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.     

Activation of extracellular signal-regulated protein kinases 5 in the spinal cord contributes to the neuropathic pain behaviors induced by CCI in rats

Abstract

Objective: To investigate whether activation and translocation of extracellular signal-regulated kinase 5 (ERK5) is involved in the induction and maintenance of neuropathic pain and observe the effects of activation and translocation of ERK5 on the expression of phosphorylated cAMP response element binding (pCREB) in the chronic neuropathic pain.

Methods: Lumbar intrathecal catheters were chronically implanted in male Sprague–Dawley rats. The left sciatic nerve was loosely ligated proximal to the sciatica's trifurcation at ～ 1.0 mm intervals with 4-0 silk sutures. The phosphorothioate-modified antisense oligonucleotides (AS-ODNs) were intrathecally administered every 12 hours, 1 day pre-chronic constriction injury (CCI) and 3 day post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal latency to a radiant heat and von Frey filaments. Expressions of phosphorylated ERK5 (pERK5), pCREB, were assessed by both Western blotting and immunohistochemical analysis.

Results: Intrathecal injection of ERK5 AS-ODN significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. CCI significantly increased the expression of pERK5 neurons in the ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. The time courses of pERK5 expression showed that the levels of both cytosol and nuclear pERK5 were increased at all points after CCI and reached a peak level on post-operative day 5. CCI significantly increased the expression of pERK5 neurons in the laminae I and II of ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. Phospho-CREB-positive neurons were distributed in all laminae of the bilateral spinal cord. Intrathecal injection AS-ODN markedly suppressed the increase of CCI-induced pERK5, pCREB expression in the spinal cord.

Conclusion: The activation of ERK5 pathways contributes to neuropathic pain in CCI rats, and the function of pERK5 may partly be accomplished via the CREB protein-dependent gene expression.     

Cell death in the superficial dorsal horn in a model of neuropathic pain

The aims of this study were to investigate the occurrence of apoptotic cell death in the dorsal horn of the adult rat spinal cord following chronic constriction injury (CCI) to the sciatic nerve and to correlate this with behavioural responses. Six groups of six rats were used as follows: 1) CCI, 2) CCI, 3) MK801 + CCI, 4) axotomy, 5) sham, and 6) naive. Group 1 animals were behaviourally tested for thermal hyperalgesia 8 days following surgery and sacrificed and the spinal cords removed and frozen. The rest of the groups underwent the same procedure 14 days following surgery. The lumbar region of the spinal cord was cryosectioned and the incidence of apoptotic cells investigated using the TUNEL technique plus Hoechst double labelling. By 8 days post-CCI, hyperalgesia had developed in the ipsilateral paw, which was still present 14 days after the injury compared to the contralateral paw and naive and sham animals. Preemptive MK-801 prevented the onset of hyperalgesia. Significant numbers of apoptotic cells were present in the ipsilateral dorsal horn of the spinal cord 8 and 14 days following CCI compared to the contralateral side and to naive and sham animals. Preemptive treatment with MK-801 reduced the extent of apoptosis resulting from CCI to the level seen in control animals. This study demonstrates that cells undergo apoptosis as a result of CCI simultaneous with the occurrence of hyperalgesia. Furthermore, MK-801 prevents the onset of hyperalgesia and reduces the extent of apoptotic cell death, suggesting, perhaps, that apoptosis contributes to the initiation/maintenance of hyperalgesia.     

Distribution of c-fos expressing dorsal horn neurons after electrical stimulation of low threshold sensory fibers in the chronically injured sciatic nerve

The distribution of proto-oncogene c-Fos protein-immunoreactive cells in the spinal cord dorsal horn was studied after electrical stimulation at Aα/Aß-fiber intensity of normal and previously injured sciatic nerves in urethane anesthetized rats. No or only occasional Fos protein-like immunoreactive cells were seen after stimulation of the normal uninjured nerve or after nerve transection without stimulation. Electrical nerve stimulation at 3, 12, and 21 days after sciatic nerve transection resulted in substantial increases in the numbers of Fos protein-like immunoreactive cell nuclei in each of Rexed's laminae I–V. Combined demonstration of Fos protein-like immunoreactivity and of glial fibrillary acidic protein-like immunoreactivity (astroglia) or OX-42 immunoreactivity (microglia), indicated that the observed Fos protein-like response was confined to neurons and not to astroglia or microglia. Combined demonstration in the spinal cord of Fos protein-like immunoreactive neurons and neurons labeled retrogradely with Fluoro-Gold from the gracile nucleus showed that some of the Fos protein-like immunoreactive neurons in Rexed's laminae III and IV contributed to the postsynaptic dorsal column pathway. The results indicate that stimulation at Aα/Aß-fiber intensity of a previously injured nerve gives rise to an abnormally increased activation pattern of postsynaptic neurons in the dorsal horn, some of which contribute to the postsynaptic dorsal column pathway.     

Increased c-fos expression in spinal lumbosacral projection neurons and preganglionic neurons after irritation of the lower urinary tract in the rat.

Chemical irritation of the lower urinary tract (LUT) induces c-fos expression in neurons in the lumbosacral (L(6) and S(1)) spinal cord. This study used axonal tracing with fluorescent dyes to identify the types of spinal neurons expressing Fos immunoreactivity (IR) after LUT irritation in the rat. Fos-IR was detected in lateral and medial superficial dorsal horn, the sacral parasympathetic nucleus (SPN) and lamina X around the central canal. Fos-IR was detected in spinal neurons projecting to supraspinal sites (brainstem and hypothalamus), in preganglionic neurons (PGN) and in unlabeled segmental interneurons. A substantial percentage (20%) of dye labeled PGN exhibited Fos-IR after LUT irritation; and a larger percentage (36%) exhibited Fos-IR after electrical stimulation of the pelvic nerve which contains afferent pathways from all of the pelvic organs. The majority (average 55%) of Fos-positive neurons projecting to supraspinal sites were also located in the region of the SPN. A selective distribution of different types of neurons was detected in this region: PGN were located ventral to the spinal projection neurons which in turn were located ventral to the majority of unidentified Fos-positive neurons. The distribution of Fos-positive PGN and projection neurons was similar in spinal intact and spinal transected animals indicating that c-fos expression was mediated by monosynaptic afferent input or input from segmental interneurons and was not due to activation of supraspinal micturition reflex pathways.     

Chronic constriction injury of sciatic nerve induces the up-regulation of descending inhibitory noradrenergic innervation to the lumbar dorsal horn of mice

Peripheral nerve injury in rodents results in hypersensitivity to mechanical and thermal stimuli accompanied by reduced antinociceptive efficacy of opioids and, in some models, sensitivity to sympathetic blockade. 2-Adrenergic receptor agonists increase in potency and efficacy after nerve injury in rodents and effectively relieve neuropathic pain in humans who do not get pain relief from opioids. However, the underlying mechanisms are unclear. It has been well known that the major noradrenergic innervation of the spinal dorsal horn originates from the locus coeruleus nucleus (LC) in the brainstem. Therefore, the aim of this study is to examine whether peripheral nerve injury that causes neuropathic pain modulates the noradrenergic innervation to the lumbar dorsal horn, in order to determine the possible anatomical substrates underlying increased potency and efficacy of noradrenergic receptor agonists in alleviating neuropathic pain. At 2 weeks after chronic constriction injury (CCI) of the sciatic nerve, a remarkable increase in tyrosine-hydroxylase (TH) and dopamine β-hydroxylase (DβH) immunoreactive (IR) axonal terminals was observed in the ipsilateral L4–L6 dorsal horn. Consistently, greater numbers of both TH- and DβH-IR neurons were detected in the ipsilateral LC. Interestingly, in the lower lumbar and upper sacral spinal dorsal horn, numerous TH-IR neurons were observed in the superficial dorsal horn (primarily lamina I). CCI of the sciatic nerve did not change the number of these TH-IR cells. These findings suggest that augmented descending inhibitory noradrenergic innervation to the dorsal horn could be one of the mechanisms underlying the increased effectiveness in the anti-allodynic effect elicited by 2-adrenergic receptor agonists.     

Changes in phosphorylation of ERK and Fos expression in dorsal horn neurons following noxious stimulation in a rat model of neuritis of the nerve root

Mechanical compression and chemical inflammation of the spinal nerve root are considered major sensory pathologies secondary to a lumbar disc herniation. In order to elucidate the dorsal horn responsiveness to noxious stimulation to the peripheral tissue in the neuritis model of the nerve root, we examined extracellular signal-regulated kinase (ERK) phosphorylation and Fos expression in spinal cord dorsal horn neurons. Male Sprague-Dawley rats received hemilaminectomies and the implantation of disc tissue that was obtained from coccygeal intervertebral discs. Three or 7 days after surgery, rats were perfused after receiving noxious mechanical stimulation of the plantar surface of the hind paw using a hemoclip, and the L4/5 spinal cord was processed for immunohistochemistry with antibodies for phospho-ERK and Fos. The number of Fos-immunoreactive (Fos-LI) neurons and phospho-ERK-immunoreactive (phospho-ERK-LI) neurons in the neuritis group after the noxious stimulation significantly increased compared to the sham-treated group at 3 and 7 days after surgery. The change in number of phospho-ERK-LI and Fos-LI neurons occurred mainly in the superficial dorsal horn. The number of Fos-LI neurons observed when the MEK inhibitor, U0126, was administered was significantly suppressed compared to the DMSO- (vehicle control) administered group. The increase in ERK phosphorylation and Fos expression in the spinal cord dorsal horn neurons indicates that responses/activation by the noxious stimulation applied to the periphery were elevated in spinal cord neurons in this neuritis model of the lumbar nerve root. Moreover, the increase in the Fos expression in the spinal cord dorsal horn may have been the result of the activation of the MAP kinase cascade.     

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.     

Increased c-fos expression in spinal lumbosacral projection neurons and preganglionic neurons after irritation of the lower urinary tract in the rat

Chemical irritation of the lower urinary tract (LUT) induces c-fos expression in neurons in the lumbosacral (L₆ and S₁) spinal cord. This study used axonal tracing with fluorescent dyes to identify the types of spinal neurons expressing Fos immunoreactivity (IR) after LUT irritation in the rat. Fos-IR was detected in lateral and medial superficial dorsal horn, the sacral parasympathetic nucleus (SPN) and lamina X around the central canal. Fos-IR was detected in spinal neurons projecting to supraspinal sites (brainstem and hypothalamus), in preganglionic neurons (PGN) and in unlabeled segmental interneurons. A substantial percentage (20%) of dye labeled PGN exhibited Fos-IR after LUT irritation; and a larger percentage (36%) exhibited Fos-IR after electrical stimulation of the pelvic nerve which contains afferent pathways from all of the pelvic organs. The majority (average 55%) of Fos-positive neurons projecting to supraspinal sites were also located in the region of the SPN. A selective distribution of different types of neurons was detected in this region: PGN were located ventral to the spinal projection neurons which in turn were located ventral to the majority of unidentified Fos-positive neurons. The distribution of Fos-positive PGN and projection neurons was similar in spinal intact and spinal transected animals indicating that c-fos expression was mediated by monosynaptic afferent input or input from segmental interneurons and was not due to activation of supraspinal micturition reflex pathways.     

Pulsed electrical stimulation protects neurons in the dorsal root and anterior horn of the spinal cord after peripheral nerve injury

Most studies on peripheral nerve injury have focused on repair at the site of injury, but very few have examined the effects of repair strategies on the more proximal neuronal cell bodies. In this study, an approximately 10-mm-long nerve segment from the ischial tuberosity in the rat was transected and its proximal and distal ends were inverted and sutured. The spinal cord was subjected to pulsed electrical stimulation at T10 and L3, at a current of 6.5 m A and a stimulation frequency of 15 Hz, 15 minutes per session, twice a day for 56 days. After pulsed electrical stimulation, the number of neurons in the dorsal root ganglion and anterior horn was increased in rats with sciatic nerve injury. The number of myelinated nerve fibers was increased in the sciatic nerve. The ultrastructure of neurons in the dorsal root ganglion and spinal cord was noticeably improved. Conduction velocity of the sciatic nerve was also increased. These results show that pulsed electrical stimulation protects sensory neurons in the dorsal root ganglia as well as motor neurons in the anterior horn of the spinal cord after peripheral nerve injury, and that it promotes the regeneration of peripheral nerve fibers.     

Chronic tooth pulp inflammation causes transient and persistent expression of Fos in dynorphin-rich regions of rat brainstem

We have analyzed central Fos immunoreactivity (Fos-IR) brainstems of adult rats after three clinically relevant dental injuries: filled dentin (DF) cavities that cause mild pulp injury and heal within 1-2 weeks; open pulp exposures (PX) that cause gradual pulp loss and subsequent periodontal lesions; and filled pulp exposures (PXF). By 1 week after DF cavities, no Fos-IR remained except for sites such as lateral-ventral periolivary nucleus (LVPO) that had Fos-IR in all rats including controls. PX injury induced (1) a delayed transient expression of Fos at 1-2 weeks at three loci (ipsilateral neurons in dorsomedial nucleus oralis, paratrigeminal nucleus, and trigeminal tract), (2) persistent ipsilateral Fos for at least 4 weeks after injury in dynorphin (Dyn)-rich regions (rostral lateral solitary nucleus, periobex dorsal nucleus caudalis), and (3) late Fos-IR at 2-4 weeks (bilateral superficial cervical dorsal horn, contralateral dorsal nucleus caudalis, contralateral rostral lateral solitary nucleus). Rats with PXF injury were examined at 2 weeks, and they had greater numbers and more extensive rostro-caudal distribution of Fos neurons than the PX group. One week after PX injury, Fos-IR neurons were found in regions with strong Dyn-IR central fibers. Co-expression of Dyn and Fos was found in some unusually large neurons of the ipsilateral rostral lateral solitary nucleus, trigeminal tract, and dorsal nucleus caudalis. Immunocytochemistry for the p75 low affinity neurotrophin receptor (p75NTR) or for calcitonin gene-related peptide (CGRP) showed no consistent change in trigeminal central endings in any Fos-reactive brainstem areas, despite the extensive structural and cytochemical reorganization of the peripheral endings of the dental neurons. The Fos responses of central neurons to tooth injury have some unusual temporal and spatial patterns in adult rats compared to other trigeminal injury models.     

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.     

Altered responses of nociceptive cat lamina I spinal dorsal horn neurons after chronic sciatic neuroma formation

The activity of lumbar spinal dorsal horn lamina I neurons with afferent drive from the sciatic nerve was studied in intact cats and in cats with acute sciactic nerve transection or chronic sciatic nerve transection with neuroma formation. The majority (51 of 75) of neurons recorded in lamina I ipsilateral to a neuroma had no receptive field and could only be identified by their responses to electrical stimulation of the sciatic nerve. The remainder could be activated by the sciatic nerve, but their responses to mechanical stimulation were irregular in comparison to the stable responses of cells recorded in control animals and to the responses of cells contralateral to chronic nerve lesions. Animals with acute nerve transections demonstrated as loss sciatic nerve-innvervated cells with receptive fields except for those cells located on the lateral edge of the dorsal horn, which had normal, proximal receptive fields and response characteristics. In addition, the characteristic somatotopy of lamina I cells was not observed in some cats with chronic neuromata. The mediolateral distribution of cell types indicated that some cells had altered receptive fields following chronic nerve transection. The data presented for lamina I neurons agrees with the observation of spinal cord plasticity first presented for cat dorsal horn cells. Since there is no evidence for a redistribution of intact afferent fibers following chronic nerve transection in adult mammals, the mechanism of altered somatotopy may involved alterations in synaptic efficacy at existing synapses.     

Activation of p38 MAP kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury

The intrathecal administration of p38 MAP kinase (p38) inhibitor has been shown to reduce hyperalgesia. In the present study, we investigated the activation of p38 in the rat dorsal root ganglion (DRG) and spinal cord following peripheral tissue inflammation and nerve injury immunohistochemically. Peripheral inflammation and chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the percentage of phosphorylated (P-) p38-immunoreactive (IR) neurons, primarily small sized ones in bilateral DRGs. In contrast, following axotomy, a significant decrease in the percentage of IR neurons was observed in ipsilateral DRGs. In addition, a marked increase was observed in the number of P-p38-IR microglia in the ipsilateral laminae I-IV and IX of the spinal cord following peripheral inflammation, CCI or axotomy. These findings suggest that p38 may play an important role in hyperalgesia and the activation of the spinal microglia.     

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.     

Temporal and spatial distribution of Fos protein in the parabrachial nucleus neurons during experimental tooth movement of the rat molar

The present study was undertaken to reveal spatio-temporal changes in the distribution of Fos-like immunoreactive (-IR) neurons in the parabrachial nucleus (PBN), one of the important relay nuclei for processing autonomic and somatosensory information from the oro-facial regions, following the induction of experimental tooth movement in rat upper molars. The experimental tooth movement was induced by the insertion of elastic rubber between the first and second upper molars. In normal animals, the PBN contained a smaller number of Fos-IR neurons. Following experimental tooth movement, the Fos-IR neurons increased in number significantly on both the ipsilateral and contralateral PBN, reaching a maximum at 4 h (about 10 times that of normal animals), and then decreased gradually. However, a significant number of Fos-IR neurons remained at 24 h post-operation. Remarkable side-by-side differences in the number of Fos-IR neurons were recognized at 1 to 4 h following the experimental tooth movement. Their number returned to normal (basal) levels at 5 days post. All subnuclei of PBN showed similar temporal changes in the number of Fos-IR neurons, this being particularly apparent in lateral PBN. Administrations of morphine (3 and 10 mg/kg, i.p.) drastically reduced the induction of Fos-IR neurons in all subnuclei of both the ipsilateral and contralateral PBN in a dose-dependent manner, and its effect was antagonized by pretreatment with naloxone (2 mg/kg, i.p.). The reduction of Fos-IR neurons by morphine pretreatment suggests that the appearance of Fos-IR neurons in the PBN may be partly due to the noxious stimulation and/or stress arising from tooth movement. The bilateral expression of Fos-IR neurons in the PBN indicates that the experimental tooth movement causes the activation of PBN neurons for the processing of somatosensory as well as autonomic information. The prolonged expression of Fos-IR neurons in all the subnuclei of bilateral PBN reflects clinical features of the transient discomfort and/or abnormal sensations, which many patients often complain about during orthodontic treatment.     

Myelinated afferents sprout into lamina II of L3–5 dorsal horn following chronic constriction nerve injury in rats

In order to investigate the consequences of chronic constriction injury (CCI) to nerve, we explored the relationship between the development of mechanical allodynia and the reorganization of primary afferent terminals in the sensory lamina of the rat spinal cord dorsal horn. Following sciatic CCI neuropathy, mechanical allodynia developed in the corresponding footpad within two weeks and persisted throughout the experimental period which extended for an additional two weeks. The neuropathy of the sciatic injury includes extensive Wallerian-like degeneration of myelinated fibers but relative sparing of unmyelinated fibers. We observed that there was no significant change in the dorsal horn termination of unmyelinated C fibers in lamina II of the dorsal horn, using nerve injections of wheat germ agglutin-horseradish peroxidase for transganglionic axonal tracing of these fibers from the nerve injury site, and no evidence of sprouting into adjacent lamina. In contrast, myelinated afferent fibers were observed to be sprouting into lamina II of the dorsal horn, as indicated by cholera toxin β-subunit-horseradish peroxidase retrograde axonal tracings. This region of the dorsal horn is associated with nociceptive-specific neurons that are not generally associated with myelinated fiber input from mechanical and proprioceptive receptors. As previously suggested in nerve transection and crush injuries, and now demonstrated in CCI neuropathy, these morphological changes may have significance in the pathogenesis of chronic mechanical allodynia.     

Myelinated afferents sprout into lamina II of L3-5 dorsal horn following chronic constriction nerve injury in rats

In order to investigate the consequences of chronic constriction injury (CCI) to nerve, we explored the relationship between the development of mechanical allodynia and the reorganization of primary afferent terminals in the sensory lamina of the rat spinal cord dorsal horn. Following sciatic CCI neuropathy, mechanical allodynia developed in the corresponding footpad within two weeks and persisted throughout the experimental period which extended for an additional two weeks. The neuropathy of the sciatic injury includes extensive Wallerian-like degeneration of myelinated fibers but relative sparing of unmyelinated fibers. We observed that there was no significant change in the dorsal horn termination of unmyelinated C fibers in lamina II of the dorsal horn, using nerve injections of wheat germ agglutin-horseradish peroxidase for transganglionic axonal tracing of these fibers from the nerve injury site, and no evidence of sprouting into adjacent lamina. In contrast, myelinated afferent fibers were observed to be sprouting into lamina II of the dorsal horn, as indicated by cholera toxin beta-subunit-horseradish peroxidase retrograde axonal tracings. This region of the dorsal horn is associated with nociceptive-specific neurons that are not generally associated with myelinated fiber input from mechanical and proprioceptive receptors. As previously suggested in nerve transection and crush injuries, and now demonstrated in CCI neuropathy, these morphological changes may have significance in the pathogenesis of chronic mechanical allodynia.     

Cholecystokinin in Mammalian Primary Sensory Neurons and Spinal Cord: In Situ Hybridization Studies in Rat and Monkey

The peptide cholecystokinin (CCK) has been suggested to be involved in nociception, but its exact localization at the level of the spinal cord and in spinal ganglia has been a controversial issue. Therefore the distribution of messenger RNA (mRNA) for CCK was studied by in situ hybridization using oligonucleotide probes on sections of adult rat lumbar dorsal root ganglia following unilateral section of the sciatic nerve and on sections of untreated monkey trigeminal ganglia, spinal cord and spinal ganglia from all levels. For comparison, calcitonin gene-related peptide (CGRP) mRNA was also studied in the monkey tissue using the same techniques. Peripheral sectioning of the sciatic nerve in the rat resulted in the appearance of detectable CCK mRNA in up to 30% of remaining ipsilateral L4 and L5 dorsal root ganglion neurons 3 weeks after surgery, with a distinct but more limited appearance also in the contralateral ganglia. No cells, or only single cells, could be seen in normal control rat ganglia. In contrast, in the normal monkey, ∼20% of dorsal root ganglion neurons, regardless of spinal level, and 10% of trigeminal ganglia neurons expressed mRNA for CCK. CGRP mRNA was expressed at detectable levels in ∼80% of these monkey dorsal root ganglion neurons. In the monkey spinal cord, CCK mRNA was detected in the dorsal horn and in motoneurons, whereas CGRP mRNA was only seen in motoneurons. The present results suggest that CCK peptides can be involved in sensory processing in the dorsal horn of the spinal cord in normal monkeys and in rats after peripheral nerve injury, adding one more possible excitatory peptide to the group of mediators in the dorsal horn.     

Peri-sciatic administration of recombinant rat TNF-alpha induces mechanical allodynia via upregulation of TNF-alpha in dorsal root ganglia and in spinal dorsal horn: the role of NF-kappa B pathway

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) and TNF receptor 1 (TNFR1) in dorsal root ganglia (DRG) and in spinal dorsal horn are upregulated after nerve injury and that many TNF-alpha-containing neurons overexpress TNFR1. In the present study, we found that peri-sciatic administration of rat recombinant TNF-alpha (rrTNF) at the concentrations of 10, 100 and 1000 pg/ml (daily for 2 days) induced mechanical allodynia in bilateral hindpaws, lasting for about 20 days. The immunoreactivity (IR) of TNF-alpha and TNFR1 in the ipsilateral (but not in the contralateral) L4 and L5 DRGs increased significantly on day 1 and day 3 after administration of rrTNF, respectively. Double immunofluorescence staining revealed that in DRGs the increased TNF-alpha-IR was mainly in neuronal cells and with a lesser extent in satellite glial cells, while the upregulation of TNFR1-IR was almost restricted at neuronal cells. TNF-alpha-IR but not TNFR1-IR also increased in bilateral lumbar spinal dorsal horn from day 3 to day 14, which was observed in astrocytes, microglias and neurons. In addition, a progressive infiltration of monocyte/macrophages and T lymphocytes in the ipsilateral L5 DRG and sciatic nerve was observed, starting on day 2 following administration of rrTNF. Intrathecal delivery of PDTC (8.2 ng in 10 microl volume), a nuclear factor-kappa B (NF-kappaB) inhibitor, 30 min before each rrTNF administration blocked mechanical allodynia completely and inhibited the upregulation of TNF-alpha-IR and TNFR1-IR substantially. The results suggest that peri-sciatic administration of rrTNF may induce mechanical allodynia by an autocrine mechanism via activation of the NF-kappaB pathway.