Expansion of spinal cord primary sensory afferent projection following combined sciatic nerve resection and saphenous nerve crush: a horseradish peroxidase study in the adult rat

Transganglionic transport of horseradish peroxidase was used to study the potential for collateral sprouting of saphenous nerve afferent fibers in the lumbar dorsal horn of the adult rat following (1) combined unilateral saphenous nerve crush and ipsilateral sciatic nerve resection, (2) unilateral saphenous nerve crush, and (3) unilateral sciatic nerve resection. The saphenous nerve on the nonlesioned contralateral side served as control. Eight weeks after the lesion(s) the animals were subjected to bilateral application of horseradish peroxidase to the saphenous nerves. The distribution of the ensuing labeling in the superficial dorsal horn was subsequently mapped. Combined saphenous nerve crush and sciatic nerve resection resulted in expansion of the saphenous nerve projection area in the dorsal horn when compared to the nonlesioned control side (mean = 13%, P less than 0.05). No expansion of the saphenous nerve projection was found following isolated saphenous nerve crush or sciatic nerve resection, respectively (P greater than 0.05). The findings indicate that in the adult rat, central processes of primary sensory neurons which are regenerating their peripheral processes can extend collateral sprouts into adjacent projection areas in the superficial dorsal horn subjected to previous deafferentation by peripheral nerve resection.     

Tissue plasminogen activator in primary afferents induces dorsal horn excitability and pain response after peripheral nerve injury

The extracellular protease cascade of plasminogen activators and plasminogen are known to regulate neuronal plasticity and extracellular matrix modification, and to be important factors involved in producing long-term potentiation in the CNS. The purpose of this study is to examine the expression of plasminogen activators in primary afferents and its role in nociceptive pathways after peripheral nerve injury. We found the induction of mRNAs for tissue type plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the rat dorsal root ganglia following sciatic nerve transection. Immunoreactivity for tPA was increased in laminae I and II of the dorsal horn and, importantly, the increase in proteolytic activity mediated by tPA was observed in the same area. As neither immunoreactivity for uPA nor uPA-mediated proteolysis was observed, we further examined the effects of tPA on dorsal horn excitability and neuropathic pain behaviour. Intrathecal injection of a specific inhibitor of tPA decreased electrical stimulation-induced Fos expression in dorsal horn neurons following axotomy, and also prevented the development of thermal hyperalgesia following partial sciatic nerve ligation. These findings suggest that the increased tPA in the dorsal horn due to mRNA expression in the dorsal root ganglia increases the dorsal horn excitability and has an important role in pain behaviour after peripheral nerve injury. The tPA-mediated hypersensitivity in dorsal horn neurons may be a novel molecular mechanism of neuropathic pain.     

Enhancement of stimulation-induced ERK activation in the spinal dorsal horn and gracile nucleus neurons in rats with peripheral nerve injury

It has been suggested that low-threshold sensory pathways have an important role in the formation and maintenance of sensory abnormalities which are observed after peripheral nerve injury. In the present study, we examined the involvement of these pathways in the development of hyperexcitability after sciatic nerve injury (SNI) by detecting the intracellular signal molecule. The rats that received a transection of the sciatic nerve 7 days before were electrically stimulated at 0.1 mA and 3 mA in the proximal region of the nerve injury site. We found a small number of phosphorylated extracellular signal-regulated kinase (pERK)-labelled neurons in laminae I-II and III-IV of the spinal dorsal horn in the control rats after 0.1 mA stimulation. By contrast, there was a marked increased of pERK-labelled neurons both in the superficial laminae and laminae III-IV after the same stimulation in the SNI rats. Enhancement of ERK activation induced by 3 mA stimulation was also observed. Immunoreactivity of pERK in gracile nucleus neurons was also dramatically increased after 0.1 mA stimulation to the injured nerve. These data suggest that the rats with peripheral nerve injury had an increased responsiveness to the low- or high-threshold peripheral stimuli in I-II, III-IV and gracile nucleus neurons. Furthermore, SNI rats that received neonatal capsaicin treatment showed a decreased number of pERK neurons after 0.1 mA stimulation in the dorsal horn and gracile nucleus neurons compared to the control rats. Thus, C-fibres may contribute to the enhanced excitability of the low-threshold sensory neurons after peripheral nerve injury.     

The time-course of Aβ-evoked c-fos expression in neurons of the dorsal horn and gracile nucleus after peripheral nerve injury

We have examined the mechanisms underlying Aβ-evoked c-fos expression in the dorsal horn and gracile nucleus following either sciatic nerve section or crush injury. The results indicate that in the spinal cord Aβ-evoked c-fos does not depend on primary afferent sprouting but is associated with the disconnection from the peripheral target since its expression in the dorsal horn reverts to normal after crush injury when regeneration occurs but persists after cut and ligation where regeneration is prevented. In contrast, however, Aβ-evoked c-fos expression in the gracile nucleus may be under some other control since its expression appears independent of peripheral nerve regeneration.     

Identity of myelinated cutaneous sensory neurons projecting to nocireceptive laminae following nerve injury in adult mice

It is widely thought that, after peripheral injury, some low‐threshold mechanoreceptive (LTMR) afferents “sprout” into pain‐specific laminae (I–II) of the dorsal horn and are responsible for chronic pain states such as mechanical allodynia. Although recent studies have questioned this hypothesis, they fail to account for a series of compelling results from single‐fiber analyses showing extensive projections from large‐diameter myelinated afferents into nocireceptive layers after nerve injury. Here we show that, in the thoracic spinal cord of naïve adult mouse, all myelinated nociceptors gave rise to terminal projections throughout the superficial dorsal horn laminae (I–II). Most (70%) of these fibers had large‐diameter axons with recurving flame‐shaped central arbors that projected throughout the dorsal horn laminae I–V. This morphology was reminiscent of that attributed to sprouted LTMRs described in previous studies. After peripheral nerve axotomy, we found that LTMR afferents with narrow, uninflected somal action potentials did not sprout into superficial laminae of the dorsal horn. Only myelinated noiceptive afferents with broad, inflected somal action potentials were found to give rise to recurving collaterals and project into superficial “pain‐specific” laminae after axotomy. We conclude that the previously undocumented central morphology of large, myelinated cutaneous nociceptors may very well account for the morphological findings previously thought to require sprouting of LTMRs. J. Comp. Neurol. 508:500–509, 2008. © 2008 Wiley‐Liss, Inc.     

Light and electron microscopic localization of B-50 (GAP43) in the rat spinal cord during transganglionic degenerative atrophy and regeneration.

Crush or transection of a peripheral nerve is known to induce transganglionic degenerative atrophy (TDA) in the segmentally related, ipsilateral Rolando substance of the spinal cord. When the lost peripheral connectivity is reestablished, the consecutive regenerative synaptoneogenesis results in restoration of the circuitry in the formerly deteriorated upper dorsal horn. Enhanced expression of the growth-associated protein (GAP43) B-50 occurs during neuronal differentiation, axon outgrowth, and peripheral nerve regeneration. This study documents changes in immunocytochemical distribution of B-50 in the regions of the lumbar spinal cord which are segmentally related to the axotomized sciatic nerve. At the light microscopic level, a weak B-50 immunoreactivity (BIR) is present in the neuropil of the upper dorsal horn of control animals. After unilateral transection and ligation of the sciatic nerve, BIR increased in the ipsilateral upper dorsal horn at 17 days postinjury, but decreased again after 24 days with respect to the contralateral side. Differences between effects of crush and transection were prominent in combined crush-cut experiments as well (i.e., after unilateral crush and contralateral transection and ligation of the sciatic nerve). Electron microscopic studies show that in the uninjured and injured spinal cord, BIR is detected in axons and axon terminals, but not all are stained. After transection of the sciatic nerve, BIR is found in afflicted primary sensory axon terminals, including those contacting substantia gelatinosa neurons and in axon terminals undergoing glial phagocytosis. The localization of BIR seen after crushing the sciatic nerve is similar. However, at 24 days after crush, BIR is detected also in axonal growth cones. In the ventral horn of control animals, synaptic boutons impinging upon motor neurons exhibited weak BIR. At 17 days after unilateral transection of the sciatic nerve, the pericellular BIR surrounding motor neurons is decreased at the ipsilateral with respect to the contralateral side, whereas 24 days after crush injury it increased considerably. Our results show that peripheral nerve injury inducing TDA also affects BIR distribution in the spinal gray matter. Successful regeneration of the peripheral nerve after crush lesion is associated with enhanced expression of B-50 in growth cones of sprouting central axons. The neuroplastic response of B-50 is in line with a function of B-50 in axonal sprouting and reactive synaptogenesis.     

Territorial and extra-territorial distribution of Fos protein in the lumbar spinal dorsal horn neurons in rats with chronic constriction nerve injuries

This study aimed to examine the relationship between temporal and spatial expression patterns of Fos protein in the spinal dorsal horn neurons and thermal hyperalgesia behaviors in rats with chronic constriction injury (CCI) to the sciatic nerve. Our results demonstrated that Fos protein expression in the spinal dorsal horn neurons at L5 segment ipsilateral and contralateral to CCI of the sciatic nerve was significantly greater than in sham rats from days 10 to 30 postoperatively (PO 10d to 30d), and was concentrated on the injury (ipsilateral) side. Unlike the short-lived expression after tissue inflammation, laminae I to VI (especially laminae III/IV) displayed a persistent greater number of Fos-like immunoreactive (Fos-LI) neurons for at least 30 days after CCI of the sciatic nerve. After the increase in laminae III/IV, Fos-LI neurons tended to gradually increase in laminae I/II and V/VI at L5 segment from PO 2d to 30d, which were correlated with the heat hyperalgesia (48 degrees C) behaviors measured by paw withdrawal latency in CCI rats but not in sham rats. Interestingly, a persistent increase of Fos-LI neurons in laminae I to VI at L5 segment of the ipsilateral and contralateral sides and at the L1 segment that was out of the normal central terminations of the sciatic nerve suggested the probable presence of territorial and extra-territorial central sensitization or inadequate central nervous system (CNS) adaptive mechanisms. These findings may partly explain why abnormal pain sensations are sometimes distributed in a pattern that does not coincide with the territories of nerves or with the posterior roots of the peripheral nerve after injury.     

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.     

Release of immunoreactive brain-derived neurotrophic factor in the spinal cord of the rat following sciatic nerve transection

Using the antibody microprobe method, the sites of spinal release of immunoreactive brain-derived neurotrophic factor (BDNF) was studied in normal rats, and rats with prior sciatic nerve transection. In normal rats, a significant basal release of immunoreactive BDNF was found in the superficial dorsal horn. Following sciatic nerve transection (performed 14 days previously), release of BDNF was found throughout the whole of the dorsal horn, extending into deeper laminae. Electrical stimulation of the ipsilateral sciatic nerve at a strength adequate to excite either A fibres (20 Hz at 2x threshold voltage) or A and C fibres (2 Hz at 20x threshold voltage) did not alter the basal release of immunoreactive BDNF in normal or in nerve-injured rats. The results suggest that BDNF is released from the central terminals of primary afferent fibres, but such release is not solely dependent upon action potential invasion of these terminals. The increased extent of release following nerve transection is consistent with the hypothesis that BDNF plays a role in the central response to peripheral nerve injury.     

Morphological characterization of dorsal horn spinal neurons in rats with unilateral constriction nerve injury: A preliminary study

Abstract

A new animal model of neuropathic pain utilizing loose ligation of a peripheral nerve has been previously reported. In addition to displaying abnormal pain symptoms such as allodynia and hyperalgesia, physiologic and morphologic changes are seen in spinal cord dorsal horn neurons. Two weeks after ligation of the right common sciatic nerve, rat dorsal horn spinal cord neurons with signs of transsynaptic changes (dark neurons) were found on the side ipsilateral to the nerve injury. A few dark neurons were also found in the contralateral dorsal horn. The distribution of dark neurons in lumbar dorsal horn was limited to the superficial laminae (l-lll). The following changes which suggest altered cellular activity were seen under the electron microscope. The nuclear envelope appeared ruffled while the mitochondria appeared normal. In additioni, the dense cytoplasm was filled with rosettes of ribosomes as well as extensively developed rough endoplasmic reticulum and distended Golgi apparatus cisternae. While dark neurons had normal appearing somatic synapses, a few appeared atypical. The altered activity of these neurons may lead to abnormal sensory experiences and may be a consequence of central changes in response to persistent peripheral nerve injury. The purpose of the present study was to assess morphologic, hence functional changes in spinal cord neurons in response to peripheral nerve constriction injury which evokes chronic pain-related behaviour. [Neurol Res 1994; 16: 297-304]     

Early decline and late restoration of spinal cord binding and transganglionic transport of isolectin B4 from Griffonia simplicifolia I after peripheral nerve transection or crush

Isolectin B4 from Griffonia simplicifolia I (B4) has a high binding affinity to a large population of unmyelinated primary sensory neurons (Wang et al., Neuroscience 62 (1994) 539-551). Using immunohistochemical techniques, binding and transganglionic transport of B4 in the spinal cord was investigated, both at short and long survival times, after sciatic nerve transection and ligation or crush in the adult rat. Nerve transection and ligation resulted in nearly complete disappearance of B4 immunolabelling in the sciatic nerve territory of the superficial dorsal horn after B4 binding, as well as after transganglionic transport of B4 by 2 weeks postinjury. Partial recovery of both B4 binding and B4 transport was found by 8 months, and nearly complete recovery by 16 months, indicating that reappearance of B4 binding is not critically dependent on peripheral reinnervation. Crush injury made by jeweller's forceps resulted in partial depletion of binding and transport by 2 weeks and a nearly complete recovery by 10 weeks. The results show that binding and transganglionic transport of B4 can be used to label dorsal horn connections of unmyelinated primary afferents during the process of regeneration after crush injury. Furthermore, as B4 binding and transport recover at long survival times in the absence of reestablished peripheral connections, the same techniques can be used to study central primary afferent connections at long survival times after nerve transection. Binding and transganglionic transport of B4 offer alternatives to the use of previous techniques such as transganglionic transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) to study central connections of fine primary afferents after injury.     

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1(TRPV1) provides the sensation of pain(nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517(300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.     

Alterations in the distribution of stimulus-evoked c-fos in the spinal cord after neonatal peripheral nerve injury in the rat

Neonatal peripheral nerve injury results in a significant rearrangement of the central terminals of surviving axotomized and adjacent intact primary afferents in the dorsal horn of the spinal cord. This study investigates the ability of these afferents to make functional contacts with dorsal horn cells, using c-fos expression as a marker of synaptic activation. Graded electrical stimulation at A- or C-fiber strength of either the neonatally axotomized sciatic nerve or the adjacent uninjured saphenous nerve was performed in adult rats. Stimulation of the contralateral uninjured nerve served as a control. Quantitative examination of the number and distribution of c-fos-labeled cells in the spinal cord laminae was performed. Electrical stimulation of the previously axotomized sciatic nerve at A-fiber intensity resulted in many labeled profiles in laminae I-V of the lumbar spinal cord on the experimental as compared to the contralateral side. Electrical stimulation of uninjured saphenous nerve or saphenous-nerve-innervated skin (using pin electrodes) at A-fiber intensity did not evoke c-fos. Stimulation of the saphenous nerve at C-fiber intensity, however, resulted in a significant increase in the number and distribution of c-fos-labeled profiles in laminae I-V on the experimental side as compared to the contralateral control side. The results show that the distribution of c-fos-expressing cells after neonatal nerve injury is compatible with the previously demonstrated distribution of sprouting of primary afferents belonging to an uninjured nerve adjacent to an injured nerve, and that the surviving axotomized afferents are capable of transmitting signals to postsynaptic cells. These findings indicate that Abeta afferent stimulation of injured but not uninjured afferents elicits c-fos expression in postsynaptic cells. This may reflect an injury-induced maintenance of a normal developmental process whereby Abeta stimulation elicits c-fos in dorsal horn neurons.     

Plasticity of acid phosphatase (FRAP) afferent terminal fields and of dorsal horn cell growth in the neonatal rat

Peripheral nerve section results in depletion of fluoride-resistant acid phosphatase (FRAP) from the nerve terminals in the dorsal horn of the spinal cord (Schoenen et al., '68) and this has been used in the past to map the termination field of individual nerves (Rustioni et al., '71; Devor and Claman, '80). In the present study we show that a similar central depletion occurs following sciatic nerve section or crush in neonatal rats. Unlike adults, however, the area of depletion is rapidly filled by sprouting of FRAP-containing afferent terminals from nearby intact peripheral nerves. The sprouting is extensive but never completely fills the depleted area. After nerve crush there is some recovery of FRAP from the sciatic nerve terminals themselves as well as from nearby nerve terminals. The source of recovered FRAP is demonstrated by resectioning or recrushing the nerves. The sprouting occurred when the sciatic was injured on day 1 but failed to take place when the injury was applied on or after day 10. Sciatic nerve section on day 1 also produces marked growth retardation of the ipsilateral dorsal horn gray matter that becomes more apparent as the rat matures. Nerve crush produces a less marked shrinkage that is slower in onset. If the nerve is crushed repeatedly, however, so that regeneration is prevented, the shrinkage is analogous to that following nerve section. No shrinkage occurs if the nerve is cut or crushed on day 10. The results show that separation of the spinal cord from its peripheral input at a critical stage in development results in disruption of the somatotopic organization of the C fibre afferent input to the dorsal horn and in slowing of growth of the dorsal horn gray matter.     

Nociceptive responses and spinal plastic changes of afferent C-fibers in three neuropathic pain models induced by sciatic nerve injury in the rat

Peripheral nerve injuries induce plastic changes on primary afferent fibers and on the spinal circuitry, which are related to the emergence of neuropathic pain. In this study we compared three models of sciatic nerve injury in the rat with different degrees of damage and impact on regeneration capability: crush nerve injury, chronic constriction injury (CCI) and spared nerve injury (SNI). All three models were characterized by means of nerve histology, in order to describe the degenerative and regenerative process of injured axons. Nociceptive responses were evaluated by mechanical and thermal algesimetry tests. Crush animals displayed higher withdrawal thresholds on the ipsilateral paw compared to the contralateral during the time of denervation, while CCI and SNI animals showed mechanical and thermal hyperalgesia. Central plasticity was evaluated by immunohistochemical labeling of non-peptidergic (IB4-positive) and peptidergic (substance P-positive) nociceptive C-fibers on L4-L6 spinal cord sections. After crush nerve injury and SNI, we observed progressive and sustained reduction of IB4 and SP immunolabeling at the sciatic projection territory in the superficial laminae of the dorsal horn, which affected only the tibial and peroneal nerves projection areas in the case of SNI. After CCI, changes on SP-immunoreactivity were not observed, and IB4-immunoreactive area decreased initially but recovered to normal levels on the second week post-injury. Thus, nociceptive responses depend on the type of injury, and the immunoreactivity pattern of afferent fibers at the spinal cord display changes less pronounced after partial than complete sciatic nerve injury. Although signs of neuropathic pain appear in all three lesion models, nociceptive responses and central plasticity patterns differ between them.     

Primary afferent origin of substance P-containing axons in the superficial dorsal horn of the rat spinal cord: depletion, regeneration and replenishment of presumed nociceptive central terminals

Substance P-like immunoreactivity (SPLI) was localized in the superficial spinal dorsal horn of the rat by means of light and electron microscopic immunocytochemical techniques. Serial immunocytochemical sections were subjected to densitometric measurements with an electronic Image Analyser, and with aid of a computer program, a two-dimensional reconstruction of the fine neuroanatomical structure of the SPLI-active regions of the lumbosacral upper superficial spinal dorsal horn was obtained. SPLI activity in the superficial dorsal horn outlines four well-marked and distinctly differing regions, called, in the mediolateral sequence, areas A, B, C, and D, plus Cajal's noyeau interstitiel ("lateral spinal nucleus" = "nucleus of the dorsolateral fascicle," L). Lumbosacral dorsal rhizotomy results in an almost complete depletion of SPLI from ipsilateral areas A, B, C, and D; it induces decreased SPLI in the area of the lateral spinal nucleus (L), ipsi- or contralaterally in an alternating fashion. Transection of the segmentally related, ipsilateral peripheral nerve induces a marked depletion of SPLI from areas A, B, and C but only a slight decrease in area D and virtually none in the area of L. Whereas a simple crush of the peripheral nerve (axocompression) induces only a slight depletion of SPLI, if any, semiautomatic densitometric analysis of serial immunocytochemical sections proves that a controlled crush injury (axocontusion) results in depletion of SPLI from the upper dorsal horn, similar to transection of the peripheral nerve. Following regeneration of the ipsilateral, segmentally related peripheral nerve, the original immunocytochemical structure of the superficial dorsal horn is re-established by SPLI-positive axonal sprouts originating from previously damaged dorsal root axons.     

GAP-43 mRNA in Rat Spinal Cord and Dorsal Root Ganglia Neurons: Developmental Changes and Re-expression Following Peripheral Nerve Injury

The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000–3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons.     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Rac1-regulated dendritic spine remodeling contributes to neuropathic pain after peripheral nerve injury

Although prior studies have implicated maladaptive remodeling of dendritic spines on wide-dynamic range dorsal horn neurons as a contributor to pain after spinal cord injury, there have been no studies on dendritic spines after peripheral nerve injury. To determine whether dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain after peripheral nerve injury, we analyzed dendritic spine morphology and functional influence in lamina IV–V dorsal horn neurons after sham, chronic constriction injury (CCI) of the sciatic nerve, and CCI treatment with NSC23766, a selective inhibitor of Rac1, which has been implicated in dendritic spine development. 10 days after CCI, spine density increased with mature, mushroom-shaped spines preferentially distributed along dendritic branch regions closer to the cell body. Because spine morphology is strongly correlated with synaptic function and transmission, we recorded the response of single units to innocuous and noxious peripheral stimuli and performed behavioral assays for tactile allodynia and thermal hyperalgesia. Wide dynamic range dorsal horn neurons of CCI animals exhibited hyperexcitable responses to a range of stimuli. They also showed reduced nociceptive thresholds in the ipsilateral hind paw. 3-day treatment with NSC23766 significantly reduced post-CCI spine dimensions and densities, and attenuated injury-induced hyperexcitability. Drug treatment reduced behavioral measures of tactile allodynia, but not for thermal hyperalgesia. Together, our results demonstrate that peripheral nerve injury induces Rac1-regulated remodeling of dendritic spines on dorsal horn neurons, and suggest that this spine remodeling contributes to neuropathic pain.     

改善微循环对大鼠坐骨神经损害血管内皮细胞生长因子表达及病理改变的影响

目的:探讨改善微循环对周围神经嵌压性损害血管内皮细胞生长因子(VEGF)表达和病理改变的影响。方法:分别检测坐骨神经嵌压后12、72h和7d背根神经节细胞VEGF水平与第4周神经干、神经节病理变化、脊髓病变神经元计数等,并运用正常对照组、模型组、前列地尔组和丁咯地尔组采用SPSS10.0统计软件进行方差分析统计处理。结果:嵌压后72hVEGF水平达到高峰,与正常对照组比较,其余各组均显著增加(P<0.01,P<0.05);前列地尔组和丁咯地尔组较模型组为优(P<0.01,P<0.05);嵌压后4周前列地尔组和丁咯地尔组的神经干、神经节病理变化、脊髓病变神经元计数均优于模型组(P<0.05)。结论:对于周围神经嵌压性损害,改善微循环可增加VEGF的表达并减轻其病理损害,从而对周围神经嵌压性损害的修复具有促进作用。