血小板活化因子在脊髓水平的疼痛调控作用
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摘要
组织损伤和炎症常引起慢性疼痛，表现为自发性疼痛、痛觉过敏和触觉异常性痛敏。近十年来，对慢性疼痛的研究取得了较大的进展，痛觉传导通路的中枢敏化被认为是慢性疼痛产生和维持的重要原因。然而，中枢敏化的机制尚未完全阐明。研究表明，在中枢敏化机制中，大量的内源性神经介质参与了痛觉信号的传导和调控。
血小板活化因子(platelet-activating factor,PAF)是免疫细胞和炎症细胞产生的一种内源性磷脂，是一种强效的炎症介质。研究发现，PAF与中枢神经系统许多生理功能和病理生理过程有关。神经细胞生成PAF，同时，神经细胞也是PAF的靶细胞，PAF在神经信号传递中发挥重要作用。近几年有研究报道表明PAF可能参与神经中枢痛觉信号的传导和调控，而PAF在痛觉信号传导和调控中的作用及相应机制仍有待进一步研究。
前列腺素类物质(prostaglandins，PGs)在炎性疼痛中的作用已得到广泛认可，随着研究的深入，PGs在其它类型疼痛如神经病理性疼痛中的作用也受到关注。目前的研究认为，在慢性疼痛的产生及维持中，中枢及外周PGs均起作用。脊髓是调控痛觉信号的重要中枢，脊髓水平的PGs在疼痛的产生及维持中占有重要地位。体外实验表明，PAF可诱导培养的大鼠星形胶质细胞释放前列腺素E_2(prostaglandin E_2，PGE_2)，这一研究提示PGE_2及其限速酶环氧酶(cyclooxygenase，COX)可能参与神经中枢PAF调控痛觉信号的机制，但目前尚无相关研究的报道。
传统的观点认为，神经胶质细胞仅对神经元起着支持和营养作用，而没有细胞之间的信号传递功能，因而认为从外周传入的痛觉信号在脊髓水平的调控和放大以及痛敏状态的产生仅与神经元及其递质有关，而与神经胶质细胞无关。近些年，越来越多的研究证据表明胶质细胞与疼痛调控有着密切关系，特别是在如炎症和神经损伤等病理状态下。胶质细胞的激活及其促炎细胞因子的释放在病理状态下的痛觉过敏的产生和疼痛持续状态中发挥重要作用。脊髓和胶质细胞大量表达PAF受体，PAF可激活体外培养的胶质细胞，并诱导胶质细胞产生神经递质和促炎性细胞因子。以上研究结果提示，胶质细胞的激活及其促炎性细胞因子的释放可能参与神经中枢PAF调控痛觉信号的机制，而目前尚无相关报道。
目前的研究认为，PAF通过与其受体相结合而发挥生物学效应，PAF有二类受体结合位点，即低结合力的细胞膜表面结合位点和高结合力的细胞内位点。抑制PAF受体可减轻大鼠炎性痛反应，而PAF受体拮抗剂用于其它疼痛模型如神经病理性疼痛的研究尚未见报道。
为了进一步探讨PAF在痛觉信号调控中的作用及其机制，本课题进行了以下实验研究：(1)鞘内注射PAF对大鼠痛阈和脊髓环氧酶表达的影响，不同选择性环氧酶抑制剂对鞘内注射PAF所致大鼠痛敏的抗伤害效应；(2)鞘内注射PAF对脊髓胶质细胞活性和促炎性细胞因子表达的影响；(3)鞘内注射PAF受体拮抗剂对SNI神经痛大鼠镇痛作用的研究。
研究方法与结果
1．鞘内注射PAF对大鼠痛阈和脊髓环氧酶表达的影响
方法雄性Sprague-Dawley大鼠48只随机分为6组，每组8只，鞘内置管：人工脑脊液(artificial cerebral spinal fluid，ACSF)组，鞘内注射ACSF 10μl；PAF组，鞘内注射PAF 5μg；生理盐水(normal saline，NS)组，尾静脉注射1．5 ml生理盐水，10 min后鞘内注射PAF 5μg；SC-560(环氧酶1选择性抑制剂，150μg/kg)组，NS-398(环氧酶2选择性抑制剂，150μg/kg)组和吲哚美辛(低选择性环氧酶抑制剂，300μg/kg)组，SC-560、NS-398和吲哚美辛分别溶解于1．5 ml生理盐水，尾静脉注射给药，10 min后鞘内注射PAF 5μg。PAF均溶解于10μl ACSF。给药前测机械缩爪阈(PWMT)和热缩爪潜伏期(PWTL)基础值，ACSF组和PAF组鞘内给药15 min后测PWMT和PWTL值，连续5 h每15 min测定一次；生理盐水组、SC-560组、NS-398组和吲哚美辛组鞘内注射PAF 30 min后测PWMT和PWTL值。各组大鼠痛阈测定结束后立即处死，取L_(4-6)段脊髓组织，RT-PCR分析检测ACSF组和PAF组大鼠脊髓环氧酶COX-1、COX-2和COX-3 mRNA表达，放射免疫分析检测各组大鼠脊髓PGE_2含量。
结果(1)疼痛行为学观察：大鼠PWMT平均基础值为39．75±1．85 g，PAF组鞘内注射5μg PAF迅速诱发大鼠触觉异常痛敏(tactile allodynia)，鞘内给药后各测痛时间点PWMT值下降，与ACSF对照组比较差异有统计学意义(P＜0．05)，触觉异常痛敏在5 h观察周期内持续存在，高峰期持续约60 min；鞘内注射PAF诱发大鼠热痛敏(thermal hyperalgesia)，大鼠PWTL平均基础值为13．04±0．20 s，鞘内给药后各测痛时间点PWTL值下降(P＜0．05)，热痛敏持续时间和高峰期与触觉异常痛敏一致。环氧酶抑制剂NS-398和吲哚美辛减轻PAF诱发的触觉异常痛敏和热痛敏，与生理盐水对照组相比，NS-398组和吲哚美辛组PWMT和PWTL值升高(P＜0．05)，而SC-560对机械缩爪阈值和热缩爪潜伏期均无明显影响。(2)脊髓COX表达RT-PCR分析：ACSF组脊髓环氧酶COX-1、COX-2和COX-3在转录水平均有表达，COX-1和COX-3 mRNA表达水平低，COX-2表达水平高(P＜0．05)；PAF组鞘内注射PAF诱导COX-2表达增强(P＜0．05)，而COX-1和COX-3表达水平未见明显改变。(3)脊髓PGE_2放射免疫分析：与ACSF组比较，PAF组鞘内注射PAF 5 h后脊髓组织PGE_2含量升高(P＜0．05)；与生理盐水组比较，COX-2选择性抑制剂NS-398和非选择性环氧酶抑制剂吲哚美辛均抑制PAF诱发的PGE_2升高(P＜0．05)，其中，NS-398组PGE_2含量低于吲哚美辛组(P＜0．05)，而COX-1选择性抑制剂SC-560组对PAF引起的PGE_2含量升高无明显影响。
2．鞘内注射PAF对脊髓胶质细胞活性和促炎性细胞因子表达的影响
方法雄性Sprague-Dawley大鼠64只随机分为6组，鞘内置管：人工脑脊液(ACSF)组，16只，鞘内注射ACSF 10μl；PAF组，16只，鞘内注射PAF 5μg；DMSO(二甲基亚砜)组，8只，腹腔注射0．1％DMSO生理盐水2 ml，2 h后鞘内注射PAF 5μg；SC-514(10 mg/g)组，SC-514(50 mg/kg)组和SC-514(100 mg/kg)组，每组8只，SC-514分别溶解于2 ml 0．1％DMSO生理盐水，腹腔注射给药，2 h后鞘内注射PAF 5μg。PAF均溶解于10μl ACSF。给药前测PWMT和PWTL基础值，鞘内给药后5 min、15 min、30 min、45 min、60 min、90min和120min各时间点测PWMT和PWTL值。各组大鼠痛阈测定结束后立即处死，取L_(4-6)段脊髓组织，免疫组织化学染色检测ACSF组和PAF组大鼠腰段脊髓GFAP和OX-42的表达，ELISA检测各组大鼠脊髓TNF-a和IL-1β的表达。
结果(1)疼痛行为学观察：PAF组鞘内注射给药迅速诱发大鼠触觉异常痛敏和热痛敏，各测痛时间点PWMT和PWTL值均下降(P＜0．05)。IKKβ选择性抑制剂SC-514预处理明显减轻PAF诱发的触觉异常痛敏和热痛敏，其作用呈剂量依赖性增强，与DMSO对照组比较，SC-514不同剂量组各测痛时间点PWMT和PWTL值均升高(P＜0．05)。(2)胶质细胞活性免疫组织化学分析：PAF组鞘内注射PAF激活星形胶质细胞和小胶质细胞，大鼠脊髓灰质区可见分别以GFAP和OX-42标记的活化星形胶质细胞和小胶质细胞，活化评分达(++)，对照组活化评分为(—)；各组脊髓灰质背角GFAP和OX-42免疫阳性反应物的光密度值分析，PAF组高于ACSF对照组，差异有统计学意义(P＜0．05)。活化的胶质细胞分布于整个脊髓灰质，在脊髓背角中主要分布在第Ⅰ～Ⅱ层。(3)促炎性细胞因子TNF-a和IL-1β表达ELISA分析：与ACSF组比较，PAF组鞘内注射PAF 2 h后脊髓促炎性细胞因子TNF-a和IL-1β表达均增强(P＜0．05)，IKKβ选择性抑制剂SC-514预处理明显抑制PAF诱导的TNF-a和IL-1β的表达增强(P＜0．05)，其抑制作用呈剂量依赖性。
3．鞘内注射PAF受体拮抗剂对SNI神经痛大鼠镇痛作用的研究
方法雄性Sprague-Dawley大鼠64只，随机分为6组：SNI组，16只，制作SNI疼痛模型；假手术组(sham组)，16只：DMSO(二甲基亚砜)组，8只，制作SNI疼痛模型，鞘内注射0．1％DMSO生理盐水5μl；BN52021(100μg)组，BN50730(100μg)组和BN52021(100μg)+BN50730(100μg)组，每组各8只，制作SNI疼痛模型，鞘内注射给药，PAF受体拮抗剂BN52021和BN50730溶解于5μl 0．1％DMSO生理盐水。DMSO组和拮抗剂治疗组每天鞘内注射给药一次，连续给药7 d。第7 d测各组大鼠PWMT值，随后处死大鼠取L_(4-6)段脊髓，免疫组织化学染色检测各组大鼠脊髓c-fos表达，放射免疫分析检测sham组和SNI组大鼠脊髓PAF含量。
结果(1)疼痛行为学观察：SNI组神经损伤诱发大鼠触觉异常痛敏，术后第7 dPWMT值下降，与假手术组比较，差异有统计学意义(P＜0．05)；鞘内注射PAF受体拮抗剂BN52021和BN50730均减轻SNI神经损伤诱发的触觉异常痛敏，与SNI组及DMSO对照组比较，三个拮抗剂组PWMT值升高(P＜0．05)，BN52021和BN50730二者联合给药与单独用药比较镇痛效应更明显(P＜0．05)。(2)脊髓c-fos表达免疫组织化学分析：Fos蛋白表达分布较广泛，位于Ⅰ～Ⅴ层，其在Ⅰ～Ⅱ层分布较为密集，FLI(Fos-likeimmunoreactivity)阳性神经元为细胞核染成棕黄色的圆形或卵圆形颗粒，胞浆未着色。与sham组比较，SNI组手术侧脊髓背角内FLI阳性神经元数目明显增加(P＜0．05)，对侧背角内仅有少量FLI阳性神经元；三个拮抗剂给药组大鼠脊髓背角各层FLI阳性神经元数目与SNI组及DMSO对照组相比明显下降(P＜0．05)。(3)脊髓PAF放射免疫分析：SNI组神经损伤后第7d脊髓L_(4-6)节段组织PAF水平升高(1．183±0．098 pg/mg)，与假手术组(0．571±0．065 pg/mg)比较，差异有统计学意义(P＜0．05)。
4．统计学方法
所有数据以均数±标准差((?)±s)表示，采用SPSS11．0统计软件包分析。两组间比较采用非配对t检验，多组间比较采用单因素方差分析，继之以Fisher's PLSD多重比较法，P＜0．05为差异有统计学意义。
研究结论
1．鞘内注射PAF诱发大鼠触觉异常痛敏和辐射热痛敏，环氧酶COX-2的激活和表达增强以及PGE_2的产生参与其机制。
2．鞘内注射PAF诱发大鼠触觉异常痛敏和辐射热痛敏，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的表达增强参与其机制；IKKβ是PAF诱发的NF-κB通路激活以及促炎性细胞因子TNF-α和IL-1β表达增强的重要激酶。
3．脊髓内源性PAF参与SNI神经损伤大鼠痛敏的中枢调控，PAF的二类结合位点均介导痛觉信号的传导和调控，PAF受体拮抗剂可应用于治疗神经病理性疼痛。
Background
Tissue damage and inflammation usually induce persistant pain, characterized asspontaneous pain, hyperalgesia, tactile allodynia and so on. Within the past decade, there isgreat development in research on persistent pain.Central sensitization in pain signalconduction pathway is viewed as important mechanism in the creation and maintenance ofpersistent pain. However, so far the mechanism of central sensitization remainsunintelligible.Years of research indicates an amount of endogenous nerve mediators areimplicated in pain signal conduction and modulation in central sensitization.
Platelet-activating factor (PAF) is an endogenous phospholipid, described in a varietyof immune and inflammatory cells and a potent mediator that participates in inflammatoryresponses.In further researches, neural cells produce PAF and also are target cells of PAF.PAF is suggested to be implicated in a variety of physiological and pathological states incentral nervous system, and play a significant role in signal conduction in nervous system.Recently, evidence suggests that PAF may play a part in pain signal conduction andmodulation in central nervous system. At present, the role and mechanism of PAF acted inpain signal conduction and modulation remain to be established further.
The role of prostaglandins (PGs) as nociceptive mediators in inflammatory pain isaccepted extensively. Along with intensive research, the role of prostaglandins in other painmodels such as nuropathic pain is recognized gradually. Prostaglandins are thought to playan important role in the creation and maintenance of persistent pain both at peripheral sitesand in central nervous system. Spinal cord is the primary center in pain signal transmission,and prostaglandins in spinal cord act as important mediators in the creation andmaintenance of persistent pain. In vitro, PAF induces prostaglandin E_2 (PGE_2) release fromrat primary astrocytes.It suggests that prostaglandins and their rate-limiting enzymes (i.e.cyclooxygenases) may be involved in pain modulation mediated by PAF in central nervoussystem.There are no reports corresponding to this subject.
Traditionally, glial cells were simply thought to be housekeepers for neurons andprovide neurochemical precursors and energy sources to neurons, not to function in signaltransmission among neural cells, and it were neurons and their transmitters rather than glialcells that modulated and amplified signals from the periphery in spinal cord and wereresponsible for hyperalgesia. However, in the last several years, we have seen exponentiallyincreasing number of research articles suggesting the role of glial cells in pain controlespecially in conditions involving inflammation and nerve injury. Activation of glial cells(e.g. astrocytes and microglia) and proinflammatory cytokines release from glial cellscontribute importantly to the development of pain hypersensitivity under pathologicalconditions. PAF receptor mRNA and functional expression are abundant in spinal cord andglial cells. In addition, PAF receptors function to stimulate the production of transmittersand proinflammatory cytokines in cultured glial cells in vitro.These researches abovesuggest the activation of glial cells and release of proinflammatory cytokines may beimplicated in pain modulation mediated by PAF in central nervous system. There are noreports corresponding to this subject.
Evidence suggests that PAF exerts cellular actions through binding to its receptors.PAF has two kinds of binding sites which are high-affinity intracellular membrane binding site and low-affinity cell surface receptor. Inhibition of platelet-activating factor receptorsattenuates the inflammatory nociceptive response in rats.Whether antagonists for PAFreceptors attenuate nociceptive response in other pain model such as neuropathic pain hasnot been reported.
To further explore the role of PAF in nociceptive modulation and its mechanism, thepresent study is to investigate: (1) Effects of platelet-activating factor administeredintrathecally on pain behaviour and cyclooxygenase expression in spinal cord in rats,antinociceptive effects of different inhibitors of cyclooxygenases on pain behaviouralresponse induced by PAF. (2) Effects of platelet-activating factor administeredintrathecally on glial activation and proinflammatory cytokines in spinal cord in rats. (3)Antinociceptive effects of PAF receptor antagonists administered intrathecally in rats withspared nerve injury.
Methods and Results
1. Effects of platelet-activating factor administered intrathecally on painbehaviour and cyclooxygenase expression in spinal cord in rats
Methods Forty-eight Sprague-Dawley rats were randomly divided into six groups, andintrathecal PE-10 catheters were placed in the spinal subarachnoid space of rats: InACSF(artificial cerebral spinal fluid)group, rats were treated with 10μl artificial cerebralspinal fluid intrathecally. In PAF group, rats were treated with 5μg PAF which wasdissolved in 10μl artificial cerebral spinal fluid. In normal saline, SC-560, NS-398 andindomethacin group, all rats were pretreated with 1.5 ml normal saline, SC-560, NS-398and indomethacin intravenously through tail vein 10 minutes prior to intrathecal PAF(5μg,dissolved in 10μl ACSF) injection respectively. The doses of SC-560, NS-398 andindomethacin dissolved in 1.5 ml normal saline were 150μg/kg, 150μg/kg and 300μg/kg respectively. Baseline paw withdrawal mechanical threshold (PWMT) and paw withdrawalthermal latency (PWTL) were measured before intravenous and intrathecal injection. InACSF and PAF group, behavioural tests were processed at 15 minutes after intrathecalinjection and repeated every 15 minutes in the following 5 hours.In normal saline, SC-560,NS-398 and indomethacin group, behavioural tests were processed at 30 minutes afterintrathecal PAF injection.The rats were euthanized immediately after nociceptivebehavioural tests were all over. RT-PCR analysis (in ACSF and PAF group) andradioimmunoassay (in each group) were used to assess the expressions of COX-1, COX-2and COX-3 mRNA and concentrations of PGE_2 in L_(4-6) spinal cord respectively.
Results (1) Nociceptive behavioural tests: The mean baseline PWMT of rats in thepresent study was 39.75±1.85 g. Intrathecal injection with 5μg PAF induced developmentof tactile allodynia rapidly in PAF group. PWMT of rats in PAF group in different timepoints reduced significantly compared with that in ACSF control group(P＜0.05).Tactileallodynia in rats existed during the whole observation period of 5 hours, and the peak timespersisted about 60 minutes.Intrathecally administered PAF also induced development ofthermal hyperalgesia rapidly, The mean baseline PWTL of rats in the present study was13.04±0.20 s. PWTL of rats in PAF group in different time points reduced significantlycompared with that in ACSF control group(P＜0.05).The time-course of thermalhyperalgesia was similar to that of tactile allodynia.The cyclooxygenase inhibitors, NS-398and indomethacin attenuated tactile allodynia and thermal hyperalgesia induced by PAF.PWMT and PWTL in NS-398 and indomethacin group increased significantly comparedwith that in normal saline control group(P＜0.05). The COX-1 inhibitor, SC-560 had nosignificant effects on both PWMT and PWTL. (2) RT-PCR analysis of cyclooxygenases inspinal cord: COX-1, COX-2 and COX-3 mRNA all expressed constitutively in spinal cordin ACSF group. Both COX-1 and COX-3 mRNA expressed lower than COX-2 mRNA inspinal cord(P＜0.05). PAF increased the expression of COX-2 mRNA(P＜0.05), and had noeffects on the expression of COX-1 and COX-3 mRNA. (3) Radioimmunoassay of PGE_2 in spinal cord: PGE_2 concentration in PAF group increased significantly at 5 hours afterPAF injection compared with that in ACSF group(P＜0.05). Compared with normal salinegroup, NS-398 and indomethacin inhibited the release of PGE_2 induced by PAF in spinalcord (P＜0.05), and the PGE_2 concentration in NS-398 group is lower than that inindomethacin group(P＜0.05). SC-560 did not affect the release of PGE_2 induced by PAF.
2. Effects of platelet-activating factor administered intrathecally on glialactivation and proinflammatory cytokine expression in spinal cord in rats
Methods Sixty-four Sprague-Dawley rats were randomly divided into six groups, andintrathecal PE-10 catheters were placed in the spinal subarachnoid space of rats: In ACSFgroup, rats were treated with 10μl artificial cerebral spinal fluid intrathecally. In PAFgroup, rats were treated with 5μg PAF which was dissolved in 10μl artificial cerebralspinal fluid intrathecally. In DMSO control group, SC-514(10 mg/kg)group, SC-514(50mg/kg)group and SC-514(100 mg/kg)group, DMSO(2 ml, the final concentration ofDMSO in normal saline was 0.1%) and SC-514 which was dissolved in 2 ml 0.1% DMSOwere injected intraperitoneally respectively 2 hours prior to intrathecal injection with 5μgPAF. Baseline PWMT and PWTL were measured before intraperitoneal and intrathecalinjection. PWMT and PWTL were measured at 5, 15, 30, 45, 60, 90 and 120 minutes afterPAF was administered intrathecally. The rats were euthanized immediately afternociceptive behavioural tests were all over. The activation of astrocyte and microglia in L_(4-6)spinal cord were assessed with immunohistochemical staining of glial fibrillary acid protein(GFAP) and OX-42 respectively in ACSF and PAF group. Proinflammatory cytokinesTNF-a and IL-1βin L_(4-6) spinal cord were analyzed with ELISA in each group.
Results (1) Nociceptive behavioural tests: Intrathecally administered PAF induceddevelopment of tactile allodynia and thermal hyperalgesia rapidly, decreased PWMT andPWTL in rats in PAF group significantly (P＜0.05). Pretreatment with SC-514dose-dependently attenuated nociceptive behavioural response induced by PAF. Both PWMT and PWTL in different time points in SC-514 groups increased significantlycompared with that in DMSO control group(P＜0.05). (2) Immunohistochemical analysisof glial activation: Intrathecally administered PAF activated astrocyte and microglia labeledrespectively with GFAP and OX-42 staining in dorsal horn of gray matter in PAF group.Activating grade of glia is (++) and (-) in PAF and control group respectively. Opticaldensity of GFAP and OX-42 immunoreactive profiles increased significantly in PAF groupcompared with that in control group (P＜0.05). Activated glia distributed throughout thespinal gray matter, and in dorsal horn, activated glia distributed mostly in laminaⅠ～Ⅱ. (3)ELISA analysis of proinflammatory cytokines: Intrathecally admini- stered PAFsignificantly increased the expressions of TNF-a and IL-1βin lumbar spinal cord at 2 hoursafter intrathecal injection (P＜0.05). Pretreatment with SC-514 dose-dependently inhibitedthe increase of TNF-a and IL-1βexpression induced by PAF in spinal cord (P＜0.05).
3. Antinociceptive effects of PAF receptor antagonists administeredintrathecally in rats with spared nerve injury
Methods Sixty-four Sprague-Dawley rats were randomly divided into six groups:sham group, SNI group, DMSO (5μl, 0.1% final concentration in normal saline) controlgroup, BN52021(100μg) group, BN50730(100μg) group and BN52021(100μg)+BN50730(100μg) group. BN52021 and BN50730, the antagonists for PAF receptors weredissolved in 5μl DMSO (the final concentration of DMSO in normal saline was 0.1%)respectively. Spared nerve injury neuropathic pain model was set up in all rats except shamgroup. Intrathecal injections with DMSO or antagonists were administered once a day in 7days in DMSO or antagonists groups. PWMT were measured at 7 days in all rats. The ratswere euthanized immediately after nociceptive behavioural tests were all over.Immunohistochemical staining (in each group),and radioimmunoassay (in ACSF and PAFgroup) were used to assess the expressions of c-fos and PAF levels in L_(4-6) spinal cordrespectively.
Results (1) Nociceptive behavioural tests: Spared nerve injury induced tactileallodynia in rats. PWMT in SNI group reduced significantly compared with that in shamgroup (P＜0.05). The antagonists BN52021 and BN50730 attenuated tactile allodynia in ratswith spared nerve injury. PWMT in three antagonist groups increased, significantlydifferent from that in SNI and DMSO group (P＜0.05). Combination treatment withBN52021 and BN50730 were more potent in antinociceptive effects than treatment withone alone (P＜0.05). (2) Immunohistochemical analysis: The expression of c-fos in spinalcord distributed extensively in laminaⅠ～Ⅴ,mostly in laminaⅠ～Ⅱ.Fos-like immuno-reactivityneurons were present dyeing deep brown on nucleus, with round or ellipticgranule-like product, and not dyeing in cytoplasm.The number of Fos-like immuno-reactivityneurons in ipsilateral dorsal horn in SNI rats increased, significantly differentfrom that in sham group(P＜0.05). The number of Fos-like immunoreactivity neurons indifferent lamina of dorsal horn in spinal cord in three antagonist groups reduced,significantly different from that in SNI and DMSO group(P＜0.05). (3) Radioimmunoassayof PAF in spinal cord: SNI induced PAF release in spinal cord in SNI group rats at 7days. PAF concentration in spinal cord in SNI group was 1.183±0.098 pg/mg, significantlydifferent from 0.571±0.065 pg/mg in sham group(P＜0.05).
Conclusions
1. Intrathecally administered PAF may induce development of tactile allodynia andthermal hyperalgesia in rats.The activation and increased expression of COX-2 andrelease of PGE_2 in spinal cord are implicated in its mechanism.
2. Intrathecally administered PAF may induce development of tactile allodynia andthermal hyperalgesia in rats.The activation of glia and NF-κB pathway, and theincreased expressions of TNF-αand IL-1βin spinal cord in rats are implicated in its mechanism. IKKβplays the major role in the activation of NF-κB pathway andexpressions of proinflammatory cytokine TNF-αand IL-1βinduced by PAF.
3. These findings suggest a role for endogenous PAF in central nociceptive modulationfor spared nerve injury induced neuropathic pain in rats.Both intracellular and cellsurface PAF binding sites are involved in nociceptive transmission and modulation inrats, and that PAF receptor antagonists might be useful for treating some patients withneuropathic pain.
血小板活化因子(platelet-activating factor,PAF)是免疫细胞和炎症细胞产生的一种内源性磷脂，是一种强效的炎症介质。研究发现，PAF与中枢神经系统许多生理功能和病理生理过程有关。神经细胞生成PAF，同时，神经细胞也是PAF的靶细胞，PAF在神经信号传递中发挥重要作用。近几年有研究报道表明PAF可能参与神经中枢痛觉信号的传导和调控，而PAF在痛觉信号传导和调控中的作用及相应机制仍有待进一步研究。
前列腺素类物质(prostaglandins，PGs)在炎性疼痛中的作用已得到广泛认可，随着研究的深入，PGs在其它类型疼痛如神经病理性疼痛中的作用也受到关注。目前的研究认为，在慢性疼痛的产生及维持中，中枢及外周PGs均起作用。脊髓是调控痛觉信号的重要中枢，脊髓水平的PGs在疼痛的产生及维持中占有重要地位。体外实验表明，PAF可诱导培养的大鼠星形胶质细胞释放前列腺素E_2(prostaglandin E_2，PGE_2)，这一研究提示PGE_2及其限速酶环氧酶(cyclooxygenase，COX)可能参与神经中枢PAF调控痛觉信号的机制，但目前尚无相关研究的报道。
传统的观点认为，神经胶质细胞仅对神经元起着支持和营养作用，而没有细胞之间的信号传递功能，因而认为从外周传入的痛觉信号在脊髓水平的调控和放大以及痛敏状态的产生仅与神经元及其递质有关，而与神经胶质细胞无关。近些年，越来越多的研究证据表明胶质细胞与疼痛调控有着密切关系，特别是在如炎症和神经损伤等病理状态下。胶质细胞的激活及其促炎细胞因子的释放在病理状态下的痛觉过敏的产生和疼痛持续状态中发挥重要作用。脊髓和胶质细胞大量表达PAF受体，PAF可激活体外培养的胶质细胞，并诱导胶质细胞产生神经递质和促炎性细胞因子。以上研究结果提示，胶质细胞的激活及其促炎性细胞因子的释放可能参与神经中枢PAF调控痛觉信号的机制，而目前尚无相关报道。
目前的研究认为，PAF通过与其受体相结合而发挥生物学效应，PAF有二类受体结合位点，即低结合力的细胞膜表面结合位点和高结合力的细胞内位点。抑制PAF受体可减轻大鼠炎性痛反应，而PAF受体拮抗剂用于其它疼痛模型如神经病理性疼痛的研究尚未见报道。
为了进一步探讨PAF在痛觉信号调控中的作用及其机制，本课题进行了以下实验研究：(1)鞘内注射PAF对大鼠痛阈和脊髓环氧酶表达的影响，不同选择性环氧酶抑制剂对鞘内注射PAF所致大鼠痛敏的抗伤害效应；(2)鞘内注射PAF对脊髓胶质细胞活性和促炎性细胞因子表达的影响；(3)鞘内注射PAF受体拮抗剂对SNI神经痛大鼠镇痛作用的研究。
研究方法与结果
1．鞘内注射PAF对大鼠痛阈和脊髓环氧酶表达的影响
方法雄性Sprague-Dawley大鼠48只随机分为6组，每组8只，鞘内置管：人工脑脊液(artificial cerebral spinal fluid，ACSF)组，鞘内注射ACSF 10μl；PAF组，鞘内注射PAF 5μg；生理盐水(normal saline，NS)组，尾静脉注射1．5 ml生理盐水，10 min后鞘内注射PAF 5μg；SC-560(环氧酶1选择性抑制剂，150μg/kg)组，NS-398(环氧酶2选择性抑制剂，150μg/kg)组和吲哚美辛(低选择性环氧酶抑制剂，300μg/kg)组，SC-560、NS-398和吲哚美辛分别溶解于1．5 ml生理盐水，尾静脉注射给药，10 min后鞘内注射PAF 5μg。PAF均溶解于10μl ACSF。给药前测机械缩爪阈(PWMT)和热缩爪潜伏期(PWTL)基础值，ACSF组和PAF组鞘内给药15 min后测PWMT和PWTL值，连续5 h每15 min测定一次；生理盐水组、SC-560组、NS-398组和吲哚美辛组鞘内注射PAF 30 min后测PWMT和PWTL值。各组大鼠痛阈测定结束后立即处死，取L_(4-6)段脊髓组织，RT-PCR分析检测ACSF组和PAF组大鼠脊髓环氧酶COX-1、COX-2和COX-3 mRNA表达，放射免疫分析检测各组大鼠脊髓PGE_2含量。
结果(1)疼痛行为学观察：大鼠PWMT平均基础值为39．75±1．85 g，PAF组鞘内注射5μg PAF迅速诱发大鼠触觉异常痛敏(tactile allodynia)，鞘内给药后各测痛时间点PWMT值下降，与ACSF对照组比较差异有统计学意义(P＜0．05)，触觉异常痛敏在5 h观察周期内持续存在，高峰期持续约60 min；鞘内注射PAF诱发大鼠热痛敏(thermal hyperalgesia)，大鼠PWTL平均基础值为13．04±0．20 s，鞘内给药后各测痛时间点PWTL值下降(P＜0．05)，热痛敏持续时间和高峰期与触觉异常痛敏一致。环氧酶抑制剂NS-398和吲哚美辛减轻PAF诱发的触觉异常痛敏和热痛敏，与生理盐水对照组相比，NS-398组和吲哚美辛组PWMT和PWTL值升高(P＜0．05)，而SC-560对机械缩爪阈值和热缩爪潜伏期均无明显影响。(2)脊髓COX表达RT-PCR分析：ACSF组脊髓环氧酶COX-1、COX-2和COX-3在转录水平均有表达，COX-1和COX-3 mRNA表达水平低，COX-2表达水平高(P＜0．05)；PAF组鞘内注射PAF诱导COX-2表达增强(P＜0．05)，而COX-1和COX-3表达水平未见明显改变。(3)脊髓PGE_2放射免疫分析：与ACSF组比较，PAF组鞘内注射PAF 5 h后脊髓组织PGE_2含量升高(P＜0．05)；与生理盐水组比较，COX-2选择性抑制剂NS-398和非选择性环氧酶抑制剂吲哚美辛均抑制PAF诱发的PGE_2升高(P＜0．05)，其中，NS-398组PGE_2含量低于吲哚美辛组(P＜0．05)，而COX-1选择性抑制剂SC-560组对PAF引起的PGE_2含量升高无明显影响。
2．鞘内注射PAF对脊髓胶质细胞活性和促炎性细胞因子表达的影响
方法雄性Sprague-Dawley大鼠64只随机分为6组，鞘内置管：人工脑脊液(ACSF)组，16只，鞘内注射ACSF 10μl；PAF组，16只，鞘内注射PAF 5μg；DMSO(二甲基亚砜)组，8只，腹腔注射0．1％DMSO生理盐水2 ml，2 h后鞘内注射PAF 5μg；SC-514(10 mg/g)组，SC-514(50 mg/kg)组和SC-514(100 mg/kg)组，每组8只，SC-514分别溶解于2 ml 0．1％DMSO生理盐水，腹腔注射给药，2 h后鞘内注射PAF 5μg。PAF均溶解于10μl ACSF。给药前测PWMT和PWTL基础值，鞘内给药后5 min、15 min、30 min、45 min、60 min、90min和120min各时间点测PWMT和PWTL值。各组大鼠痛阈测定结束后立即处死，取L_(4-6)段脊髓组织，免疫组织化学染色检测ACSF组和PAF组大鼠腰段脊髓GFAP和OX-42的表达，ELISA检测各组大鼠脊髓TNF-a和IL-1β的表达。
结果(1)疼痛行为学观察：PAF组鞘内注射给药迅速诱发大鼠触觉异常痛敏和热痛敏，各测痛时间点PWMT和PWTL值均下降(P＜0．05)。IKKβ选择性抑制剂SC-514预处理明显减轻PAF诱发的触觉异常痛敏和热痛敏，其作用呈剂量依赖性增强，与DMSO对照组比较，SC-514不同剂量组各测痛时间点PWMT和PWTL值均升高(P＜0．05)。(2)胶质细胞活性免疫组织化学分析：PAF组鞘内注射PAF激活星形胶质细胞和小胶质细胞，大鼠脊髓灰质区可见分别以GFAP和OX-42标记的活化星形胶质细胞和小胶质细胞，活化评分达(++)，对照组活化评分为(—)；各组脊髓灰质背角GFAP和OX-42免疫阳性反应物的光密度值分析，PAF组高于ACSF对照组，差异有统计学意义(P＜0．05)。活化的胶质细胞分布于整个脊髓灰质，在脊髓背角中主要分布在第Ⅰ～Ⅱ层。(3)促炎性细胞因子TNF-a和IL-1β表达ELISA分析：与ACSF组比较，PAF组鞘内注射PAF 2 h后脊髓促炎性细胞因子TNF-a和IL-1β表达均增强(P＜0．05)，IKKβ选择性抑制剂SC-514预处理明显抑制PAF诱导的TNF-a和IL-1β的表达增强(P＜0．05)，其抑制作用呈剂量依赖性。
3．鞘内注射PAF受体拮抗剂对SNI神经痛大鼠镇痛作用的研究
方法雄性Sprague-Dawley大鼠64只，随机分为6组：SNI组，16只，制作SNI疼痛模型；假手术组(sham组)，16只：DMSO(二甲基亚砜)组，8只，制作SNI疼痛模型，鞘内注射0．1％DMSO生理盐水5μl；BN52021(100μg)组，BN50730(100μg)组和BN52021(100μg)+BN50730(100μg)组，每组各8只，制作SNI疼痛模型，鞘内注射给药，PAF受体拮抗剂BN52021和BN50730溶解于5μl 0．1％DMSO生理盐水。DMSO组和拮抗剂治疗组每天鞘内注射给药一次，连续给药7 d。第7 d测各组大鼠PWMT值，随后处死大鼠取L_(4-6)段脊髓，免疫组织化学染色检测各组大鼠脊髓c-fos表达，放射免疫分析检测sham组和SNI组大鼠脊髓PAF含量。
结果(1)疼痛行为学观察：SNI组神经损伤诱发大鼠触觉异常痛敏，术后第7 dPWMT值下降，与假手术组比较，差异有统计学意义(P＜0．05)；鞘内注射PAF受体拮抗剂BN52021和BN50730均减轻SNI神经损伤诱发的触觉异常痛敏，与SNI组及DMSO对照组比较，三个拮抗剂组PWMT值升高(P＜0．05)，BN52021和BN50730二者联合给药与单独用药比较镇痛效应更明显(P＜0．05)。(2)脊髓c-fos表达免疫组织化学分析：Fos蛋白表达分布较广泛，位于Ⅰ～Ⅴ层，其在Ⅰ～Ⅱ层分布较为密集，FLI(Fos-likeimmunoreactivity)阳性神经元为细胞核染成棕黄色的圆形或卵圆形颗粒，胞浆未着色。与sham组比较，SNI组手术侧脊髓背角内FLI阳性神经元数目明显增加(P＜0．05)，对侧背角内仅有少量FLI阳性神经元；三个拮抗剂给药组大鼠脊髓背角各层FLI阳性神经元数目与SNI组及DMSO对照组相比明显下降(P＜0．05)。(3)脊髓PAF放射免疫分析：SNI组神经损伤后第7d脊髓L_(4-6)节段组织PAF水平升高(1．183±0．098 pg/mg)，与假手术组(0．571±0．065 pg/mg)比较，差异有统计学意义(P＜0．05)。
4．统计学方法
所有数据以均数±标准差((?)±s)表示，采用SPSS11．0统计软件包分析。两组间比较采用非配对t检验，多组间比较采用单因素方差分析，继之以Fisher's PLSD多重比较法，P＜0．05为差异有统计学意义。
研究结论
1．鞘内注射PAF诱发大鼠触觉异常痛敏和辐射热痛敏，环氧酶COX-2的激活和表达增强以及PGE_2的产生参与其机制。
2．鞘内注射PAF诱发大鼠触觉异常痛敏和辐射热痛敏，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的表达增强参与其机制；IKKβ是PAF诱发的NF-κB通路激活以及促炎性细胞因子TNF-α和IL-1β表达增强的重要激酶。
3．脊髓内源性PAF参与SNI神经损伤大鼠痛敏的中枢调控，PAF的二类结合位点均介导痛觉信号的传导和调控，PAF受体拮抗剂可应用于治疗神经病理性疼痛。
Background
Tissue damage and inflammation usually induce persistant pain, characterized asspontaneous pain, hyperalgesia, tactile allodynia and so on. Within the past decade, there isgreat development in research on persistent pain.Central sensitization in pain signalconduction pathway is viewed as important mechanism in the creation and maintenance ofpersistent pain. However, so far the mechanism of central sensitization remainsunintelligible.Years of research indicates an amount of endogenous nerve mediators areimplicated in pain signal conduction and modulation in central sensitization.
Platelet-activating factor (PAF) is an endogenous phospholipid, described in a varietyof immune and inflammatory cells and a potent mediator that participates in inflammatoryresponses.In further researches, neural cells produce PAF and also are target cells of PAF.PAF is suggested to be implicated in a variety of physiological and pathological states incentral nervous system, and play a significant role in signal conduction in nervous system.Recently, evidence suggests that PAF may play a part in pain signal conduction andmodulation in central nervous system. At present, the role and mechanism of PAF acted inpain signal conduction and modulation remain to be established further.
The role of prostaglandins (PGs) as nociceptive mediators in inflammatory pain isaccepted extensively. Along with intensive research, the role of prostaglandins in other painmodels such as nuropathic pain is recognized gradually. Prostaglandins are thought to playan important role in the creation and maintenance of persistent pain both at peripheral sitesand in central nervous system. Spinal cord is the primary center in pain signal transmission,and prostaglandins in spinal cord act as important mediators in the creation andmaintenance of persistent pain. In vitro, PAF induces prostaglandin E_2 (PGE_2) release fromrat primary astrocytes.It suggests that prostaglandins and their rate-limiting enzymes (i.e.cyclooxygenases) may be involved in pain modulation mediated by PAF in central nervoussystem.There are no reports corresponding to this subject.
Traditionally, glial cells were simply thought to be housekeepers for neurons andprovide neurochemical precursors and energy sources to neurons, not to function in signaltransmission among neural cells, and it were neurons and their transmitters rather than glialcells that modulated and amplified signals from the periphery in spinal cord and wereresponsible for hyperalgesia. However, in the last several years, we have seen exponentiallyincreasing number of research articles suggesting the role of glial cells in pain controlespecially in conditions involving inflammation and nerve injury. Activation of glial cells(e.g. astrocytes and microglia) and proinflammatory cytokines release from glial cellscontribute importantly to the development of pain hypersensitivity under pathologicalconditions. PAF receptor mRNA and functional expression are abundant in spinal cord andglial cells. In addition, PAF receptors function to stimulate the production of transmittersand proinflammatory cytokines in cultured glial cells in vitro.These researches abovesuggest the activation of glial cells and release of proinflammatory cytokines may beimplicated in pain modulation mediated by PAF in central nervous system. There are noreports corresponding to this subject.
Evidence suggests that PAF exerts cellular actions through binding to its receptors.PAF has two kinds of binding sites which are high-affinity intracellular membrane binding site and low-affinity cell surface receptor. Inhibition of platelet-activating factor receptorsattenuates the inflammatory nociceptive response in rats.Whether antagonists for PAFreceptors attenuate nociceptive response in other pain model such as neuropathic pain hasnot been reported.
To further explore the role of PAF in nociceptive modulation and its mechanism, thepresent study is to investigate: (1) Effects of platelet-activating factor administeredintrathecally on pain behaviour and cyclooxygenase expression in spinal cord in rats,antinociceptive effects of different inhibitors of cyclooxygenases on pain behaviouralresponse induced by PAF. (2) Effects of platelet-activating factor administeredintrathecally on glial activation and proinflammatory cytokines in spinal cord in rats. (3)Antinociceptive effects of PAF receptor antagonists administered intrathecally in rats withspared nerve injury.
Methods and Results
1. Effects of platelet-activating factor administered intrathecally on painbehaviour and cyclooxygenase expression in spinal cord in rats
Methods Forty-eight Sprague-Dawley rats were randomly divided into six groups, andintrathecal PE-10 catheters were placed in the spinal subarachnoid space of rats: InACSF(artificial cerebral spinal fluid)group, rats were treated with 10μl artificial cerebralspinal fluid intrathecally. In PAF group, rats were treated with 5μg PAF which wasdissolved in 10μl artificial cerebral spinal fluid. In normal saline, SC-560, NS-398 andindomethacin group, all rats were pretreated with 1.5 ml normal saline, SC-560, NS-398and indomethacin intravenously through tail vein 10 minutes prior to intrathecal PAF(5μg,dissolved in 10μl ACSF) injection respectively. The doses of SC-560, NS-398 andindomethacin dissolved in 1.5 ml normal saline were 150μg/kg, 150μg/kg and 300μg/kg respectively. Baseline paw withdrawal mechanical threshold (PWMT) and paw withdrawalthermal latency (PWTL) were measured before intravenous and intrathecal injection. InACSF and PAF group, behavioural tests were processed at 15 minutes after intrathecalinjection and repeated every 15 minutes in the following 5 hours.In normal saline, SC-560,NS-398 and indomethacin group, behavioural tests were processed at 30 minutes afterintrathecal PAF injection.The rats were euthanized immediately after nociceptivebehavioural tests were all over. RT-PCR analysis (in ACSF and PAF group) andradioimmunoassay (in each group) were used to assess the expressions of COX-1, COX-2and COX-3 mRNA and concentrations of PGE_2 in L_(4-6) spinal cord respectively.
Results (1) Nociceptive behavioural tests: The mean baseline PWMT of rats in thepresent study was 39.75±1.85 g. Intrathecal injection with 5μg PAF induced developmentof tactile allodynia rapidly in PAF group. PWMT of rats in PAF group in different timepoints reduced significantly compared with that in ACSF control group(P＜0.05).Tactileallodynia in rats existed during the whole observation period of 5 hours, and the peak timespersisted about 60 minutes.Intrathecally administered PAF also induced development ofthermal hyperalgesia rapidly, The mean baseline PWTL of rats in the present study was13.04±0.20 s. PWTL of rats in PAF group in different time points reduced significantlycompared with that in ACSF control group(P＜0.05).The time-course of thermalhyperalgesia was similar to that of tactile allodynia.The cyclooxygenase inhibitors, NS-398and indomethacin attenuated tactile allodynia and thermal hyperalgesia induced by PAF.PWMT and PWTL in NS-398 and indomethacin group increased significantly comparedwith that in normal saline control group(P＜0.05). The COX-1 inhibitor, SC-560 had nosignificant effects on both PWMT and PWTL. (2) RT-PCR analysis of cyclooxygenases inspinal cord: COX-1, COX-2 and COX-3 mRNA all expressed constitutively in spinal cordin ACSF group. Both COX-1 and COX-3 mRNA expressed lower than COX-2 mRNA inspinal cord(P＜0.05). PAF increased the expression of COX-2 mRNA(P＜0.05), and had noeffects on the expression of COX-1 and COX-3 mRNA. (3) Radioimmunoassay of PGE_2 in spinal cord: PGE_2 concentration in PAF group increased significantly at 5 hours afterPAF injection compared with that in ACSF group(P＜0.05). Compared with normal salinegroup, NS-398 and indomethacin inhibited the release of PGE_2 induced by PAF in spinalcord (P＜0.05), and the PGE_2 concentration in NS-398 group is lower than that inindomethacin group(P＜0.05). SC-560 did not affect the release of PGE_2 induced by PAF.
2. Effects of platelet-activating factor administered intrathecally on glialactivation and proinflammatory cytokine expression in spinal cord in rats
Methods Sixty-four Sprague-Dawley rats were randomly divided into six groups, andintrathecal PE-10 catheters were placed in the spinal subarachnoid space of rats: In ACSFgroup, rats were treated with 10μl artificial cerebral spinal fluid intrathecally. In PAFgroup, rats were treated with 5μg PAF which was dissolved in 10μl artificial cerebralspinal fluid intrathecally. In DMSO control group, SC-514(10 mg/kg)group, SC-514(50mg/kg)group and SC-514(100 mg/kg)group, DMSO(2 ml, the final concentration ofDMSO in normal saline was 0.1%) and SC-514 which was dissolved in 2 ml 0.1% DMSOwere injected intraperitoneally respectively 2 hours prior to intrathecal injection with 5μgPAF. Baseline PWMT and PWTL were measured before intraperitoneal and intrathecalinjection. PWMT and PWTL were measured at 5, 15, 30, 45, 60, 90 and 120 minutes afterPAF was administered intrathecally. The rats were euthanized immediately afternociceptive behavioural tests were all over. The activation of astrocyte and microglia in L_(4-6)spinal cord were assessed with immunohistochemical staining of glial fibrillary acid protein(GFAP) and OX-42 respectively in ACSF and PAF group. Proinflammatory cytokinesTNF-a and IL-1βin L_(4-6) spinal cord were analyzed with ELISA in each group.
Results (1) Nociceptive behavioural tests: Intrathecally administered PAF induceddevelopment of tactile allodynia and thermal hyperalgesia rapidly, decreased PWMT andPWTL in rats in PAF group significantly (P＜0.05). Pretreatment with SC-514dose-dependently attenuated nociceptive behavioural response induced by PAF. Both PWMT and PWTL in different time points in SC-514 groups increased significantlycompared with that in DMSO control group(P＜0.05). (2) Immunohistochemical analysisof glial activation: Intrathecally administered PAF activated astrocyte and microglia labeledrespectively with GFAP and OX-42 staining in dorsal horn of gray matter in PAF group.Activating grade of glia is (++) and (-) in PAF and control group respectively. Opticaldensity of GFAP and OX-42 immunoreactive profiles increased significantly in PAF groupcompared with that in control group (P＜0.05). Activated glia distributed throughout thespinal gray matter, and in dorsal horn, activated glia distributed mostly in laminaⅠ～Ⅱ. (3)ELISA analysis of proinflammatory cytokines: Intrathecally admini- stered PAFsignificantly increased the expressions of TNF-a and IL-1βin lumbar spinal cord at 2 hoursafter intrathecal injection (P＜0.05). Pretreatment with SC-514 dose-dependently inhibitedthe increase of TNF-a and IL-1βexpression induced by PAF in spinal cord (P＜0.05).
3. Antinociceptive effects of PAF receptor antagonists administeredintrathecally in rats with spared nerve injury
Methods Sixty-four Sprague-Dawley rats were randomly divided into six groups:sham group, SNI group, DMSO (5μl, 0.1% final concentration in normal saline) controlgroup, BN52021(100μg) group, BN50730(100μg) group and BN52021(100μg)+BN50730(100μg) group. BN52021 and BN50730, the antagonists for PAF receptors weredissolved in 5μl DMSO (the final concentration of DMSO in normal saline was 0.1%)respectively. Spared nerve injury neuropathic pain model was set up in all rats except shamgroup. Intrathecal injections with DMSO or antagonists were administered once a day in 7days in DMSO or antagonists groups. PWMT were measured at 7 days in all rats. The ratswere euthanized immediately after nociceptive behavioural tests were all over.Immunohistochemical staining (in each group),and radioimmunoassay (in ACSF and PAFgroup) were used to assess the expressions of c-fos and PAF levels in L_(4-6) spinal cordrespectively.
Results (1) Nociceptive behavioural tests: Spared nerve injury induced tactileallodynia in rats. PWMT in SNI group reduced significantly compared with that in shamgroup (P＜0.05). The antagonists BN52021 and BN50730 attenuated tactile allodynia in ratswith spared nerve injury. PWMT in three antagonist groups increased, significantlydifferent from that in SNI and DMSO group (P＜0.05). Combination treatment withBN52021 and BN50730 were more potent in antinociceptive effects than treatment withone alone (P＜0.05). (2) Immunohistochemical analysis: The expression of c-fos in spinalcord distributed extensively in laminaⅠ～Ⅴ,mostly in laminaⅠ～Ⅱ.Fos-like immuno-reactivityneurons were present dyeing deep brown on nucleus, with round or ellipticgranule-like product, and not dyeing in cytoplasm.The number of Fos-like immuno-reactivityneurons in ipsilateral dorsal horn in SNI rats increased, significantly differentfrom that in sham group(P＜0.05). The number of Fos-like immunoreactivity neurons indifferent lamina of dorsal horn in spinal cord in three antagonist groups reduced,significantly different from that in SNI and DMSO group(P＜0.05). (3) Radioimmunoassayof PAF in spinal cord: SNI induced PAF release in spinal cord in SNI group rats at 7days. PAF concentration in spinal cord in SNI group was 1.183±0.098 pg/mg, significantlydifferent from 0.571±0.065 pg/mg in sham group(P＜0.05).
Conclusions
1. Intrathecally administered PAF may induce development of tactile allodynia andthermal hyperalgesia in rats.The activation and increased expression of COX-2 andrelease of PGE_2 in spinal cord are implicated in its mechanism.
2. Intrathecally administered PAF may induce development of tactile allodynia andthermal hyperalgesia in rats.The activation of glia and NF-κB pathway, and theincreased expressions of TNF-αand IL-1βin spinal cord in rats are implicated in its mechanism. IKKβplays the major role in the activation of NF-κB pathway andexpressions of proinflammatory cytokine TNF-αand IL-1βinduced by PAF.
3. These findings suggest a role for endogenous PAF in central nociceptive modulationfor spared nerve injury induced neuropathic pain in rats.Both intracellular and cellsurface PAF binding sites are involved in nociceptive transmission and modulation inrats, and that PAF receptor antagonists might be useful for treating some patients withneuropathic pain.
引文
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