热痛敏的影响及可能机制.方法：30只雄性SD大鼠建立大鼠坐骨神经镜像痛模型,随机分为3组（n=10）：Ⅰ组鞘内注射生理盐水,Ⅱ组鞘内注射氯胺酮25 μg,Ⅲ组鞘内注射氯胺酮50 μg,分别测定鞘内给药前（T1）、给药后2 h（T2）、4 h（T3）、6 h（T4）的大鼠热缩足反射潜伏期（TWL）、运动功能.每组大鼠在给药6h后处死,取出腰段脊髓背角,石蜡切片,采用免疫组化检测单核细胞趋化蛋白-1（MCP-1）的表达.结果：鞘内给药后,Ⅱ组、Ⅲ组在各时间点与Ⅰ组及给药前相比,两侧TWL明显延长,比较有显著差异（P＜0.01）.Ⅰ组MCP-1平均积分光密度值（IOD）表达明显高于Ⅱ组和Ⅲ组（P＜0.01）,鞘内注射氯胺酮后大鼠运动功能不受明显影响.结论：鞘内注射氯胺酮可抑制镜像痛大鼠的热痛觉过敏,该作用可能与抑制脊髓背角的MCP-1表达有关.     

鞘内注射μ、δ阿片受体激动剂对大鼠骨癌痛的影响

目的: 比较鞘内及RVM区注射MOR和DOR激动剂对大鼠骨癌痛及神经病理痛的痛行为学的影响，探讨MOR和DOR在骨癌痛发病机制中的作用。方法：雌性Wistar大鼠，随机分为14组：BCP+NS组、BCP+DAMGO（1、5、10μg）组、BCP+DPDPE（0.1、0.5、1μg）组、SNI+NS组、SNI+DAMGO（0.5、1、5μg）组、SNI+ DPDPE（0.1、0.5、1μg）组。注药20min后，观察不同剂量药物对大鼠触诱发痛及机械痛觉过敏反应的影响。结果：在骨癌痛大鼠，鞘内注射DAMGO 1μg 仅轻微升高大鼠双侧后爪对von fray的缩爪阈值；DAMGO 5、10μg可显著升高大鼠双侧后爪对von fray的缩爪阈值，明显缩短术侧后爪对针刺的缩爪持续时间（P＜0.05）；DPDPE（0.1、0.5、1μg）对骨癌痛大鼠双侧后爪的触诱发痛和术侧后爪的机械痛觉过敏反应呈剂量依赖性的抑制作用；在SNI模型大鼠，DAMGO和DPDPE均呈剂量依赖性的抑制大鼠的触诱发痛和机械痛觉过敏反应（P＜0.05）。结论: 鞘内给予μ、δ阿片受体选择性激动剂对大鼠骨癌痛及神经病理性疼痛均有明显的镇痛作用，所需的剂量在骨癌痛大于神经病理性疼痛。     

预先鞘内给予氯诺昔康对福尔马林致痛大鼠行为学的影响

目的：探讨预先鞘内给予氯诺昔康对福尔马林致痛大鼠行为学的影响。方法：（1）采用序贯法测定预先鞘内给予氯诺昔康对福尔马林致痛大鼠镇痛作用的ED如值。（2）预先鞘内给予氯诺昔康对福尔马林致痛大鼠行为学的影响：雄性SD大鼠24只，随机分为对照组与实验组（鞘内氯诺昔康组IT组），对照组又分为鞘内生理盐水组（NS组）与肌注氯诺昔康组（IM组）。NS组鞘内注射NS20μl后10min足底注射5％福尔马林100μl，IT组与IM组分别鞘内或肌注氯诺昔康120μg（根据ED50值及95％Cl确定）后10min足底注射5％福尔马林100μl。完成处理后，记录缩腿、舔爪时间。结果：（1）预先鞘内给予氯诺昔康对福尔马林致痛大鼠镇痛作用ED50值为114．6μg，95％Cl为103．0～127．4μg。（2）IT组第二相缩腿、舔爪累计时间显著短于NS组与IM组（P〈0．001）；IM组与NS组第一、第二相缩腿、舔爪累计时间无统计学差异（P〉0．05）。结论：预先鞘内给予小剂量氯诺昔康对福尔马林致痛大鼠有镇痛作用。     

N-甲基D-天门冬氨酸受体2B亚型反义寡核苷酸鞘内注射对骨癌痛模型大鼠痛行为的改善作用

目的:在大鼠骨癌痛模型上观察鞘内注射N-甲基-D-天冬氨酸受体2B亚基(NR2B)反义寡核苷酸的镇痛效果.方法:将含2 × 105个SHZ-88大鼠乳腺癌细胞注射到SD大鼠左侧股骨远端骨髓腔,制作骨癌痛模型(T组).同时设假手术组(S组,n=8).所有大鼠均于接种前1 d,接种后第3、5、7、10、14天检测痛行为学指标.骨癌痛模型制作成功的T组大鼠在接种后第14天,进一步分为鞘内注射反义寡核苷酸(TA组)、正义寡核苷酸(TS组)、生理盐水(TN组),分别鞘内注射相应剂寡核苷酸和溶媒,鞘内注射后第1、3、5天再进行行为学检测.并在最后一次行为学测定后将大鼠处死,取其L4～6脊髓组织,检测术侧的NR2BmRNA.结果:与S组和术前基础值相比.造模后第7天,T组大鼠自由行走痛行为评分显著增高(P＜0.05),机械痛缩足阈值(PWT)显著降低(P＜0.05);在鞘内给药后第3、5天,TA组大鼠自由行走痛行为评分显著降低(P＜0.05),PWT增高(P＜0.05);而TS组和TN组在鞘内注射前后无显著变化(P＞0.05);TA、TS组及TN组L4～6脊髓组织的NR2B mRNA表达均高于S组(P＜0.05,P＜0.01);鞘内注射后5 d,TA组L4～6脊髓组织的NR2B mRNA表达低于TS组和TN组降低(P＜0.01).结论:鞘内注射NR2B反义寡核苷酸能下调脊髓NR2B mRNA表达.有效改善骨癌痛大鼠的痛行为学反应.     

靶向抑制脊髓5-羟色胺能通路对大鼠骨癌痛的影响

目的:探讨靶向抑制脊髓5-羟色胺能通路对大鼠骨癌痛的影响。方法:雌性SD大鼠70只,体重160~180 g,随机分组。骨癌痛组大鼠于胫骨骨髓腔注射Walker 256大鼠乳腺癌细胞悬液10μl制备癌痛模型。假手术组于骨髓腔注射D-hank's液10μl。大鼠造模后第10天行鞘内置管,第14天鞘内注射生理盐水或5-羟色胺选择性神经毒素5,7-DHT。鞘内给药前1天和给药后5天每天测定机械痛阈。鞘内给药后第3天取脊髓背角组织采用高效液相色谱法检测5-羟色胺含量。大鼠测痛结束后行胫骨X线摄片,观察肿瘤导致的骨破坏程度。结果:X线片显示骨癌痛大鼠术侧胫骨可见肿瘤生长导致的骨皮质大片缺损。与鞘内给予生理盐水的骨癌痛组大鼠相比,骨癌痛组大鼠鞘内给予5,7-DHT后机械性痛觉超敏显著缓解(P<0.05),缩爪持续时间明显缩短(P<0.05)。与鞘内给予等体积生理盐水的大鼠相比,鞘内给予60μg的5,7-DHT可以显著的降低脊髓背角中5-羟色胺含量。结论:本实验证实了鞘内注射5,7-DHT选择性消除脊髓5-羟色胺可显著缓解骨癌痛大鼠的疼痛,说明脊髓背角中5-羟色胺含量的增加参与了大鼠骨癌痛的维持。     

Cav3.2通道对背根节慢性压迫痛大鼠脊髓CaMKⅡ表达的影响

目的:观察Cav3.2通道对背根节慢性压迫痛大鼠脊髓CaMKⅡ表达的影响,探讨Cav3.2-CaMKⅡ通路在神经病理性疼痛中的作用。方法:雄性SD大鼠60只,体重250±20 g,随机分为5组,每组12只(其中行为学实验8只,免疫印迹实验4只)。分别是正常对照组(C组);模型组(CCD组);生理盐水组(NS组);错义寡聚核苷酸组(MS-Cav3.2组);反义寡聚核苷酸组(AS-Cav3.2组)。分别于鞘内置管前(T1),鞘内置管后3 d(T2),鞘内给药后1 d(T3)、4 d(T4),CCD模型制备后5d(T5)、10 d(T6)、15 d(T7)检测大鼠机械缩腿阈值(mechanical withdrawal threshold,MWT)和热缩足潜伏期(thermal withdrawal latency,TWL)。并于CCD模型制备后5 d,各组取4只大鼠处死,取脊髓腰膨大,采用免疫印迹法检测Cav3.2及CaMKⅡ的表达。结果:与C组比较,CCD组大鼠在模型制备后第5 d、10 d、15 d时MWT及TWL均显著降低。鞘内注射NS和Cav3.2错义寡聚核苷酸对CCD大鼠MWT及TWL无影响,鞘内注射Cav3.2反义寡聚核苷酸可增加CCD大鼠MWT和TWL,减轻大鼠机械痛敏和热痛敏。C组大鼠脊髓Cav3.2和CaMKⅡ蛋白均有表达。大鼠在制备CCD模型后5 d Cav3.2及CaMKⅡ蛋白表达均显著增加。鞘内注射NS及Cav3.2错义寡聚核苷酸对Cav3.2及CaMKⅡ蛋白表达无影响,鞘内注射Cav3.2反义寡聚核苷酸则可显著抑制Cav3.2及CaMKⅡ蛋白的表达。结论:抑制脊髓Cav3.2通道的表达可降低慢性背根节压迫痛大鼠脊髓CaMKⅡ的表达。     

Development of tactile allodynia and thermal hyperalgesia by intrathecally administered platelet-activating factor in mice

Platelet-activating factor (PAF) is a potent inflammatory lipid mediator in peripheral tissues. However, its role in mediation of nociception in central nervous system is unknown. In the present study, whether PAF plays some role in pain transduction in the spinal cord was studied in mice. Intrathecal injection of PAF induced tactile pain, tactile allodynia at as low as 10 fg to 1 pg with a peak response at 100 fg, while lyso-PAF was without effect in the range of doses. Tactile allodynia induced by PAF was blocked by a PAF receptor antagonists, TCV-309, WEB 2086 and BN 50739. The expression of PAF receptor mRNA by RT-PCR was observed in DRG and spinal cord in mice. ATP P2X receptor antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5-triphosphate, NMDA receptor antagonist, MK 801 and nitric oxide synthetase inhibitor, 7-nitroindazole blocked the PAF-induced tactile allodynia. PAF-induced tactile allodynia and thermal hyperalgesia disappeared in neonatally capsaicin-treated adult mice, while tactile allodynia but not thermal hyperalgesia induced by intrathecally injected alpha,beta-methylene ATP, a P2X receptor agonist, was capsaicin-insensitive. The present study demonstrated that PAF is a potent inducer of tactile allodynia and thermal hyperalgesia at the level of the spinal cord. PAF-evoked tactile allodynia is suggested to be mediated by ATP and the following NMDA and NO cascade through capsaicin-sensitive fiber, different from exogenously injected alpha,beta-methylene ATP which is insensitive to capsaicin treatment.     

Glycinergic mediation of tactile allodynia induced by platelet-activating factor (PAF) through glutamate-NO-cyclic GMP signalling in spinal cord in mice

Our previous study showed that intrathecal (i.t.) injection of platelet-activating factor (PAF) induced tactile allodynia, suggesting that spinal PAF is a mediator of neuropathic pain. The present study further examined the spinal molecules participating in PAF-induced tactile allodynia in mice. I.t. injection of L-arginine, NO donor (5-amino-3-morpholinyl-1,2,3-oxadiazolium (SIN-1) or 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18)) or cGMP analog (8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate; pCPT-cGMP) induced tactile allodynia. PAF- and glutamate- but not SIN-1- or pCPT-cGMP-induced tactile allodynia was blocked by an NO synthase inhibitor. NO scavengers and guanylate cyclase inhibitors protected mice against the induction of allodynia by PAF, glutamate and SIN-1, but not by pCPT-cGMP. cGMP-dependent protein kinase (PKG) inhibitors blocked the allodynia induced by PAF, glutamate, SIN-1 and pCPT-cGMP. To identify signalling molecules through which PKG induces allodynia, glycine receptor alpha3 (GlyR alpha3) was knocked down by spinal transfection of siRNA for GlyR alpha3. A significant reduction of GlyR alpha3 expression in the spinal superficial layers of mice treated with GlyR alpha3 siRNA was confirmed by immunohistochemical and Western blotting analyses. Functional targeting of GlyR alpha3 was suggested by the loss of PGE(2)-induced thermal hyperalgesia and the enhancement of allodynia induced by bicuculline, a GABA(A) receptor antagonist in mice after GlyR alpha3 siRNA treatment. pCPT-cGMP, PAF, glutamate and SIN-1 all failed to induce allodynia after the knockdown of GlyR alpha3. These results suggest that the glutamate-NO-cGMP-PKG pathway in the spinal cord may be involved in the mechanism of PAF-induced tactile allodynia, and GlyR alpha3 could be a target molecule through which PKG induces allodynia.     

Cyclooxygenase-1 in the spinal cord plays an important role in postoperative pain

Cyclooxygenase-2 (COX-2) activity in the spinal cord plays a key role in sensitization to sensory stimuli during acute inflammation. In contrast, intrathecal administration of COX-2 specific inhibitors has minimal analgesic effects in an incisional model of postoperative pain. We investigated the role of COX isoforms in this model by examining the expression of COX-1 and the effect of intrathecal COX inhibitors. A 1cm longitudinal incision was made through skin, fascia and muscles of the plantar aspect of the left paw in male rats, and withdrawal threshold to von Frey filaments measured. Rats were perfused at 1, 2, 3, 5, and 7 days after incision, and COX-1 immunohistochemistry was performed on L3 to S2 spinal cord and gracile nucleus sections. Other rats received intrathecally the COX-1 preferring inhibitor, ketorolac, the specific COX-1 inhibitor, SC-560, the COX-2 inhibitor, NS-398 or vehicle 1 day after surgery. Withdrawal threshold was measured at intervals up to 5 days later. COX-1 immunoreactivity increased in glia in the ipsilateral L4-L6 spinal dorsal horn and ipsilateral gracile nucleus after incision. Mechanical allodynia peaked on postoperative day 1, and COX-1 immunoreactivity increased on day 1, peaked on day 2, and declined thereafter. Ketorolac and SC-560 dose-dependently increased withdrawal threshold in this model, but NS-398 had no effect. These results suggest that COX-1 plays an important role in spinal cord pain processing and sensitization after surgery. Increased COX-1 activity could precede the up-regulation of COX-1 protein, and spinally administered specific COX-1 inhibitors may be useful to treat postoperative pain.     

Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats

Neuropathic pain is often associated with the appearance of pain in regions not related to the injured nerve. One mechanism that may underlie neuropathic pain is abnormal, spontaneous afferent drive which may contribute to NMDA-mediated central sensitization by the actions of glutamate and by the non-opioid actions of spinal dynorphin. In the present study, injuries to lumbar or sacral spinal nerves elicited elevation in spinal dynorphin content which correlated temporally and spatially with signs of neuropathic pain. The increase in spinal dynorphin content was coincident with the onset of tactile allodynia and thermal hyperalgesia. Injury to the lumbar (L(5)/L(6)) spinal nerves produced elevated spinal dynorphin content in the ipsilateral dorsal spinal quadrant at the L(5) and L(6) spinal segments and in the segments immediately adjacent. Lumbar nerve injury elicited ipsilateral tactile allodynia and thermal hyperalgesia of the hindpaw. In contrast, S(2) spinal nerve ligation elicited elevated dynorphin content in sacral spinal segments and bilaterally in the caudal lumbar spinal cord. The behavioral consequences of S(2) spinal nerve ligation were also bilateral, with tactile allodynia and thermal hyperalgesia seen in both hindpaws. Application of lidocaine to the site of S(2) ligation blocked thermal hyperalgesia and tactile allodynia of the hindpaws suggesting that afferent drive was critical to maintenance of the pain state. Spinal injection of antiserum to dynorphin A((1-17)) and of MK-801 both blocked thermal hyperalgesia, but not tactile allodynia, of the hindpaw after S(2) ligation. These data suggest that the elevated spinal dynorphin content consequent to peripheral nerve injury may drive sensitization of the spinal cord, in part through dynorphin acting directly or indirectly on the NMDA receptor complex. Furthermore, extrasegmental increases in spinal dynorphin content may partly underlie the development of extraterritorial neuropathic pain.     

Allodynia and hyperalgesia in diabetic rats are mediated by GABA and depletion of spinal potassium-chloride co-transporters

Diabetic rats show behavioral indices of painful neuropathy that may model the human condition. Hyperalgesia during the formalin test in diabetic rats is accompanied by the apparently paradoxical decrease in spinal release of excitatory neurotransmitters and increase in the inhibitory neurotransmitter GABA. Decreased expression of the potassium-chloride co-transporter, KCC2, in the spinal cord promotes excitatory properties of GABA. We therefore measured spinal KCC2 expression and explored the role of the GABA(A) receptor in rats with painful diabetic neuropathy. KCC2 protein levels were significantly reduced in the spinal cord of diabetic rats, while levels of NKCC1 and the GABA(A) receptor were unchanged. Spinal delivery of the GABA(A) receptor antagonist bicuculline reduced formalin-evoked flinching in diabetic rats and also dose-dependently alleviated tactile allodynia. GABA(A) receptor-mediated rate-dependent depression of the spinal H reflex was absent in the spinal cord of diabetic rats. Control rats treated with the KCC2 blocker DIOA, mimicked diabetes by showing increased formalin-evoked flinching and diminished rate- dependent depression. The ability of bicuculline to alleviate allodynia and formalin-evoked hyperalgesia in diabetic rats is consistent with a reversal of the properties of GABA predicted by reduced spinal KCC2 and suggests that reduced KCC2 expression and increased GABA release contribute to spinally mediated hyperalgesia in diabetes.     

Intrathecal protease-activated receptor stimulation produces thermal hyperalgesia through spinal cyclooxygenase activity

Activation of protease-activated receptors (PARs) in non-neural tissue results in prostaglandin production. Because PARs are found in the spinal cord and increased prostaglandin release in the spinal cord causes thermal hyperalgesia, we hypothesized that activation of these spinal PARs would stimulate prostaglandin production and cause a cyclooxygenase-dependent thermal hyperalgesia. PARs were activated using either thrombin or peptide agonists derived from the four PAR subtypes, delivered to the lumbar spinal cord. Dialysis experiments were conducted in conscious, unrestrained rats using loop microdialysis probes placed in the lumbar intrathecal space. Intrathecal thrombin stimulated release of prostaglandin E (PGE)(2) but not aspartate or glutamate. Intrathecal delivery of the PAR 1-derived peptide SFLLRN-NH(2) and the PAR 2-derived peptide SLIGRL both stimulated PGE(2) release; PAR 3-derived TFRGAP and PAR 4-derived GYPGQV were inactive. Intrathecal thrombin had no effect upon formalin-induced flinching or tactile sensitivity but resulted in a thermal hyperalgesia. Intrathecal SFLLRN-NH(2) and SLIGRL both produced thermal hyperalgesia. Consistent with their effects on spinal PGE(2), hyperalgesia from these peptides was blocked by pretreatment with the cyclooxygenase inhibitor ibuprofen. SLIGRL-induced hyperalgesia was also blocked by the selective inhibitors SC 58,560 [5-(4-fluorophenyl)-1-[4-(methylsulfonyl)phenyl]-3-(trifluoromethyl)-1H-pyrazole; cyclooxygenase (COX) 1] and SC 58,125 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; COX 2]. These data indicate that activation of spinal PAR 2 and possibly PAR 1 results in the stimulation of the spinal cyclooxygenase cascade and a prostaglandin-dependent thermal hyperalgesia.     

Ethanol withdrawal-associated allodynia and hyperalgesia: age-dependent regulation by protein kinase C epsilon and gamma isoenzymes.

Ethanol (EtOH) withdrawal increases sensitivity to painful stimuli in adult rats. In this study, withdrawal from a single, acute administration of EtOH dose-dependently produced mechanical allodynia and thermal hyperalgesia in postnatal day 7 (P7) rats. In contrast, P21 rats exhibited earlier and more prolonged mechanical allodynia but not thermal hyperalgesia. For both P7 and P21 rats, blood and spinal cord EtOH levels peaked at 30 minutes after administration, with P7 rats achieving overall higher spinal cord concentrations. Protein kinase C (PKC) has been implicated in mediating pain responses. Inhibitory PKC- and gamma-specific peptides attenuated mechanical allodynia and thermal hyperalgesia in P7 rats, whereas only the PKCgamma inhibitor prevented mechanical allodynia in P21 rats. Immunoreactive PKC in dorsal root ganglion and PKCgamma in lumbar spinal cord increased at 6 hours after EtOH administration in P7 rats. In P21 rats, the density of PKC immunoreactivity remained unchanged, whereas the density of PKCgamma immunoreactivity increased and translocation occurred. These studies demonstrate developmental differences in neonatal nociceptive responses after withdrawal from acute EtOH and implicate a role for specific PKC isozymes in EtOH withdrawal-associated allodynia and hyperalgesia. PERSPECTIVE: This study examines age-specific nociceptive responses after ethanol exposure by using 2 different ages of rats. The results suggest that ethanol age-dependently alters sensitivity to mechanical and thermal stimuli via specific protein kinase C isozymes. These results begin to ascertain the mechanisms that produce abnormal pain after alcohol exposure.     

Nerve injury-induced tactile allodynia is mediated via ascending spinal dorsal column projections

Peripheral nerve injury produces signs of neuropathic pain including tactile allodynia and thermal hyperalgesia, sensory modalities which may be associated with different neuronal pathways. Studies of spinally-transected, nerve-injured rats have led to suggestions that thermal hyperalgesia may be mediated predominately through local spinal circuitry whereas ascending input to supraspinal sites is critical to the manifestation of tactile allodynia. Here, the nature of ascending spinal input mediating tactile allodynia was explored using selective spinal lesions. Male Sprague-Dawley rats received L(5)/L(6) spinal nerve ligation (SNL) and ipsilateral or contralateral (relative to the SNL side) lesions including spinal hemisections and bilateral and unilateral dorsal column lesions. The rats were maintained in a sling and monitored for tactile allodynia by measuring withdrawal thresholds to probing with von Frey filaments 24 h after the hemisection. Rats receiving dorsal column lesions demonstrated no motor deficits while rats receiving spinal hemisection showed paralysis of the paw which nevertheless responded to strong noxious stimulation. Spinal hemisection ipsilateral, but not contralateral, to SNL completely abolished tactile allodynia while maintaining spinal nocifensive reflexes to noxious pinch. Bilateral and ipsilateral dorsal column lesions blocked tactile allodynia while contralateral dorsal column lesions did not. Administration of lidocaine into the nucleus gracilis ipsilateral to SNL also blocked tactile allodynia, but did not alter thermal hyperalgesia in SNL rats or increase thermal nociceptive responses in sham-operated rats. Lidocaine microinjected into the contralateral nucleus gracilis produced no changes in responses to tactile or thermal stimuli in either group. These results indicate that tactile allodynia after peripheral nerve injury is dependent upon inputs to supraspinal sites. Furthermore, it is apparent that afferent signals interpreted as tactile allodynia course through the ipsilateral dorsal columns and are relayed through the nucleus gracilis. This neuronal pathway is consistent with the interpretation that tactile allodynia pursuant to peripheral nerve injury is transmitted to the central nervous system by means of large diameter, myelinated fibers.     

Spinal EP receptors mediating prostaglandin E2-induced mechanical hyperalgesia, thermal hyperalgesia, and touch-evoked allodynia in rats.

Intrathecal administration of prostaglandin E(2) (PGE(2)) produces mechanical hyperalgesia, thermal hyperalgesia, and touch-evoked allodynia in rats. Experiments were conducted to examine the effects of intrathecal administration of relatively selective PGE(2) receptor (EP receptor) agonists to establish which spinal EP receptors mediate these behavioral effects of spinally administered PGE(2). Administration of either sulprostone (EP(3) receptor agonist) or PGE(1) alcohol (EP(4) receptor agonist) produced marked mechanical and thermal hyperalgesia and touch-evoked allodynia. Neither 17-phenyl trinor PGE(2) (EP(1) receptor agonist) nor butaprost (EP(2) receptor agonist) produced any significant changes in behavioral response thresholds to mechanical or thermal stimuli. However, 17-phenyl trinor PGE(2) (EP(1) receptor agonist) did produce marked touch-evoked allodynia. These data suggest that in rats activation of spinal EP(3) and EP(4) receptors by PGE(2) is important for development of both mechanical and thermal hyperalgesia as well as for touch-evoked allodynia. PGE(2)-induced allodynia also appears to involve activation of spinal EP(1) receptors.