Effect of multiple dorsal rhizotomies on calcitonin gene-related peptide-like immunoreactivity in the lumbosacral dorsal spinal cord of the cat: a radioimmunoassay analysis

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was measured by radioimmunoassay in the cat lumbosacral dorsal spinal cord following unilateral dorsal rhizotomy of 5 consecutive dorsal roots. The dorsal rhizotomies greatly reduced but did not eliminate the CGRP-LI from the ipsilateral rhizotomized segments. The amount of CGRP-LI remaining in the rhizotomized segments was greatest in the most caudal segment (846 +/- 311 pmoles/g tissue) and decreased below 300 pmoles/g tissue in the remaining segments. When these values were compared to the intact contralateral side, the percent CGRP remaining ranged from 65% in the sacral segments to less than 20% in the lumbar segments. Rostral to the rhizotomized segments there was a gradual return of CGRP-LI to control levels within 3 segments. Small diameter primary afferent fibers are the only known source of CGRP within the dorsal spinal cord. These results suggest that the most likely origin of the CGRP that remained in the rhizotomized lumbar segments was the rostrally and caudally projecting branches of ipsilateral primary afferents that entered the spinal cord through intact dorsal roots caudal and rostral to the transected roots. These results support the hypothesis that small diameter primary afferents project several segments in the cat spinal cord.     

Reduced numbers of calcitonin gene-related peptide-(CGRP-) and tachykinin-immunoreactive sensory neurones associated with greater enkephalin immunoreactivity in the dorsal horn of a mutant rat with hereditary sensory neuropathy

Summary The mutilated foot rat is a mutant with autosomal recessive sensory neuropathy and frequent mutilation of the hindlimbs. Decreased numbers of dorsal root ganglion cells and diminished sensitivity to painful stimuli are characteristics of these animals. By use of immunocytochemistry, changes in the distributions of peptides involved in sensory and/or autonomic regulation, i.e. calcitonin generelated peptide (CGRP), tachykinins, enkephalin and neuropeptide Y in spinal cord, dorsal root ganglia and skin of these animals, were studied. In comparison with normal litter-mate controls, the dorsal horn of mutilated foot rats contained substantially fewer CGRP and tachykinin-immunoreactive fibres but more fibres immunoreactive for enkephalin. Many enkephalin-immunoreactive cell bodies were also found in the dorsal horn of the mutants, by contrast none were visible in control animals. Neuropeptide Y immunoreactivity was, however, unchanged in the spinal cord of the mutants. In the dorsal root ganglia of the mutants, the number of CGRPor tachykinin-immunoreactive cells and their proportion to total neuronal numbers were significantly less in comparison with normal controls. The diameter range of CGRP- and tachykinin-immunoreactive cells shifted from small (15–25 m) to medium size (25–45 m) as revealed by frequency distribution histograms. The skin from the affected foreand hindlimbs of the mutant rats, in keeping with fewer CGRP- and tachykinin-immunoreactive cells in the dorsal root ganglia, contained substantially less fibres immunoreactive for CGRP and tachykinins; a difference that was not seen in skin of unaffected areas (whiskers and snout). By contrast, neuropeptide Y-immunoreactive fibres showed a normal distribution around blood vessels and sweat glands of mutilated foot rats. The data suggest that diminished pain perception in the mutilated foot rat is related to loss of peptide-containing sensory neurones. Furthermore, the intraspinal increase of enkephalinergic neurones in the dorsal horn, concomitant with the decreased number of primary sensory neurones, may also play a contributory rôle in reducing pain thresholds.     

Calcitonin gene-related peptide 8-37 inhibits the evoked discharge frequency of wide dynamic range neurons in dorsal horn of the spinal cord in rats.

The present study was performed to explore the effect of calcitonin gene-related peptide 8-37 (CGRP8-37) on the electrical stimulation-evoked discharge frequency of wide dynamic range (WDR) neurons in the dorsal horn of the spinal cord in rats. The discharge frequencies of WDR neurons were evoked by transdermic electrical stimulation applied on the ipsilateral hindpaw. CGRP8-37 was applied directly on the dorsal surface of the L3 to L5 spinal cord. After the administration of 3 nmol of CGRP8-37, the evoked discharge frequency of WDR neurons decreased significantly, an effect lasting more than 30 min. The results indicate that CGRP receptors play an important role in the transmission of presumed nociceptive information in the dorsal horn of the spinal cord.     

Oxytocin prevents neuronal network pain-related changes on spinal cord dorsal horn in vitro

Recently, oxytocin (OT) has been studied as a potential modulator of endogenous analgesia by acting upon pain circuits at the spinal cord and supraspinal levels. Yet the detailed action mechanisms of OT are still undetermined. The present study aimed to evaluate the action of OT in the spinal cord dorsal horn network under nociceptive-like conditions induced by the activation of the N-methyl-d-aspartate (NMDA) receptor and formalin injection, using calcium imaging techniques. Results demonstrate that the spontaneous Ca²⁺-dependent activity of the dorsal horn cells was scarce, and the coactivity of cells was mainly absent. When NMDA was applied, high rates of activity and coactivity occurred in the dorsal horn cells; these rates of high activity mimicked the activity dynamics evoked by a neuropathic pain condition. In addition, although OT treatment increased activity rates, it was also capable of disrupting the conformation of coordinated activity previously consolidated by NMDA treatment, without showing any effect by itself. Altogether, our results suggest that OT globally prevents the formation of coordinated patterns previously generated by nociceptive-like conditions on dorsal horn cells by NMDA application, which supports previous evidence showing that OT represents a potential therapeutic alternative for the treatment of chronic neuropathic pain.     

Calcitonin gene-related peptide-receptor component protein expression in the uterine cervix, lumbosacral spinal cord, and dorsal root ganglia

The neuropeptide calcitonin gene-related peptide (CGRP) may play a role in neurogenic inflammation, tissue remodeling of the uterine cervix, promoting vasodilation, parturition, and processing of sensory information in the spinal cord. CGRP-immunoreactive nerves of the cervix and spinal cord have been studied but cellular identification of the CGRP receptor has received little attention. CGRP-receptor component protein (CGRP-RCP) is a small protein associated with the CGRP receptor; thus, immunostaining for the CGRP-RCP can be used to identify sites of the CGRP receptor. We determined sites of CGRP-RCP immunoreactivity relative to the presence of CGRP-ir nerve fibers in the female rat uterine cervix, spinal cord, and dorsal root ganglia. CGRP-RCP immunoreactivity was expressed in the dorsal horn of the spinal cord, venules of the uterine cervix, and perikarya of sensory neurons in dorsal root ganglia. CGRP-immunoreactive fibers were adjacent to CGRP-RCP-immunoreactive vessels in the cervix and among CGRP-RCP-immunoreactive structures in the dorsal horn of the spinal cord. This suggests CGRP-RCP is associated with structures innervated by CGRP nerves and these interactions may be changed in tissues in response to an appropriate stimulus.     

Metformin attenuates increase of synaptic number in the rat spinal dorsal horn with painful diabetic neuropathy induced by type 2 diabetes: a stereological study

In our previous study, we have shown that number of synapses in the L5 segment of spinal dorsal horn increased significantly in a rat model of painful diabetic neuropathy (PDN) induced by high-dose of streptozotocin (an animal model of type 1 diabetes). The aims of this study were: (1) to determine whether high fat diet/low dose streptozotocin-diabetes, a rat model for type 2 diabetes, related PDN was also associated with this synaptic plasticity, (2) to reveal the range of this synaptic plasticity change occurred (in the whole length of spinal dorsal horn or only in the L5 lumbar segment of spinal dorsal horn) and (3) to discover whether treatment with metformin had effect on this synaptic plasticity. Male adult Sprague–Dawley rats were randomly allocated into the control group (n?=?7), the PDN group (n?=?6) and the PDN treated with metformin (PDN?+?M) group (n?=?7), respectively. 28 days after medication, synaptic and neuronal numbers in the whole length of spinal dorsal horn or in 1 mm length of the L5 segment of spinal dorsal horn were estimated by the optical disector (a stereological technique). Compared to the control group and the PDN?+?M group, number of synapses in the L5 segment of spinal dorsal horn increased significantly in the PDN group (P?<?0.05). There was no significant change between the control group and the PDN?+?M group in terms of the parameters in the L5 segment of the spinal dorsal horn (P?>?0.05). Parameters of the whole length of spinal dorsal horn showed no significant changes (P?>?0.05). Our results suggest that high fat diet/low dose streptozotocin diabetes related PDN is also associated with a numerical increase of synapses in the L5 segment of spinal dorsal horn but not in the whole length of spinal dorsal horn. Furthermore, the analgesic effect of metformin against PDN is related to its inhibition of numerical increase of synaptic number in the rat spinal dorsal horn.     

Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system

Using an antiserum directed against human calcitonin gene-related peptide (hCGRP), which fully cross reacts with rat CGRP, a sensitive radioimmunoassay was developed. The antiserum was characterized by displacement curve characteristics and high performance liquid chromatography. The assay was applied to rat brain tissue and the concentration of CGRP for 48 microdissected brain areas is presented. Highest levels (1000–4500 fmol/mg protein) were found in the central amygdaloid, caudate putamen, and spinal trigeminal nerve nucleus and tract, substantia gelatinosa, and the dorsal horn of the spinal cord. Moderate levels (200–600 fmol/mg protein) were found in the bed nucleus of the stria terminalis, the subfornical organ, the paraventricular, arcuate, dorsomedial, dorsal parabrachial, ambiguus and tractus solitarii nuclei and in the median eminence. These results coincide with those previously obtained by immunohistochemistry. The widespread distribution in the brain suggests involvement of CGRP in a variety of behavioral functions.     

针刺对鼠部分背根切断模型的背根神经节和脊髓背角内生长相关蛋白GAP43表达的影响

制备大鼠备用根模型 （切断单侧腰骶背根Ｌ２, ３, ４, ６, 及Ｓ１, ２, 保留Ｌ５ 背根）, 用免疫组织化学和原位杂交方法研究生长相关蛋白ＧＡＰ４３ 在相应节段背根神经节和脊髓背角表达的变化及针刺对其表达的影响。结果发现, 切断一侧Ｌ２, ３, ４, ６, 及Ｓ１, ２ 背根后, 它们对应的背根神经节内ＧＡＰ４３ 表达与正常对照组和假手术组相比无显著性差别, 而备用根神经节Ｌ５ 的ＧＡＰ４３ 表达较正常对照组和假手术组明显增强; 手术侧Ｌ５ 水平脊髓背角与正常对照组相比ＧＡＰ４３ 阳性信号加强。针刺后可促进手术侧Ｌ５ 神经节内ＧＡＰ４３ 表达增加; Ｌ５ 水平脊髓背角ＧＡＰ４３ 阳性信号也进一步加强这表明在一定条件下, 神经系统损伤可诱发中枢神经系统ＧＡＰ４３ 介导的可塑性变化, 针刺可通过ＧＡＰ４３ 对神经的可塑性起调节作用。     

Transient receptor potential vanilloid 1, calcitonin gene-related peptide, and substance P mediate nociception in acute pancreatitis

The mechanism of pancreatitis-induced pain is unknown. In other tissues, inflammation activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to liberate CGRP and substance P (SP) in peripheral tissues and the dorsal horn to cause neurogenic inflammation and pain, respectively. We evaluated the contribution of TRPV1, CGRP, and SP to pancreatic pain in rats. TRPV1, CGRP, and SP were coexpressed in nerve fibers of the pancreas. Injection of the TRPV1 agonist capsaicin into the pancreatic duct induced endocytosis of the neurokinin 1 receptor in spinal neurons in the dorsal horn (T10), indicative of SP release upon stimulation of pancreatic sensory nerves. Induction of necrotizing pancreatitis by treatment with L-arginine caused a 12-fold increase in the number of spinal neurons expressing the proto-oncogene c-fos in laminae I and II of L1, suggesting activation of nociceptive pathways. L-arginine also caused a threefold increase in spontaneous abdominal contractions detected by electromyography, suggestive of referred pain. Systemic administration of the TRPV1 antagonist capsazepine inhibited c-fos expression by 2.5-fold and abdominal contractions by 4-fold. Intrathecal, but not systemic, administration of antagonists of CGRP (CGRP(8-37)) and SP (SR140333) receptors attenuated c-fos expression in spinal neurons by twofold. Thus necrotizing pancreatitis activates TRPV1 on pancreatic sensory nerves to release SP and CGRP in the dorsal horn, resulting in nociception. Antagonism of TRPV1, SP, and CGRP receptors may suppress pancreatitis pain.     

Effect of aging on the substance P receptor,NK–1, in the spinal cord of rats with peripheral nerve injury

Substance P (SP) levels in the spinal cords of very old rats are less than the levels in younger rats (Bergman et al., 1996). After injury to a peripheral nerve in young rats, immunoreactivity (ir) to the SP receptor, NK–1 (neurokinin-1), increases in the spinal cord ipsilateral to the injury and the increases are correlated with the development of thermal hyperalgesia (Goff et al., 1998). Thus we postulated that aged rats might display an increased sensitivity to thermal stimulation before peripheral nerve injury and that they might respond differently to injury than do younger rats. To test this hypothesis, we used the Bennett and Xie model (1988) of chronic constriction injury (CCI) to the sciatic nerve to induce a neuropathic pain condition. We investigated the effect of age on changes in NK-1 ir in superficial layers of the dorsal horn and on numbers of NK ir cells in deeper laminae at the L4-L5 levels of the spinal cord after CCI. NK-1 receptors were tagged immunohistochemically and their distribution quantified by use of computer-assisted image analysis. NK-1 ir changes were related to alterations in thermal and tactile sensitivity that developed after CCI in young, mature and aged (4-6, 14-16, and 24-26 months) Fischer F344 BNF1 hybrid rats. No differences in thermal or tactile sensitivity of young and aged rats were seen in the absence of nerve injury. After injury, aged rats developed thermal hyperalgesia and tactile allodynia more slowly than did the younger rats. NK-1 receptor ir and numbers of NK-1 ir cells in the dorsal horn increased with time post-injury in all three groups. NK-1 ir increases were correlated with the development of thermal hyperalgesia in those rats that displayed hyperalgesia. However, some rats developed an increased threshold to thermal stimuli (analgesia) and that also was correlated with increases in NK-1 ir. Thus NK-1 ir extent, while correlated with thermal sensitivity in the absence of injury, is not a specific marker for disturbances in one particular sensory modality; rather it increases with peripheral nerve injury per se.     

大鼠脊髓诱发电位与脊髓损伤的关系——Ⅲ.脊髓局部损毁后电位的变化及电位来源分析

本文描述了大鼠脊髓L_1节段后柱、后索、侧索和前角的诱发电位及其损伤后的变化,并观察了切断L_4、L_5脊神经背、腹根与横断高位颈髓对电位的影响,以进行行电位来源分析。结果可见,上述四个区域的诱发电位基本由早反应三相波和晚反应组成。分别电解损毁这些部位后,电位波幅均普遍降低,晚期反应较早反应降低明显。后柱或后索受损对电位影响最大。局部损毁后可见L_1及T_(13)水平的硬膜上电位改变明显,尤其晚反应减弱、波峰平坦。反应时值与潜伏时未见明显改变。切断L_4脊神经背、腹根后、电位基本消失。去大脑对电位未见明显影响。结果表明,刺激坐骨神经诱发的脊髓电位起源于低位腰段传入神经和脊髓内多通路的兴奋传导,在一定程度上受腹根逆行活动的影响,与大脑及脊髓下行传导束活动无直接联系。脊髓诱发电位的幅度与波形改变可作为脊髓损伤的判断指标之一。     

Role of calcitonin gene-related peptide and its antagonist on the evoked discharge frequency of wide dynamic range neurons in the dorsal horn of the spinal cord in rats.

The present study was performed to explore the effect of calcitonin gene-related peptide (CGRP) and its antagonist CGRP8-37 on the evoked discharge frequency of wide dynamic range (WDR) neurons in the dorsal horn of the spinal cord in rats. Recording was performed with a multibarrelled glass micropipette and the chemicals were delivered by iontophoresis. The discharge of WDR neurons was evoked by transdermic electrical stimulation applied on the ipsilateral hindpaw. (1) Iontophoretic application of CGRP at an ejection current of 100 nA increased the discharge frequency of WDR neurons significantly. (2) Iontophoretic application of CGRP8-37 at an ejection current of 80 or 160 nA induced significant decreases in the discharge frequency of WDR neurons, but not at 40 nA. (3) Iontophoretic application of CGRP8-37 not only antagonized the CGRP-induced increase in the evoked discharge frequency of WDR neurons but also induced a significant decrease in the evoked discharge frequency of WDR neurons compared to basal levels. The results indicate that CGRP and its receptors play a facilitary role on the transmission and/or modulation of nociceptive information in the dorsal horn of the spinal cord in rats.     

Neurokinin A, calcitonin gene-related peptide, and dynorphin A (1-8) in spinal dorsal horn contribute to descending inhibition evoked by nociceptive afferent pathways: an immunocytochemical study

Immunocytochemical technique was used to compare the contents of neurokinin A (NKA), calcitonin gene-related peptide (CGRP), and dynorphin A (1-8) (DynA) on two sides of the lumbar dorsal horn of rats in which the unilateral thoracic dorsalateral funiculus (DLF) was transected while formalin (0.2 ml, 0.5%) was injected equally into two hindpaws. The results showed that all the NKA-like, CGRP-like, and DynA (1-8)-like immunoreactivities were significantly lower in the superficial laminae of the dorsal horn on the side ipsilateral to the lesioned DLF than that on the side with intact DLF. This implies that peripheral noxious inputs activate the supraspinal descending inhibitory systems which in turn modulate the transmission of noxious message at the spinal level by changing the release of related neuropeptides.     

去甲肾上腺素在脊髓背角的镇痛机制

疹髓背角浅层是传递和调制外周伤害性信息的关键部位。起源于脑干的去甲肾上腺素（NA）能纤维终止脊髓背角,它们释放的NA具有抑制初级传入末梢释放谷氨酸和P物质、增加Ⅱ层（胶状质）抑制性神经活性物质释放的作用。此外,形态学研究提示NA可能直接抑制Ⅰ/Ⅲ层向丘脑传递伤害性信息的投射神经元。NA可能通过以上途径,实现对外周伤害性信息传递的调制而发挥镇痛作用。     

不同频率电针刺激对神经痛模型大鼠脊髓背角P物质的影响

目的:观察P物质(Substance P,SP)在慢性坐骨神经压迫损伤(chronic constriction injury,CCI)模型脊髓中表达的变化,探讨电针镇痛的机制是否与脊髓背角中SP表达的变化有关。方法:选择32只雄性、体重180-200 g的SD大鼠,并将其随机均分为4组(n=8)。空白组(Con组)为正常痛阈值大鼠;假电针组(CCI+A组)在损伤的坐骨神经旁置入电针,但无电流刺激;2 Hz组和100Hz组分别给予相应频率电流刺激30 min。在实验开始前和术后1、4、7、14、20、22天记录大鼠的热缩足反射潜伏期(Paw Withdrawal Latency,PWL)和机械刺激缩足反射阈值(Paw Withdrawal Threshold,PWT)。免疫组化方法检测脊髓背角SP的表达。结果:术后20天,电针治疗后,100 Hz组和2 Hz组PWT分别为(7.33±1.42)g和(7.80±1.42)g,均显著高于假电针组(2.60±1.46)g,差异有统计学意义(P0.05)。100 Hz组在术后20天后和2 Hz组在术后14天后PWL值均显著高于假电针组,差异有统计学意义(P0.05)。免疫组化显示:2 Hz组和100 Hz组大鼠脊髓背角中P物质阳性细胞显著低于假电针组(P0.05)。结论:坐骨神经旁电针刺激能够显著减轻CCI模型大鼠热痛觉及机械痛觉过敏,其机制可能与抑制脊髓背角SP的表达有关。     

Decreased Expression and Role of GRK6 in Spinal Cord of Rats After Chronic Constriction Injury

Nerve injury and inflammation can both induce neuropathic pain via the production of pro-inflammatory cytokines. In the process, G protein-coupled receptors (GPCRs) were involved in pain signal transduction. GPCR kinase (GRK) 6 is a member of the GRK family that regulates agonist-induced desensitization and signaling of GPCRs. However, its expression and function in neuropathic pain have not been reported. In this study, we performed a chronic constriction injury (CCI) model in adult male rats and investigated the dynamic change of GRK6 expression in spinal cord. GRK6 was predominantly expressed in the superficial layers of the lumbar spinal cord dorsal horn neurons and its expression was decreased bilaterally following induction of CCI. The changes of GRK6 were mainly in IB4 and P substrate positive areas in spinal cord dorsal horn. And over-expression of GRK6 in spinal cord by lentivirus intrathecal injection attenuated the pain response induced by CCI. In addition, the level of TNF-α underwent the negative pattern of GRK6 in spinal cord. And neutralized TNF-α by antibody intrathecal injection up-regulated GRK6 expression and attenuated the mechanical allodynia and heat hyperalgesia in CCI model. All the data indicated that down-regulation of neuronal GRK6 expression induced by cytokine may be a potential mechanism that contributes to increasing neuronal signaling in neuropathic pain.     

CGRP受体拮抗剂CGRP8-37对甲醛炎性痛大鼠自发痛反应及脊髓后角NOS表达和NO含量的影响

目的:探讨CGRP受体拮抗剂CGRP8-37对甲醛炎性痛大鼠自发痛反应及脊髓后角NOS表达和NO含量的影响.方法:大鼠足底注射甲醛制造炎性痛模型;计数缩足反射次数反映自发痛程度;NADPH-d组织化学法观察脊髓后角NOS表达;硝酸还原酶法测定NO-3/NO-2含量以反映NO含量.结果:足底注射甲醛后,动物出现自发痛反应行为.足底注射甲醛后24 h,双侧脊髓后角NOS表达及NO含量明显增加.预先鞘内注射CGRP8-37可使甲醛诱导的自发性缩足反射次数明显减少,并可明显抑制甲醛炎性痛诱导的脊髓后角NOS表达及NO含量的增加.结论:甲醛炎性痛时,脊髓后角CGRP受体激活可促进NOS活性表达及NO的产生.     

The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn

The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1-expressing post-synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1-expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter-2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1-expressing primary afferents but did not affect TRPV1- and vesicular glutamate transporter-2-expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in almost all the lamina II neurons tested in control rats. In RTX-treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non-NMDA receptor antagonist. In RTX-treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post-synaptic currents of lamina II neurons in RTX-treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1-expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord.