天麻素对小鼠炎性痛脊髓背角自发放电抑制作用

目的:探究天麻素(gastrodin or GAS)对炎性痛小鼠诱致的脊髓背角I层神经元自发放电的影响。方法:成年小鼠10只,一侧后足底皮下注射完全弗氏佐剂(complete Freund’s adjuvant,CFA)制备炎性痛模型后,于6、12、24、48、96、168 h分别观察机械性缩足反射阈值(paw withdrawal mechanical threshold,PWMT)和热刺激缩足反射潜伏期(paw withdrawal thermal latency,PWTL)的变化。选取出生14~18 d小鼠,按照上述相同方法建立炎性痛模型,24 h后制备脊髓薄片(300μm),孵育1 h后用全细胞膜片钳技术通过灌流系统给予GAS(300μmol/L),并探究其对炎性痛脊髓背角I层神经元自发放电的影响。结果:(1)在成年小鼠炎性痛模型中,CFA诱致的痛觉过敏可持续1~2周,在24 h达到高峰;(2)在出生14~18 d小鼠CFA注射24 h后,脊髓背角I层神经元出现显著的自发放电增强现象;(3)GAS可以显著抑制脊髓背角I层神经元自发放电的频率,对幅度无显著作用。结论:GAS对炎症导致的脊髓背角I层神经元自发放电增强具有显著的抑制作用,进而降低神经元的兴奋性,从而达到缓解炎性痛的效果。     

Reduction in inflammation-induced sensitization of dorsal horn neurons by transcutaneous electrical nerve stimulation in anesthetized rats

Transcutaneous electrical nerve stimulation (TENS) is utilized to treat a variety of painful conditions. Inflamed animals present with an increased response to noxious stimuli, i.e., hyperalgesia, at the site of injury (primary hyperalgesia) and outside the site of injury (secondary hyperalgesia). Further, following acute inflammation, dorsal horn neurons show an increased responsiveness to peripherally applied stimuli, which has been termed sensitization. Previous studies demonstrate a reduction in dorsal horn neuron activity following TENS treatment in normal animals and a reduction in primary and secondary hyperalgesia in acutely inflamed animals. The purpose of this study was to examine the effects of TENS on dorsal horn neurons sensitized by acute inflammation. Extracellular recordings from wide dynamic range (WDR), high threshold (HT) and low threshold (LT) dorsal horn neurons in anesthetized rats were assessed for spontaneous activity, responses to innocuous and noxious mechanical stimulation and receptive field size. Responses were measured before and 3 h after induction of inflammation, and immediately and 1 h after application of either high (100 Hz) or low (4 Hz) frequency TENS (motor intensity, pulse duration = 100 microseconds). TENS was applied to the inflamed paw to encompass the receptive field of the neuron for 20 min. WDR and HT dorsal horn neurons sensitized to mechanical stimulation after induction of inflammation. Application of either high or low frequency TENS to the inflamed paw reduced both innocuous and noxious evoked responses of WDR and HT dorsal horn neurons immediately and 1 h after treatment with TENS. Comparison of responses after TENS with baseline responses showed that the evoked responses in the majority of WDR and HT cells returned to or fell below baseline responses. TENS had no effect on responses of LT neurons. In summary, central neuron sensitization is reduced by TENS and may underlie the reduction in hyperalgesia observed after treatment with TENS.     

Spinal effects of the calmodulin inhibitor calmidazolium on dorsal horn neurons in the rat

Drugs able to inhibit calmodulin activation can prevent some consequences of the rise in intracellular calcium. It has recently been shown that intrathecal injection of calmodulin inhibitors induce analgesia in rats. We study here the effect induced by the calmodulin inhibitor, calmidazolium, on the activity of dorsal horn neurons driven by noxious and non-noxious stimuli. Extracellular recordings of convergent (n = 12), low-threshold mechanoreceptive (n = 5) and proprioceptive (n = 5) units were made in the presence of calmidazolium. Calmidazolium (600 micrograms) reduced the noxious (50 degrees C) heat-evoked responses obtained in convergent neurons. On the contrary, the non-noxious tactile responses obtained in low-threshold mechanoreceptive neurons as well as the joint movement-evoked responses obtained in proprioceptive units remained unmodified. We conclude that calmidazolium can block nociceptive processing in the spinal cord and that this fact can help to explain the analgesic effects that intrathecal W-7 and calmidazolium induce in behavioral tests.     

Responses of spinal dorsal horn neurons to foot movements in rats with a sprained ankle

Acute ankle injuries are common problems and often lead to persistent pain. To investigate the underlying mechanism of ankle sprain pain, the response properties of spinal dorsal horn neurons were examined after ankle sprain. Acute ankle sprain was induced manually by overextending the ankle of a rat hindlimb in a direction of plantarflexion and inversion. The weight-bearing ratio (WBR) of the affected foot was used as an indicator of pain. Single unit activities of dorsal horn neurons in response to plantarflexion and inversion of the foot or ankle compression were recorded from the medial part of the deep dorsal horn, laminae IV-VI, in normal and ankle-sprained rats. One day after ankle sprain, rats showed significantly reduced WBRs on the affected foot, and this reduction was partially restored by systemic morphine. The majority of deep dorsal horn neurons responded to a single ankle stimulus modality. After ankle sprain, the mean evoked response rates were significantly increased, and afterdischarges were developed in recorded dorsal horn neurons. The ankle sprain-induced enhanced evoked responses were significantly reduced by morphine, which was reversed by naltrexone. The data indicate that movement-specific dorsal horn neuron responses were enhanced after ankle sprain in a morphine-dependent manner, thus suggesting that hyperactivity of dorsal horn neurons is an underlying mechanism of pain after ankle sprain.     

Inflammation induced increase of fluoride resistant acid phosphatase (FRAP) activity in the spinal dorsal horn in rats

Fluoride-resistant acid phosphatase (FRAP) has been suggested as an enzymatic marker for nociceptive primary afferent terminals in the spinal dorsal horn, however there has not been demonstrated a direct functional relation between FRAP activity and an increased nociceptive transmission. For this purpose, we quantitated FRAP activity in the spinal dorsal horn of the rat in a heat-induced cutaneous inflammatory model. Male Sprague-Dawley rats anaesthetised with thiopental were separated in two groups where the left hindpaw was submerged during 60 s either in water at room temperature (control group) or in water at 60 degrees C (inflammation group) which induce in this group a progressive hindpaw inflammation. After 8 h, the lumbar enlargement of the spinal cord was extracted, cut in slices and 1 mm micropunch fragments were obtained from the right and left dorsal horn. The activity of FRAP was determined using the Gomori colorimetric method and corrected by the protein concentrations. FRAP activity in the left dorsal horn was statistically higher than right dorsal horn in the inflammation group (3.05+/-0.54 versus 1.91+/-0.23 u/g per l; P<0.05). Also, FRAP activity from the left dorsal horn of the control and inflammation groups show a significant increase in the last group (3.05+/-0.54 versus 2.17+/-0.23 u/g per l; P<0.05). This results demonstrate that FRAP is not only an enzymatic marker for neuronal and fibre integrity of nociceptive primary afferents but also it is associated to the nociceptive afferent activation.     

不同剂量的一氧化氮对福尔马林炎性痛大鼠脊髓背角Bcl-2/Bax表达的影响

目的:探讨多次应用不同剂量的一氧化氮(NO)对福尔马林炎性痛中脊髓背角神经元Bcl-2、Bax表达的影响。方法:连续4 d给大鼠各进行鞘内注射不同剂量的一氧化氮前体左旋精氨酸(L-arginine,L-Arg)10μg/d(低L-Arg组)、250μg/d(高L-Arg组)或一氧化氮合酶(nitric oxide synthase,NOS)抑制剂Nω-硝基-L-精氨酸甲酯(Nω-nitro-L-arginine methylester,L-NAME)2700μg/d(L-NAME组),鞘内注射生理盐水(NS组)作为对照,各给药1次/d。随后于大鼠右后肢脚掌皮下注射福尔马林(2%,100μl),4 h后分别做免疫组化和Western Blot检测脊髓背角Bcl-2和Bax蛋白的表达。结果:免疫组化结果显示,Bcl-2和Bax表达分布于两侧脊髓背角,但主要位于脊髓背角浅层,各组在注射福尔马林同侧均明显高于对侧。Western Blot结果显示Bcl-2和Bax蛋白表达含量的比值,在各实验组与对照组比较,低L-Arg组Bcl-2/Bax比值明显升高,而高L-Arg组和L-NAME组Bcl-2/Bax比值均明显降低。bcl-2为抑制细胞凋亡的基因,而bax为促进细胞凋亡的基因。结论:在炎性痛模型中,低剂量的NO以促进抑凋亡基因的表达为主,而高剂量的NO以及NO生成不足均以促进促凋亡基因的表达为主,进而影响炎性痛的发生。     

Evidence that adenosine mediates the depression of spinal dorsal horn neurons induced by peripheral vibration in the cat

Nociceptive neurons in the dorsal horn of the cat spinal cord are depressed by vibration applied to the ipsilateral hind limb. The present study investigated the pharmacological properties of this depression because of the possibility that it represents the neural basis at the spinal level for the analgesic effects of vibration in humans. Experiments were done in cats anesthetized with sodium pentobarbital and acutely spinalized at the first lumbar level. Extracellular recordings were made from nociceptive neurons in the lower lumbar segments. The depression of these neurons induced by vibration to the hindlimb was attenuated by administration of the P1-purinergic (adenosine) receptor antagonist, caffeine (20-60 mg/kg i.v.); the maximum attenuation was 100%. Effects of caffeine began within 2 min after the start of injection (1-3 min injection period), were greatest in the 10 min period after the end of injection and lasted for up to 2 hr. Importantly, another P1-purinergic receptor antagonist, which does not cross the blood-brain barrier, 8-sulphophenyltheophylline (8-16 mg/kg), had no effect on the depression when given intravenously (n = 5); however, when administered by iontophoresis 8-sulphophenyltheophylline blocked the depression in 2 of 6 units. Dipyridamole (1.0-2.0 mg/kg i.v.), an inhibitor of adenosine uptake, potentiated the depression in 2 of 5 cases. These results prompt us to suggest that depression induced by vibration may be mediated by adenosine via the activation of P1-purinergic receptors. On the other hand, the GABAA antagonist, bicuculline, failed to attenuate vibration-induced depression when administered either intravenously (0.2-0.4 mg/kg; n = 5) or by iontophoresis (n = 10) and the glycine antagonist, strychnine (0.2-0.6 mg/kg; n = 3) and the opiate antagonist, naloxone (0.1-0.4 mg/kg; n = 4) were similarly ineffective. These findings suggest that vibration-induced depression of these units occurs without involvement of bicuculline-sensitive GABA receptors, strychnine-sensitive glycine receptors and naloxone-sensitive opiate receptors. In view of the fact that vibration-induced depression is evoked synaptically, this study is the first to demonstrate in the central nervous system a synaptic response which is mediated by adenosine. In addition, we suggest that the analgesic effects of vibration in humans may be mediated at the spinal level by activation of P1-purinergic receptors.     

Synaptic activation of dorsal horn neurons by selective C-fibre excitation with capsaicin in the mouse spinal cord in vitro.

Low doses (0.2-0.8 microM) of capsaicin were used to achieve selective excitation of C-fibres and the consequent synaptic activation of dorsal horn neurons (laminae I-VI) in the spinal cord of the 12-20-day-old mouse, maintained in vitro. Most dorsal horn cells were activated by application of capsaicin to dorsal root ganglia. The response consisted of a long-lasting membrane depolarization with increased regenerative (synaptic) activity in 79% of the cells, and in a further 7% only an increased synaptic activity was evoked. These effects of capsaicin were completely blocked by removing extracellular calcium ions from the superfusate to the spinal cord, or by the addition of 1 microM tetrodotoxin, suggesting a presynaptic origin of the capsaicin action. Only 67% of cells excited by capsaicin were sensitive to exogenous substance P. The excitatory amino acid antagonists, kynurenic acid (50-100 microM) or (-)-2-amino-5-phosphonovaleric acid (10-20 microM) completely blocked the capsaicin-evoked response in deep dorsal horn cells, indicating the involvement of excitatory amino acid receptors in the synaptic pathway. However, in superficial dorsal horn neurons these antagonists attenuated, but never completely abolished, the capsaicin-evoked depolarization. The kynurenic acid-resistant component of the capsaicin-evoked excitation in superficial dorsal horn cells suggests the involvement of non-amino acid excitatory transmitters--possibly neuropeptides--in the synaptic transmission. Activation of primary afferents by high-intensity electrical stimulation of the dorsal roots induced a prolonged (0.5-3 s) postsynaptic excitation in the majority of deep dorsal horn cells. The duration of the synaptic response was significantly reduced by (-)-2-amino-5-phosphonovaleric acid. Following repeated application of capsaicin, desensitization of the capsaicin-evoked synaptic activation of dorsal horn cells occurred. This effect was paralleled with the loss of the prolonged (-)-2-amino-5-phosphonovaleric acid-sensitive phase of the excitatory postsynaptic potential evoked by the high-intensity electrical stimulation of dorsal roots. This observation suggested that activation of the N-methyl-D-aspartate receptors in the dorsal horn can be activated by small-calibre capsaicin-sensitive fibres. In summary, our data suggest that the selective activation of C-fibre afferents with capsaicin produces synaptic activity in the dorsal horn which has a strong excitatory amino acid component as well as a non-excitatory amino acid, possibly peptidergic, component.     

Differential input to dorsal horn dorsal spinocerebellar tract neurons in mid- and low-lumbar segments from upper cervical spinal cord in the cat

Previous studies showed that ipsilaterally projecting dorsal horn dorsal spinocerebellar tract (dh-DSCT) neurons located outside Clarke's column in mid- and caudal-lumbar segments of the spinal cord receive different afferent inputs. Here, we examined, using extracellular recordings in anaesthetized cats, whether there are also input differences to these populations of dh-DSCT neurons from: (a) the spinocervical tract (SCT), estimated by stimulation of the ipsilateral dorsolateral funiculus at cervical cord C3 and rostral C1, below and above the termination of SCT axons in the lateral cervical nucleus (LCN), and (b) descending/ascending fibres activated by electrical stimulation at rostral C1. Seventy percent (21/30) of the lower-lumbar (L6-L7) dh-DSCT neurons received significantly greater excitation from C3 than rostral C1, whereas only 17% (5/30) of the mid-lumbar (L5) dh-DSCT neurons had greater responses from C3 than rostral C1. Inhibition of background activity was seen in 30% of the lower-lumbar neurons, but only in 3% of mid-lumbar neurons. These findings suggest that lower-lumbar dh-DSCT neurons are much more likely, than mid-lumbar ones, to be influenced by the SCT and by systems descending from the brain, LCN and/or ascending systems. The experiments provide further evidence for differences in input to the subpopulations of dh-DSCT neurons.     

Paradoxical effects of a selective cyclooxygenase-2 inhibitor,etodolac, on proliferative changes of forestomach in alloxan-induced diabetic rats

A single intravenous injection of alloxan, a non-genotoxic diabetogenic chemical, induces proliferative changes in forestomach mucosa of rats, and some lesions progress to squamous cell carcinoma accompanied with inflammatory change. The present study was conducted to examine the effects of a selective cyclooxygenase-2 (COX-2) inhibitor, etodolac, on the proliferative changes of forestomach mucosa in alloxan-induced diabetic rats. Alloxan-induced diabetic rats were fed a diet containing 0.01% etodolac (AL+Et group) and standard diet (AL group). They were sacrificed after 25 and 50 weeks of feeding, respectively. Squamous cell hyperplasia of forestomach was completely suppressed by etodolac after 25 weeks. After 50 weeks of treatment, the proliferative changes in forestomach developed in all rats of the AL+Et group, but in only 55.6% of the rats in the AL group. The severity of proliferative lesions was much enhanced in the AL+Et group compared to the AL group, and was parallel to the inflammatory changes in individual cases. Ulceration and erosion were more severe in the AL+Et group. These findings demonstrate that etodolac suppresses proliferative and inflammatory changes with COX-2 expression of forestomach in the early stage, but enhances them after 50 weeks.     

大鼠坐骨神经分支选择性损伤后脊髓背角毛细血管和星形胶质细胞的反应

目的探讨成年大鼠坐骨神经分支选择性结扎切断(SNI)后,腰段脊髓背角内毛细血管和星形胶质细胞的反应。方法成年雄性SD大鼠40只,随机分为光镜组(n=30)和电镜组(n=10)。光镜组又分为对照组、假手术组和SNI手术组。SNI手术为分别结扎切断左侧胫神经和腓总神经,保留腓肠神经,存活20d。部分动物在术后经尾静脉给予30g/L伊文思蓝,切取脊髓L4平面,荧光显微镜观察伊文思蓝的渗出;另一部分在术后进行常规固定取材,切片进行抗鼠IgG和GFAP的免疫组织化学反应。电镜组(对照组和SNI后20d组),脊髓切片进行常规抗大鼠IgG的免疫电镜处理。结果 1.SNI后20d,可见明显的伊文思蓝渗出红色光斑;2.SNI后GFAP阳性细胞变为反应型,数量明显增加,毛细血管壁及其周围的IgG颗粒也显著增加。3.电镜观察:SNI术后20d,IgG阳性颗粒增多,内皮细胞观察到下列变化:(1)内皮细胞向管腔内伸出许多微细的突起,有的含有IgG阳性颗粒。(2)内皮细胞的管腔侧胞膜上有许多"内吞"小凹,凹内可见IgG阳性颗粒,胞质内有含IgG阳性颗粒的小泡,在反管腔侧胞膜上有"胞吐"现象。(3)内皮细胞紧密连接出现间隙,间隙内有IgG颗粒。(4)胞质内出现囊泡,有的泡内显示IgG阳性颗粒。结论 SNI后脊髓背角内毛细血管内皮细胞发生明显的反应,大鼠IgG和伊文思蓝可经过血脊髓屏障渗出,星形胶质细胞被激活。     

Responses of medullary dorsal horn neurons to corneal stimulation by CO(2) pulses in the rat

Corneal-responsive neurons were recorded extracellularly in two regions of the spinal trigeminal nucleus, subnucleus interpolaris/caudalis (Vi/Vc) and subnucleus caudalis/upper cervical cord (Vc/C1) transition regions, from methohexital-anesthetized male rats. Thirty-nine Vi/Vc and 26 Vc/C1 neurons that responded to mechanical and electrical stimulation of the cornea were examined for convergent cutaneous receptive fields, responses to natural stimulation of the corneal surface by CO(2) pulses (0, 30, 60, 80, and 95%), effects of morphine, and projections to the contralateral thalamus. Forty-six percent of mechanically sensitive Vi/Vc neurons and 58% of Vc/C1 neurons were excited by CO(2) stimulation. The evoked activity of most cells occurred at 60% CO(2) after a delay of 7-22 s. At the Vi/Vc transition three response patterns were seen. Type I cells (n = 11) displayed an increase in activity with increasing CO(2) concentration. Type II cells (n = 7) displayed a biphasic response, an initial inhibition followed by excitation in which the magnitude of the excitatory phase was dependent on CO(2) concentration. A third category of Vi/Vc cells (type III, n = 3) responded to CO(2) pulses only after morphine administration (>1.0 mg/kg). At the Vc/C1 transition, all CO(2)-responsive cells (n = 15) displayed an increase in firing rates with greater CO(2) concentration, similar to the pattern of type I Vi/Vc cells. Comparisons of the effects of CO(2) pulses on Vi/Vc type I units, Vi/Vc type II units, and Vc/C1 corneal units revealed no significant differences in threshold intensity, stimulus encoding, or latency to sustained firing. Morphine (0.5-3.5 mg/kg iv) enhanced the CO(2)-evoked activity of 50% of Vi/Vc neurons tested, whereas all Vc/C1 cells were inhibited in a dose-dependent, naloxone-reversible manner. Stimulation of the contralateral posterior thalamic nucleus antidromically activated 37% of Vc/C1 corneal units; however, no effective sites were found within the ventral posteromedial thalamic nucleus or nucleus submedius. None of the Vi/Vc corneal units tested were antidromically activated from sites within these thalamic regions. Corneal-responsive neurons in the Vi/Vc and Vc/C1 regions likely serve different functions in ocular nociception, a conclusion reflected more by the difference in sensitivity to analgesic drugs and efferent projection targets than by the CO(2) stimulus intensity encoding functions. Collectively, the properties of Vc/C1 corneal neurons were consistent with a role in the sensory-discriminative aspects of ocular pain due to chemical irritation. The unique and heterogeneous properties of Vi/Vc corneal neurons suggested involvement in more specialized ocular functions such as reflex control of tear formation or eye blinks or recruitment of antinociceptive control pathways.     

Differential processing of noxious colonic input by thoracolumbar and lumbosacral dorsal horn neurons in the rat

Previous studies suggest the lumbosacral (LS) spinal cord processes acute colorectal stimuli whereas the thoracolumbar (TL) and LS spinal segments process inflammatory stimuli. In this study, the effects of colorectal distention (CRD) on TL and LS dorsal horn neuronal activity were recorded in Nembutal-anesthetized male rats both with and without colonic inflammation. Both single cells (before and after inflammation) and populations (multiple cells from noninflamed or inflamed rats) were studied. CRD-responsive neurons had excitatory Abrupt (on-off with stimulus) or Sustained (prolonged after discharge) responses or were Inhibited by CRD. In noninflamed rats, a significantly greater percentage of LS neurons (63% Abrupt, 27% Sustained) were excited by CRD than TL neurons (61% Abrupt, 3% Sustained). The remaining cells were Inhibited (10% LS, 36% TL). LS Abrupt neurons had lower thresholds and greater response magnitudes to CRD compared with TL Abrupt neurons. After colonic inflammation, TL neurons became more excitable: the percentage of Inhibited neurons decreased, the response magnitude of Abrupt neurons increased, and the threshold decreased. In contrast, in single-cell recordings, the response of LS Sustained neurons increased, whereas LS Abrupt neurons decreased. These data suggest that in noninflamed rats, the net response to CRD of TL visceroceptive spinal sensory neurons is less than that of LS neurons. Colonic inflammation increases the net response of TL neurons and differentially modulates the response of LS neurons. These differences may contribute to the functional dichotomy between the TL and LS spinal segments in processing acute and inflammatory colorectal pain.     

体视学研究坐骨神经慢性限制性损伤对大鼠脊髓背角内突触数量的可塑性改变及COX-2抑制剂的作用

目的:探讨坐骨神经慢性限制性损伤(CCI)所致神经病理性疼痛是否伴有脊髓背角神经元和突触数量的可塑性变化以及帕瑞昔布干预的作用.方法:正常成年SD大鼠随机分为假手术组、CCI组及帕瑞昔布组.术后28d取第5腰段脊髓作石蜡包埋切片,分别用尼氏染色和突触素的免疫组织化学显色显示神经元和突触,采用体视学新技术--光学体视框估计脊髓背角内神经元和突触的数量.结果:与对侧未手术侧相比,CCI组手术侧单位长度脊髓背角内的突触数及突触数与神经元数之比分别增加了86%、98%;帕瑞昔布组手术侧单位长度脊髓背角内的突触数及突触数与神经元数之比分别增加了78%、68%.与假手术组手术侧相比,CCI组手术侧单位长度脊髓背角内的突触数及突触数与神经元数之比分别增加了78%、73%;帕瑞昔布组则分别增加了81%、71%.结论:CCI所致神经病理性疼痛伴有脊髓背角内突触数量增加的可塑性变化,COX-2抑制剂帕瑞昔布对CCI致突触数量的增加无作用.     

选择性环氧合酶-2抑制剂对结肠癌细胞的生长抑制作用及机制探讨

目的：研究NS-398（一种选择性环氧合酶-2抑制剂）对结肠癌HT-29细胞的生长抑制作用并探讨其机制。 方法： 通过MTT法检测细胞的增殖情况，通过流式细胞术检测细胞凋亡率和细胞周期，通过RT-PCR检测bcl-2 mRNA和bax mRNA表达，通过共聚焦激光扫描显微镜观察细胞骨架成分F-actin的变化。 结果： NS-398对结肠癌HT-29细胞生长的抑制具有时间和剂量依赖性。流式细胞术结果显示NS-398可剂量依赖地诱导HT-29细胞凋亡，使其停滞于G0/G1期。经不同浓度的NS-398处理72 h后，bcl-2 mRNA 在HT-29细胞的表达降低，bcl-2／bax比值降低。细胞骨架成分F-actin主要分布在HT-29细胞核周围，呈环状结构，NS-398作用后细胞核周围的环状结构消失。 结论： NS-398可显著抑制结肠癌细胞的体外生长并诱导其凋亡，这与下调bcl-2／bax比值以及细胞骨架的破坏有关。     

Differential effects of MK-801, a N-methyl-D-aspartate non-competitive antagonist, on the dorsal horn neuron hyperactivity and hyperexcitability in neuropathic rats

N-methyl-D-aspartate (NMDA) involvement in altered spinal neuron activity following peripheral nerve injury has been investigated in rats with chronic constriction of the sciatic nerve. Extracellular single neuron recordings were performed, in anesthetized, paralyzed rats, from the sciatic spinal cord segments (lumbar, L5-L6) ipsilateral to the constriction, and the effect of iontophoresized MK-801, an NMDA receptor non-competitive antagonist, was tested on baseline hyperactivity and hyperresponsiveness to noxious stimulation. The results show that baseline activity was unaffected whereas the noxious evoked responses were significantly modified, there being amplitude reduction and after-discharges suppression. The different role of NMDA in the abnormal pain states related to the abnormal neuronal activities is discussed.     

GABAA-Receptor blockade reverses the injury-induced sensitization of nociceptor-specific (NS) neurons in the spinal dorsal horn of the rat

Single-unit electrical activity was recorded from 80 nociceptor-specific (NS) neurons in the dorsal horn of the lumbar spinal cord of pentobarbital anesthetized rats. Their responses to low- and high-intensity mechanical stimulation of their receptive fields (RFs) were recorded before and after the application of irritant agents [capsaicin (CAP) or mustard oil (MO)] to the RF. Before the applications of the irritants the neurons responded only to high-intensity stimuli, but after this procedure 20 of 28 neurons tested were sensitized, i.e., gave increased responses to high-intensity stimuli and showed novel responses to low-intensity mechanical stimulation as well as an Abeta-fiber afferent drive. CAP was more likely to induce sensitization than MO and the majority of sensitized neurons were located in the superficial dorsal horn. No relationship was found between the magnitude of the response to the sensitizing agent and the presence or absence of sensitization. Cumulative doses of two gamma-aminobutyric acid type A (GABA(A))-receptor antagonists, picrotoxin and bicuculline, were administered systemically or applied directly over the spinal cord. The GABA(A) antagonists reversed the sensitization of the neurons by reducing the novel low-threshold responses. These results show that NS neurons in the spinal dorsal horn can be sensitized by a sustained afferent discharge in peripheral nociceptors and that this sensitization can be reduced or reversed by low doses of GABA(A)-receptor antagonists. This provides evidence for a mechanism in which an enhanced GABAergic transmission can lead to hyperexcitability and sensitization of NS neurons in the dorsal horn.     

Different effects of spinally applied prostaglandin D2 on responses of dorsal horn neurons with knee input in normal rats and in rats with acute knee inflammation

Prostaglandin D(2) (PGD(2)) is the most produced prostanoid in the CNS of mammals, and in behavioral experiments it has been implicated in the modulation of spinal nociception. In the present study we addressed the effects of spinal PGD(2) on the discharge properties of nociceptive spinal cord neurons with input from the knee joint using extracellular recordings in vivo, both in normal rats and in rats with acute inflammation in the knee joint. Topical application of PGD(2) to the spinal cord of normal rats did not influence responses to mechanical stimulation of the knee and ankle joint except at a high dose. Specific agonists at either the prostaglandin D(2) receptor 1 (DP1) or the prostaglandin D(2) receptor 2 (DP2) receptor had no effect on responses to mechanical stimulation of the normal knee. By contrast, in rats with inflamed knee joints either PGD(2) or a DP1 receptor agonist decreased responses to mechanical stimulation of the inflamed knee and the non-inflamed ankle thus reducing established inflammation-evoked spinal hyperexcitability. Vice versa, spinal application of an antagonist at DP1 receptors increased responses to mechanical stimulation of the inflamed knee joint and the non-inflamed ankle joint suggesting that endogenous PGD(2) attenuated central sensitization under inflammatory conditions, through activation of DP1 receptors. Spinal application of a DP2 receptor antagonist had no effect. The conclusion that spinal PGD(2) attenuates spinal hyperexcitability under inflammatory conditions is further supported by the finding that spinal coapplication of PGD(2) with prostaglandin E(2) (PGE(2)) attenuated the PGE(2)-induced facilitation of responses to mechanical stimulation of the normal joint.     

Antifibrotic potential of a selective COX-2 inhibitor (celecoxib) on liver fibrosis in rats

Hepatic stellate cells (HSCs) play a critical role in the fibrogenesis of the liver, so they are the target cells of antifibrotic therapy. Activated HSCs, but not quiescent HSCs, express cyclooxygenase-2 (COX-2). The present study was designed to investigate the possible prophylactic and therapeutic effects of a selective COX-2 inhibitor (celecoxib) on liver fibrosis induced by thioacetamide (TAA) in rats. Forty-two male albino rats were divided into five groups: group I, negative control; group II, model of fibrosis; group III, preventive model before induction of fibrosis where celecoxib was given for 4 weeks before TAA; group IV, preventive model at the time of induction of fibrosis where celecoxib was given concomitantly with TAA for 6 weeks; group V, therapeutic model treated after induction of fibrosis. Liver function tests, serum TGF-β1 (ELISA), and histopathological examination of liver sections were performed. Both Metavir and Ishak fibrosis scoring systems were used for the evaluation of fibrosis. Groups III, IV, and V showed significant amelioration of liver function tests and a decrease in serum TGF-β1 as compared to the fibrosis model group (II). Histopathological examination showed the mildest degree of fibrosis in group III. Celecoxib had a hepatoprotective and therapeutic effect against liver damage produced by TAA but with different degrees. The highest efficacy of celecoxib was in the preventive group (III) before time of induction of liver fibrosis, followed by the therapeutic group (V) and then the preventive group (IV) at time of induction of liver fibrosis.