Therapeutic and prophylactic effects of macrophage-derived small extracellular vesicles in the attenuation of inflammatory pain

Small extracellular vesicles (sEVs) derived from antigen-presenting cells such as macrophages can induce therapeutically relevant immune responses. Anti-inflammatory miRNAs are elevated in sEVs secreted by RAW 264.7 mouse macrophages after lipopolysaccharide (LPS) stimulation. We observed uptake of these sEVs by primary mouse cortical neurons, microglia and astrocytes followed by downregulation of proinflammatory miRNA target genes in recipient cells. Pre-treating primary microglia with these sEVs decreased pro-inflammatory gene expression. A single intrathecal injection of sEVs derived from LPS stimulated RAW 264.7 cells attenuated mechanical hyperalgesia in the complete Freund’s adjuvant (CFA) mouse model of inflammatory pain and formalin induced acute pain. Importantly, sEVs did not alter the normal pain threshold in control mice. RNA sequencing of dorsal horn of the spinal cord showed sEVs-induced modulation of immune regulatory pathways. Further, a single prophylactic intrathecal injection of sEVs two weeks prior, attenuated CFA-induced pain hypersensitivity and was ineffective in formalin model. This indicates that prophylactic sEVs administration can be beneficial in attenuating chronic pain without impacting responses to the protective physiological and acute inflammatory pain. Prophylactic administration of sEVs could form the basis for a safe and novel vaccine-like therapy for chronic pain or as an adjuvant, potentially reducing the dose of drugs needed for pain relief.     

Induction of type IV collagen and other basement-membrane-associated proteins after spinal cord injury of the adult rat may participate in formation of the glial scar.

We investigated the spatial and temporal expression of basement-membrane-forming and neurite-outgrowth-supporting matrix proteins after a unilateral dorsal root injury combined with a collagen I/laminin-1 graft and a stab wound lesion to the dorsal horn of the adult rat spinal cord. Ten days after injury, the gamma1 laminin was induced in the reactive glia. At this early stage, the glial cells failed to express type IV collagen and the alpha1 laminin. One month after injury, reactive astrocytes in the dorsal horn of the lesioned side expressed gamma1 laminin, type IV collagen, and the alpha1 laminin whereas astrocytes of the normal spinal cord or the uninjured contralateral dorsal horn were negative. Both astrocytes and neurons of the ipsilateral ventral horn were induced to express laminin-1 and gamma1 laminin. Astrocytes of the ipsilateral ventral horn also expressed type IV collagen. Simultaneously with the changes in expression of the extracellular matrix proteins, the expression pattern of basic fibroblast growth factor (FGF-2) was markedly altered after spinal cord injury. In normal and contralateral spinal cord, FGF-2 was expressed in nerve fibers, but its expression changed from neuronal into glial in the ipsilateral spinal cord within 1 month after injury. Four months after injury, expression of both type IV collagen and the alpha1 laminin had declined, but the astrocytes at the injury site continued expressing the gamma1 laminin. Cultured astrocytes were negative for type IV collagen, but several cytokines, including IL-1beta and TGFbeta1, induced expression of type IV collagen in the astrocytes. These factors also increased deposition of type IV collagen matrix in the glial cultures. These results indicate that type IV collagen and the alpha1 laminin are induced in reactive astrocytes after spinal cord injury in vivo. Induction of type IV collagen in astrocytes in vitro by cytokines indicates that blood-borne or local factors at the injury site may induce the spinal cord glial expression of type IV collagen in vivo. Simultaneous expression of laminin-1 and alpha1 laminin with type IV collagen is known to lead to production of basement membranes. This may hamper the neurite-outgrowth-promoting potential of the gamma1 laminin by initiating formation of the glial scar.     

Inflammatory pain reduces correlated activity in the dorsal column nuclei

Persistent pain can result in sensitization of neurons in the spinal cord dorsal horn and produce physiological changes in sites such as the thalamus, that receive projections from the dorsal horn. Although the dorsal column nuclei receive both primary afferent input and projections from the dorsal horn, their participation in persistent pain states is relatively unexplored, perhaps because they play a limited role in acute, cutaneous nociception. We have used a model of inflammatory pain to examine the physiological properties of dorsal column nucleus neurons during persistent pain. We used this model in order to minimize direct damage to large myelinated primary afferents that project directly to the dorsal column nuclei. Inflammation was produced by injection of complete Freund's adjuvant into one hindpaw in rats, and neurons in the gracile nucleus were recorded 2-8 days later. Inflammation resulted in increased responsiveness to nociceptive (pinch) stimulation and increased incidence of afterdischarge firing 2-3 days after injection. Spontaneous activity was increased 6-8 days after injection. Inflammation decreased the strength of correlated firing in neuron pairs that shared common inputs, but did not affect the strength of monosynaptic interactions between neurons. These results suggest that the dorsal column nuclei can participate in persistent pain processes. Based on their anatomical connections, the dorsal column nuclei may contribute to thalamic changes during persistent pain as well as to supraspinal centers that modulate pain transmission in the spinal cord.     

Effects of inflammation on the ultrastructural localization of spinal cord dorsal horn group I metabotropic glutamate receptors

Inflammatory pain is thought to induce functional plasticity of spinal dorsal horn neurons and may produce changes in glutamate receptor expression. Plasticity of group I metabotropic glutamate receptors (mGluR1 and mGluR5) is important in various neuronal systems, and these receptors are also known to modulate nociceptive neurotransmission in the spinal dorsal horn. The present study aimed at determining whether persistent inflammatory pain produces alterations in intracellular and plasma membrane-associated mGluR1alpha and mGluR5 in spinal cord dorsal horn. Persistent inflammation was induced in male Long Evans rats by a unilateral intraplantar injection of 100 muL of complete Freund's adjuvant (CFA). Three days after the CFA injection thermal withdrawal latencies were obtained prior to processing of transverse spinal cord sections for preembedding immunogold labeling after incubation in primary antibody for mGluR1alpha or mGluR5. Using electron microscopy, we quantified immunogold-labeled mGluR1alpha and mGluR5 profiles, located in lamina V and I-II, respectively, of both CFA-treated rats and untreated control rats. Compared to untreated rats, CFA-treated rats had a significant increase in the number of plasma membrane-associated mGluR5 immunogold-labeled particles in lamina I-II neurons of the spinal cord. Although no changes to mGluR1alpha expression were found in CFA-treated rats, plasma membrane-associated mGluR1alpha was significantly closer to the synapse. Therefore, in CFA-treated rats there was a specific increase in the ratio of plasma membrane-associated versus intracellular immunogold-labeled particles for mGluR5, and lateral movement of mGluR1alpha toward the synapse, indicating that peripheral inflammation-induced trafficking of group I mGluRs in spinal dorsal horn neurons may be an important factor in the development of plastic changes associated with inflammation-induced chronic pain.     

Spatial and temporal activation of spinal glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats

In the spinal cord, neuron and glial cells actively interact and contribute to neurofunction. Surprisingly, both cell types have similar receptors, transporters and ion channels and also produce similar neurotransmitters and cytokines. The neuroanatomical and neurochemical similarities work synergistically to maintain physiological homeostasis in the normal spinal cord. However, in trauma or disease states, spinal glia become activated, dorsal horn neurons become hyperexcitable contributing to sensitized neuronal-glial circuits. The maladaptive spinal circuits directly affect synaptic excitability, including activation of intracellular downstream cascades that result in enhanced evoked and spontaneous activity in dorsal horn neurons with the result that abnormal pain syndromes develop. Recent literature reported that spinal cord injury produces glial activation in the dorsal horn; however, the majority of glial activation studies after SCI have focused on transient and/or acute time points, from a few hours to 1 month, and peri-lesion sites, a few millimeters rostral and caudal to the lesion site. In addition, thoracic spinal cord injury produces activation of astrocytes and microglia that contributes to dorsal horn neuronal hyperexcitability and central neuropathic pain in above-level, at-level and below-level segments remote from the lesion in the spinal cord. The cellular and molecular events of glial activation are not simple events, rather they are the consequence of a combination of several neurochemical and neurophysiological changes following SCI. The ionic imbalances, neuroinflammation and alterations of cell cycle proteins after SCI are predominant components for neuroanatomical and neurochemical changes that result in glial activation. More importantly, SCI induced release of glutamate, proinflammatory cytokines, ATP, reactive oxygen species (ROS) and neurotrophic factors trigger activation of postsynaptic neuron and glial cells via their own receptors and channels that, in turn, contribute to neuronal-neuronal and neuronal-glial interaction as well as microglia-astrocytic interactions. However, a systematic review of temporal and spatial glial activation following SCI has not been done. In this review, we describe time and regional dependence of glial activation and describe activation mechanisms in various SCI models in rats. These data are placed in the broader context of glial activation mechanisms and chronic pain states. Our work in the context of work by others in SCI models demonstrates that dysfunctional glia, a condition called "gliopathy", is a key contributor in the underlying cellular mechanisms contributing to neuropathic pain.     

Up-regulation and time course of protein kinase C immunoreactivity during persistent inflammation of the rat spinal cord☆

BACKGROUND： It has been reported that activation and/or translocation of protein kinase C （PKC） is related to hyperalgesia, and changes in PKC expression in the dorsal horn of spinal cord take place during inflammatory pain. OBJECTIVE： To observe PKC changes in the dorsal horn of spinal cord using immunohistochemistry and to measure the time-course during persistent pain produced by chemical stimulation with a right hind-paw injection of formalin. DESIGN： Randomized controlled animal experiment. SETTING： Institute of Basic Medical Science, Hebei Medical University MATERIALS： The present experiment was performed at the Department of Pathophysiology, Institute of Basic Medical Science, Hebei Medical University between September 2000 and June 2002. Forty-two Sprague-Dawley rats, weighing 260-280 g, irrespective of gender, were provided by the Center of Animal Experimentation at Hebei Medical University. PKC antibody was provided by Sigma, USA. Immunohistochemistry kits were purchased from Zhongshan Biotechnology Company, Beijing. HPIAS-1000 definition multicolor system was provided by Qianping Wuxiang Project Company of Tongji Medical University. Animal use during experimentation was consistent with the standards of Animal Ethics Committee. METHODS： Sprague-Dawley rats were divided randomly into control （n = 6） and experimental groups （n = 36）. Experimental rats were given an intracutaneous injection of 5% formalin into the planta surface of the right hind-paw. Animals with inflammatory pain were anesthetized and sacrificed to obtain the L5 spinal region at 1, 3, 12 hours, 1, 3, and 7 days after formalin treatment, with 6 rats in each time group. The spinal cords at the L5 region were collected from the control group following sodium chloride injections into the planta surface of the right hind-paw, identical to the experimental group. MAIN OUTCOME MEASURES： Pain reaction of experimental rats after formalin treatment. PKC-positive neurons, and distribution of PKC-immunoreactive particles, i     

关节炎大鼠脊髓背角内脑源性神经营养因子和促肾上腺皮质激素释放因子表达增加(英文)

研究表明 ,外周感染可导致脑源性神经营养因子 (BDNF)的过度表达 ,BDNF可影响其他神经递质的合成。采用免疫组化和原位杂交的方法观察了完全福氏佐剂所致的关节炎大鼠脊髓内 BDNF及其功能性受体酪氨酸蛋白激酶 B(trk B)的表达和促肾上腺皮质激素释放因子 (CRF) m RNA的水平。实验发现 ,在皮下注射完全福氏佐剂后 4h,脊髓腰膨大同侧背角中的 BDNF免疫活性神经元和 CRF m RNA阳性神经元数升高 ,在 2 4 h时达到峰值 ,在 7d时仍维持在较高水平。实验提示 ,关节炎大鼠脊髓内的 BDNF和 CRF可能参与了慢性痛反应。     

Tumor necrosis factor-mediated downregulation of spinal astrocytic connexin43 leads to increased glutamatergic neurotransmission and neuropathic pain in mice

Spinal cord astrocytes are critical in the maintenance of neuropathic pain. Connexin 43 (Cx43) expressed on spinal dorsal horn astrocytes modulates synaptic neurotransmission, but its role in nociceptive transduction has yet to be fully elaborated. In mice, Cx43 is mainly expressed in astrocytes, not neurons or microglia, in the spinal dorsal horn. Hind paw mechanical hypersensitivity was observed beginning 3 days after partial sciatic nerve ligation (PSNL), but a persistent downregulation of astrocytic Cx43 in ipsilateral lumbar spinal dorsal horn was not observed until 7 days post-PSNL, suggesting that Cx43 downregulation mediates the maintenance and not the initiation of nerve injury-induced hypersensitivity. Downregulation of Cx43 expression by intrathecal treatment with Cx43 siRNA also induced mechanical hypersensitivity. Conversely, restoring Cx43 by an adenovirus vector expressing Cx43 (Ad-Cx43) ameliorated PSNL-induced mechanical hypersensitivity. The sensitized state following PSNL is likely maintained by dysfunctional glutamatergic neurotransmission, as Cx43 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of glutamate receptor antagonists MK801 and CNQX, but not neurokinin-1 receptor antagonist CP96345 or the Ca²⁺ channel subunit α₂δ₁ blocker gabapentin. The source of this dysfunctional glutamatergic neurotransmission is likely decreased clearance of glutamate from the synapse rather than increased glutamate release into the synapse. Astrocytic expression of glutamate transporter GLT-1, but not GLAST, and activity of glutamate transport were markedly decreased in mice intrathecally injected with Cx43-targeting siRNA but not non-targeting siRNA. Glutamate release from spinal synaptosomes prepared from mice treated with either Cx43-targeting siRNA or non-targeting siRNA was unchanged. Intrathecal injection of Ad-Cx43 in PSNL mice restored astrocytic GLT-1 expression. The cytokine tumor necrosis factor (TNF) has been implicated in the induction of central sensitization, particularly through its actions on astrocytes, in the spinal cord following peripheral injury. Intrathecal injection of TNF in naïve mice induced the downregulation of both Cx43 and GLT-1 in spinal dorsal horn, as well as hind paw mechanical hypersensitivity, as observed in PSNL mice. Conversely, intrathecal treatment of PSNL mice with the TNF inhibitor etanercept prevented not only mechanical hypersensitivity but also the downregulation of Cx43 and GLT-1 expression in astrocytes. The current findings indicate that spinal astrocytic Cx43 are essential for the maintenance of neuropathic pain following peripheral nerve injury and suggest modulation of Cx43 as a novel target for developing analgesics for neuropathic pain.     

Upregulation of Chemokine CXCL12 in the Dorsal Root Ganglia and Spinal Cord Contributes to the Development and Maintenance of Neuropathic Pain Following Spared Nerve Injury in Rats

Emerging evidence indicates that CXCL12/CXCR4 signaling is involved in chronic pain. However, few studies have systemically assessed its role in direct nerve injury-induced neuropathic pain and the underlying mechanism. Here, we determined that spared nerve injury(SNI)increased the expression of CXCL12 and its cognate receptor CXCR4 in lumbar 5 dorsal root ganglia(DRG)neurons and satellite glial cells. SNI also induced longlasting upregulation of CXCL12 and CXCR4 in the ipsilateral L4–5 spinal cord dorsal horn, characterized by CXCL12 expression in neurons and microglia, and CXCR4 expression in neurons and astrocytes. Moreover, SNIinduced a sustained increase in TNF-a expression in the DRG and spinal cord. Intraperitoneal injection(i.p.) of the TNF-a synthesis inhibitor thalidomide reduced the SNI-induced mechanical hypersensitivity and inhibited the expression of CXCL12 in the DRG and spinal cord.Intrathecal injection(i.t.) of the CXCR4 antagonist AMD3100, both 30 min before and 7 days after SNI,reduced the behavioral signs of allodynia. Rats given an i.t.or i.p. bolus of AMD3100 on day 8 of SNI exhibited attenuated abnormal pain behaviors. The neuropathic pain established following SNI was also impaired by i.t. administration of a CXCL12-neutralizing antibody. Moreover,repetitive i.t. AMD3100 administration prevented the activation of ERK in the spinal cord. The mechanical hypersensitivity induced in na?¨ve rats by i.t. CXCL12 was alleviated by pretreatment with the MEK inhibitor PD98059. Collectively, our results revealed that TNF-a might mediate the upregulation of CXCL12 in the DRG and spinal cord following SNI, and that CXCL12/CXCR4 signaling via ERK activation contributes to the development and maintenance of neuropathic pain.     

Nitric oxide and c-Jun N-terminal kinase are involved in the development of dark neurons induced by inflammatory pain

Dark neurons, whose morphological characteristics are consistent with those of cells undergoing apoptosis, are generated in vivo as an acute or delayed consequence of several pathological situations and lesions. The present study was designed to evaluate whether inflammatory pain induced by injection of formalin to the rat hind paw lead to the formation of dark neurons in the dorsal horn of the lumbar spinal cord in rat. Since nitric oxide (NO) and c-Jun N-terminal Kinase (JNK) pathway are involved in the mechanisms of pain generation and degenerative neuronal alteration, their roles were also considered. The methods used spectrophotometrical analysis of the serum nitrite (metabolite of NO) and histological procedures for detection of dark neurons, following induction of inflammatory pain. According to the results, injection of formalin led to an increase of the serum nitrite level in both concentration and time-dependent manners. Visual inspections of the lumbar spinal cord sections showed that, on day 5, following chronic injections of 5% formalin, numbers of dark neurons were significantly increased. Acute and chronic administration of 1% or 2.5% formalin did not induce any remarkable neuronal alterations in the dorsal horn of the lumbar spinal cord. Daily intrathecal administration of quercetin (inhibitor of JNK pathway) 100 microg/rat, or 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; NO scavenger) 30 mug/rat before injection of 5% formalin led to a reliable reduction in the number of dark neurons. These results indicate that induction of inflammatory pain for longer periods may result in a serious central disorder. Pretreatment with neutralizers or inhibitors of NO and JNK may exert a neuroprotective effect in this regard.     

Differentiation and migration of astrocytes in the spinal cord following dorsal root injury in the adult rat

Nerve fibre degeneration in the spinal cord is accompanied by astroglial proliferation. It is not known whether these cells proliferate in situ or are recruited from specific regions harbouring astroglial precursors. We found cells expressing nestin, characteristic of astroglial precursors, at the dorsal surface of the spinal cord on the operated side from 30 h after dorsal root injury. Nestin-expressing cells dispersed to deeper areas of the dorsal funiculus and dorsal horn on the operated side during the first few days after injury. Injection of bromodeoxyuridine (BrdU) 2 h before the end of the experiment, at 30 h after injury, revealed numerous BrdU-labelled, nestin-positive cells in the dorsal superficial region. In animals surviving 20 h after BrdU injection at 28 h postlesion, cells double-labelled with BrdU and nestin were also found in deeper areas. Labeling with BrdU 2 h before perfusion showed proliferation of microglia and radial astrocytes in the ventral and lateral funiculi on both sides of the spinal cord 30 h after injury. Nestin-positive cells coexpressed the calcium-binding protein Mts1, a marker for white matter astrocytes, in the dorsal funiculus, and were positive for glial fibrillary acidic protein (GFAP), but negative for Mts1 in the dorsal horn. One week after injury the level of nestin expression decreased and was undetectable after 3 months. Taken together, our data indicate that after dorsal root injury newly formed astrocytes in the degenerating white and grey matter first appear at the dorsal surface of the spinal cord from where some of them subsequently migrate ventrally, and differentiate into white- or grey-matter astrocytes.     

Upregulation of brain-derived neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats

Selective motor nerve injury by lumbar 5 ventral root transection (L5 VRT) induces neuropathic pain, but the underlying mechanisms remain unknown. Previously, increased expression and secretion of brain-derived neurotrophic factor (BDNF) had been implicated in injury-induced neuropathic pain in the sensory system. In this study, as a step to examine potential roles of BDNF in L5 VRT-induced neuropathic pain, we investigated BDNF gene and protein expression in adult rats with L5 VRT. L5 VRT induced a dramatic upregulation of BDNF mRNA in intact sensory neurons in the ipsilateral L5 dorsal root ganglia (DRG), in non-neuronal cells in the ipsilateral sciatic nerve, and in motoneurons in the ipsilateral spinal cord. L5 VRT also induced de novo synthesis of BDNF mRNA in spinal dorsal horn neurons and in glial cells in the white matter of the ipsilateral spinal cord. Consistent with the mRNA expression pattern, BDNF protein was also mainly upregulated in all populations of sensory neurons in the ipsilateral L5 DRG and in spinal neurons and glia. Quantitative analysis by ELISA showed that the BDNF content in the DRG and sciatic nerve peaked on day 1 and remained elevated 14 days after L5 VRT. These results suggest that increased BDNF expression in intact primary sensory neurons and spinal cord may be an important factor in the induction of neuropathic pain without axotomy of sensory neurons.     

Activation of the bile acid receptors TGR5 and FXR in the spinal dorsal horn alleviates neuropathic pain

Aims

Beyond digestion, bile acids have been recognized as signaling molecules with broad paracrine and endocrine functions by activating plasma membrane receptor (Takeda G protein-coupled receptor 5, TGR5) and the nuclear farnesoid X receptor (FXR). The present study investigated the role of bile acids in alleviating neuropathic pain by activating TGR5 and FXR.

Method

Neuropathic pain was induced by spared nerve injury (SNI) of the sciatic nerve. TGR5 or FXR agonist was injected intrathecally. Pain hypersensitivity was measured with Von Frey test. The amount of bile acids was detected using a bile acid assay kit. Western blotting and immunohistochemistry were used to assess molecular changes.

Results

We found that bile acids were downregulated, whereas the expression of cytochrome P450 cholesterol 7ahydroxylase (CYP7A1), a rate-limiting enzyme for bile acid synthesis, was upregulated exclusively in microglia in the spinal dorsal horn after SNI. Furthermore, the expression of the bile acid receptors TGR5 and FXR was increased in glial cells and GABAergic neurons in the spinal dorsal horn on day 7 after SNI. Intrathecal injection of either TGR5 or FXR agonist on day 7 after SNI alleviated the established mechanical allodynia in mice, and the effects were blocked by TGR5 or FXR antagonist. Bile acid receptor agonists inhibited the activation of glial cells and ERK pathway in the spinal dorsal horn. All of the above effects of TGR5 or FXR agonists on mechanical allodynia, on the activation of glial cells, and on ERK pathway were abolished by intrathecal injection of the GABA_A receptor antagonist bicuculline.

Conclusion

These results suggest that activation of TGR5 or FXR counteracts mechanical allodynia. The effect was mediated by potentiating function of GABA_A receptors, which then inhibited the activation of glial cells and neuronal sensitization in the spinal dorsal horn.     

Change of vanilloid receptor 1 expression in dorsal root ganglion and spinal dorsal horn during inflammatory nociception induced by complete Freund's adjuvant in rats

The present study aimed to systematically observe the change of vanilloid receptor 1 (VR1) during inflammatory nociception induced by intraplantar injection of complete Freund's adjuvant (CFA) into the left hind paw in rats. Hot plate latency (HPL) was used to evaluate resulting thermal hyperalgesia and immunohistochemistry to observe VR1 expression in dorsal root ganglion and spinal cord dorsal horn. Results showed that HPL decreased from day 1 to day 28 after CFA injection, with shortest at day 14. VR1 expression correspondingly increased from day 1 to day 21 with peak at day 14, and returning to the control level at day 28. A shift of VRI expression from small to medium DRG neurons over the observation period was seen. These results suggest that VR1 could play an important role in the early stage, but not the late stage, of CFA inflammatory nociception.     

Lentiviral-mediated targeted transgene expression in dorsal spinal cord glia: tool for the study of glial cell implication in mechanisms underlying chronic pain development

Activated glial cells in the dorsal spinal cord take an important part in the development of pain after peripheral nerve injury. Our understanding of mechanisms involved in functional changes of spinal glia remains incomplete. Excepting drugs that completely disrupt glial function, pharmacological studies fail to target glia and to modify locally its function in order to really discriminate the role of neuronal versus glial cells in chronic pain. We developed an intraspinal gene transfer approach using pseudotyped lentiviral-derived vector targeting highly preferentially glial cells. Single microinjection of vector expressing EGFP under a CMV promoter control (LV-EGFP) allowed vector diffusion along a rostro-caudal axis but strictly restricted to the grey matter of the ipsilateral dorsal spinal cord. EGFP transgene was mainly expressed in astrocytes and microglial cells whereas less than 9% of cells containing EGFP were neurons. Notably, LV-EGFP administration and EGFP overexpression in glial cells did neither modify glial activity, nor alter animal's nociceptive or locomotor behaviors. Targeted modulation of the expression of gene of interest in glial cells, closely restricted to a particular region of the spinal cord, may thus represent an interesting approach to refine the understanding of mechanisms by which spinal glial cells participate in pain processing.     

Transient expression of GABA immunoreactivity in the developing rat spinal cord.

The development of GABAergic neurons in the spinal cord of the rat has been investigated by immunocytochemical staining of frozen sections with anti-gamma-aminobutyric acid (GABA) antiserum. In the cervical cord, GABA-immunoreactive fibers first appeared at embryonic day (E) 13 in the presumptive white matter within the ventral commissure, ventral funiculus, and dorsal root entrance zone, and in the ventral roots. There were no GABA-immunoreactive cell bodies detected at this age. By E14, motoneurons, the earliest generated spinal cells, were the first cell population to become GABA-immunoreactive at the cell body level. Thereafter, GABA-immunoreactive neurons increased progressively in number and extended from ventral to dorsal regions. GABA-immunoreactive relay neurons within lamina I of the dorsal horn were initially detected at E17. Interneurons in the substantia gelatinosa, the latest generated cells in the spinal cord, were also the last to express the GABA immunoreactivity at E18. Immunoreactive neurons peaked in intensity and extent at E18 and 19. GABA immunoreactivity was only detectable in neurons within the intermediate and marginal zones 1-3 days after they withdrew from the cell cycle. This contrasts to glutamate decarboxylase immunoreactivity, which is detected in precursor cells in the ventricular zone prior to, or during, withdrawal from the cell cycle. Toward the end of gestation, GABA immunoreactivity declined in intensity and extent. This regression began in the ventral horn of the cervical region and ended in the dorsal horn of the lumbosacral region. During the first week after birth, immunoreactivity in motoneurons and in many other neurons within the ventral horn, intermediate gray, and deeper layers of the dorsal horn disappeared, and only in those neurons predominantly within the superficial layers of the dorsal horn did it persist into adulthood. Thus, the expression and regression of GABA immunoreactivity in the spinal cord followed ventral-to-dorsal, rostral-to-caudal, and medial-to-lateral gradients. These observations indicate that the majority of embryonic spinal neurons pass through a stage of transient expression of GABA immunoreactivity. The functional significance of this transient expression is unknown, but it coincides with the period of intense neurite growth of motoneurons, sensory neurons, and interneurons, and of neuromuscular junction formation, suggesting that the transient presence of GABA may play an important role in the differentiation of sensorimotor neuronal circuits.     

Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion‐derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.     

Expression of metabotropic glutamate receptor mRNAs in the human spinal cord: implications for selective vulnerability of spinal motor neurons in amyotrophic lateral sclerosis

To elucidate the relevance of metabotropic glutamate receptors (mGluRs) to the selective vulnerability of motor neurons in the spinal cord in patients with amyotrophic lateral sclerosis (ALS), we investigated the distribution of mRNAs coding mGluR1-5 in the normal human spinal cord. The mRNAs for mGluR1, 4 and 5 were observed in the spinal gray matter, whereas mGluR2 mRNA was absent in the spinal cord and mGluR3 mRNA was displayed only on glial cells in the white matter. Signals for mGluR1 and mGluR5 were enriched in the dorsal horn, while mGluR4 mRNA was abundant in the ventral horn. Since agonists to group I mGluRs (mGluR 1 and 5) have been demonstrated to have neuroprotective effects on spinal motor neurons, less expression of mRNAs coding mGluR1 and mGluR5 in the ventral horn than in the dorsal horn may be implicated in the selective susceptibility of spinal motor neurons in ALS.