Characteristics of spinal dorsal horn neurons after partial chronic deafferentation by dorsal root transection

Unilateral transections of 1-3 lumbar dorsal roots were performed in 13 adult cats to investigate the effect of partial deafferentation on dorsal horn neurons. Eleven to 45 days after deafferentation various parameters of spontaneous and evoked activity of 169 neurons were measured and compared to the data of 168 neurons from previous experiments recorded under identical experimental conditions except that these animals had not been deafferented. Eighty-six of the units encountered were located in the segment of transected dorsal root(s) and 82 in the caudally adjacent segment. No significant differences could be observed in the functional properties of these two samples of units. Most parameters measured indicate that either no change at all in responsiveness or signs of decreased excitability occurred in the partially deafferented neurons compared to units recorded in control animals. Discharges evoked by noxious skin heating indicate a linear relationship between discharge frequency and skin temperature. This kind of encoding curve could also be measured during a reversible cold block of the spinal cord at segment L1. The mean encoding curves before and during spinal blockade were not different in deafferented compared to corresponding curves measured in control animals. The only finding that could be interpreted as an indication for increased excitability of partially deafferented neurons was that the mean frequency of spontaneous discharges of a subsample of heat-sensitive neurons was higher in deafferented compared to control animals. Possible mechanisms are discussed.     

Nitrous oxide inhibits glutamatergic transmission in spinal dorsal horn neurons

The effects of nitrous oxide (N2O) are thought to be mediated by several pharmacological pathways at different levels of the central nervous system. Here, we focus on excitatory glutamatergic transmission in the superficial dorsal horn of the spinal cord with respect to its importance for the nociceptive processing. The effects of 50% N2O on electrically evoked and spontaneous excitatory glutamatergic transmission and on the response to exogenous administration of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) receptor agonists were examined in lamina II neurons of adult rat spinal cord slices using the whole-cell patch-clamp technique. Peak amplitudes of Adelta- and C-fiber evoked monosynaptic NMDA- and AMPA-receptor-mediated excitatory postsynaptic currents (EPSCs) were decreased in the presence of N2O. N2O reduced the peak amplitude and integrated area of exogenous NMDA- and AMPA-induced currents. Moreover N2O changed the distribution of miniature EPSC amplitude, but not frequency distribution in most neurons. N2O inhibits glutamatergic transmission in the superficial dorsal horn by modulating the NMDA- and AMPA-receptors. Our findings raise the possibility that the antinociceptive effect of N2O may be directly mediated at the level of the spinal cord.     

Modulation of presynaptic activity by phosphorylation in cultured rat spinal dorsal horn neurons.

Phosphorylation, in particular by protein kinase C (PKC), modulates spinal sensory transmission and nociceptive behaviors. Whereas PKC's postsynaptic actions are well established, its presynaptic effects in spinal sensory neurons are mostly inferred from postsynaptic recordings. Here we first show that the amphipathic styryl dye FM 1-43 can be used to image presynaptic activity in cultured spinal dorsal horn cultures and then test whether PKC modulates presynaptic activity in cultured spinal dorsal horn neurons. Pretreatment with the broad-spectrum kinase inhibitor staurosporine (2 micromol/L) inhibited dye release. Bisindolylmaleimide I, a PKC inhibitor, potentiated dye release at low doses (200 nmol/L and 1 micromol/L), while inhibiting it at a higher dose (5 micromol/L). Activating PKC with phorbol dibutyrate (0.5 micromol/L) induced an increase in exocytosis, which is partially blocked by bisindolylmaleimide I. These results indicate that styryl dyes can be used to observe presynaptic regulation of spinal dorsal horn neurons, and that PKC acts presynaptically to modulate spinal sensory transmission. PERSPECTIVE: With dye imaging technique, we demonstrate here that PKC presynaptically regulates sensory transmission in spinal dorsal horn neurons. In combination with conventional whole-cell patch-clamp recording technique, the present study provides a new methodology for studying spinal sensory transmission and modulation and facilitates our understanding of pain mechanism.     

氯胺酮对甲醛致痛诱导大鼠脊髓背角神经元兴奋性的抑制

背景：氯胺酮是否可通过影响脊髓水平的伤害性信息的传递而发挥抗伤害作用尚不清楚；一氧化氮在脊髓水平主要参与痛觉过敏的形成和发展，可诱导Fos表达，但其是否参与了氯胺酮对痛信号的转导或调控的机制不明。 目的：观察大鼠脊髓对甲醛痛刺激的反应及氯胺酮的影响。 设计：均衡随机的动物实验。 单位：徐州医学院附属医院麻醉科和江苏省麻醉学重点实验室。 材料：实验于2000-01／03在徐州医学院江苏省麻醉学重点实验室进行。取SD大鼠30只，用均衡随机方法分为6组。甲醛组6只，甲醛＋氯胺酮组6只，氯胺酮＋甲醛组6只，氯胺酮组6只。甲醛＋生理盐水组3只，生理盐水组3只，各组雌雄比例相同。 方法：④甲醛组：体积分数为0．05的甲醛200μL一侧前爪掌心皮下注射，刺激1h。②甲醛＋氯胺酮组：甲醛痛刺激10min后腹腔注射100mg／kg氯胺酮1h。③氯胺酮＋甲醛组：腹腔注射氯胺酮10min，后再行甲醛痛刺激1h。④氯胺酮组：腹腔注射同等剂量氯胺酮1h。⑤甲醛＋生理盐水组：甲醛痛刺激10min后腹腔注射等容（10mL／kg）的生理盐水1h。⑥生理盐水组：腹腔注射等容生理盐水1h。 主要观察指标：①各组大鼠行为学表现。②取脊髓切片，用c-fos基因免疫组化法和NADPH-d组化技术染色，观察大鼠脊髓背角4层（Ⅰ～Ⅱ层，Ⅲ～Ⅳ层，Ⅴ～Ⅵ层，Ⅶ～Ⅹ层）切片Fos样免疫阳性神经元（FLI）和FLI／NOS双标记神经元的数目变化。 结果：30只大鼠全部进入结果分析。①行为学变化：甲醛组及甲醛＋生理盐水组大鼠注射甲醛后，出现痛反应；注射氯胺酮的大鼠，注射后数分钟内翻正反射消失，无明显的痛行为表现，而呈持续睡眠状态。至灌注时翻正反射仍未恢复。②FLI神经元表达：甲醛组及甲醛＋生理盐水组大鼠注射侧脊髓背角出现大量FLI阳性神经元，主要分布在脊髓背角Ⅰ～Ⅱ层；氯胺酮＋甲醛组、甲醛＋氯胺酮组大鼠脊髓FLI细胞的分布与甲醛组及甲醛＋生理盐水组基本相似，但FLI阳性细胞数量显著减少（P〈0．01）；氯胺酮组和生理盐水组大鼠脊髓未见或偶见FLI阳性细胞。③FLI／NOS双标记神经元表达：氯胺酮＋甲醛组、甲醛＋氯胺酮组脊髓背角Ⅰ～Ⅱ层双标记神经元数目显著少于甲醛组及甲醛＋生理盐水组[（1&;#177;1），（1&;#177;1），（7&;#177;3），（8&;#177;3）个／切片，P〈0．01]，氯胺酮组和生理盐水组无表达。 结论：同侧相应脊髓节段的某些神经元参与了化学性致痛信息的传导和调控，氯胺酮通过抑制这些神经元的活动而产生抗伤害作用；此作用与抑制脊髓内一氧化氮合酶阳性神经元的活性有关。     

氯胺酮对甲醛致痛诱导大鼠脊髓背角神经元兴奋性的抑制

背景:氯胺酮是否可通过影响脊髓水平的伤害性信息的传递而发挥抗伤害作用尚不清楚;一氧化氮在脊髓水平主要参与痛觉过敏的形成和发展,可诱导Fos表达,但其是否参与了氯胺酮对痛信号的转导或调控的机制不明。目的:观察大鼠脊髓对甲醛痛刺激的反应及氯胺酮的影响。设计:均衡随机的动物实验。单位:徐州医学院附属医院麻醉科和江苏省麻醉学重点实验室。材料:实验于2000-01/03在徐州医学院江苏省麻醉学重点实验室进行。取SD大鼠30只,用均衡随机方法分为6组熏甲醛组6只,甲醛 氯胺酮组6只,氯胺酮 甲醛组6只,氯胺酮组6只,甲醛 生理盐水组3只,生理盐水组3只,各组雌雄比例相同。方法:①甲醛组:体积分数为0.05的甲醛200μL一侧前爪掌心皮下注射,刺激1h。②甲醛 氯胺酮组:甲醛痛刺激10min后腹腔注射100mg/kg氯胺酮1h。③氯胺酮 甲醛组:腹腔注射氯胺酮10min后再行甲醛痛刺激1h。④氯胺酮组:腹腔注射同等剂量氯胺酮1h。⑤甲醛 生理盐水组:甲醛痛刺激10min后腹腔注射等容(10mL/kg)的生理盐水1h。⑥生理盐水组:腹腔注射等容生理盐水1h。主要观察指标:①各组大鼠行为学表现。②取脊髓切片,用c-fos基因免疫组化法和NADPH-d组化技术染色,观察大鼠脊髓背角4层(Ⅰ～Ⅱ层,Ⅲ～Ⅳ层,Ⅴ～Ⅵ层,Ⅶ～Ⅹ层)切片Fos样免疫阳性神经元(FLI)和FLI/NOS双标记神经元的数目变化。结果:30只大鼠全部进入结果分析。①行为学变化:甲醛组及甲醛 生理盐水组大鼠注射甲醛后,出现痛反应;注射氯胺酮的大鼠,注射后数分钟内翻正反射消失,无明显的痛行为表现,而呈持续睡眠状态,至灌注时翻正反射仍未恢复。②FLI神经元表达:甲醛组及甲醛 生理盐水组大鼠注射侧脊髓背角出现大量FLI阳性神经元,主要分布在脊髓背角Ⅰ～Ⅱ层;氯胺酮 甲醛组、甲醛 氯胺酮组大鼠脊髓FLI细胞的分布与甲醛组及甲醛 生理盐水组基本相似,但FLI阳性细胞数量显著减少(P<0.01);氯胺酮组和生理盐水组大鼠脊髓未见或偶见FLI阳性细胞。③FLI/NOS双标记神经元表达:氯胺酮 甲醛组、甲醛 氯胺酮组脊髓背角Ⅰ～Ⅱ层双标记神经元数目显著少于甲醛组及甲醛 生理盐水组眼(1±1),(1±1),(7±3),(8±3)个/切片,P<0.01演,氯胺酮组和生理盐水组无表达。结论:同侧相应脊髓节段的某些神经元参与了化学性致痛信息的传导和调控,氯胺酮通过抑制这些神经元的活动而产生抗伤害作用;此作用与抑制脊髓内一氧化氮合酶阳性神经元的活性有关。     

Interferon-gamma induces characteristics of central sensitization in spinal dorsal horn neurons in vitro

Hyperexcitability of spinal dorsal horn neurons, also known as 'central sensitization', is a component of pain associated with pathological conditions in the nervous system. The aim of the present study was to analyze if the pro-inflammatory cytokine, interferon-gamma (IFN-gamma), which can be released for extended periods of time in the nervous system during inflammatory and infectious events, can alter synaptic activity in dorsal horn neurons and thereby contribute to such hyperexcitability. Treatment of cultured dorsal horn neurons with IFN-gamma for 2 weeks resulted in a significantly reduced clustering of alpha-amino-3-hydroxy-5-methylisoxazole (AMPA) receptor subunit 1 (GluR1) that was dependent on nitric oxide. The neurons displayed an increased frequency and amplitude of excitatory postsynaptic currents (EPSCs) upon IFN-gamma treatment. Treated dorsal horn neurons also exhibited increased responsiveness to stimulation of dorsal root ganglia (DRG) axons in a two-compartment model. Furthermore, disinhibition by the GABA(A) receptor antagonist picrotoxin (PTX) significantly increased EPSC frequency and induced bursting in untreated cultures but did not significantly increase the frequency in treated neurons, which displayed bursting even without PTX. GABA(A) agonists reduced activity more strongly in treated cultures and immunochemical staining for GABA(A) receptors showed no difference from controls. Since GluR1-containing AMPA receptors (AMPARs) occur predominantly on inhibitory neurons in the dorsal horn, we suggest that the IFN-gamma-mediated increase in spontaneous activity and responsiveness to DRG axon stimulation, decrease in sensitivity to PTX and tendency for EPSC bursting result from a reduced expression of GluR1 on these neurons and not from a reduction in active GABA(A) receptors in the network. IFN-gamma thereby likely causes disinhibition of synaptic activity and primary afferent input in the dorsal horn, which consequently results in central sensitization.     

Control of glycinergic input to spinal dorsal horn neurons by distinct muscarinic receptor subtypes revealed using knockout mice

Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA(B) receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABA(B) receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.     

Systemic morphine inhibits dorsal horn projection neurons through spinal cholinergic system independent of descending pathways

Cholinergic circuitry and muscarinic receptors within the spinal cord have been proposed to contribute to the analgesic effects of systemic morphine. In this study, we determined whether the descending pathways are involved in the inhibitory effect of systemic morphine on dorsal horn projection neurons mediated by activation of the spinal cholinergic system. Single-unit activity of dorsal horn projection neurons was recorded in anesthetized rats. The neuronal responses to mechanical stimuli applied to the receptive field were determined before and after intravenous injection of morphine. The inhibitory effect of intravenous morphine on dorsal horn neurons was also tested before and after topical spinal application of the muscarinic antagonist atropine in both intact and spinally transected rats. Intravenous injection of 2.5 mg/kg morphine significantly inhibited the evoked response of dorsal horn neurons in both intact and spinally transected rats. Spinal topical application of the mu opioid antagonist H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) completely blocked the effect of morphine on dorsal horn neurons. In addition, spinal application of 10 microM atropine significantly attenuated the effect of systemic morphine. In rats subjected to cervical spinal transection, atropine produced a similar attenuation of the inhibitory effect of systemic morphine on dorsal horn neurons. Data from this electrophysiological study suggest that systemic morphine inhibits ascending dorsal horn neurons through stimulation of spinal mu opioid receptors. Furthermore, activation of the local spinal cholinergic circuitry and muscarinic receptors is involved in the inhibitory effect of systemic morphine on dorsal horn projection neurons independent of descending pathways.     

Effects of spinal kappa-opioid receptor agonists on the responsiveness of nociceptive superficial dorsal horn neurons.

Spinal cord application of the kappa-opioid receptor agonists dynorphin (50 nmol) or (1S,2S)U-50,488H (0.19-1.9 mumol) produced changes in the excitability of some superficial dorsal horn nociceptive neurons. One-third of the cells exhibited expansion of their receptive fields as defined using mechanical stimuli following a spinal kappa agonist (dynorphin or U-50,488H); receptive field expansions were of the same order as those observed immediately after a conditioning electrical stimulus applied to a peripheral nerve. In addition, spinal U-50,488H produced changes in mechanical and thermal thresholds of the majority of superficial dorsal horn neurons. These changes were dose-dependent. Facilitation of responses occurred at lower doses and inhibition occurred primarily at higher doses, but these effects were not reversed by subsequent administration of naloxone. The data are consistent with the hypothesis that one action of increases in spinal dynorphin levels due to peripheral inflammation, tissue injury or nerve damage, is to contribute to enhanced neuronal excitability in superficial dorsal horn neurons.     

Effects of glutamate receptor antagonists on spinal dorsal horn neurons during zymosan-induced inflammation in rats.

These experiments examined the effects of spinal administration of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or the metabotropic glutamate receptor antagonist DL-2-amino-3-phosphonoproprionic acid (AP3) on responses of spinal dorsal horn neurons evoked by thermal and mechanical stimuli applied to the rat hindpaw in either an inflamed or noninflamed state. Administration of APV, DNQX, or AP3 decreased heat-evoked neuronal discharges of wide dynamic range (WDR) neurons that were previously augmented by zymosan-induced inflammation. APV and DNQX also decreased heat-evoked discharges of WDR neurons that were previously unaffected by saline injection. Administration of either APV or DNQX, but not AP3, decreased heat-evoked neuronal discharges of nociceptive-specific (NS) neurons in both zymosan- and saline-injected rats. These data suggest that NMDA and non-NMDA receptors contribute to spinal processing of thermal stimuli in both the inflamed and noninflamed state, whereas metabotropic glutamate receptors might serve a role that is unique to WDR neurons in the inflamed state. Only DNQX consistently increased mechanical response thresholds and decreased slopes of the mechanical stimulus response functions (SRFs) of NS and WDR neurons, but this effect was observed in both inflamed and noninflamed states. These data suggest that spinal processing of mechanical stimuli is preferentially mediated by glutamate acting at non-NMDA receptors in either the inflamed or noninflamed state.     

Effects of sumatriptan on rat medullary dorsal horn neurons

This study examined the cellular actions of the anti-migraine drug sumatriptan, on neurons in the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis. Sumatriptan inhibited the miniature EPSC (mEPSC) rate in a dose dependent fashion, with an EC(50) of 250 nM. Sumatriptan (3 microM) inhibited the mEPSC rate by 36%, without altering the mEPSC amplitude. This effect was partially reversed by the 5HT(1D) specific antagonist BRL15572 (10 microM). In contrast, the 5HT(1B) agonist CP93129 (10 microm) did not alter the mEPSC rate. Furthermore, sumatriptan (3 microM) decreased the amplitude of electrically evoked EPSCs (eEPSC) by 40%. After incubating the slices in ketanserin (an antagonist which shows selectivity for 5HT(1D) over 5HT(1B) receptors) sumatriptan had little effect on eEPSC amplitude. In control conditions paired stimuli resulted in paired pulse depression (PPD; the ratio eEPSC(2)/eEPSC(1)=0.7+/-0.01), whilst in the presence of sumatriptan the PPD was blocked (ratio eEPSC(2)/eEPSC(1)=0.9+/-0.1). Sumatriptan produced no post-synaptic membrane current and had no significant effect on membrane conductance over a range of membrane potentials (-60 to -130 mV). RT-PCR experiments revealed the presence of mRNA for both 5HT(1D) and 5HT(1B) receptor subtypes in the trigeminal ganglia and subnucleus caudalis. These data suggest that sumatriptan acts pre-synaptically on trigeminal primary afferent central terminals to reduce the probability of release of glutamate, and that this action is mediated through 5HT(1D) receptors.     

Presynaptic inhibition by baclofen of miniature EPSCs and IPSCs in substantia gelatinosa neurons of the adult rat spinal dorsal horn

Intrathecal administration of baclofen, a GABA(B)-receptor agonist, affects pain behavior induced by formalin in a biphasic manner; baclofen at low doses enhances pain while producing antinociception at high doses. This may be due to the fact that baclofen modulates each of excitatory and inhibitory transmission in the dorsal horn of the spinal cord with a distinct sensitivity, resulting in a biphasic action on pain transmission. To address this issue, we examined the actions of baclofen on miniature excitatory (glutamatergic) and inhibitory (GABAergic) postsynaptic currents (mEPSCs and mIPSCs, respectively) in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell voltage-clamp technique. Baclofen reduced the frequency of both mEPSC and mIPSC without a change in their amplitudes. These actions were dose-dependent in a concentration range of 0.1-100 microM; the effective concentrations for the half-inhibition of mEPSC and mIPSC frequency were 4.44+/-0. 60 microM (n=7) and 4.31+/-0.77 microM (n=6), respectively. These results indicate that each of glutamatergic and GABAergic nerve terminals in the SG is endowed with the GABA(B) receptor, the activation of which depresses the release of neurotransmitter from the terminal; this provides a cellular basis for the modulation of pain by baclofen. It is suggested from a similar affinity for baclofen of the GABA(B) receptors on both terminals that the baclofen-induced biphasic action on pain behaviors cannot be accounted for by only its presynaptic actions in the SG and that other actions such as an inhibitory action of baclofen on postsynaptic neurons also have to be taken into consideration.     

Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy

EphrinB–EphB receptor signaling plays diverse roles during development, but recently has been implicated in synaptic plasticity in the matured nervous system and in pain processes. The present study investigated the correlation between expression of ephrinB and EphB receptor proteins and chronic constriction injury (CCI) of the sciatic nerve and dorsal rhizotomy (DR) in dorsal root ganglion (DRG) and spinal cord (SC); and interaction of CCI and DR on expression of these signals. Adult, male Sprague–Dawley rats were employed and thermal sensitivity was determined in the sham operated CCI and DR rats. Western blot and immunobiochemistry analysis and immunofluorescence staining techniques were used to detect the expression and location of the ephrinB–EphB receptor proteins in DRG and SC. The results showed that expression of ephrinB1 and EphB1 receptor proteins was significantly upregulated in DRG and SC in a time‐dependent manner corresponding to the development of thermal hyperalgesia after CCI. The increased expression is predominately located in the medium‐ and small‐sized DRG neurons, the superficial layers of spinal dorsal horn (DH) neurons, and the IB4 positive nociceptive terminals. DR increases ephrinB1 in DRG, not SC and EphB receptor in SC, not DRG. DR suppressed CCI‐induced upregulation of ephrinB1 in SC and EphB1 receptor in DRG and SC. These findings indicate that ephrinB–EphB receptor activation and redistribution in DRG and DH neurons after nerve injury could contribute to neuropathic pain. This study may also provide a new mechanism underlying DR‐induced analgesia in clinic.     

部分背根切断猫脊髓Ⅱ板层和背核c-jun的表达与脊髓可塑性的关系

目的:探讨即早基因c-jun蛋白在部分背根切断猫脊髓Ⅱ板层、背核(L3)表达的时空变化,以了解c-jun与脊髓可塑性的关系。方法:取成年健康雄性猫10只。随机分为2组:假手术组、单侧部分切断背根手术7d组(手术组),每组5只动物。取各组脊髓L3,L5,L6节段连续切片,片厚20μm,分别用兔抗c-jun(1∶200)抗体行免疫组化ABC法染色。观察并测量各组术侧脊髓Ⅱ板层(L3,L5,L6)和背核(L3)中c-jun蛋白的分布、含量及时空变化。结果:c-jun在假手术猫脊髓Ⅱ板层和背核的神经元和胶质细胞中均有表达。部分背根切断术后,c-jun阳性神经元数均较假手术组相应节段显著增加:L3,L5,L6II板层犤(17±3),(6±1)个;(13±2),(8±2)个;(11±2),(7±3)个犦,两组比较,差异有显著性意义(t=-7.778,-3.953,P<0.01;t=-2.481,P<0.05);L3背核,两组比较,差异有显著性意义(t=-5.000,P<0.01)。结论:部分背根切断术后c-jun在脊髓Ⅱ板层和背核表达增加,提示c-jun与脊髓Ⅱ板层和背核可塑性有关。     

Enhancement of NMDA receptor phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in neuropathic rats

NR1 is an essential component of functional NMDA receptors and can be activated by phosphorylation. It is suggested that phosphorylation of NR1 (pNR1) contributes to central sensitization after intradermal capsaicin injection. The present study investigates whether increases of spinal pNR1 are correlated to central sensitization and thus pain behaviors in neuropathic pain. Neuropathic rats were produced by L5 spinal nerve ligation, mechanical thresholds of the paw were measured, and then the L4/5 spinal cords and the nucleus gracilis (NG) were removed and immunostained for pNR1. The results showed that the number of pNR1-immunoreactive neurons was significantly increased in the ipsilateral cord, at 3, 7, and 28 days after nerve ligation and these increases coincide with mechanical allodynia. The increase of pNR1-immunoreactive neurons in the NG was observed only at 28 days after the nerve ligation. Western blot analyses confirmed the significant increase of pNR1 protein in spinal dorsal horn after nerve ligation. A protein kinase A inhibitor, H89, moderately reversed mechanical allodynia in 7 day neuropathic rats. Many pNR1-immunoreactive neurons were identified as projection neurons by retrograde tracer. The data suggest that PKA mediated NMDA receptor phosphorylation plays an important role in spinal nerve ligation induced neuropathic pain.     

鞘内注射吗啡对坐骨神经慢性压迫性损伤大鼠腰段脊髓后角神经元中c-fos蛋白表达的影响

目的 坐骨神经慢性压迫性损伤(CCI)可以引起腰段脊髓后角中c-fos的表达.μ-阿片受体长期以来被认为与脊髓中的镇痛机制有关,而吗啡作为μ-阿片受体激动剂被应用于病理性疼痛的治疗中.本实验通过给坐骨神经CCI大鼠模型鞘内注射吗啡,观察其对脊髓后角中c-fos表达的影响.方法 23只Sprague-Dawley大鼠随机分为三组.其中B、C两组大鼠接受右侧坐骨神经外周结扎手术,而A组为假手术组.9 d后,A、C两组大鼠接受鞘内注射吗啡同时给B组大鼠鞘内注射生理盐水.注射后6 d,解剖三组大鼠并取出L3～L5节段的脊髓制成40μm的冰冻切片.标本在室温下进行荧光免疫染色后制成玻片.使用激光共焦点显微镜下观察各脊髓切片标本双侧c-fos的染色情况.结果 三组大鼠手术侧同侧的脊髓后角中c-fos阳性神经元较对侧明显增多.但是c组大鼠脊髓后角同侧的c-fos阳性神经元较B组少.结论 μ-阿片受体激动剂能明显减少CCI大鼠腰段脊髓后角中c-fos的表达.     

不同程度腰椎椎管狭窄对脊髓背角神经肽的影响

目的:探讨不同程度的腰椎椎管狭窄时,相应节段脊髓背角中P物质(SP)和血管活性肠多肽(VIP)等神经肽含量和分布的变化特点,以及该变化的临床意义。方法:以家犬建立腰椎椎管狭窄的动物模型。实验分为A组:正常组;B组:对照组;C组:25%狭窄组;D组:50%狭窄组等4组,每组6只。分别检测各组脊髓背角SP、VIP的表达及含量变化。结果:脊髓背角中SP含量25%狭窄组各时相点差异无显著性意义(P>0.05)。50%狭窄组各时相点均非常显著升高(P<0.01)。脊髓背角中VIP含量25%狭窄组术后1,4周时较正常组、对照组有显著升高(P<0.05)。50%狭窄组在各时相点均显著(P<0.05)或非常显著(P<0.01)升高。结论:椎管狭窄造成的马尾神经压迫性损害可引起脊髓背角SP、VIP显著升高;脊髓背角中SP含量的升高随狭窄程度的增加而增加。VIP含量的升高不随狭窄程度的增加而增加。SP、VIP等神经肽类物质在腰椎椎管狭窄引起的下腰部和下肢疼痛的机制中扮演了重要的角色。     

脊髓背角细胞外信号调节激酶磷酸化参与调控2,4-二硝基氟苯诱导的小鼠慢性痒

目的：探讨脊髓背角细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）磷酸化对2,4-二硝基氟苯（2,4-dinitrofluorobenzene,DNFB）诱导的小鼠慢性痒的调控作用。方法：8～12周ICR雄性小鼠随机分为DNFB诱导组和丙酮对照组。DNFB组小鼠分别于面颊部和背部反复涂抹1.5% DNFB溶液,建立类似特异性皮炎的慢性痒模型。分别观察慢性痒模型建立后小鼠的自发性行为,以及鞘内注射MEK抑制剂U0126后小鼠自发性慢性痒行为的变化。免疫组织化学染色法分析小鼠脊髓组织磷酸化ERK（phosphrylated ERK,pERK）的表达情况。结果：面颊部诱导模型中,DNFB组小鼠的搔抓次数明显高于丙酮对照组（P<0.05）,而擦涂行为无差异,证实DNFB诱发的是痒觉;颈背部诱导模型中,DNFB组小鼠表现出剧烈的搔抓行为,而丙酮对照组的搔抓行为不明显。背部慢性痒模型建立后,分别在第1、3、7和14天鞘内注射MEK抑制剂U0126,小鼠的搔抓行为受到明显抑制（P<0.05）,且脊髓背角pERK的表达水平也明显降低（P<0.05）。pERK活化主要表达于小鼠脊髓背角Ⅰ～Ⅱ层,且与神经元标志物NeuN共标,与星形胶质细胞标志物GFAP和小胶质细胞标志物Iba1不共标。结论：脊髓背角浅层神经元ERK磷酸化可能参与调控DNFB诱导的慢性痒。     

Expression of 5-HT1A receptor mRNA in rat lumbar spinal dorsal horn neurons after peripheral inflammation

The present study observed the expression of the 5-hydroxytryptamine (5-HT) (1A) receptor mRNA in the lumbar spinal dorsal horn neurons following carrageenan inflammation using in situ hybridization (ISH). We also studied the co-localization of 5-HT(1A) receptor mRNA and gamma-amino butyric acid (GABA) or enkephalin (ENK) immunoreactivities using a combined fluorescent ISH and immunofluorescent histochemical double-staining technique. The finding of this study demonstrated that 5-HT(1A) receptor mRNA was widely distributed in the spinal dorsal horn with the highest density in laminae III-VI. Following carrageenan-induced inflammation, the 5-HT(1A) receptor mRNA expression in all layers of ipsilateral dorsal horn was significantly enhanced, and the peak occurred after 8h. Furthermore, the number of 5-HT(1A) receptor mRNA and GABA or ENK immunoreactive double-labeled cells was also markedly increased 8h after carrageenan injection. These findings suggested that following peripheral inflammation, the synthesis of 5-HT(1A) receptor was increased in the lumbar spinal dorsal horn neurons, especially in spinal GABA and ENK neurons.