Tramadol relieves thermal hyperalgesia in rats with chronic constriction injury of the sciatic nerve

The present study was designed to test whether tramadol is effective in the control of neuropathic pain in rats. Chronic constriction injury (CCI) of the sciatic nerve was induced over the left hind limb in male Sprague-Dawley rats. Identical surgery was performed on the opposite side except that the sciatic nerve was not ligated (sham surgery). Paw withdrawal latency (PWL) to heat was tested for each hind paw 1 day before surgery and on the 4th day after surgery to ensure the development of thermal hyperalgesia. In the acute treatment groups, saline or tramadol was administered subcutaneously at doses of 10, 20 or 30 mg/kg, and PWLs were measured 30, 60, 90, 120, 150 and 180 min after treatment. In the semi-chronic treatment groups, continuous systemic administration of tramadol 40 mg/kg/day or saline for 7 days was provided at a uniform rate via osmotic mini pumps. Tramadol reversed PWL in a dose-dependent manner in the acute treatment groups. PWLs were significantly reversed at 2 days after tramadol infusion, and this effect was sustained throughout the remainder of the treatment period in comparison with the saline group. Tramadol also resulted in a decreased sensitivity to thermal stimulus on the sham limb both in acute and semi-chronic administration. We conclude that both acute and semi-chronic tramadol treatment relieves thermal hyperalgesia effectively in rats with CCI of the sciatic nerve. This indicates that tramadol shows promise as a potential treatment for relief of neuropathic pain in humans.     

Effects of intrathecal lidocaine on hyperalgesia and allodynia following chronic constriction injury in rats

The present study investigated the effects of different doses of intrathecal lidocaine on established thermal hyperalgesia and tactile allodynia in the chronic constriction injury model of neuropathic pain, defined the effective drug dose range, the duration of pain‐relief effects, and the influence of this treatment on the body and tissues. Male Sprague–Dawley rats were divided into five groups and received intrathecal saline or lidocaine (2, 6.5, 15, and 35mg/kg) 7 days after loose sciatic ligation. Respiratory depression and hemodynamic instability were found to become more severe as doses of lidocaine increased during intrathecal therapy. Two animals in the group receiving 35mg/kg lidocaine developed pulmonary oedema and died. Behavioral tests indicated that 6.5, 15, and 35mg/kg intrathecal lidocaine showed different degrees of reversal of thermal hyperalgesia, and lasted for 2–8 days, while 2mg/kg lidocaine did not. The inhibition of tactile allodynia was only observed in rats receiving 15 and 35mg/kg lidocaine, and the anti‐allodynic effects were identical in these two groups. Histopathologic examinations on the spinal cords revealed mild changes in rats receiving 2–15mg/kg lidocaine. However, lesions were severe after administration of 35mg/kg lidocaine. These findings indicate that intrathecal lidocaine has prolonged therapeutic effects on established neuropathic pain. The balance between sympathetic and parasympathetic nervous activities could be well preserved in most cases, except for 35mg/kg. Considering the ratio between useful effects and side effects, doses of 15mg/kg are suitable for intrathecal injection for relief of neuropathic pain.     

2-氨基-5-磷酰基戊酸酯对大鼠坐骨神经慢性压迫性损伤的机械痛敏和热痛敏的影响

目的:建立大鼠坐骨神经慢性压迫性损伤(CCI)动物模型,腹腔注射N-甲基-D-天冬氨酸(NMDA)受体竞争性拮抗剂2-氨基-5-磷酰基戊酸酯(AP5)后观察腹腔用药对大鼠坐骨神经慢性压迫性损伤(CCI)动物模型机械痛敏和热痛敏的影响.方法:48只成年Wistar大鼠,雌雄不拘,随机分成3组,即Ⅰ组,假手术组(Sham),8只大鼠;Ⅱ组,CCI组,8只大鼠;Ⅲ组,治疗组,32只大鼠再分为4个亚组,Ⅲa组(CCI+NS),mb组[CCI+AP50.1mg/(kg·d)]、Ⅷc组(CCI+AP50.2 mg/kg)、Ⅲd组(CCI+AP50.5 mg/kg),每个亚组8只大鼠.48只大鼠分别于术前(0 d)及术后1、3、5、7、14、21 d测量每组大鼠的机械性缩足反射阈值(MWT)和热缩腿潜伏期(TWL).结果:CCI组从术后第3天开始直到本实验观察的术后第21天,MWT和TWL均明显降低,与假手术组Sham相比具有显著性(P＜0.01);CCI加生理盐水组大鼠与CCI组大鼠的MWT和TWL相比无明显改变(P＞0.05);腹腔注射AP5各个剂量的CCI组在术后5～21 d的MWT和TWL明显增加,与给药前和生理盐水组相比具有显著性(P＜0.01).结论:腹腔注射AP5有明显减轻大鼠CCI模型的机械性痛敏和热痛敏的作用.     

坐骨神经慢性挤压伤模型大鼠行为学及形态学变化

目的:观察大鼠坐骨神经慢性挤压伤模型所引起的行为学及病理形态学变化。方法:实验于2006-02/05在解放军军事医学科学院生物工程研究所完成。①SD大鼠24只,随机分为3组,坐骨神经结扎组12只制备右侧坐骨神经慢性挤压伤模型,正常对照组6只不干预,假手术组6只手术但不结扎,观察处理后42d内自发痛、触诱发痛、热刺激及冷刺激痛觉过敏等行为学变化。②SD大鼠54只,随机分为正常对照组、假手术组及坐骨神经结扎后3,7,14,21,28,35,42d组9组,处理同前,相应时间点处死大鼠观察坐骨神经和脊髓组织大体形态、苏木精-伊红染色和轴突髓鞘染色观察脊髓组织病理变化,电镜观察超微结构变化。结果:78只大鼠进入结果分析。①行为学变化:坐骨神经结扎组大鼠术后3d即出现明显的自发性疼痛,机械刺激痛阈值从术前的(17.33±5.42)g降至(3.28±1.37)g;热刺激爪退缩阈值从(12.13±2.37)s缩短至(10.43±1.65)s;冷刺激抬足次数从(3.50±1.09)次增加至(14.75±2.34)次。术后7￣14d这些阈值的变化逐渐达高峰,并持续至观察期结束(42d)。②坐骨神经结扎大鼠坐骨神经结扎部位及其远端轴突水肿,部分脱髓鞘。结论:大鼠坐骨神经慢性挤压伤模型可以产生明显的、稳定的自发性疼痛和诱发性疼痛等类似临床神经痛的症状和体征;其局部病理变化与症状相符。     

Effect of intrathecal octreotide on thermal hyperalgesia and evoked spinal c-Fos expression in rats with sciatic constriction injury

This study was designed to determine whether intrathecal octreotide (sandostatin), a synthetic octapeptide derivative of somatostatin, relieved thermal hyperalgesia and reduced the evoked spinal c-Fos expression in rats with chronic constriction injury (CCI) of the sciatic nerve. Intrathecal catheters were implanted in rats 7 days before CCI of the sciatic nerve over the left hind limb. After confirmation of the development of thermal hyperalgesia by decreased paw withdrawal latencies (PWL) to heat stimulation 7 days after CCI, intrathecal sandostatin at 20, 40, and 80 microg was administered, respectively. Rats in the control group received saline injections intrathecally. PWLs were evaluated at 30, 60, 120, 180, and 240 min after drug administration. Detection of Fos-like immunoreactivity (Fos-LI) neurons in the dorsal horn of the spinal cord following drug administration was performed after mechanical stimulation (stroking of the hind paws) on the 14th day after CCI. The reduction of PWL was attenuated significantly in the groups that received intrathecal sandostatin at 20, 40, and 80 g when compared with the saline group. However, PWL did not return to pre-CCI values in all groups. In the 40 microg group, PWL returned up to 76% of pre-CCI values 120 min after drug administration. Stroking of the hind paw in CCI-treated (ipsilateral) limbs induced a significantly greater expression of spinal Fos-LI neurons than that of non-CCI treated (contralateral) limbs in each group. The number of Fos-LI neurons in animals receiving intrathecal sandostatin was dose-dependently reduced. Expression of Fos-LI neurons in the 80 microg group was nearly completely inhibited. These data suggest that intrathecal sandostatin significantly relieved thermal hyperalgesia behaviorally but with limited effects and dose-dependently reduced spinal Fos-LI neurons expression evoked by stroking stimulation, which may reflect mechanical allodynia in rats with sciatic constriction injury. This implies that intrathecal sandostatin was effective in the treatment of neuropathic pain.     

Effect of NGF and anti-NGF on neuropathic pain in rats following chronic constriction injury of the sciatic nerve

The systemic administration of anti-nerve growth factor (NGF) antibodies can prevent local sensory hypersensitivity and block nociceptive fibers from sprouting into denervated adult rat skin. However, in the case of chronic constriction injury (CCI) in a rat, there is evidence that NGF reverses some effects of axotomy and alleviates thermal hyperalgesia. It is with this in mind that we investigated the influence of local anti-NGF and NGF on neuropathic pain and collateral sprouting caused by CCI. In our study, we looked at the effects to the ligated nerves after 30 consecutive days of local injections of anti-NGF and NGF. A high-dose of anti-NGF (1800 ng) was found to eradicate heat and cold hyperalgesia during postoperative days 16-28 and from days 8 to 34 after CCI, respectively. Our results show that a low-dose anti-NGF (18 ng) only mildly alleviates heat hyperalgesia but not cold hyperalgesia. There is evidence that a rebound phenomenon occurs for a short period of time after the anti-NGF injections cease. Results show that anti-NGF injections, whether in a high or low dose, significantly reduces the severity of autotomy or prevents the spread of collateral sprouting from the saphenous nerve into the sciatic innervation territory. In contrast, when a NGF (0.75 ng/g body weight) was applied to the ligated nerve immediately after the ligation, heat and cold hyperalgesia were eradicated during postoperative days 4-68 and from days 4 to 28, respectively. The results show that the effect of anti-NGF is delayed at the onset, is short in duration, and is dependent on the dosage. However, anti-NGF but not NGF blocked collateral sprouting and decreased the severity of autotomy, suggesting that anti-NGF may be a better potential alternative analgesic for the treatment of neuropathic pain in humans. The different initiation times to abolish thermal hyperalgesia by anti-NGF (delayed onset) and NGF (early onset) suggests that alterations in neurotrophic factors contribute to the development of behavioral hyperalgesia via a complex mechanism in CCI rats.     

Extraction of cyclic AMP for the determination in the competitive protein binding assay

The changes in free calcium, total calcium, albumin, protein, pH, lactate, sodium, magnesium, and potassium values during and following venous stasis (3 min at an external pressure of 100 mmHg) with and without forearm exercise have been measured. The pattern of changes observed with time were dependent on the presence or absence of forearm exercise. Without exercise significant changes are only observed at l min following stasis and then the increases are only moderate (free calcium 2.0%, total calcium 2.4%, albumin 6.9%, and protein 5.9%). However, when the forearm is exercised, larger increases (free calcium 8.6%, total calcium 8.4%, albumin and protein 12.4%) were observed for all parameters following stasis. These increases took 1 to 3 min to return to baseline for most parameters. Only potassium and magnesium went below baseline during the recovery period. It is recommended that when a tourniquet is used to aid in obtaining venous blood samples for these analytes, exercise of the forearm be avoided. If forearm exercise is unavoidable, then the sample should be obtained 1 to 3 min after release of the tourniquet.     

Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain

GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABA(A) and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA-immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA- and/or glycine-immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham-operated and nai;ve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I-III of the ipsilateral dorsal horn that showed GABA- or glycine-immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham-operated or nai;ve animals. In addition, we did not see any evidence for alterations of GABA- or glycine-immunostaining in the neuropil of laminae I-III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.     

cAMP反应元件结合蛋白在海洛因依赖大鼠杏仁核及海马的表达

目的：观察cAMP反应元件结合蛋白在海洛因依赖SD大鼠杏仁核及海马的表达。方法：实验于2004-12在贵阳医学院生理教研室完成。选择健康SD大鼠20只，随机分成海洛因依赖组和生理盐水对照组，每组10只。海洛因（纯度61．48％，贵州省公安厅提供）。海洛因依赖组按剂量逐日递增原则，2次／d，连续9d皮下注射海洛因。对照组以同样方法注射等量生理盐水。第10天用纳洛酮催促戒断，确定海洛因依赖模型的建立。采用SABC免疫组化染色观察两组杏仁和海马cAMP反应元件结合蛋白的表达情况，结合计算机图像分析系统，比较两组杏仁和海马cAMP反应元件结合蛋白阳性细胞数和平均吸光度值。结果：纳入大鼠20只，实验组在建立模型过程中有1只死亡，另外随机选取大鼠补充，最终进入结果分析大鼠保持为20只。①SABC免疫组化染色发现，海洛因依赖组大鼠杏仁核cAMP反应元件结合蛋白阳性细胞数比对照组显著增多[（7．69&;#177;0．86），（6．66&;#177;1．23），P〈0．05]。②两组大鼠杏仁核cAMP反应元件结合蛋白阳性细胞平均吸光度接近，差异无显著性[（215．56&;#177;4．17），（215．23&;#177;1．68），P〉0．05]。③海洛因依赖组大鼠海马cAMP反应元件结合蛋白阳性细胞平均吸光度显著低于对照组，差异有显著性[（218．84&;#177;2．54），（222．27&;#177;2．67），P〈0．05]。④两组大鼠海马cAMP反应元件结合蛋白阳性细胞数接近，差异无显著性[（17.34&;#177;1．90），（17．66&;#177;1．53），P〉0．05]。结论：cAMP反应元件结合蛋白在海洛因依赖大鼠的杏仁核及海马的表达增强，提示cAMP反应元件结合蛋白参与海洛因依赖的形成，可能与成瘾记忆有关。     

cAMP反应元件结合蛋白在海洛因依赖大鼠杏仁核及海马的表达

目的:观察cAMP反应元件结合蛋白在海洛因依赖SD大鼠杏仁核及海马的表达。方法:实验于2004-12在贵阳医学院生理教研室完成。选择健康SD大鼠20只,随机分成海洛因依赖组和生理盐水对照组,每组10只。海洛因(纯度61.48%,贵州省公安厅提供)。海洛因依赖组按剂量逐日递增原则,2次/d,连续9d皮下注射海洛因。对照组以同样方法注射等量生理盐水。第10天用纳洛酮催促戒断,确定海洛因依赖模型的建立。采用SABC免疫组化染色观察两组杏仁和海马cAMP反应元件结合蛋白的表达情况,结合计算机图像分析系统,比较两组杏仁和海马cAMP反应元件结合蛋白阳性细胞数和平均吸光度值。结果:纳入大鼠20只,实验组在建立模型过程中有1只死亡,另外随机选取大鼠补充,最终进入结果分析大鼠保持为20只。①SABC免疫组化染色发现,海洛因依赖组大鼠杏仁核cAMP反应元件结合蛋白阳性细胞数比对照组显著增多[(7.69±0.86),(6.66±1.23),P<0.05]。②两组大鼠杏仁核cAMP反应元件结合蛋白阳性细胞平均吸光度接近,差异无显著性[(215.56±4.17),(215.23±1.68),P>0.05]。③海洛因依赖组大鼠海马cAMP反应元件结合蛋白阳性细胞平均吸光度显著低于对照组,差异有显著性[(218.84±2.54),(222.27±2.67),P<0.05]。④两组大鼠海马cAMP反应元件结合蛋白阳性细胞数接近,差异无显著性[(17.34±1.90),(17.66±1.53),P>0.05]。结论:cAMP反应元件结合蛋白在海洛因依赖大鼠的杏仁核及海马的表达增强,提示cAMP反应元件结合蛋白参与海洛因依赖的形成,可能与成瘾记忆有关。     

Long-term changes of c-Fos expression in the rat spinal cord following chronic constriction injury.

The expression of c-Fos protein has been used as a relative marker of nociceptive neuronal activity in the spinal cord following various noxious stimuli. Experiments were conducted to examine c-Fos expression in lumbar spinal cord (L3-L6) following chronic constriction injury (CCI) in relation to nociceptive behavior over longer survival period up to 28 days. Development of mechanical allodynia was observed in the ipsilateral hind paw of CCI rats at day 3 and lasted up to 28 days. In contrast, the spinal c-Fos expression in CCI rats appeared in a biphasic manner. The highest number of c-Fos positive neurons occurred during the first week, followed by a decline at 7 and 14 days and reappearance at day 28 following injury. The early increase of c-Fos expression correlated with allodynia development, however, at longer survival period (28 days) c-Fos positivity become comparable in both CCI and sham groups despite their obvious behavior differences. Our results suggest that, at least in the CCI model, the c-Fos protein expression should not be considered as a reliable index of pain sensation disorders.     

Calcitonin ameliorates enhanced arterial contractility after chronic constriction injury of the sciatic nerve in rats

In addition to its regulatory effect on bone mass, calcitonin has been shown to relieve pain and alleviate peripheral circulatory disturbance in patients with Raynaud's syndrome and complex regional pain syndrome. In the present study, we investigated whether calcitonin ameliorates diminished blood flow and enhanced arterial contraction in response to noradrenaline in chronic constriction injury (CCI) of the sciatic nerve in rats. Following surgically induced CCI, laser Doppler flowmetry studies showed a significant decrease in plantar skin blood flow of the ipsilateral hind paw compared to the contralateral side. A subcutaneous bolus injection of elcatonin (20 U/kg), a synthetic derivative of eel calcitonin, significantly improved decreased skin blood flow in the ipsilateral side. In vitro analysis of plantar arteries isolated from the ipsilateral hind paw 7-13 days after the CCI procedure showed higher sensitivity to noradrenaline than the plantar arteries from the contralateral side. Elcatonin (0.1-10 nm) significantly reduced noradrenaline-induced contraction in the arteries of the ipsilateral side, whereas it had little effect on those of the contralateral side. These results suggest that calcitonin selectively ameliorates enhanced arterial contractility in CCI neuropathic rats, thus leading to its alleviating effect on peripheral circulatory disturbance.     

GW406381, a novel COX-2 inhibitor, attenuates spontaneous ectopic discharge in sural nerves of rats following chronic constriction injury

There are several lines of evidence to suggest that cyclooxygenase-2 (COX-2) plays an important role in the generation and maintenance of neuropathic pain states following peripheral nerve injury. However, COX-2 inhibitors are generally ineffective in reversing mechanical allodynia and hyperalgesia in models of neuropathic hypersensitivity. Here, we have investigated the effects of GW406381, a novel COX-2 inhibitor, on mechanical allodynia, hyperalgesia and generation of spontaneous ectopic discharge in rats following chronic constriction injury (CCI) of the sciatic nerve and compared it with rofecoxib. GW406381 (5mg/kg, 5 days of treatment) significantly reversed the CCI-induced decrease in paw withdrawal thresholds (PWTs), assessed using both von Frey hair and paw pressure tests, whereas an equi-effective dose of rofecoxib (5mg/kg, 5 days of treatment) in inflammatory pain models was ineffective. In rats treated with GW406381, the proportion of fibres showing spontaneous activity was significantly lower (15.58%) than that in the vehicle (32.67%)- and rofecoxib (39.66%)-treated rats. Ibuprofen, a non-selective COX inhibitor, at 5mg/kg, orally dosed three times a day for 5 days did not significantly affect the PWTs in CCI rats. In na?ve rats, GW406381 did not significantly change the PWTs. These results illustrate that COX-2 may indeed play an important role in the maintenance of neuropathic pain following nerve injury, but that only certain COX-2 inhibitors, such as GW406381, are effective in this paradigm. Whilst the mechanisms underlying this differential effect of GW406381 are not clear, differences in drug/enzyme kinetic interactions may be a key contributing factor.     

Critical role of the rostral ventromedial medulla in early spinal events leading to chronic constriction injury neuropathy in rats

Neuropathic pain is a major clinical problem, and several animal models have been developed to investigate its mechanisms and its treatment. In this report, the role of the rostral ventromedial medulla (RVM) in the early events of the chronic constriction injury (CCI) model was investigated in behavioral and electrophysiological experiments. Placing the 4 CCI ligatures around the sciatic nerve induced large discharges and residual ongoing activity in spinal nociceptive neurons. Two weeks after CCI ligation, the rats showed behavioral hyperalgesia and allodynia as well as increased ongoing activity and responsiveness of spinal nociceptive neurons to innocuous and noxious stimuli. Blockade of excitatory synapses in the RVM by a kynurenate microinjection (2 nmol in 0.5 muL) 5 minutes before placement of the sciatic ligatures had no immediate effect on spinal neuronal activity but largely prevented the activation of spinal neurons. In kynurenate microinjected rats, behavioral hyperalgesia and allodynia developed slowly and incompletely, which corresponded with an incompletely developed hyperexcitability of spinal neurons. To the best of our knowledge, these results show for the first time that the initial response to nerve damage requires facilitation from the RVM. PERSPECTIVE: The present and previous findings indicate that descending facilitation from brainstem nuclei critically contributes to the spinal hyperexcitability that underlies neuropathic pain. The present results indicate that this contribution begins at the very moment the nerve is damaged and should be prevented and treated accordingly.