Inflammation in sympathetic ganglia proximal to sciatic nerve transection in rats

Comparison was made between recruitment of T-lymphocytes and macrophages into lumbar sympathetic ganglia (SGs) and dorsal root ganglia (DRGs) following sciatic nerve transection in rats. In both control and lesioned SGs, resident (ED2+) macrophages expressed less major histocompatibility complex class II (MHC II), but MHC II+ macrophage density was higher, than in equivalent DRGs. The influx of T-cells was larger and the influx and activation of macrophages were more sustained in SGs than in DRGs. Only two of the five subtypes of macrophage that invade lesioned DRGs were recruited to SGs. While some MHC II+ cells phagocytosed dead sympathetic neurones, most phagocytes in SGs lacked a macrophage marker. The different patterns of response between ganglia may provide clues about macrophage involvement in neuronal death and hyperexcitability after peripheral nerve lesions.     

Distinct sprouting responses of sympathetic and peptidergic sensory axons proximal to a sciatic nerve transection in guinea pigs and rats

Sprouting of sympathetic and peptidergic sensory neurones proximal to nerve lesions may reflect upregulation of growth factors around damaged dorsal root ganglion (DRG) cells. Axons containing noradrenaline or calcitonin gene-related peptide were visualized in DRGs and spinal roots of guinea pigs and rats. After sciatic transection in rats, varicose terminals of both types appeared around large DRG somata. These neurones were surrounded by proliferated satellite cells expressing glial fibrillary acidic protein (GFAP) and p75. This did not occur in guinea pigs. Instead, sympathetic axons grew through the DRG and centrally along the dorsal roots (which contained p75-positive glia), avoiding the DRG somata. Thus the glial reaction to peripheral injury differs between species such that, in guinea pigs, the environment in the spinal roots rather than in the DRGs favours sympathetic sprouting.     

Changing pattern of c-FOS expression in spinal cord neurons after electrical stimulation of the chronically injured sciatic nerve in the rat.

Immunocytochemical technique was used to study the distribution of c-FOS protein immunoreactive cells in the spinal cord and gracile nuclei 2 h after electrical stimulation of the sciatic nerve in ketamine/xylazine/acepromazine-anesthetized adult rats. Quantitative examination of the c-fos-labeled cells in the spinal cord laminae was made in unoperated and sham operated controls, after sciatic nerve transection without electrical stimulation, and after electrical stimulation at C-fiber or A alpha/beta-fiber intensity, both in normal animals and at various survival times after chronic sciatic nerve injury (transection and ligation) or crush. Unoperated animals showed very few c-fos-labeled cells, and sham operated controls showed labeled cells located mainly outside the sciatic nerve projection territory. A small increase in number of c-fos protein positive cells was seen after sciatic nerve transection without electrical stimulation. Stimulation of the normal sciatic nerve at C-fiber intensity resulted in c-fos protein-positive cells within the sciatic projection territory in the ipsilateral dorsal horn. Labeled cells were seen in all spinal cord laminae except lamina IX, with the vast majority in lamina I and outer lamina II. No labeled cells were seen in the gracile nucleus. Stimulation at A alpha/beta fiber intensity resulted in no or only a very small number of c-fos-positive neurons. Electrical stimulation of the injured sciatic nerve at C-fiber intensity, using the uninjured contralateral side as control, resulted in significant decreases in c-fos-immunoreactive cells in lamina I plus the outer portion of lamina II at 12 and 39 days survival after injury. A non-significant decrease was seen in these laminae also after 21 days. Significant increases were seen in laminae III and IV at 21 days. Decreases in laminae V, VI and more ventral laminae were significant at 21 and 39 days after injury. At longer survival times, the difference between the normal and injured side seen weeks after injury tended to disappear. Stimulation at A alpha/beta fiber intensity 21 days after injury resulted in increases in the numbers of labeled cells in ipsilateral laminae II, III and IV and in the gracile nucleus. Sciatic nerve stimulation after crush injury resulted in more variable side differences, with tendencies for the same alterations as those noted after chronic transection-ligation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term changes in the distribution of galanin in dorsal root ganglia after sciatic or spinal nerve transection in rats

The neuropeptide galanin is upregulated in primary afferent and sympathetic neurones and might be involved in the development of sympathetic perineuronal baskets ("rings") following nerve injury. Galanin, calcitonin gene-related peptide and tyrosine hydroxylase have been examined immunohistochemically in dorsal root ganglia and associated roots at times up to one year after transection of either sciatic or L5 spinal nerves in adult rats. Small diameter somata containing calcitonin gene-related peptide (with or without galanin) were reduced in number, whereas galanin (and, at later times, calcitonin gene-related peptide) appeared in medium to large diameter cells after both types of lesion. Galanin also appeared in axons in grey rami and somata in lumbar paravertebral ganglia. Within dorsal root ganglia, galanin-positive axons formed perineuronal rings of two types: (i) smooth coiled axons surrounded small (< 30 microm diameter) somata from which they probably arose; these were rare after 12 weeks, particularly after a spinal nerve lesion; and (ii) varicose terminals encircled medium to large galanin-positive somata; some arose from brightly immunofluorescent somata nearby and took nearly a year to disappear. About 30% of varicose galanin-positive rings had associated calcitonin gene-related peptide-positive terminals (partly colocalized) whereas nearly 45% had associated tyrosine hydroxylase-positive terminals (partly colocalized). Synaptophysin was present in swollen axons and in some varicosities of all types.We conclude that, after peripheral nerve lesions, varicose perineuronal rings around large diameter dorsal root ganglion cells may be formed by axotomized primary afferent neurones (some containing calcitonin gene-related peptide) and sympathetic neurones, both of which contain upregulated galanin. Exocytosis from the varicosities may modify the excitability of mechanosensitive somata. Small galanin-positive somata disappear over several months after both lesions as calcitonin gene-related peptide reappears in medium to large neurones.     

Differential galanin upregulation in dorsal root ganglia and spinal cord after graded single ligature nerve constriction of the rat sciatic nerve

Single ligature nerve constriction (SLNC) is a newly developed animal model for the study of neuropathic pain. SLNC of the rat sciatic nerve induces pain-related behaviors, as well as changes in the expression of neuropeptide tyrosine and the Y(1) receptor in lumbar dorsal root ganglia (DRGs) and spinal cord. In the present study, we have analyzed the expression of another neuropeptide, galanin, in lumbar DRGs and spinal cord after different degrees of constriction of the rat sciatic nerve. The nerve was ligated and reduced to 10-30, 40-80 or 90% of its original diameter (light, medium or strong SLNCs). At different times after injury (7, 14, 30, 60 days), lumbar 4 and 5 DRGs and the corresponding levels of the spinal cord were dissected out and processed for galanin-immunohistochemistry. In DRGs, SLNC induced a gradual increase in the number of galanin-immunoreactive (IR) neurons, in direct correlation with the degree of constriction. Thus, after light SLNC, a modest upregulation of galanin was observed, mainly in small-sized neurons. However, following medium or strong SLNCs, there was a more drastic increase in the number of galanin-IR neurons, involving also medium and large-sized cells. The highest numbers of galanin-IR neurons were detected 14 days after injury. In the dorsal horn of the spinal cord, medium and strong SLNCs induced a marked ipsilateral increase in galanin-like immunoreactivity in laminae I-II. These results show that galanin expression in DRGs and spinal cord is differentially regulated by different degrees of nerve constriction and further support its modulatory role on neuropathic pain.     

Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin

The temporal course of changes in peptide expression in the dorsal root ganglia L4 and L5 and in the dorsal horn of the spinal cord has been studied in rats subjected to a sciatic nerve transection at a mid-thigh level following different survival times. Galanin-, substance P-, vasoactive intestinal polypeptide-, peptide histidine-isoleucine- and calcitonin gene-related peptide-like immunoreactivities have been studied both by immunohistochemistry and radioimmunoassay. Galanin messenger ribonucleic acid has also been studied by in situ hybridization in the dorsal root ganglia of normal and lesioned animals. In addition, a group of animals with a sciatic nerve crush was studied to compare possible differences in peptide expression after both types of lesions. The results show that the transection induces an increase in the number of cell bodies expressing galanin-like immunoreactivity in the ganglia, and that the galanin levels rise about 120-fold after three and 14 days of survival. This increase reflected increased synthesis of the peptide, since there was a rise in the galanin messenger ribonucleic acid already at 24 h post-lesion, which was maintained for at least 60 days. In the spinal cord there was an increase of staining in the midportion of the outer layers of the dorsal horn that corresponded to fibers thought to arise from cells of the dorsal root ganglia affected by the transection. Also a depletion of substance P-like and an increase in vasoactive intestinal polypeptide- and peptide histidine-isoleucine-like immunoreactivities in the dorsal root ganglia were confirmed. These changes were shown to be rapidly detectable and were paralleled by similar changes in the dorsal horn of the spinal cord. For calcitonin gene-related peptide the immunohistochemistry was inconclusive, and the radioimmunoassay showed no detectable changes. After nerve crush a transient increase in the number of galanin immunoreactive neurons was observed, as well as a decrease in the number of neurons showing substance P-like immunoreactivity. These changes were most noticeable between six and 14 days of survival. After this, peptide expression seemed to return slowly to normal, that is by day 45 post-crush only a few cells showed galanin-like, and many sensory neurons expressed substance P-like immunoreactivity. The results demonstrate that when primary sensory neurons are peripherally lesioned they respond in a complex manner, altering their normal production of peptides by increasing or decreasing their synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effects of endogenous brain-derived neurotrophic factor on the survival of axotomized sensory neurons in dorsal root ganglia: a possible role for the p75 neurotrophin receptor

After peripheral nerve injury, axotomized sensory neurons in dorsal root ganglia (DRG) undergo apoptosis and up-regulate brain-derived neurotrophic factor (BDNF). We tested whether endogenous BDNF plays any role in the survival of axotomized sensory neurons using in vitro and in vivo models. In the in vitro model, treatment with BDNF antibody significantly reduced apoptosis of sensory neurons in DRG explants from both adult and neonate rats and adult mice cultured for 48 h. Consistently, exogenous BDNF increased the percentage of apoptotic neurons in the DRGs from mice. The effects of the BDNF antibody and BDNF were not seen in DRGs from p75NTR(-/-) mice. In the in vivo model, sciatic nerve transection in neonatal rats decreased the total number of neurons in the injured DRG and treatment with antiserum to BDNF significantly exaggerated the loss of DRG neurons. Numbers of sensory neurons expressing BDNF and p75NTR in cultured DRGs increased but that expressing TrkB decreased. In contrast, sciatic nerve transection in vivo reduced the numbers of neurons expressing both p75NTR and TrkB but increased the numbers of cells expressing BDNF, 1 and 7 days after the surgery. These results suggest that BDNF may have differential effects on the survival of sensory neurons depending on the expression of p75NTR. While endogenous BDNF induced apoptosis of axotomized sensory neurons through p75NTR in vitro where more neurons expressed p75NTR, it prevented apoptosis in vivo where fewer neurons expressed p75NTR after sciatic nerve transection.     

Injured primary sensory neurons switch phenotype for brain-derived neurotrophic factor in the rat.

Peripheral nerve injury results in plastic changes in the dorsal root ganglia and spinal cord, and is often complicated with neuropathic pain. The mechanisms underlying these changes are not known. We have now investigated the expression of brain-derived neurotrophic factor in the dorsal root ganglia with histochemical and biochemical methods following sciatic nerve lesion in the rat. The percentage of neurons immunoreactive for brain-derived neurotrophic factor in the ipsilateral dorsal root ganglia was significantly increased as early as 24 h after the nerve lesion and the increase lasted for at least two weeks. The level of brain-derived neurotrophic factor messenger RNA was also significantly increased in the ipsibut not contralateral dorsal root ganglia. Both neurons and satellite cells in the lesioned dorsal root ganglia synthesized brain-derived neurotrophic factor messenger RNA after the nerve lesion. There was a dramatic shift in size distribution of positive neurons towards large sizes seven days after sciatic nerve lesion. Morphometric analysis and retrograde tracing studies showed that no injured neurons smaller than 600 microm2 were immunoreactive for brain-derived neurotrophic factor, whereas the majority of large injured neurons were immunoreactive in the ipsilateral dorsal root ganglia seven days postlesion. The brain-derived neurotrophic factor-immunoreactive nerve terminals in the ipsilateral spinal cord were reduced in the central region of lamina II, but increased in more medial regions or deeper into laminae III/IV. These studies indicate that sciatic nerve injury results in a differential regulation of brain-derived neurotrophic factor in different subpopulations of sensory neurons in the dorsal root ganglia. Small neurons switched off their normal synthesis of brain-derived neurotrophic factor, whereas larger ones switched to a brain-derived neurotrophic factor phenotype. The phenotypic switch may have functional implications in neuronal plasticity and generation of neuropathic pain after nerve injury.     

Injury-induced reduction of acidic fibroblast growth factor levels in the distal parts of rat sciatic nerve.

Acidic fibroblast growth factor (aFGF) level in sciatic nerve after lesioning was measured by enzyme immunoassay to determine if aFGF functions as a neurotrophic factor like nerve growth factor (NGF). Whereas the NGF level increased in distal segments, the aFGF level there decreased after transection or crushing and recovered to the original level by 10 weeks after crushing. The amount of aFGF mRNA in the sciatic nerve was extremely low to supply the high level of protein found in the sciatic nerve. Sympathetic ganglia, dorsal root ganglia, and spinal cord, which contain neuronal cell bodies extending their axons into the sciatic nerve, showed a greater or similar level of aFGF as sciatic nerve. These results imply that aFGF is synthesized in neuronal cell bodies and distributed anterogradely into their axons. Difference of injury-induced changes in levels between aFGF and NGF suggests distinct mechanisms of the effects elicited from these factors on regeneration of the sciatic nerve.     

SOCS3通过抑制STAT3抑制受损伤大鼠初级感觉神经元生长

目的通过慢病毒转导SOCS3和作用相反的突变型SOCS3(mSOCS3),体外研究SOCS3在成年鼠的初级感觉神经元再生中的作用。方法将慢病毒载体质粒pRRL-SOCS3-IRES-GFP,pRRL-mSOCS3-IRES-GFP和pRRL-STAT3ER-IRES-GFP分别转染293T细胞,包装慢病毒载体并测定滴度。完全切断大鼠左侧坐骨神经,术后饲养8-128h,在不同时间点摘取双侧L5背根神经节。通过实时PCR和原位杂交检测DRGs中SOCS3 mRNA的存在;通过背根神经节分离神经元培养,分别将三种慢病毒载体感染神经元细胞,采用免疫荧光染色法观察神经元细胞核质反应和突起的长度。结果大鼠左侧坐骨神经损伤后,SOCS3 mRNA的表达在背根神经节神经元中明显增加,外源性SOCS3能阻止神经元中STAT3的磷酸化及核移位,mSOCS3增强了突起生长。结论 SOCS3可通过抑制STAT3抑制轴突生长。     

p27kip1和Skp2在损伤后大鼠坐骨神经中的表达变化

为了探讨损伤后周围神经p27kip1和S期激酶相关蛋白2(Skp2)的定位表达和变化,本实验将成年SD大鼠随机分为正常对照组、夹伤组和切断组,运用Western blot结合免疫组织化学及免疫荧光双标,在大鼠坐骨神经损伤时,对p27kip1和Skp2表达的影响进行了研究。结果表明:(1)坐骨神经夹伤后,p27kip1蛋白表达先逐渐下降,后又逐渐上升;坐骨神经切断后,远侧段p27kip1蛋白表达持续下降,而近侧段p27kip1蛋白表达在切断后6h明显下降,后又逐渐升高至正常水平,而Skp2表达变化与之相反;(2)免疫组织化学染色结果显示,坐骨神经切断后1w,远侧段从断端到末端,p27kip1阳性信号逐渐增加,而Skp2阳性信号逐渐减弱;(3)免疫荧光双标显示,正常和损伤坐骨神经的雪旺氏细胞中都有p27kip1和Skp2表达。以上结果提示:周围神经损伤后影响雪旺氏细胞中p27kip1和Skp2的表达变化,为进一步研究它们在周围神经损伤和修复中的作用机制奠定基础。     

Mesenchymal stem cells in a polycaprolactone conduit promote sciatic nerve regeneration and sensory neuron survival after nerve injury

Despite the fact that the peripheral nervous system is able to regenerate after traumatic injury, the functional outcomes following damage are limited and poor. Bone marrow mesenchymal stem cells (MSCs) are multipotent cells that have been used in studies of peripheral nerve regeneration and have yielded promising results. The aim of this study was to evaluate sciatic nerve regeneration and neuronal survival in mice after nerve transection followed by MSC treatment into a polycaprolactone (PCL) nerve guide. The left sciatic nerve of C57BL/6 mice was transected and the nerve stumps were placed into a biodegradable PCL tube leaving a 3-mm gap between them; the tube was filled with MSCs obtained from GFP+ animals (MSC-treated group) or with a culture medium (Dulbecco's modified Eagle's medium group). Motor function was analyzed according to the sciatic functional index (SFI). After 6 weeks, animals were euthanized, and the regenerated sciatic nerve, the dorsal root ganglion (DRG), the spinal cord, and the gastrocnemius muscle were collected and processed for light and electron microscopy. A quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers in the group that received, within the nerve guide, stem cells. The number of neurons in the DRG was significantly higher in the MSC-treated group, while there was no difference in the number of motor neurons in the spinal cord. We also found higher values of trophic factors expression in MSC-treated groups, especially a nerve growth factor. The SFI revealed a significant improvement in the MSC-treated group. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase enzyme, suggesting an improvement of reinnervation and activity in animals that received MSCs. Immunohistochemistry documented that some GFP+ -transplanted cells assumed a Schwann-cell-like phenotype, as evidenced by their expression of the S-100 protein, a Schwann cell marker. Our findings suggest that using a PCL tube filled with MSCs is a good strategy to improve nerve regeneration after a nerve transection in mice.     

Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy.

The effects of peripheral axotomy (sciatic nerve transection) on the presence and distribution of neuropeptide Y (NPY) in rat dorsal root ganglion (DRG) and spinal grey matter were examined using immunocytochemistry. In normal rats and on the sham-operated side of experimental rats, NPY-like immunoreactivity (NPYir) was observed in all laminae of the lumbar spinal cord, with an especially dense concentration of immunostained axons and axonal varicosities in laminae I-II of the dorsal horn. There was no detectable NPYir in L4-L5 DRG cells from normal rats or from the sham-operated side of experimental rats. At 14 days after axotomy, there was a large ipsilateral increase in the density of NPYir axons and varicosities in the lumbar spinal cord on the side of the nerve injury; this was especially apparent in laminae III-V. In the same rats, NPYir was observed in many small, medium, and large neurons in the L4-L5 DRGs on the side of the severed nerve.     

Neurochemical characterization of neuronal populations expressing protein kinase C gamma isoform in the spinal cord and gracile nucleus of the rat

Protein kinase C gamma (PKCgamma) is widely distributed throughout the CNS and is thought to play a role in long term hyper-excitability in nociceptive neurones. Here, we provide the first report of PKCgamma cells in the dorsal column nuclei of the adult rat. Retrograde labeling of PKCgamma cells from the thalamus with choleragenoid revealed that 25% of the PKCgamma positive gracile cells projected to the thalamus. Further, we have characterized the distribution of PKCgamma within gracile nucleus in terms of colocalization with various neurotransmitter receptors or enzymes and calcium binding proteins, and compared this with PKCgamma colocalization in cells of laminae I-III of the spinal cord. We show that approximately 90% of the PKCgamma cells in the gracile nucleus and 60% in the dorsal horn were immuno-positive for the AMPA receptor subunit glutamate 2/3 (GluR2/3). Little coexpression was seen with neurokinin 1 receptor, nitric oxide synthase (NOS) and the AMPA receptor subunit GluR1, markers of distinct neuronal subpopulations. In the spinal cord, a quarter of PKCgamma cells expressed calbindin, but very few cells did so in the gracile nucleus. Electrical stimulation at c-fiber strength of the normal or injured sciatic nerve was used to induce c-fos as a marker of postsynaptic activation in the spinal cord and gracile nucleus. Quantitative analysis of the number of PKCgamma positive gracile cells that expressed also c-fos increased from none to 24% after injury, indicating an alteration in the sensory activation pattern in these neurones after injury. C-fos was not induced in inner lamina II following c-fiber electrical stimulation of the intact or axotomized sciatic nerve, indicating no such plasticity at the spinal cord level. As dorsal column nuclei cells may contribute to allodynia after peripheral nerve injury, pharmacological modulation of PKCgamma activity may therefore be a possible way to ameliorate neuropathic pain after peripheral nerve injury.     

Bone marrow stromal cells induce changes in pain behavior after sciatic nerve constriction

Peripheral nerve injury, i.e. a single ligature nerve constriction (SLNC), triggers neuropathic pain. Bone marrow stromal cells (MSCs) have been observed to migrate to the injured tissues and mediate functional recovery following brain, spinal cord and peripheral nerve lesions. We have recently shown MSC selective migration to the ipsilateral lumbar (L3-6) dorsal root ganglia (DRGs) after a sciatic nerve SLNC. In this study, we have analyzed the thermal and mechanical sensitivities of animals subjected to a SLNC of the sciatic nerve and an ipsilateral intraganglionic MSC injection, using the von Frey and Choi tests. Control animals were subjected to the nerve lesion either alone or followed by the administration of phosphate-buffered saline (PBS) or bone marrow non-adherent mononuclear cells (BNMCs). All the animals were tested both before surgery and after 1, 3, 7, 14, 21, 28 and 56 days. Animals subjected to the sciatic nerve constriction developed ipsilateral mechanical and thermal allodynia already 3 days after the lesion. The allodynic responses were maintained even after 56 days. MSC administration prevented the generation of mechanical allodynia and reduced the number of allodynic responses to cold stimuli. On the contrary, the injection of either PBS or BNMCs could not counteract allodynia. These results suggest that MSCs may modulate pain generation after sciatic nerve constriction. The underlying mechanisms by which MSCs exert their actions on pain behavior need to be clarified.     

Expression of neuronal isoform of nitric oxide synthase in spinal neurons of neonatal rats after sciatic nerve transection.

Motoneuron death induced by sciatic nerve transection in neonatal rats has been related to induction of the neuronal isoform of nitric oxide synthase (nNOS), a diaphorase of which one of the cofactors is nicotinamide adenine dinucleotide phosphate (NADPH). We transected the sciatic nerve of neonatal rats (P2) and examined nNOS expression by immunostaining in neurons of the sciatic pool and of other spinal levels on the 5th day after surgery. No correspondence was observed between the surviving motoneurons and nNOS positive cells. The appearance and distribution of nNOS positive neurons at all spinal levels and laminae were similar to those of adult animals. These results are at variance with previous studies which showed correlation between motoneuron loss after axotomy and number of NADPH-diaphorase positive motoneurons after sciatic transection.     

Profile of adult rat sensory neuron loss, apoptosis and replacement after sciatic nerve crush

Following permanent transection of the adult rat sciatic nerve, sensory neuron apoptosis in the contributing L4 and L5 dorsal root ganglia can be observed for at least 6 months afterwards. To establish the profile of any sensory neuron apoptosis and loss over time when axonal regeneration is allowed, serial sections of L4 and L5 ganglia were examined and the neurons counted using a stereological technique 1, 2 and 3 months after crushing the right sciatic nerve at mid-thigh level. Our results show that an identical degree of sensory neuron loss and apoptosis occurs 1 month after crush as at 1 month after permanent transection. However, at 3 months no neurons undergoing apoptosis could be observed and no significant loss could be detected in the ipsilateral ganglia when compared to unoperated controls. One explanation was a neuronal replacement mechanism, which was investigated by administering bromodeoxyuridine to rats for 1 month after sciatic nerve transection or crush, prior to detection using immunohistochemistry on sections of their ganglia after 2 months. The presence of bromodeoxyuridine in the nuclei of occasional cells that would be counted as neurons on the basis of size and morphology indicates that a process of apparent neurogenesis may underlie the profile of sensory neuron loss after axotomy.     

Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury

Upon noxious stimulation, substance P (SP) is released from primary afferent fibers into the spinal cord where it interacts with the SP receptor (SPR). The SPR is located throughout the dorsal horn and undergoes endocytosis after agonist binding, which provides a spatial image of SPR-containing neurons that undergo agonist interaction. Under normal conditions, SPR internalization occurs only in SPR+ cell bodies and dendrites in the superficial dorsal horn after noxious stimulation. After nerve transection and inflammation, SPR immunoreactivity increases, and both noxious as well as nonnoxious stimulation produces SPR internalization in the superficial and deep dorsal horn. We investigated the primary afferent fibers that contribute to enhanced SPR internalization in the spinal cord after nerve transection and inflammation. Internalization evoked by electrical stimulation of the sciatic nerve was examined in untreated animals, at 14 days after sciatic nerve transection or sham surgery and at 3 days after hindpaw inflammation. Electrical stimulation was delivered at intensities to excite Abeta fibers only, Abeta and Adelta fibers or A and C fibers as determined by the compound action potential recorded from the tibial nerve. Electrical stimuli were delivered at a constant rate of 10 Hz for a duration of 5 min. Transection of the sciatic nerve and inflammation produced a 33.7 and 32.5% increase in SPR and immunoreactivity in lamina I, respectively. Under normal conditions, stimulation of Adelta or C fibers evoked internalization that was confined to the superficial dorsal horn. After transection or inflammation, there was a 20-24% increase in the proportion of SPR+ lamina I neurons that exhibited internalization evoked by stimulation of Adelta fibers. The proportion of lamina I SPR+ neurons that exhibited internalization after stimulation of C-fibers was not altered by transection or inflammation because this was nearly maximal under normal conditions. Moreover, electrical stimulation sufficient to excite C fibers evoked SPR internalization in 22% of SPR+ lamina III neurons after nerve transection and in 32-36% of SPR+ neurons in lamina III and IV after inflammation. Stimulation of Abeta fibers alone never evoked internalization in the superficial or deep dorsal horn. These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord after nerve transection and inflammation and suggest that recruitment of neurons that possess the SPR contributes to hyperalgesia.     

Dynamic changes in Robo2 and Slit1 expression in adult rat dorsal root ganglion and sciatic nerve after peripheral and central axonal injury

Robos are transmembrane receptors that mediate Slit signaling to repel growth cone outgrowth and neural migration in the developing central nervous system. Their distribution and function in the peripheral nervous system remains unclear. In the present study, we examined expression of Slit1 and Robo2 in adult rat dorsal root ganglion (DRG), spinal cord and sciatic nerve after peripheral nerve injury (axotomy). In control rats, Slit1 and Robo2 mRNA and protein were expressed at basic levels in the L5 and L6 DRGs. Sciatic transection resulted in a significant up-regulation of both Robo2 and Slit1 mRNA and protein (p<0.05 versus control). The peak of Slit1 and Robo2 expression occurred at days 7 and 14, respectively, and returned to control levels at days 28 and 21 post-axotomy, respectively. By contrast, injury to the central axons of the DRG by dorsal rhizotomy did not up-regulate Slit1 and Robo2 expression. Robo2 staining was stronger in small diameter neurons than in large diameter neurons in control DRG. Interestingly, post-axotomy, Robo2 immunostaining increased in the large diameter neurons and the number of Robo2 positive large diameter neurons increased significantly relative to controls. Non-neuronal cells surrounding the primary sensory neurons, including the satellite cells, were Slit1-positive, and Slit1 protein was expressed in the myelin sheath and non-neural cells in both intact and degenerating sciatic nerve axons. Sciatic nerve transection also led to an accumulation of Slit1 protein in peripheral region of the traumatic neuroma. In conclusion, we report an altered expression and redistribution of Robo2 and Slit1 in the DRG and sciatic nerve trunk after peripheral axotomy. Our results indicate that Slit1 and Robo2 likely play an important role in regeneration after peripheral nerve injury.