From neuroanatomy to gene therapy: searching for new ways to manipulate the supraspinal endogenous pain modulatory system

The endogenous pain modulatory system is a complex network of brain areas that control nociceptive transmission at the spinal cord by inhibitory and facilitatory actions. The balance between these actions ensures effective modulation of acute pain, while during chronic pain the pronociceptive effects appear to prevail. The mechanisms underlying this imbalance were studied as to the role of two medullary components of the pain modulatory system: the dorsal reticular nucleus and the caudal ventrolateral medulla, which function primarily as pronociceptive and antinociceptive centres, respectively. Both areas are connected with the spinal dorsal horn by closed reciprocal loops. In the spino-dorsal reticular nucleus loop, the ascending branch is strongly inhibited by spinal GABAergic neurons, which may act as a buffering system of the dorsal reticular nucleus-centred amplifying effect. In the spino-caudal ventrolateral medulla loop, the ascending branch is under potent excitation of substance P (SP) released from primary afferents, which is likely to trigger the intense descending inhibition detected in acute pain. During chronic pain, the activity in the lateral reticular formation of the caudal ventrolateral medulla changes, so that the action of the caudal ventrolateral medulla upon SP-responsive spinal neurons shifts from inhibitory to excitatory. The mechanisms of this modulatory shift are unknown but probably relate to the decreased expression of micro-opioid, delta-opioid and GABAB receptors. Normalizing receptor expression in the caudal ventrolateral medulla or controlling noci-evoked activity at the dorsal reticular nucleus or caudal ventrolateral medulla by interfering with neurotransmitter release is now possible by the use of gene therapy, an approach that stands out as a unique tool to manipulate the supraspinal endogenous pain control system.     

Proteomic analysis of the dorsal spinal cord in the mouse model of spared nerve injury-induced neuropathic pain

Peripheral nerve injury often causes neuropathic pain and is associated with changes in the expression of numerous proteins in the dorsal horn of the spinal cord. To date, proteomic analysis method has been used to simultaneously analyze hundreds or thousands of proteins differentially expressed in the dorsal horn of the spinal cord in rats or dorsal root ganglion of rats with certain type of peripheral nerve injury. However, a proteomic study using a mouse model of neuropathic pain could be attempted because of abundant protein database and the availability of transgenic mice. In this study, whole proteins were extracted from the ipsilateral dorsal half of the 4th–6th lumbar spinal cord in a mouse model of spared nerve injury (SNI)-induced neuropathic pain. In-gel digests of the proteins size-separated on a polyacrylamide gel were subjected to reverse-phase liquid-chromatography coupled with electrospray ionization ion trap tandem mass spectrometry (MS/MS). After identifying proteins, the data were analyzed with subtractive proteomics using ProtAn, an in-house analytic program. Consequently, 15 downregulated and 35 upregulated proteins were identified in SNI mice. The identified proteins may contribute to the maintenance of neuropathic pain, and may provide new or valuable information in the discovery of new therapeutic targets for neuropathic pain.     

Noradrenergic pain modulation

Norepinephrine is involved in intrinsic control of pain. Main sources of norepinephrine are sympathetic nerves peripherally and noradrenergic brainstem nuclei A1-A7 centrally. Peripheral norepinephrine has little influence on pain in healthy tissues, whereas in injured tissues it has variable effects, including aggravation of pain. Its peripheral pronociceptive effect has been associated with injury-induced expression of novel noradrenergic receptors, sprouting of sympathetic nerve fibers, and pronociceptive changes in the ionic channel properties of primary afferent nociceptors, while an interaction with the immune system may contribute in part to peripheral antinociception induced by norepinephrine. In the spinal cord, norepinephrine released from descending pathways suppresses pain by inhibitory action on alpha-2A-adrenoceptors on central terminals of primary afferent nociceptors (presynaptic inhibition), by direct alpha-2-adrenergic action on pain-relay neurons (postsynaptic inhibition), and by alpha-1-adrenoceptor-mediated activation of inhibitory interneurons. Additionally, alpha-2C-adrenoceptors on axon terminals of excitatory interneurons of the spinal dorsal horn possibly contribute to spinal control of pain. At supraspinal levels, the pain modulatory effect by norepinephrine and noradrenergic receptors has varied depending on many factors such as the supraspinal site, the type of the adrenoceptor, the duration of the pain and pathophysiological condition. While in baseline conditions the noradrenergic system may have little effect, sustained pain induces noradrenergic feedback inhibition of pain. Noradrenergic systems may also contribute to top-down control of pain, such as induced by a change in the behavioral state. Following injury or inflammation, the central as well as peripheral noradrenergic system is subject to various plastic changes that influence its antinociceptive efficacy.     

Rostral ventromedial medulla control of spinal sensory processing in normal and pathophysiological states

Complex networks of pathways project from various structures in the brain to modulate spinal processing of sensory input in a top-down fashion. The rostral ventromedial medulla (RVM) in the brainstem is one major final common output of this endogenous modulatory system and is involved in the relay of sensory information between the spinal cord and brain. The net output of descending neurons that exert inhibitory and facilitatory effects will determine whether neuronal activity in the spinal cord is increased or decreased. By pharmacologically blocking RVM activity with the local anesthetic lignocaine, and then measuring evoked responses of dorsal horn neurons to a range of applied peripheral stimuli, our aim was to determine the prevailing descending influence operating in normal anesthetized animals and animals with experimental neuropathic pain. The injection of 0.8 microl 2% lignocaine into the RVM caused a reduction in deep dorsal horn neuronal responses to electrical and natural stimuli in 64% of normal animals and in 81% of spinal-nerve-ligated (SNL) animals. In normal animals, responses to noxious input were predominantly reduced, while in SNL animals, reductions in spinal cord activity induced by intra-RVM lignocaine further included responses to non-noxious stimuli. This suggests that in terms of activity at least, if not number, descending facilitations are the predominant RVM influence that impacts the spinal cord in normal animals. Moreover, the increase in the proportion of neurons showing a post-lignocaine reduction in dorsal horn activity in SNL rats suggests that the strength of these facilitatory influences increases after neuropathy. This predominant inhibitory spinal effect following the injection of lignocaine into the RVM may be due to blockade of facilitatory On cells.     

大鼠坐骨神经受压和解压后背根节和脊髓内神经元nNOS表达的变化

目的 :观察坐骨神经受压及解压后大鼠腰段背根节和脊髓内神经元型一氧化氮合酶 (nNOS)表达的变化 ,借以探讨外周神经源性痛的发病和影响机制。方法 :大鼠随机分为压迫组、解压组和对照组 ,采用聚乙烯管压迫坐骨神经的动物模型 ,用免疫细胞化学方法并结合计算机图像分析进行研究。结果 :与对照组比较 ,压迫组和解压组腰4～ 6背根节中nNOS的表达显著增加 ,相应节段脊髓背角的表达则明显降低 ;解压组与压迫组比较 ,背根节中nNOS的表达明显减少 ,而脊髓背角的已经下调的nNOS表达则回升 ,但仍然低于对照组水平。结论 :NO可能与神经源性痛时在中枢和外周的痛觉敏感性形成和神经系统长时程改变有关。     

5-HT_(1A)受体亚型mRNA在坐骨神经分支选择性损伤模型大鼠脊髓和背根节中的表达变化(英文)

坐骨神经分支选择性损伤 (SNI)模型是一种新型神经病理痛模型。本实验用 SD雄性大鼠 ,分支结扎并切断左侧坐骨神经干的胫神经和腓总神经 ,保留腓肠神经分支 ,右侧仅暴露坐骨神经。术后 1、2、3、4、7、14、2 1和 2 8d,用 RT-PCR的方法对 5 -HT1 A受体 m RNA在腰髓的背角和背根神经节 (DRG)的表达水平进行检测。结果显示 ,5 -HT1 A受体 m RNA在损伤侧腰髓背角内的表达水平于 1d后开始升高 ,7d时达高峰 ,随后逐渐下降 ,但仍高于正常水平。其表达水平在对侧脊髓背角内没有明显变化。在损伤侧 DRG内 ,5 -HT1 A受体 m RNA的表达水平于 1d后开始增高 ,4d时达高峰 ,随后开始下降 ,但仍维持较高的表达水平 ;而损伤对侧 DRG内的 5 -HT1 A受体 m RNA的表达没有变化。上述结果提示 5 -HT1 A受体亚型可能在脊髓及外周伤害性信息的传递和调节中发挥着重要作用 ,本研究的结果为进一步了解 5 -HT1 A受体在神经病理性痛中的作用机制提供了依据。     

Fast synaptic inhibition in spinal sensory processing and pain control

The two amino acids GABA and glycine mediate fast inhibitory neurotransmission in different CNS areas and serve pivotal roles in the spinal sensory processing. Under healthy conditions, they limit the excitability of spinal terminals of primary sensory nerve fibers and of intrinsic dorsal horn neurons through pre- and postsynaptic mechanisms, and thereby facilitate the spatial and temporal discrimination of sensory stimuli. Removal of fast inhibition not only reduces the fidelity of normal sensory processing but also provokes symptoms very much reminiscent of pathological and chronic pain syndromes. This review summarizes our knowledge of the molecular bases of spinal inhibitory neurotransmission and its organization in dorsal horn sensory circuits. Particular emphasis is placed on the role and mechanisms of spinal inhibitory malfunction in inflammatory and neuropathic chronic pain syndromes.     

Anatomical localization and expression pattern for the NMDA-2D receptor subunit in a rat model of neuropathic pain

The N-methyl-d-aspartate receptor (NMDAR) has been implicated in the etiology of chronic pain. In this regard, this study sought to characterize the localization and expression pattern for the NMDAR-2D subunit in a rat model of neuropathic pain. To this end, one group of rats, 3 weeks post-dorsal root rhizotomy (DRR) and a second group, 3 weeks post-spinal nerve ligation (SNL) and sham surgery, were generated. Dorsal root ganglia (DRG) and/or lumbar spinal cord were excised from DRR, naïve, SNL and sham rats. Both immunohistochemical and real-time PCR analysis confirmed discrete NMDAR-2D subunit expression within the DRG and dorsal horn. However, no overt differences in staining intensity or expression were noted between DRG and spinal cord sections obtained from the different surgical groups. Results also demonstrated that the NMDAR-2D subunit was present within Neu N+ cells in the spinal cord and DRG, but excluded from cells labeled with the astrocytic marker, GFAP, and the microglial maker, OX-42. Lastly, the NMDAR-2D subunit was not co-expressed within neurokinin-1 (NK-1)+ or neurofilament-52 (N-52)+ neurons, but the antibody did co-label a number of isolectin B4+ (IB4) DRG cells. Together, these findings seem to suggest that the NMDAR-2D receptor subunit is present within the cell body region of a population of small diameter sensory afferents and post-synaptically within second order dorsal horn neurons. Although these data suggest that the NMDAR-2D subunit is well poised anatomically to modulate pain neurotransmission, the expression pattern for this subunit is not altered in rats demonstrating the presence of neuropathic-like pain behavior.     

The medullary dorsal reticular nucleus as a pronociceptive centre of the pain control system

The endogenous pain control system has long been considered as engaged in pain depression through the commitment of multiple descending actions that reduce the response capacity of spinal dorsal horn nociceptive neurones. Such a pure inhibitory antinociceptive nature was lately questioned by the observation of pronociceptive effects from areas classically regarded as antinociceptive. The thereby raised hypothesis of a more versatile functional arrangement that dynamically adjusts the pain modulatory effect to multiple conditions by balancing several excitatory and inhibitory actions found strong support on the recent discovery of a medullary area particularly dedicated to pain facilitation.Lesioning the medullary dorsal reticular nucleus (DRt) depresses nociceptive responses to acute and inflammatory pain, whereas stimulation produces the inverse effect. The decrease in formalin-induced pain behaviour following DRt lesioning is accompanied by a decrease of spinal noxious-evoked c-fos neuronal activation. DRt blocking by lidocaine results in a decrease of the nociceptive activity of spinal dorsal horn neurones, whereas stimulation by glutamate has the opposite effect. A reciprocal disynaptic putative excitatory circuit that links the DRt and the spinal dorsal horn and conveys nociceptive input through the ascending branch was described, indicating that the DRt pain facilitating action is mediated by a reverberating spino-DRt circuit that promotes the enhancement of the response capacity of spinal neurones to noxious stimulation.The demonstration of a primary pronociceptive centre in the endogenous pain control system brings new important data to the emerging concept of pain modulation as a dynamic and flexible process that integrates nociceptive processing by balancing multiple excitatory and inhibitory actions as the way of adapting to the various unsteady pain determinants.     

Activation of peripheral P2X receptors is sufficient to induce central sensitization in rat medullary dorsal horn nociceptive neurons

Central sensitization and purinergic receptor mechanisms have been implicated as important processes in acute and chronic pain conditions following injury or inflammation of peripheral tissues. This study has documented that application of the P2X(1,2/3,3) receptor agonist αβ-meATP (100mM) to the rat tooth pulp induces central sensitization in medullary dorsal horn nociceptive neurons that is reflected in significant increases in mechanoreceptive field size and responses to noxious stimuli and decreased mechanical activation threshold. Furthermore, these responses can be blocked by pulp application of the P2X(1,2/3,3) antagonist TNP-ATP and also attenuated by medullary application of TNP-ATP. These results suggest that activation of P2X(1,2/3,3) receptors in orofacial tissues plays a critical role in producing central sensitization in medullary dorsal horn nociceptive neurons.     

Nitric oxide contributes to both spinal nociceptive transmission and its descending inhibition in rats: an immunocytochemical study

The present study was designed to determine if nitric oxide (NO) was involved both in the dorsal horn responses to the primary nociceptive inputs and the descending inhibitory action on these responses. The first part of the experiments showed that when formalin was injected into one hindpaw, the nociceptive c-fos expression in the lumbar dorsal horn ipsilateral to the injection was suppressed dose-dependently by intrathecal (i.t.) administration of N^ω-nitro-l-arginine (l-NNA), a nitric oxide synthase inhibitor. In the second part of the study, the formalin injection was carried out into two hindpaws of the rats with a sectioned dorsal quadrant at the thoracic spinal level, in these rats, there was a significant suppression of c-fos expression in the dorsal horn on the side with intact dorsal quadrant, reasonably owing to the preservation of the spinally descending inhibitory fibers from the supraspinal level; furthermore, this suppression could be canceled following i.t. l-NNA administration. The results suggest that endogenous NO not only facilitates the perception of nociceptive inputs at the spinal level but also enhances the descending inhibition upon the spinal nociception.     

Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury

The activation of glial cells in the CNS has been suggested to be involved in abnormal pain sensation after peripheral nerve injury. Previous studies demonstrated phosphorylation of p38 mitogen-activated protein kinase (MAPK) in spinal cord glial cells after peripheral nerve injury, and such phosphorylation has been suggested to be involved in the development of neuropathic pain. The aim of this study was to examine the dorsal column nuclei for phosphorylation of p38 MAPK following peripheral nerve injury and to explore a possibility of its contribution to neuropathic pain. Immunohistochemical labeling for phosphorylated p38 (p-p38) MAPK was performed in histological sections of the rat spinal cord and medulla oblongata after the fifth lumbar (L5) spinal nerve ligation (SNL). The number of p-p38 MAPK-immunoreactive (IR) cells was significantly increased in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury at days 3-21 after SNL. Double immunofluorescence labeling with cell-specific markers revealed that p-p38 MAPK-IR cells co-expressed OX-42, suggesting their microglial identity. Increased immunofluorescence labeling for OX-42 indicated that microglial cells were activated by SNL in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury. Continuous infusion of a p38 MAPK inhibitor into the cisterna magna for 14 days beginning on the day of SNL suppressed the development of tactile allodynia, but not thermal hyperalgesia induced by nerve injury. These results demonstrate that SNL activates p38 MAPK pathway in microglia in the gracile nucleus as well as in the spinal cord dorsal horn. Activation of p38 MAPK in medullary microglia may contribute to the pathogenesis of neuropathic pain.     

Spinal dis-inhibition in inflammatory pain

Inflammatory diseases and neuropathic insults trigger signaling cascades, which frequently lead to intense and long-lasting pain syndromes in affected patients. Such pain syndromes are characterized not only by an increased sensitivity to painful stimuli (hyperalgesia), but also by a qualitative change in the sensory perception of other, tactile stimuli (allodynia) and the occurrence of spontaneous pain in the absence of any sensory input. Long-term potentiation (LTP)-like changes in synaptic transmission between nociceptive C-fibers and spino-periaqueductal grey projection neurons as well as a loss of inhibitory control by GABAergic and glycinergic spinal dorsal horn neurons have repeatedly been proposed as underlying principles. While considerable evidence supports a significant contribution of C-fiber LTP to hyperalgesia, such monosynaptic plasticity cannot explain the occurrence of allodynia and spontaneous pain. In this review, we focus on mechanisms of synaptic dis-inhibition in inflammatory pain and propose that pathologically heightened pain sensitivity can be reversed by restoring synaptic inhibition with drugs that target specific spinal GABAA receptor subtypes.     

Upregulation of neuregulin-1 reverses signs of neuropathic pain in rats

Background: Peripheral nerve injury can result in neuropathic pain, a chronic condition of unclear cause often poorly responsive to current treatments. One possibility is that nerve injury disrupts large A-fiber-mediated inhibition of C-fiber-evoked responses in spinal dorsal horn neurons, leading to central sensitization. A recent study provided a potential molecular mechanism; large dorsal root ganglion (DRG) neurons secrete neuregulin-1 (NRG1), which binds to erbB4 receptors on interneurons and promotes GABA release to inhibit C-fiber-evoked nociceptive transmission. Thus, reduced NRG1 expression following nerve injury could induce chronic pain by disinhibition. We examined if DRG expression of NRG1 is in fact reduced in a rat model of neuropathic pain and if exogenous NRG1 alleviates behavioral signs of this condition. Methods: Three neuropathic pain models were established in rats: spared nerve injury of the tibial and common peroneal nerves (SNI model), intraplantar injection of complete Freund’s adjuvant (CFA model), and subcutaneous formalin injection. NRG1 expression was assessed by immunofluorescent staining, hyperalgesia by paw withdrawal threshold to von Frey filament stimulation, and pain-like behavior by spontaneous flinching. Results: NRG1 protein immunoreactivity was reduced in the rat DRG after SNI. Intrathecal administration of neuregulin-1beta 1 (NRG1-1), a 62 amino acid NRG1 mimetic, transiently increased paw withdrawal threshold in SNI model and reduced flinching in the formalin injection model. Conclusion: Our results are consistent with a model of neuropathic pain whereby peripheral nerve injury reduces NRG1-mediated inhibition of nociceptive signaling. Modulating NRG1 may have therapeutic potential for treating neuropathic pain.     

Projections from the mesopontine tegmental anesthesia area to regions involved in pain modulation

Pentobarbital microinjected into a restricted locus in the upper brainstem induces a general anesthesia-like state characterized by atonia, loss of consciousness, and pain suppression as assessed by loss of nocifensive response to noxious stimuli. This locus is the mesopontine tegmental anesthesia area (MPTA). Although anesthetic agents directly influence spinal cord nociceptive processing, antinociception during intracerebral microinjection indicates that they can also act supraspinally. Using neuroanatomical tracing methods we show that the MPTA has multiple descending projections to brainstem and spinal areas associated with pain modulation. Most prominent is a massive projection to the rostromedial medulla, a nodal region for descending pain modulation. Together with the periaqueductal gray (PAG), the MPTA is the major mesopontine input to this region. Less dense projections target the PAG, the locus coeruleus and pericoerulear areas, and dorsal and ventral reticular nuclei of the caudal medulla. The MPTA also has modest direct projections to the trigeminal nuclear complex and to superficial layers of the dorsal horn. Double anterograde and retrograde labeling at the light and electron microscopic levels shows that MPTA neurons with descending projections synapse directly on spinally projecting cells of rostromedial medulla. The prominence of the MPTA's projection to the rostromedial medulla suggests that, like the PAG, it may exert antinociceptive actions via this bulbospinal relay.     

Neuropathic pain: role for presynaptic T-type channels in nociceptive signaling

Pain is an important clinical problem and, in its chronic form, may be a disabling condition. Most currently available therapies are insufficient and/or accompanied by serious side effects. Recent studies have implicated the Ca_V3.2 isoform of T-type Ca channels in nociceptive signaling. Ca_V3.2 channels are located in the somas of dorsal root ganglion cells and in the central endings of these cells in the dorsal horn of the spinal cord. These channels can support the development and maintenance of both physiological (nociceptive) and pathological (neuropathic) pain. In this review, we summarize the most recent evidence linking the presynaptic Ca_V3.2 channels to the etiology of neuropathic pain disorders. In particular, we focus on data linking plasticity of Ca_V3.2 channels with neuropathic pain disorders associated with mechanical peripheral nerve injury and with diabetic peripheral neuropathy. We also discuss the development of potential pain therapies aimed at these channels.     

Influences of surgical decompression on the dorsal horn after chronic constriction injury: changes in peptidergic and delta-opioid receptor (+) nerve terminals

To understand plastic changes in the dorsal horn related to neuropathic pain, we developed a model of decompression in rats with chronic constriction injury (CCI) and investigated corresponding changes in the dorsal horn. At postoperative week 4 (POW 4) of CCI, rats were divided into a decompression group, in which ligatures were removed, and a CCI group, in which ligatures remained. Spinal cords were immunostained for substance P (SP), the delta-opioid receptor (DOR), and calcitonin gene-related peptide (CGRP). Areas of immunoreactive nerve terminals in the dorsal horn were quantified and expressed as the dorsal horn index (immunoreactive areas of the operated side compared with those of the contralateral side). At POW 4, dorsal horn indexes of all of these molecules were significantly reduced in both groups to similar degrees (0.36-0.43). At POW 8, neuropathic pain behaviors had completely disappeared in the decompression group with significant reversal of the dorsal horn indexes compared with the CCI group (0.81+/-0.02 vs. 0.58+/-0.09, P < 0.001 for SP and 0.75+/-0.04 vs. 0.55+/-0.03, P < 0.001 for DOR). In the CCI group, neuropathic pain behaviors became normalized at POW 12 with corresponding changes in dorsal horn indexes for both SP and DOR similar to those of the decompression group. In contrast, changes in the dorsal horn indexes of CGRP were similar in both the CCI and decompression groups throughout the experimental period. These findings suggest that CCI and decompression cause different patterns in peptidergic and DOR (+) nerve terminals in the dorsal horn.