Intra- and Extraneuronal Changes of Immunofluorescence Staining for TNF- and TNFR1 in the Dorsal Root Ganglia of Rat Peripheral Neuropathic Pain Models

1. Several lines of evidence suggest that cytokines and their receptors are initiators of changes in the activity of dorsal root ganglia (DRG) neurons, but their cellular distribution is still very limited or controversial. Therefore, the goal of present study was to investigate immunohistochemical distribution of TNF-α and TNF receptor-1 (TNFR1) proteins in the rat DRG following three types of nerve injury.2. The unilateral sciatic and spinal nerve ligation as well as the sciatic nerve transection were used to induce changes in the distribution of TNF-α and TNFR1 proteins. The TNF-α and TNFR1 immunofluorescence was assessed in the L4-L5 DRG affected by nerve injury for 1 and 2 weeks, and compared with the contralateral ones and those removed from naive or sham-operated rats. A part of the sections was incubated for simultaneous immunostaining for TNF-α and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-α and TNFR1 in the naive, ipsi- and contralateral DRG following nerve injury.3. The ipsilateral L4-L5 DRG and their contralateral counterparts of the rats operated for nerve injury displayed an increased immunofluorescence (IF) for TNF-α and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFα IF was increased bilaterally in the satellite glial cells (SGC) and contralaterally in the neuronal nuclei following sciatic and spinal nerve ligature. The neuronal bodies and their SGC exhibited bilaterally enhanced IF for TNF-α after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFα IF. The ED-1 positive macrophages were frequently located near the neuronal bodies to occupy a position of the satellites.4. The sciatic and spinal nerve ligature resulted in an increased TNFR1 IF in the neuronal bodies of both ipsi- and contralateral DRG. The sciatic nerve ligature for 1 week induced a rise in TNFR1 IF in the contralateral DRG neurons and their SGC to a higher level than in the ipsilateral ones. In contrast, the sciatic nerve ligature for 2 weeks caused a similar increase of TNFR1 IF in the neurons and their SGC of both ipsi- and contralateral DRG. The spinal nerve ligature or sciatic nerve transection resulted in an increased TNFR1 IF located at the surface of the ipsilateral DRG neurons, but dispersed IF in the contralateral ones. In addition, the SGC of the contralateral in contrast to ipsilateral DRG displayed a higher TNFR1 IF.5. Our results suggest more sources of TNF-α protein in the ipsilateral and contralateral DRG following unilateral nerve injury including macrophages, SGC and primary sensory neurons. In addition, the SGC and macrophages, which became to be satellites, are well positioned to regulate activity of the DRG neurons by production of TNF-α molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-α effect on the primary sensory neurons can be mediated following nerve injury.     

Spatio-temporal changes of SDF1 and its CXCR4 receptor in the dorsal root ganglia following unilateral sciatic nerve injury as a model of neuropathic pain

There is a growing evidence that chemokines and their receptors play a role in inducing and maintaining neuropathic pain. In the present study, unilateral chronic constriction injury (CCI) of rat sciatic nerve under aseptic conditions was used to investigate changes for stromal derived factor-1 (SDF1) and its CXCR4 receptor in lumbal (L4–L5) and cervical (C7–C8) dorsal root ganglia (DRG) from both sides of naïve, CCI-operated and sham-operated rats. All CCI-operated rats displayed mechanical allodynia and thermal hyperalgesia in hind paws ipsilateral to CCI, but forepaws exhibited only temporal changes of sensitivity not correlated with alterations in SDF1 and CXCR4 proteins. Naïve DRG displayed immunofluorescence for SDF1 (SDF1-IF) in the satellite glial cells (SGC) and CXCR4-IF in the neuronal bodies with highest intensity in small- and medium-sized neurons. Immunofluorescence staining and Western blot analysis confirmed that unilateral CCI induced bilateral alterations of SDF1 and CXCR4 proteins in both L4–L5 and C7–C8 DRG. Only lumbal DRG were invaded by ED-1+ macrophages exhibiting SDF1-IF while elevation of CXCR4-IF was found in DRG neurons and SGC but not in ED-1+ macrophages. No attenuation of mechanical allodynia, but reversed thermal hyperalgesia, in ipsi- and contralateral hind paws was found in CCI-operated rats after i.p. administration of CXCR4 antagonist (AMD3100). These results indicate that SDF1/CXCR4 changes are not limited to DRG associated with injured nerve but that they also spread to DRG non-associated with such nerve. Functional involvement of these alterations in DRG non-associated with injured nerve in neuropathic pain remains to be elucidated.     

A heterogeneous immunofluorescence staining for laminin-1 and related basal lamina molecules in the dorsal root ganglia following constriction nerve injury

The bodies of primary sensory neurons and their satellite glial cells (SGCs) are limited by the basal laminae from extracellular matrix of the dorsal root ganglia (DRG). The basal laminae displayed uniform immunofluorescence staining for laminin-1 in the sections of rat intact (naive) DRG. A proximal or distal ligature of the spinal nerves resulted in a heterogeneous immunostaining for laminin-1 around neuron-SGC units in the sections of the corresponding DRG. The pattern of irregular laminin-1 immunofluorescence was more extensive in the ipsilateral than the contralateral DRG of the operated rats. The immunofluorescence for laminin-1 exactly coincided with binding of Concanavalin-A as well as immunostaining for type IV collagen in both naive DRG and DRG affected by nerve ligature. Nidogen immunostaining decreased or fully disappeared at the surface of the SGCs consistently with immunofluorescence staining for laminin-1, but retained or increased in the endothelial cells and ED-1 positive cells invaded the DRG affected by nerve ligature. The results indicate an alteration of the content of basal laminae surrounding the bodies of primary sensory neurons and their SGSs following nerve constriction injury. A modulation of the basal laminae may be related with other cellular and molecular alterations related with peripheral neuropathic pain, for example, expansion of sympathetic sprouts.     

Glial cell and macrophage reactions in rat spinal ganglion after peripheral nerve lesions: an immunocytochemical and morphometric study

Following peripheral nerve injury perineuronal satellite cell reaction in the corresponding spinal ganglion is observed. The mechanisms underlying the glial responses to axon injury remain unknown. In an immunocytochemical and morphometric study we investigated satellite cell and macrophage responses in the rat L4 and L5 dorsal root ganglia (DRG) during the seven days immediately after unilateral sciatic nerve crush or transection. Nerve lesion induced a significant increase of glial fibrillary acidic protein-immunoreactive (GFAP-IR) cells in the ipsilateral L4-L5 DRGs. The number of ED1-positive macrophages significantly increased as well. We found no significant differences between the increases provoked by the two types of nerve lesion, but the macrophage activation was detected earlier after nerve transection than after crush. No correlation was detected between satellite cells and macrophages reactions over the 7 day period we examined. These findings support the idea that intercellular neuron-glial diffusible signals play a major role in DRG glial cell response to peripheral nerve lesion.     

Bilateral activation of STAT3 by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and its nuclear translocation in primary sensory neurons following unilateral sciatic nerve injury

Unilateral sciatic nerve compression (SNC) or complete sciatic nerve transection (CSNT), both varying degrees of nerve injury, induced activation of STAT3 bilaterally in the dorsal root ganglia (DRG) neurons of lumbar (L4-L5) as well as cervical (C6–C8) spinal cord segments. STAT3 activation was by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and was followed by their nuclear translocation. This is the first evidence of STAT3(S727) activation together with the well-known activation of STAT3(Y705) in primary sensory neurons upon peripheral nerve injury. Bilateral activation of STAT3 in DRG neurons of spinal segments anatomically both associated as well as non-associated with the injured nerve indicates diffusion of STAT3 activation inducers along the spinal cord. Increased levels of IL-6 protein in the CSF following nerve injury as well as activation and nuclear translocation of STAT3 in DRG after intrathecal injection of IL-6 shows that this cytokine, released into the subarachnoid space can penetrate the DRG to activate STAT3. Previous results on increased bilateral IL-6 synthesis and the present manifestation of STAT3 activation in remote DRG following unilateral sciatic nerve injury may reflect a systemic reaction of the DRG neurons to nerve injury.     

Anti-allodynic effects of intrathecally and intracerebroventricularly administered 26RFa, an intrinsic agonist for GRP103, in the rat partial sciatic nerve ligation model

26RFa and QRFP are endogenous ligands of GPR103. 26RFa binding sites are widely distributed in the brain and the spinal cord where they are involved in processing pain. In the present study, the effects of intrathecal and intracerebroventricular applications of 26RFa on the level of mechanical allodynia induced by partial sciatic nerve ligation were examined in rats. The level of mechanical allodynia was measured using von Frey filaments. Intrathecal and intracerebroventricular injection of 26RFa attenuated the level of mechanical allodynia. 26RFa has been reported to activate not only GPR103 but also neuropeptide FF2 receptor and the effect of intrathecally and intracerebroventricularly administered 26RFa was not antagonized by BIBP3226, an antagonist of neuropeptide FF receptor. Immunohistochemical examination revealed that QRFP-like immunoreactivity (QRFP-LI) was expressed mainly in the small to medium sized neurons in the L5 dorsal root ganglion (DRG) and that partial sciatic nerve injury increased the percentage of QRFP-LI positive neurons. 7 days after the nerve injury, QRFP-LI positive neurons in the L5 DRG ipsilateral to the partial sciatic nerve injury were larger than those in the L5 DRG ipsilateral to the sham operation. These data suggest that (1) exogenously applied 26RFa modulates nociceptive transmission at the spinal and the supraspinal brain in the neuropathic pain model, (2) the mechanism 26RFa uses to produce an anti-allodynic effect may be mediated by the activation of GPR103, and (3) partial sciatic nerve ligation affects the expression of QRFP-LI in the dorsal root ganglion.     

NMDA and strychnine diversely modulate spinal dorsal horn noxious responsiveness in normal rats: potential significance to sensory disorders in neuropathic pain

Changes in neuronal excitability due to increase in excitatory transmitters and/or removal of local inhibition underlie central neuron sensitization and altered responsiveness related to painful sensory disorders. To distinguish the contribution of each of the two mechanisms, they have been mimicked separately in intact rats, by iontophoretically applying excitatory (NMDA) and disinhibitory (the glycine antagonist strychnine) substances during dorsal horn neuron recording. Wide dynamic range (WDR) neurons were extracellularly recorded at the L5-L6 lumbar level in anesthetized and paralyzed rats and an analysis was made, before and during the substance application, of the characteristics of the response to noxious stimuli applied to areas supplied by the ipsilateral sciatic nerve and the contralateral sciatic and saphenous nerves ("inappropriate" areas). The results show that the neuronal response properties were modified differently during the NMDA-induced hyperexcitability and strychnine-induced release of inhibition. Both manipulations brought about the unmasking of responses to previously ineffective, noxious stimuli applied to the contralateral sciatic and saphenous nerve areas, and the enhancement of the responses to noxious stimulation of the ipsilateral sciatic nerve area. However, it was only during the increased excitation induced by NMDA that the neurons exhibited hyperresponsiveness, with long-lasting afterdischarge, to noxious stimulation of the ipsi- and contralateral areas. Such response features resemble those described in sensitized neurons in neuropathic rats and associated with behavioral signs of hyperalgesia. This suggests, by inference, a crucial contribution of the NMDA-induced increased excitability to the expression of neuronal sensitization related to this painful sensory disorder.     

NMDA and strychnine diversely modulate spinal dorsal horn noxious responsiveness in normal rats: potential significance to sensory disorders in neuropathic pain

Changes in neuronal excitability due to increase in excitatory transmitters and/or removal of local inhibition underlie central neuron sensitization and altered responsiveness related to painful sensory disorders. To distinguish the contribution of each of the two mechanisms, they have been mimicked separately in intact rats, by iontophoretically applying excitatory (NMDA) and disinhibitory (the glycine antagonist strychnine) substances during dorsal horn neuron recording. Wide dynamic range (WDR) neurons were extracellularly recorded at the L5-L6 lumbar level in anesthetized and paralyzed rats and an analysis was made, before and during the substance application, of the characteristics of the response to noxious stimuli applied to areas supplied by the ipsilateral sciatic nerve and the contralateral sciatic and saphenous nerves ("inappropriate" areas). The results show that the neuronal response properties were modified differently during the NMDA-induced hyperexcitability and strychnine-induced release of inhibition. Both manipulations brought about the unmasking of responses to previously ineffective, noxious stimuli applied to the contralateral sciatic and saphenous nerve areas, and the enhancement of the responses to noxious stimulation of the ipsilateral sciatic nerve area. However, it was only during the increased excitation induced by NMDA that the neurons exhibited hyperresponsiveness, with long-lasting afterdischarge, to noxious stimulation of the ipsi- and contralateral areas. Such response features resemble those described in sensitized neurons in neuropathic rats and associated with behavioral signs of hyperalgesia. This suggests, by inference, a crucial contribution of the NMDA-induced increased excitability to the expression of neuronal sensitization related to this painful sensory disorder.     

Intrathecal NGF Administration Reduces Reactive Astrocytosis and Changes Neurotrophin Receptors Expression Pattern in a Rat Model of Neuropathic Pain

Nerve growth factor (NGF), an essential peptide for sensory neurons, seems to have opposite effects when administered peripherally or directly to the central nervous system. We investigated the effects of 7-days intrathecal (i.t.) infusion of NGF on neuronal and glial spinal markers relevant to neuropathic behavior induced by chronic constriction injury (CCI) of the sciatic nerve. Allodynic and hyperalgesic behaviors were investigated by Von Frey and thermal Plantar tests, respectively. NGF-treated animals showed reduced allodynia and thermal hyperalgesia, compared to control animals. We evaluated on lumbar spinal cord the expression of microglial (ED-1), astrocytic (GFAP and S-100β), and C- and Aδ-fibers (SubP, IB-4 and Cb) markers. I.t. NGF treatment reduced reactive astrocytosis and the density of SubP, IB4 and Cb positive fibers in the dorsal horn of injured animals. Morphometric parameters of proximal sciatic nerve stump fibers and cells in DRG were also analyzed in CCI rats: myelin thickness was reduced and DRG neurons and satellite cells appeared hypertrophic. I.t. NGF treatment showed a beneficial effect in reversing these molecular and morphological alterations. Finally, we analyzed by immunohistochemistry the expression pattern of neurotrophin receptors TrkA, pTrkA, TrkB and p75^NTR. Substantial alterations in neurotrophin receptors expression were observed in the spinal cord of CCI and NGF-treated animals. Our results indicate that i.t. NGF administration reverses the neuro-glial morphomolecular changes occurring in neuropathic animals paralleled by alterations in neurotrophin receptors ratio, and suggest that NGF is effective in restoring homeostatic conditions in the spinal cord and maintaining analgesia in neuropathic pain.     

Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury

Peripheral nerve injury can induce spinal microglial/astrocyte activation. Substances released by activated glial cells excite spinal nociceptive neurons. Pharmacological disruption of glial activation or antagonism of substances released by activated glia prevent or reverse pain hypersensitivity. It is not known, however, what causes spinal cord glia to shift from a resting to an activated state. In an attempt to understand the potential role of monocyte chemoattractant protein-1 (MCP-1) in triggering spinal glial activation and its contribution to the development of neuropathic pain, we investigated the effect of peripheral nerve injury on MCP-1 expression in dorsal root ganglia (DRG) and the spinal cord, and established its temporal relationship with activation of spinal microglia and astrocytes. We observed that MCP-1 was induced by chronic constriction of the sciatic nerve in DRG sensory neurons, spinal cord motor neurons and in the superficial dorsal horn, ipsilateral to the injury. Neuronal MCP-1 induction was followed by surrounding microglial activation. After peaking at day 7 after injury, MCP-1 levels began to decline rapidly and had returned to baseline by day 150. In contrast, microglial activation peaked by day 14 and declined afterwards to reach a lower, yet significantly raised level beyond day 22 and remained increased until the end of the test period. Astrocyte activation became detectable later, progressed more slowly and also remained increased until the end of the test period, in parallel with a decreased nociceptive threshold. Our results suggest that neuronal MCP-1 may serve as a trigger for spinal microglial activation, which participates in the initiation of neuropathic pain. Delayed, sustained astrocyte activation may participate with microglia in the persistent phase of pain hypersensitivity.     

Axotomy-induced ornithine decarboxylase activity in the mouse dorsal root ganglion is inhibited by the vinca alkaloids

Vinca alkaloids were used to study the role of retrograde axon transport (RT) in activating neuron perikaryal repair response to nerve transection. Mouse lumbar dorsal root ganglia (DRG) (L₄-L₆) were excised 48 hours after unilateral transection of the sciatic nerve and ornithine decarboxylase (ODC) activity determined. ODC activity in DRG ipsilateral to nerve transection was increased 10–20 fold over contralateral values. Typical ODC activities in ipsilateral and contralateral DRG samples were 6.18±1.4 and 0.31±0.09 pmol¹⁴CO₂ released/h/3DRG, respectively. Systemic administration of single doses of either vincristine (1 mg/kg) or vinblastine (5 mg/kg) immediately prior to axotomy attenuated ODC induction in ipsilateral DRG by 39% and 47%, respectively. A direct inhibition of ODC activity in the DRG appears unlikely since only high concentrations of vinblastine (0.5–1.0 mM) were able to inhibit ODC activity in vitro. We suggest vinca alkaloids inhibit ODC induction as a consequence of distupting retrograde axonal transport. Interruption of this intracellular communication mechanism may be etiologically linked to the distal axon degeneration which follows repetitive exposure to vinca alkaloids and other agents that induce toxic axonal neuropathy.     

ErbB transmembrane tyrosine kinase receptors are expressed by sensory and motor neurons projecting into sciatic nerve.

Adult spinal cord motor and dorsal root ganglion (DRG) sensory neurons express multiple neuregulin-1 (NRG-1) isoforms that act as axon-associated factors promoting neuromuscular junction formation and Schwann cell proliferation and differentiation. NRG-1 isoforms are also expressed by muscle and Schwann cells, suggesting that motor and sensory neurons are themselves acted on by NRG-1 isoforms produced by their peripheral targets. To test this hypothesis, we examined the expression of the NRG-1 receptor subunits erbB2, erbB3, and erbB4 in rat lumbar DRG and spinal cord. All three erbB receptors are expressed in these tissues. Sciatic nerve transection, an injury that induces Schwann cell expression of NRG-1, alters erbB expression in DRG and cord. Virtually all DRG neurons are erbB2- and erbB3-immunoreactive, with erbB4 also detectable in many neurons. In spinal cord white matter, erbB2 and erbB4 antibodies produce dense punctate staining, whereas the erbB3 antibody primarily labels glial cell bodies. Spinal cord dorsal and ventral horn neurons, including alpha-motor neurons, exhibit erbB2, erbB3, and erbB4 immunoreactivity. Spinal cord ventral horn also contains a population of small erbB3+/S100beta+/GFAP- cells (GFAP-negative astrocytes or oligodendrocytes). We conclude that sensory and motor neurons projecting into sciatic nerve express multiple erbB receptors and are potentially NRG-1 responsive.     

Role of Thy-1 in in vivo and in vitro neural development and regeneration of dorsal root ganglionic neurons

We have examined the expression of Thy-1, an abundant glycosylphosphatidylinositol (GPI)-anchored glycoprotein, in dorsal root ganglia (DRG) and associated nerve fascicles, during postnatal development and following a nerve crush. The expression levels of Thy-1 in DRG neurons, dorsal roots, and central processes in spinal cord were rather low at postnatal day 2, and gradually increased as DRG neurons matured. During early development, the expression of Thy-1 within DRG neurons was low and equally distributed between plasma membrane and cytosol. With maturation, the staining intensities of Thy-1 in both the plasma membrane and the cytosol of DRG neurons became increased. We also studied Thy-1 expression in the regeneration of mature DRG neurons following the crush injury of sciatic nerve. Two days after the crush injury, Thy-1 expression dramatically decreased in the DRG neurons on the lesion side. Between 4 and 7 days after the injury, the expression of Thy-1 gradually increased and returned to a normal level 1 week after the sciatic nerve crush. The time course of the up-regulation of Thy-1 expression during regeneration matched that of the recovery of sensory functions, such as pain withdraw reflex, placing reflex, and the score of Basso-Beattie-Bresnahan Locomotor Rating Scale. Taken together, our results suggest that Thy-1 expression is developmentally regulated and is closely associated with the functional maturation of DRG neurons during both postnatal development and nerve regeneration. Furthermore, perturbation of Thy-1 function with anti-Thy-1 antibodies promoted neurite outgrowth from primary cultured DRG neurons, again confirming the inhibitory role of Thy-1 on neurite outgrowth.     

Non-specific cholinesterase activity in mouse spinal ganglia. The usefulness of histochemical study and image analysis for simple characterization of neuron subclasses

Non-specific cholinesterase (ChE) activity was studied histochemically at light and electron microscopical levels in dorsal root ganglia (DRG) of adult mice. The reaction staining and diameter of neuron cells perykaria were measured by using an image analysis system. The methodological approach enable to distinguish 8 subclasses of primary sensory neurons. The proportion of individual subclasses was mapping in three subsequent cervical, thoracal and lumbar DRG. The populations of small-sized neurons increased towards lumbar level similarly as medium and small neurons exhibiting high ChE reactivity. The variations in ChE-containing neurons among DRG from different area may reflect differences in modality-specific primary sensory neurons at each spinal cord level. In addition, the effect of 3 week sciatic nerve transection on the percentage of the subclasses in L4-L6 DRG has been investigated. The number of large neurons was reduced and a decrease of ChE reactivity in medium-size neurons was found in DRG on the operated side. Thus, the present results demonstrate a selective affectation of primary sensory neurons in mouse DRG by the peripheral nerve transection. Different amounts of the reaction product corresponding with ChE activity were found in the nuclear envelope and the cisternae of rough endoplasmic reticulum.     

Complete sciatic nerve transection induces increase of neuropeptide Y-like immunoreactivity in primary sensory neurons and spinal cord of frogs

Neuropeptide Y (NPY) was immunohistochemically investigated in the frog spinal cord and dorsal root ganglia after axotomy. In normal ganglia, moderate NPY-like immunoreactivity (NPY-IR) prevailed in large and medium cells. In the spinal cord, the NPY-IR was densest in the dorsal part of the lateral funiculus. Other fibers and neurons NPY-IR were observed in the dorsal and ventral terminal fields and mediolateral band. NPY-IR fibers were also found in the ventral horn and in the ventral and lateral funiculi. The sciatic nerve transection increased the NPY-IR in large and medium neurons of the ipsilateral and contralateral dorsal root ganglia at 3 and 7 days, but no clear change was found at 15 days. In the spinal cord, there was a bilateral increase in the NPY-IR of the dorsal part of the lateral funiculus. In the ipsilateral side, the NPY-IR was increased at 3 and 7 days but was decreased at 15 days. In the contralateral side, a significant reduction at 15 days occurred. These findings seem to favor the role of NPY in the modulation of pain-related information in frogs, suggesting that this role of NPY may have appeared early in vertebrate evolution.     

Progranulin contributes to endogenous mechanisms of pain defense after nerve injury in mice

Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain.     

Endothelin as a putative sensory neuropeptide in the guinea-pig: different properties in comparison with calcitonin gene-related peptide

Both endothelin-(ET) and calcitonin gene-related peptide- (CGRP) like immunoreactivity (-LI) were present in a variety of organs and neuronal tissue of the guinea-pig as determined by radioimmunoassay (RIA). Neuronal tissues like dorsal root ganglia (DRG) contained by far the highest levels of both ET- (65 +/- 11 pmol/g) and CGRP-LI (34 +/- 5 pmol/g). The tissue levels of ET-LI remained unchanged after 6-hydroxydopamine and capsaicin-pretreatment, while CGRP-LI was markedly reduced after capsaicin. Chromatographic characterization revealed that the main portion of ET-LI in the DRG, right atrium and lung corresponded to synthetic ET-1. Immunohistochemical studies showed the presence of ET-LI in a few neurons of intact DRG and many neurons in DRG cell-cultures, partly co-existing with CGRP-LI. In the neuronal cells of DRG cultures the ratio between the ET- and CGRP-LI was 1:27 compared to 2:1 in intact DRG. 24 h after ligation of the sciatic or vagal nerves no accumulation of ET-LI was observed above the ligation, while CGRP-LI was increased 4-5-fold. Transection (10 days) of the sciatic nerve caused a 85-95% depletion of CGRP-LI in the distal skin, gastrocnemius muscle and trunk below the transection site, while in the proximal portion of the nerve CGRP-LI increased. No effects on ET-LI in these tissues were observed after sciatic nerve transfection. In release experiments on DRG cell cultures. Langendorff heart preparations or perfused guinea-pig lungs, potassium (60 mM), capsaicin or antidromic nerve stimulation evoked a clear-cut increase in the supernatant levels of CGRP-LI, suggesting release, while no effect on the ET-LI concentration was observed in the effluent. Furthermore, anoxia failed to influence the outflow of ET-LI from the heart and lung. It is concluded that ET-1-LI is present in high levels in spinal ganglia and ET-LI occurs in afferent cell-bodies, but in comparison with CGRP, ET shows remarkable inertness upon various experimental conditions including no evidence for axonal transport, loss after denervation or release. The neuronal ET-LI seems to increase under culture conditions, however. The possible function for the high content of ET-LI in the intact guinea-pig peripheral nervous system remains to be elucidated and may mainly be related to a non-neuronal pool considering the relatively low content of ET-LI compared to CGRP in cultured DRG cells.     

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.