Behavioral characteristics and c‐Fos expression in the medullary dorsal horn in a rat model for orofacial cancer pain

It is well known that patients with orofacial cancer suffer from cancer‐induced pain which produces feeding difficulties. To understand the mechanisms of pain associated with orofacial cancer, we have recently created a model for rat orofacial cancer by inoculation with Walker carcinosarcoma 256B‐cells into the vibrissal pads. The present study used both behavioral and immunohistochemical techniques to investigate changes in pain‐related and ingestive behavior, along with c‐Fos expression in the medullary dorsal horn which is a site for processing orofacial pain. The tumor mass grew gradually and contacted the nerve trunks within days after the inoculation of tumor cells. Physical difficulties in ingestion were observed after day 10 post‐inoculation and facial grooming periods were prolonged. Sensitivities of the inoculated vibrissal pads to mechanical and thermal stimuli were increased on days 4 and 7, suggesting the development of mechanical allodynia and thermal hyperalgesia. Although hyposensitivity to mechanical and thermal stimulation was observed in the inoculated region after day 10, hyperalgesia developed on the margin of the tumor, suggesting that the hypersensitive region spread with growth of tumor mass. In the medullary dorsal horn, the levels of c‐Fos immunoreactivity of the ipsilateral side increased significantly on days 4, 7 and 10, supporting the behavioral observations. These results indicate that the rat model shows symptoms similar to those in patients with orofacial cancer, for example, induction of feeding disorder and neuropathic pain.     

A selective increase in Fos expression in spinal dorsal horn neurons following graded thermal stimulation in rats with experimental mononeuropathy

In order to clarify the central mechanisms of thermal hyperalgesia produced by peripheral nerve injury, Fos protein-like immunoreactive (Fos-LI) cells in spinal dorsal horn neurons were studied in rats with chronic constriction nerve injury (CCI) following graded thermal stimulation of the hind paw. The graded thermal stimuli (cold: 5, 10 and 15°C, heat: 42, 46 and 54°C) were applied to the planter surface of the operated hind paw 14 days after CCI or sham operation, and the number of Fos-LI cells in the spinal dorsal horn was quantified. Many Fos-LI cells were expressed in the superficial laminae of the spinal dorsal horn both in sham-operated and CCI rats following thermal stimulation. Fos-LI cells were mainly restricted to the medial half of the superficial laminae of the spinal dorsal horn, and were sparsely distributed in the deeper laminae. The number of Fos-LI cells in the superficial laminae (laminae I–II) of the dorsal horn was significantly higher in CCI rats after stimulation at 10 and 46°C, but not at the other stimulating temperatures (5, 15, 42, and 54°C) as compared to that in sham-operated rats. In laminae III–IV, the number of Fos-LI cells was significantly higher at all stimulus temperatures in CCI rats when compared to the sham-operated rats. No distribution difference of Fos-LI cells was observed between CCI and sham-operated rats in laminae V–VI. Thus, in the spinal dorsal horn of the CCI rats, there was a selective increase in thermal stimulus-induced Fos-LI cells in the superficial dorsal horn after stimulating at near noxious threshold intensities and a non-selective increase in Fos-LI cells in laminae III-IV after both noxious and innocuous thermal stimuli. The increase of Fos-LI cells in the superficial laminae may be related to hypersensitivity to noxious stimuli while the increase of Fos-LI cells in laminae III–IV may be related to an increased sensitivity to both noxious and innocuous stimuli that leads to increased reflex activity following nerve injury.     

Chronic pain and sensorimotor deficits following peripheral nerve injury

Following upper limb peripheral nerve transection and surgical repair (PNIr) patients frequently exhibit sensory and motor deficits, but only some develop chronic neuropathic pain. Thus, the sensorimotor outcome of PNIr may be impacted by individual factors. Therefore, our aims were to determine if patients with chronic neuropathic pain (PNI-P) following PNIr (1) are distinguished from patients without pain (PNI-NP) and healthy controls (HCs) by the psychological factors of pain catastrophizing, neuroticism or extraversion, and (2) exhibit more severe sensorimotor deficits than patients who did not develop chronic pain (PNI-NP). Thirty-one patients with complete median and/or ulnar nerve transection (21 PNI-NP, 10 PNI-P) and 21 HCs completed questionnaires to assess pain characteristics, pain catastrophizing, neuroticism and extraversion and underwent sensorimotor evaluation. Nerve conduction studies revealed incomplete sensorimotor peripheral recovery based on abnormal sensory and motor latency and amplitude measures in transected nerves. The patients also had significant deficits of sensory function (two-point discrimination and vibration, touch, and warmth detection), sensorimotor integration, and fine motor dexterity. Compared to PNI-NP patients, PNI-P patients had higher vibration detection thresholds, performed worse on sensory-motor integration tasks, had greater motor impairment, and showed more impaired nerve conduction. Furthermore, PNI-P patients had reduced cold pain tolerance, elevated pain intensity and unpleasantness during the cold pressor test, and they scored higher on neuroticism and pain-catastrophizing scales. These data demonstrate that chronic neuropathic pain following PNIr is associated with impaired nerve regeneration, profound sensorimotor deficits and a different psychological profile that may be predictive of poor recovery after injury.     

The effects of ibuprofen and the neurokinin‐1 receptor antagonist GR205171A on Fos expression in the ferret trigeminal nucleus following tooth pulp stimulation

We have developed a model to study central changes following inflammation of the tooth pulp in the ferret and have examined Fos expression in the trigeminal nucleus following stimulation of non‐inflamed and inflamed tooth pulps. The aim of this study was to establish the ability of this model to predict analgesic efficacy in clinical studies of inflammatory pain. We addressed this by assessing the effects of the neurokinin‐1 receptor antagonist GR205171A and ibuprofen on Fos expression following stimulation of the inflamed pulp and comparing this with known analgesic efficacy. Adult ferrets were prepared under anaesthesia to allow tooth pulp stimulation, recording from the digastric muscle and intravenous injections at a subsequent experiment. In some animals pulpal inflammation was induced, by introducing human caries into a deep buccal cavity. After 5 days, animals were reanaesthetised, treated with vehicle, GR205171A or ibuprofen and the teeth were stimulated at ten times the threshold of the jaw‐opening reflex. Stimulation of all tooth pulps induced ipsilateral Fos in trigeminal subnuclei caudalis and oralis. GR205171A had no significant effect on Fos expression in the trigeminal nucleus of animals with either non‐inflamed or inflamed tooth pulps. Ibuprofen reduced Fos expression in the trigeminal nucleus and this effect was most marked in animals with pulpal inflammation. These results differ from those previously described using a range of other animal models, but agree with known clinical efficacy of neurokinin‐1 receptor antagonists and ibuprofen. Therefore this model is likely to be of use in accurately predicting the analgesic efficacy of novel compounds.     

Brain derived nerve growth factor induces spinal noradrenergic fiber sprouting and enhances clonidine analgesia following nerve injury in rats

Many treatments for neuropathic pain activate or augment norepinephrine release in the spinal cord, yet these treatments are less effective against acute nociceptive stimuli. We previously showed in mice that peripheral nerve injury results in sprouting of spinal noradrenergic fibers, possibly reflecting the substrate for this shift in drug efficacy. Here, we tested whether such sprouting also occurs in rats after nerve injury and examined one signal for such sprouting. Ligation of L5 and L6 spinal nerves unilaterally in rats resulted in hypersensitivity to tactile stimulation of the ipsilateral paw, and sprouting of noradrenergic fibers in the dorsal horn of the lumbar spinal cord. Brain derived nerve growth factor (BDNF) content increased in L4-L6 dorsal root ganglia ipsilateral to injury and in lumbar spinal cord following nerve injury, and intrathecal infusion of BDNF antiserum prevented spinal noradrenergic sprouting. This treatment also prevented the increased analgesic efficacy of intrathecal clonidine observed after nerve injury. Intraspinal injection of BDNF in non-injured rats mimicked the sprouting of spinal noradrenergic fibers seen after nerve injury. These results suggest that increased BDNF synthesis and release drives spinal noradrenergic sprouting following nerve injury, and that this sprouting may paradoxically increase the capacity for analgesia in the setting of neuropathic pain from drugs which utilize or mimic the noradrenergic pathway.     

Effects of pre-emptive drug treatment on astrocyte activation in the cuneate nucleus following rat median nerve injury

In this study, we examined the relationship between astrocyte activation in the cuneate nucleus (CN) and behavioral hypersensitivity after chronic constriction injury (CCI) of the median nerve. In addition, we also examined the effects of pre-emptive treatment with a number of drugs on astrocyte activation and hypersensitivity development in this model. Using immunohistochemistry and immunoblotting, little glial fibrillary acidic protein (GFAP; an astrocyte marker) immunoreactivity was detected in the CN of the normal rats. As early as 3 days after CCI, there was a significant increase in GFAP immunoreactivity in the lesion side of CN, and this reached a maximum at 7 days, and was followed by a decline. Counting of GFAP-immunoreactive astrocytes revealed that astrocytic hypertrophy, but not proliferation, contributes to increased GFAP immunoreactivity. Furthermore, microinjection of the glial activation inhibitor, fluorocitrate, into the CN at 3 days after CCI attenuated injury-induced behavioral hypersensitivity in a dose-dependent manner. These results suggest that median nerve injury-induced astrocytic activation in the CN modulated the development of behavioral hypersensitivity. Animals received MK-801 (glutamate N-methyl-d-aspartate (NMDA) receptor antagonist), clonidine (α₂-adrenoreceptor agonist), tetrodotoxin (TTX, sodium channel blocker) or lidocaine (local anesthetic) 30 min prior to median nerve CCI. Pre-treatment with MK-801, TTX, and 2% lidocaine, but not clonidine, attenuated GFAP immunoreactivity and behavioral hypersensitivity following median nerve injury. In conclusion, suppressing reactions to injury, such as the generation of ectopic discharges and activation of NMDA receptors, can decrease astrocyte activation in the CN and attenuate neuropathic pain sensations.     

Chronic blockade of interleukin‐1 (IL‐1) prevents and attenuates neuropathic pain behavior and spontaneous ectopic neuronal activity following nerve injury

Neuropathic pain is a chronic pain state resulting from peripheral nerve injury, characterized by hyperalgesia and allodynia. We have reported that mice with genetic impairment of IL‐1 signaling display attenuated neuropathic pain behavior and ectopic neuronal activity. In order to substantiate the role of IL‐1 in neuropathic pain, WT mice were implanted subcutaneously with osmotic micropumps containing either IL‐1ra or vehicle. Two days following the implantation, two models of neuropathic pain were used; partial nerve injury (spinal nerve transection, SNT), or complete nerve cut (spinal neuroma model). Mechanosensitivity was assessed seven consecutive days following SNT, and on day 7 recordings of spontaneous ectopic activity were performed. In the spinal nerve neuroma model, autotomy scores were recorded up to 35 days. Vehicle‐treated mice developed significant allodynia and autotomy, and clear ectopic activity (4.1±1.1% of the axons); whereas IL‐1ra‐treated mice did not display allodynic response, displayed delayed onset of autotomy and markedly reduced severity of autotomy scores, and displayed reduced spontaneous activity (0.8±0.4% of the axons). To test whether IL‐1 is involved in maintenance of mechanical allodynia, a separate group of WT mice was treated with a single injection of either saline or IL‐1ra four days following SNT, after the allodynic response was already manifested. Whereas saline‐treated mice displayed robust allodynia, acute IL‐1ra treatment induced long‐lasting attenuation of the allodynic response. The results support our hypothesis that IL‐1 signaling plays an important role in neuropathic pain and in the ectopic neuronal activity that underling its development.     

Analgesic treatment with pregabalin does not prevent persistent pain after peripheral nerve injury in the rat

Reducing the risk of chronic postoperative pain through preventive analgesia is an attractive therapeutic concept. Because peripheral nerve lesions are a major cause of chronic pain after surgery, we tested in rats whether analgesic treatment with pregabalin (PGB) has the capacity to mitigate the development of persistent neuropathic pain-like behavior. Starting on the day of spared nerve injury or 1 week later, we treated rats with a continuous intrathecal infusion of PGB (300 or 900 μg/24 hours) or vehicle for up to 28 days. Rats receiving early PGB treatment had almost normal withdrawal thresholds for punctate mechanical stimuli and were clearly less sensitive to pinprick or cold stimulation. The responses to punctate mechanical and cold stimulation were still reduced for a brief period after the infusion was terminated, but the difference from vehicle-treated rats was minor. Essentially, the analgesic effect of PGB was limited to the duration of the infusion, whether analgesia started at the time of surgery or with a delay of 1 week, independently of the length of the treatment. PGB did not suppress the activation of spinal microglia, indicating that analgesia alone does not eliminate certain pain mechanisms even if they depend, at least partially, on nociceptive input. Unexpectedly, intrathecal infusion of PGB did not inhibit the nerve injury-induced accumulation of its binding target, the voltage-gated calcium channel subunit α2δ1, at primary afferent terminals in the spinal cord. Interference with the synaptic trafficking of α2δ1 is not required to achieve analgesia with PGB.     

Interleukin‐6 induces microglial CX3CR1 expression in the spinal cord after peripheral nerve injury through the activation of p38 MAPK

Peripheral nerve injury leading to neuropathic pain induces the upregulation of interleukin (IL)‐6 and microglial CX3CR1 expression, and activation of p38 mitogen‐activated protein kinase (MAPK) in the spinal cord. Here, we investigated whether IL‐6 regulates CX3CR1 expression through p38 MAPK activation in the spinal cord in rats with chronic constriction injury (CCI) of the sciatic nerve. Similar temporal changes in the expression of IL‐6, phosphorylated p38 MAPK and CX3CR1 were observed following CCI. The increases in CX3CR1 expression, p38 MAPK activation and pain behavior after CCI were suppressed by blocking IL‐6 action with a neutralizing antibody, while they were enhanced by supplying exogenous recombinant rat IL‐6 (rrIL‐6). rrIL‐6 also induced increases in spinal CX3CR1 expression, p38 MAPK activation and pain behavior in naïve rats without nerve injury. Furthermore, treatment with the p38 MAPK‐specific inhibitor, SB203580, suppressed the increase in CX3CR1 expression induced by CCI or rrIL‐6 treatment. Finally, blocking CX3CR1 or p38 MAPK activation prevented the development of mechanical allodynia and thermal hyperalgesia induced by CCI or rrIL‐6 treatment. These results suggest a new mechanism of neuropathic pain, in which IL‐6 induces microglial CX3CR1 expression in the spinal cord through p38 MAPK activation, enhancing the responsiveness of microglia to fractalkine in the spinal cord, thus playing an important role in neuropathic pain after peripheral nerve injury.     

Multimodal therapeutic assessment of peripheral nerve stimulation in neuropathic pain: Five case reports with a 20‐year follow‐up

Neuropathic pain following peripheral nerve lesion is highly resistant to conventional pain treatments but may respond well to direct electrical peripheral nerve stimulation (PNS). In the 1980 s, we treated a series of 11 peripheral neuropathic pain patients with PNS. A first outcome assessment, conducted after a 52‐month follow‐up, revealed that the majority of the patients were significantly improved. Here, we present the results of a second and more comprehensive follow‐up, conducted after more than 20 years of PNS usage. Of the six patients still using PNS, five participated in a multimodality assessment of the long‐term efficacy of PNS. Home evaluations showed reduced pain ratings and improved quality‐of‐life during active periods of stimulation. Quantitative sensory testing confirmed the neuropathic character of the pain complaints. PNS had no significant overall effect on tactile detection, cool, warmth, cold pain and heat pain thresholds. Laser‐evoked potentials showed an enlarged N2–P2 complex during active PNS. Positron Emission Tomography revealed that PNS decreased activation in the pain matrix at rest and during thermal stimulation. PNS led to increased blood flow not only in primary somatosensory cortex, but also in anterior cingulate and insular cortices, suggesting that besides activation of the dorsal column lemniscal system, other mechanisms may play a role in its analgesic effects. These data show that PNS can provide truly long‐term pain relief in carefully selected patients and they provide some objective quantitative data in support of this. They encourage the planning of future prospective studies in a larger cohort of patients.     

Genetic analysis of neuropathic pain-like behavior following peripheral nerve injury suggests a role of the major histocompatibility complex in development of allodynia

Neuropathic pain is a common consequence of damage to the nervous system. We here report a genetic analysis of development of neuropathic pain-like behaviors after unilateral photochemically-induced ischemic sciatic nerve injury in a panel of inbred rat strains known to display different susceptibility to autoimmune neuroinflammation. Pain behavior was initially characterized in Dark-Agouti (DA; RT1(av1)), Piebald Virol Glaxo (PVG; RT1(c)), and in the major histocompatibility complex (MHC)-congenic strain PVG-RT1(av1). All strains developed mechanical hypersensitivity (allodynia) following nerve injury. However, the extent and duration of allodynia varied significantly among the strains, with PVG displaying more severe allodynia compared to DA rats. Interestingly, the response of PVG-RT1(avRT1) was similar to that of DA, suggesting regulation by the MHC locus. This notion was subsequently confirmed in an F2 cohort derived from crossing of the PVG and PVG-RT1(av1)strains, where allodynia was reduced in homozygous or heterozygous carriers of the RT1(av1) allele in comparison to rats homozygous for the RT1(c) allele. These results indicate that certain allelic variants of the MHC could influence susceptibility to develop and maintain neuropathic pain-like behavior following peripheral nerve injury in rats.     

Somatosensory function in patients with and without pain after traumatic peripheral nerve injury

Why traumatic injuries to the peripheral nervous system infrequently result in neuropathic pain is still unknown. The aim of this study was to examine the somatosensory system in patients with traumatic peripheral nerve injury with and without pain to try to unravel possible links to mechanisms underlying development and maintenance of pain. Eighteen patients with spontaneous ongoing pain and 16 patients without pain after unilateral partial peripheral traumatic nerve injury were studied. In the area of partial denervation and in the corresponding contralateral area perception thresholds to warmth, cold, light touch, pressure pain, cold‐ and heat pain were assessed as were pain intensities at suprathreshold heat pain stimulation. Comparing sides patients with pain reported allodynia to cold (p = 0.03) and pressure (p = 0.016) in conjunction with an increase in the perception threshold to non‐painful warmth (p = 0.024) on the injured side. Pain‐free patients reported hypoesthesia to light touch (p = 0.002), cold (p = 0.039) and warmth (p = 0.001) on the injured side. There were no side differences in stimulus–response functions using painful heat stimuli in any of the groups. In addition, no significant difference could be demonstrated in any sensory modality comparing side‐to‐side differences between the two groups. In conclusion, increased pain sensitivity to cold and pressure was found on the injured side in pain patients, pointing to hyperexcitability in the pain system, a finding not verified by a more challenging analysis of side‐to‐side differences between patients with and without pain.     

IL‐1beta in the trigeminal subnucleus caudalis contributes to extra‐territorial allodynia/hyperalgesia following a trigeminal nerve injury

It has been reported that the whisker pad (WP) area, which is innervated by the second branch of the trigeminal nerve, shows allodynia/hyperalgesia following transection of the mental nerve (MN: the third branch of the trigeminal nerve). However, the mechanisms of this extra‐territorial pain induction still remain unclear. Glia and cytokines are known to facilitate perception of noxious input, raising a possibility that these non‐neuronal elements are involved in the induction and spread of allodynia/hyperalgesia at non‐injured skin territory. One day after MN transection, tactile allodynia/hyperalgesia developed on the ipsilateral WP area, which is in the non‐injured skin territory. The tactile allodynia/hyperalgesia lasted for more than 56 days. In response to MN transection, astrocytes and microglia appeared to be in an activated state, and interleukin (IL)‐1beta was up‐regulated in astrocytes in the trigeminal subnucleus caudalis (Vc). Allodynia/hyperalgesia at WP area induced by MN transection was attenuated dose‐dependently by IL‐1 receptor antagonist IL‐1ra (i.t., 0.05, 0.5, and 5pg/rat). Fos‐like immunoreactive (Fos‐Li) neurons were observed in the Vc after non‐noxious mechanical stimulation of the WP area in the rats with MN transection. Administration of IL‐1ra also attenuated the number of Fos‐Li neurons dose‐dependently. Administration of a noncompetitive antagonist of NMDA receptors MK‐801 (i.t., 5μg/rat) reversed allodynia/hyperalgesia. IL‐1 receptor type I (IL‐1RI) was localized in Fos‐ and phospho NR1‐immunoreactive neurons. These results suggest that IL‐1beta in the Vc plays an important role in the development of extra‐territorial tactile allodynia/hyperalgesia after MN transection.     

Effects of local lidocaine treatment before and after median nerve injury on mechanical hypersensitivity and microglia activation in rat cuneate nucleus

This study examined the relationship between microglia activation in the cuneate nucleus (CN) and behavioral hypersensitivity after chronic constriction injury (CCI) of the median nerve. We also investigated effects of local lidocaine pre‐ and post‐treatment on microglia activation and development of hypersensitivity in this model. By immunohistochemistry and immunoblotting, little immunoreactivity of OX‐42, a microglia activation marker, was detected in the CN of normal rats. As early as 1 day after CCI, there was a significant increase in OX‐42 immunoreactivity in the lesion side of CN, which reached a maximum at 14 days. Microinjection of minocycline, a microglia activation inhibitor, into the CN 1 day after CCI attenuated injury‐induced behavioral hypersensitivity in a dose‐dependent manner. Furthermore, the animals received 1%, 2% or 5% lidocaine 15 min prior to median nerve CCI (pre‐treatment), 5 h (early post‐treatment) or 1 day (late post‐treatment) after median nerve CCI. Pre‐treatment and early post‐treatment with 2% and 5% lidocaine, but not 1% lidocaine, attenuated OX‐42 immunoreactivity and behavioral hypersensitivity following median nerve injury. Late post‐treatment with 1%, 2%, or 5% lidocaine failed to decrease OX‐42 immunoreactivity and mechanical hypersensitivity in CCI rats. In conclusion, median nerve injury‐induced microglia activation in the CN modulated development of behavioral hypersensitivity. High‐concentration lidocaine was effective in decreasing microglia activation in the CN and in attenuating neuropathic pain sensations at the early stage following nerve injury, when microglia had not yet been activated.     

Clonidine maintains intrathecal self-administration in rats following spinal nerve ligation

Clonidine is approved for spinal administration against neuropathic pain, and reverses both spontaneous and elicited pain in humans following spinal administration. Rodent studies that seek to model pharmacology in pain states have historically relied on reflexive withdrawal from noxious stimuli as the primary endpoint. Drug self-administration studies have face validity in the drug abuse field for modeling drug abuse in humans, however, this methodology has not been applied to address issues related to drug seeking behaviors that may be relevant for other human populations, such as patients with neuropathic pain. Rats without spinal nerve ligation (SNL) failed to acquire intrathecal clonidine self-administration over 10 days of access. Rats were found to self-administer intrathecal infusions of clonidine following SNL in a stable and dose-responsive manner, however, and clonidine was self-administered throughout the day with 66% of total drug intake occurring during the dark cycle. Substitution of clonidine with saline or with clonidine and the alpha2-adrenoceptor antagonist idazoxan resulted in extinction of responding in SNL animals. Food reinforcement was initially decreased in SNL rats self-administering clonidine compared to normal animals, however, tolerance developed to this effect of clonidine in SNL rats after 5 days. These data demonstrate that drug self-administration can be applied to questions other than drug abuse, and provides an additional measure for development of novel therapeutic strategies for chronic pain treatment.     

P-selectin is required for neutrophils and macrophage infiltration into injured site and contributes to generation of behavioral hypersensitivity following peripheral nerve injury in mice

Growing evidence suggests that leukocyte extravasation is initiated by the interaction of selectins with their ligands; as well as an essential role for P-selectin in the initial recruitment of inflammatory cells to sites of inflammation. In this study, P-selectin-deficient (P-sel−/−) mice were used to test the hypothesis that lack of P-selectin would attenuate the recruitment of inflammatory cells to the site of inflammation, thereby modulating pain in a murine chronic neuropathic pain model. Nociceptive sensitization and the microenvironment of the peripheral injury site were studied in wild-type (P-sel+/+) and P-selectin-deficient (P-sel−/−) mice after partial sciatic nerve ligation (PSNL). Variables measured included myeloperoxidase (MPO) activity, several inflammatory cell infiltration profiles, cytokines, and endogenous opioid peptide expression in damaged nerves. Results indicate that behavioral hypersensitivity, MPO activity, and infiltration of neutrophils and macrophages were attenuated in P-sel−/− mice after PSNL. Proinflammatory cytokines, tumor necrosis factor α, and interleukin (IL)-6, were reduced in damaged nerves following PSNL; however, several antiinflammatory cytokines – IL-1Ra, IL-4, and IL-10 – were significantly increased in P-sel−/− mice. In addition, endogenous opioid peptides mRNA was significantly lower in P-sel−/− mice compared with P-sel +/+ mice. The current results demonstrated that the absence of P-selectin in mice leads to an altered microenvironment that attenuated behavioral hypersensitivity. The specific role of P-selectin could have been a result of decreased neutrophils, as well as the accumulation of macrophages at the site of injury, which may subsequently modulate the inflammatory cytokine expression and impact behavioral hypersensitivity within the injured nerve.     

Low-dose methotrexate reduces peripheral nerve injury-evoked spinal microglial activation and neuropathic pain behavior in rats

Peripheral nerve injuries that provoke neuropathic pain are associated with microglial activation in the spinal cord. We have investigated the characteristics of spinal microglial activation in three distinct models of peripheral neuropathic pain in the rat: spared nerve injury (SNI), chronic constriction injury, and spinal nerve ligation. In all models, dense clusters of cells immunoreactive for the microglial marker CD11b formed in the ipsilateral dorsal horn 7 days after injury. Microglial expression of ionised calcium binding adapter molecule 1 (Iba1) increased by up to 40% and phosphorylation of p38 mitogen-activated protein kinase, a marker of microglial activity, by 45%. Expression of the lysosomal ED1-antigen indicated phagocytic activity of the cells. Unlike the peripheral nerve lesions, rhizotomy produced only a weak microglial reaction within the spinal gray matter but a strong activation of microglia and phagocytes in the dorsal funiculus at lumbar and thoracic spinal cord levels. This suggests that although degeneration of central terminals is sufficient to elicit microglial activation, it does not account for the inflammatory response in the dorsal horn after peripheral nerve injury. Early intrathecal treatment with low-dose methotrexate, beginning at the time of injury, decreased microglial activation, reduced p38 phosphorylation, and attenuated pain-like behavior after SNI. In contrast, systemic or intrathecal delivery of the glucocorticoid dexamethasone did not inhibit the activation of microglia or reduce pain-like behavior. We confirm that microglial activation is crucial for the development of pain after nerve injury, and demonstrates that suppression of this cellular immune response is a promising approach for preventing neuropathic pain.     

An increase in spinal cord noradrenaline is a major contributor to the antihyperalgesic effect of antidepressants after peripheral nerve injury in the rat

Antidepressants are often used for the treatment of neuropathic pain. Clinical studies suggest that the efficacy of serotonin (5-HT) and noradrenaline (NA) reuptake inhibitors (SNRIs) for neuropathic pain is greater than that of selective 5-HT reuptake inhibitors (SSRIs). In the present study, we determined the efficacy and mechanisms involved in the antihyperalgesic effects of milnacipran, an SNRI, compared with paroxetine, an SSRI, and maprotiline, a selective NA reuptake inhibitor, using a rat model of neuropathic pain. Male Sprague-Dawley rats underwent spinal nerve ligation (SNL), and the withdrawal threshold to paw pressure was measured. Intraperitoneal injection of milnacipran (3-30mg/kg) produced a dose-dependent antihyperalgesic effect. The effect was reversed by intrathecal injection of the α(2)-adrenoceptor antagonist idazoxan (30μg), but not by various 5-HT receptor antagonists. Paroxetine produced an antihyperalgesic effect only at the highest dose tested (10mg/kg). This effect was reversed by intrathecal injection of both idazoxan and ondansetron (30μg), a 5-HT3 receptor antagonist. Maprotiline produced an antihyperalgesic effect (10 and 30mg/kg), and the effect was reversed by intrathecal idazoxan. In microdialysis studies, NA and 5-HT concentrations in the spinal dorsal horn were increased after injection of either milnacipran or paroxetine, and only NA was increased after maprotiline. Furthermore, the NA content in the spinal cord of SNL rats was greater than that in normal animals. These findings suggest that an increase in NA in the spinal cord plays an important role in the antihyperalgesic effects of not only NA reuptake inhibitors but also SSRIs.