Partial Sciatic Nerve Ligation Rats Research Articles

Evaluation of [18F]F-DPA PET for Detecting Microglial Activation in the Spinal Cord of a Rat Model of Neuropathic Pain

PurposeRecent studies have linked activated spinal glia to neuropathic pain. Here, using a positron emission tomography (PET) scanner with high spatial resolution and sensitivity, we evaluated the feasibility and sensitivity of N,N-diethyl-2-(2-(4-([18F]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a] pyrimidin-3-yl)acetamide ([18F]F-DPA) imaging for detecting spinal cord microglial activation after partial sciatic nerve ligation (PSNL) in rats.ProceduresNeuropathic pain was induced in rats (n = 20) by PSNL, and pain sensation tests were conducted before surgery and 3 and 7 days post-injury. On day 7, in vivo PET imaging and ex vivo autoradiography were performed using [18F]F-DPA or [11C]PK11195. Ex vivo biodistribution and PET imaging of the removed spinal cord were carried out with [18F]F-DPA. Sham-operated and PK11195-pretreated animals were also examined.ResultsMechanical allodynia was confirmed in the PSNL rats from day 3 through day 7. Ex vivo autoradiography showed a higher lesion-to-background uptake with [18F]F-DPA compared with [11C]PK11195. Ex vivo PET imaging of the removed spinal cord showed [18F]F-DPA accumulation in the inflammation site, which was immunohistochemically confirmed to coincide with microglia activation. Pretreatment with PK11195 eliminated the uptake. The SUV values of in vivo [18F]F-DPA and [11C]PK11195 PET were not significantly increased in the lesion compared with the reference region, and were fivefold higher than the values obtained from the ex vivo data. Ex vivo biodistribution revealed a twofold higher [18F]F-DPA uptake in the vertebral body compared to that seen in the bone from the skull.Conclusions[18F]F-DPA aided visualization of the spinal cord inflammation site in PSNL rats on ex vivo autoradiography and was superior to [11C]PK11195. In vivo [18F]F-DPA PET did not allow for visualization of tracer accumulation even using a high-spatial-resolution PET scanner. The main reason for this result was due to insufficient SUVs in the spinal cord region as compared with the background noise, in addition to a spillover from the vertebral body.

Analysis of the proportion and neuronal activity of excitatory and inhibitory neurons in the rat dorsal spinal cord after peripheral nerve injury

The dorsal spinal cord contains projection neurons that transmit somatosensory information to the brain and interneurons which then modulate neuronal activity of these projection neurons and/or other interneurons. Interneurons can be subdivided into two groups: excitatory and inhibitory neurons. While inhibitory interneurons are thought to play a crucial role in analgesia, it is unclear whether they are involved in neuropathic pain. In the present study, we aimed to assess the proportion and neuronal activity of excitatory/inhibitory neurons in the dorsal spinal cord using a neuropathic pain model in rats. Following partial sciatic nerve ligation (PSNL), rats showed significant mechanical hyperalgesia, and subsequent immunohistochemical studies were conducted in laminae I–III of the dorsal spinal cord. We found that the number of FosB-immunoreactive cells was significantly higher; there was no change in the percentage of Pax2 positive/negative neurons in NeuN positive neurons; Pax2 negative neurons, but not Pax2 positive neurons, were predominantly activated in PSNL rats; and the immunofluorescence intensity of the calcium channel α2δ1 subunit was significantly higher. These results indicate that while peripheral nerve injury might not affect the proportion of excitatory and inhibitory neurons, it predominantly activates excitatory neurons in laminae I–III of the rat dorsal spinal cord.

Nerve injury elevates functional Cav3.2 channels in superficial spinal dorsal horn.

Cav3 channels play an important role in modulating chronic pain. However, less is known about the functional changes of Cav3 channels in superficial spinal dorsal horn in neuropathic pain states. Here, we examined the effect of partial sciatic nerve ligation (PSNL) on either expression or electrophysiological properties of Cav3 channels in superficial spinal dorsal horn. Our in vivo studies showed that the blockers of Cav3 channels robustly alleviated PSNL-induced mechanical allodynia and thermal hyperalgesia, which lasted at least 14 days following PSNL. Meanwhile, PSNL triggered an increase in both mRNA and protein levels of Cav3.2 but not Cav3.1 or Cav3.3 in rats. However, in Cav3.2 knockout mice, PSNL predominantly attenuated mechanical allodynia but not thermal hyperalgesia. In addition, the results of whole-cell patch-clamp recordings showed that both the overall proportion of Cav3 current-expressing neurons and the Cav3 current density in individual neurons were elevated in spinal lamina II neurons from PSNL rats, which could not be recapitulated in Cav3.2 knockout mice. Altogether, our findings reveal that the elevated functional Cav3.2 channels in superficial spinal dorsal horn may contribute to the mechanical allodynia in PSNL-induced neuropathic pain model.

Involvement of GABAB receptor in the antihypersensitive effect in anterior cingulate cortex of partial sciatic nerve ligation model

The role of the GABAB receptor in the anterior cingulate cortex (ACC) of neuropathic pain is unclear. Injection of a GABAB receptor antagonist CGP35348 into the ACC induced mechanical hypersensitivity in normal rats. Activation of the GABAB receptor injected by a GABAB receptor agonist baclofen into the ACC attenuated mechanical hypersensitivity in partial sciatic nerve ligation (PSNL) rats. Co-microinjection of CGP35348 with a muscarinic M1 receptor agonist McN-A-343 into the ACC significantly inhibited McN-A-343-induced antihypersensitivity in PSNL rats. These results suggest that the GABAB receptor in the ACC contributes to mechanical hypersensitivity and is involved in muscarinic M1 receptor-mediated antihypersensitivity.

Binding Characteristics and Analgesic Effects of Mirogabalin, a Novel Ligand for the α2δ Subunit of Voltage-Gated Calcium Channels.

Mirogabalin ([(1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl]acetic acid), a novel ligand for the α2δ subunit of voltage-gated calcium channels, is being developed to treat pain associated with diabetic peripheral neuropathy and postherpetic neuralgia. In the present study, we investigated the in vitro binding characteristics and in vivo analgesic effects of mirogabalin compared with those of pregabalin, a standard α2δ ligand. Mirogabalin showed potent and selective binding affinities for the α2δ subunits, while having no effects on 186 off-target proteins. Similar to pregabalin, mirogabalin did not show clear subtype selectivity (α2δ-1 vs. α2δ-2) or species differences (human vs. rat). However, in contrast to pregabalin, mirogabalin showed greater binding affinities for human α2δ-1, human α2δ-2, rat α2δ-1, and rat α2δ-2 subunits; further, it had a slower dissociation rate for the α2δ-1 subunit than the α2δ-2 subunit. Additionally, in experimental neuropathic pain models, partial sciatic nerve ligation rats and streptozotocin-induced diabetic rats, mirogabalin showed more potent and longer lasting analgesic effects. In safety pharmacological evaluations, mirogabalin and pregabalin inhibited rota-rod performance and locomotor activity in rats; however, the safety indices of mirogabalin were superior to those of pregabalin. In conclusion, mirogabalin shows potent and selective binding affinities for the human and rat α2δ subunits, and slower dissociation rates for the α2δ-1 subunit than the α2δ-2 subunit. It shows potent and long-lasting analgesic effects in rat models of neuropathic pain, and wider safety margins for side effects of the central nervous system. These properties of mirogabalin can be associated with its unique binding characteristics.

The effects of co-administration of pregabalin and vitamin E on neuropathic pain induced by partial sciatic nerve ligation in male rats.

This study was performed to evaluate the effect of pregabalin co-administration with vitamin E in Partial Sciatic Nerve Ligation (PSNL)-induced neuropathic pain in rats. Male Wistar rats were randomly allocated as control, sham, and PSNL groups (n=8). PSNL was induced by tight ligation of the sciatic nerve with a copper wire. On day 14th, the PSNL and sham operated rats received either pregabalin (1, 3, and 30mg/kg), vitamin E (100 and 200mg/kg), or their combination intraperitoneally. An antinociceptive effect was evaluated as latency times and Maximum possible Effect Percent (%MPE) using tail-flick test. Locomotor activity was evaluated by open-field test before PSNL surgery and then twice at the 14th days (before and after drug injection). Ligated nerves were removed on the 28th days after surgery for histological examinations. The time course of latency times and %MPE showed significant decrease in PSNL but not in sham and control groups. Pregabalin (3 and 30mg/kg) and vitamin E (100 and 200mg/kg) caused significant increases in latency time in PSNL (but not sham) group compared to control group. Vitamin E 200mg/kg increased significantly %MPE in PSNL group compared to sham group. In addition, the %MPE following combination treatment of pregabalin (30mg/kg) and vitamin E (100mg/kg) was significantly higher than both vitamin E and control group. Also combination of pregabalin with 100mg/kg of vitamin E reversed Wallerian degeneration of sciatic nerve and the inflammatory responses to almost similar to sham group. Pregabalin and vitamin E did not affect locomotor activity. Our results showed antinociceptive effects of both vitamin E and pregabalin alone or in combination in PSNL rats and also neuroprotective properties without affecting locomotor activity.

Inhibition of the regulator of G protein signalling RGS4 in the spinal cord decreases neuropathic hyperalgesia and restores cannabinoid CB1 receptor signalling.

Regulators of G protein signalling (RGS) are major determinants of metabotropic receptor activity, reducing the lifespan of the GTP-bound state of G proteins. Because the reduced potency of analgesic agents in neuropathic pain may reflect alterations in RGS, we assessed the effects of CCG 63802, a specific RGS4 inhibitor, on pain hypersensitivity and signalling through cannabinoid receptors, in a model of neuropathic pain. The partial sciatic nerve ligation (PSNL) model in male Sprague Dawley rats was used to measure paw withdrawal thresholds to mechanical (von Frey hairs) or thermal (Hargreaves method) stimuli, during and after intrathecal injection of CCG 63802. HEK293 cells expressing CB1 receptors and conditional expression of RGS4 were used to correlate cAMP production and ERK phosphorylation with receptor activation and RGS4 action. Treatment of PSNL rats with CCG 63802, twice daily for 7 days after nerve injury, attenuated thermal hyperalgesia during treatment. Spinal levels of anandamide were higher in PSNL animals, irrespective of the treatment. Although expression of CB1 receptors was unaffected, HU210-induced CB1 receptor signalling was inhibited in PSNL rats and restored after intrathecal CCG 63802. In transfected HEK cells expressing CB1 receptors and RGS4, inhibition of cAMP production, a downstream effect of CB1 receptor signalling, was blunted after RGS4 overexpression. RGS4 expression also attenuated the CB1 receptor-controlled activation of ERK1/2. Inhibition of spinal RGS4 restored endogenous analgesic signalling pathways and mitigated neuropathic pain. Signalling through CB1 receptors may be involved in this beneficial effect.

The analgesic effect of tramadol in animal models of neuropathic pain and fibromyalgia

(±)-Tramadol hydrochloride (tramadol) is a widely used analgesic for the treatment of cancer pain and chronic pain. Although many animal studies have shown antinociceptive effects of tramadol in both acute and chronic pain, little is known about the effect of tramadol in putative animal models of fibromyalgia. In this study, we compared the antiallodynic effects of oral administration of tramadol in two kinds of rat chronic pain models, neuropathic pain induced by partial sciatic nerve ligation (PSL) and reserpine-induced myalgia (RIM). In PSL rats, the threshold for responses induced by tactile stimulation with von Frey filaments was significantly decreased seven days after the operation, suggesting that the operation induced tactile allodynia. Orally administered tramadol showed a potent and dose-dependent antiallodynic effect on PSL-induced allodynia. In RIM rats, the threshold was significantly decreased five days after reserpine treatment. Orally administered tramadol also attenuated reserpine-induced tactile allodynia. To explore the mechanism of the antiallodynic effect of tramadol in RIM rats, we investigated the effect of the opioid antagonist naloxone on the tramadol-induced analgesic effect in these rats. The effect of tramadol was partially antagonized by naloxone, suggesting that the opioid receptor is involved at least in part in the antiallodynic effect of tramadol in RIM rats. These data indicate that orally administered tramadol produced improvement in both PSL rats and RIM rats at similar doses and provide evidence that the opioid system is partly involved in the analgesic effect of tramadol in RIM rats.

11C]PK11195 PET imaging of spinal glial activation after nerve injury in rats

The role of glial activation has been implicated in the development and persistence of neuropathic pain after nerve injury by recent studies. PK11195 binding to the translocator protein 18kDa (TSPO) has been shown to be enhanced in activated microglia. This study was designed to assess PK11195 imaging in spinal microglia during activation after nerve injury. The development of neuropathic pain was induced by partial sciatic nerve ligation (PSL). PSL rats on days 7 and 14 after nerve injury were subjected to imaging with a small-animal positron emission tomography/computed tomography (PET/CT) scanner using [(11)C]PK11195 to detect spinal microglial activation by means of noninvasive in vivo imaging. Spinal [(3)H]PK11195 autoradiography was performed to confirm the results of [(11)C]PK11195 PET in PSL rats. Quantitative RT-PCR of CD11b and GFAP mRNA, and the immunohistochemistry of Iba1 and GFAP were investigated to detect activated microglia and astrocytes. Mechanical allodynia was observed in the ipsilateral paw of PSL rats from day 3 after nerve injury and stably persisted from days 7 to 14. PET/CT fusion images clearly showed large amounts of accumulation of [(11)C]PK11195 in the lumbar spinal cord on days 7 and 14 after nerve injury. [(11)C]PK11195 enhanced images were restricted to the L3-L6 area of the spinal cord. The standardized uptake value (SUV) of [(11)C]PK11195 was significantly increased in the lumbar spinal cord compared to that of the thoracic region. Increased specific binding of [(11)C]PK11195 to TSPO in the spinal cord of PSL rats was confirmed by competition studies using unlabeled (R, S)-PK11195. Increased [(3)H]PK11195 binding was also observed in the ipsilateral dorsal horn of the L3-L6 spinal cord on days 7 and 14 after nerve injury. CD11b mRNA and Iba1 immunoreactive cells increased significantly on days 7 and 14 after nerve injury by PSL. However, changes in GFAP mRNA and immunoreactivity were slight in the ipsilateral side of PSL rats. In the present study, we showed that glial activation could be quantitatively imaged in the spinal cord of neuropathic pain rats using [(11)C]PK11195 PET, suggesting that high resolution PET using TSPO-specific radioligands might be useful for imaging to assess the role of glial activation, including neuroinflammatory processes, in neuropathic pain patients.

Gamma Knife Irradiation of Injured Sciatic Nerve Induces Histological and Behavioral Improvement in the Rat Neuropathic Pain Model

We examined the effects of gamma knife (GK) irradiation on injured nerves using a rat partial sciatic nerve ligation (PSL) model. GK irradiation was performed at one week after ligation and nerve preparations were made three weeks after ligation. GK irradiation is known to induce immune responses such as glial cell activation in the central nervous system. Thus, we determined the effects of GK irradiation on macrophages using immunoblot and histochemical analyses. Expression of Iba-1 protein, a macrophage marker, was further increased in GK-treated injured nerves as compared with non-irradiated injured nerves. Immunohistochemical study of Iba-1 in GK-irradiated injured sciatic nerves demonstrated Iba-1 positive macrophage accumulation to be enhanced in areas distal to the ligation point. In the same area, myelin debris was also more efficiently removed by GK-irradiation. Myelin debris clearance by macrophages is thought to contribute to a permissive environment for axon growth. In the immunoblot study, GK irradiation significantly increased expressions of βIII-tubulin protein and myelin protein zero, which are markers of axon regeneration and re-myelination, respectively. Toluidine blue staining revealed the re-myelinated fiber diameter to be larger at proximal sites and that the re-myelinated fiber number was increased at distal sites in GK-irradiated injured nerves as compared with non-irradiated injured nerves. These results suggest that GK irradiation of injured nerves facilitates regeneration and re-myelination. In a behavior study, early alleviation of allodynia was observed with GK irradiation in PSL rats. When GK-induced alleviation of allodynia was initially detected, the expression of glial cell line-derived neurotrophic factor (GDNF), a potent analgesic factor, was significantly increased by GK irradiation. These results suggested that GK irradiation alleviates allodynia via increased GDNF. This study provides novel evidence that GK irradiation of injured peripheral nerves may have beneficial effects.

Prostaglandin E2 contributes to the synthesis of brain-derived neurotrophic factor in primary sensory neuron in ganglion explant cultures and in a neuropathic pain model

Brain-derived neurotrophic factor (BDNF) exists in small to medium size neurons in adult rat dorsal root ganglion (DRG) and serves as a modulator at the first synapse of the pain transmission pathway in the spinal dorsal horn. Peripheral nerve injury increases BDNF expression in DRG neurons, an event involved in the genesis of neuropathic pain. In the present study, we tested the hypothesis that prostaglandin E2 (PGE2) over-produced in injured nerves contributes to the up-regulation of BDNF in DRG neurons. Two weeks after partial sciatic nerve ligation (PSNL), BDNF levels in the ipsilateral L4–L6 DRG of injured rats were significantly increased compared to the contralateral side. Perineural injection of a selective cyclooxygenase (COX2) inhibitor or a PGE2 EP4 receptor antagonist not only dose-dependently relieved PSNL elicited mechanical hypersensitivity, but also suppressed the increased BDNF levels in DRG neurons. PSNL shifted BDNF expression in the ipsilateral DRG from small to medium and larger size injured neurons. BDNF is mainly co-expressed with the EP1 and EP4 while moderately with the EP2 and EP3 receptor subtypes in naïve and PSNL rats. PSNL also shifted the expression of EP1–4 receptors to a larger size population of DRG neurons. In DRG explant cultures, a stabilized PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) or the agonists of EP1 and EP4 receptors significantly increased BDNF levels and the phosphorylated protein kinase A (PKA), extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) and cAMP response element binding protein (CREB). The EP1 and EP4 antagonists, a sequester of nerve growth factor (NGF), the inhibitors of PKA and MEK as well as CREB small interfering RNA suppressed dmPGE2-induced BDNF. Taken together, EP1 and EP4 receptor subtypes, PKA, ERK/MAPK and CREB signaling pathways as well as NGF are involved in PGE2-induced BDNF synthesis in DRG neurons. Injured nerve derived-PGE2 contributes to BDNF up-regulation in DRG neurons following nerve injury. Facilitating the synthesis of BDNF in primary sensory neurons is a novel mechanism underlying the role of PGE2 in the genesis of neuropathic pain.

Nicotinic acetylcholine receptors expressed in the ventralposterolateral thalamic nucleus play an important role in anti-allodynic effects

Much interest is currently being focused on the anti-nociceptive effects mediated by nicotinic acetylcholine (nACh) receptors, including their location and mechanism of action. The purpose of this study was to elucidate these issues using 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5IA), a nACh receptor agonist, and [(125)I]5IA. We partially ligated the sciatic nerve of Sprague-Dawley rat to induce neuropathic pain [Seltzer's partial sciatic nerve ligation (PSL) model]. We then examined the changes in nACh receptor density in the CNS using [(125)I]5IA autoradiography and the involvement of nACh receptors in anti-nociceptive effects in the region where changes occurred. Autoradiographic studies showed that the accumulation of [(125)I]5IA and the number of nACh receptors in the thalamus of PSL rats were increased about twofold compared with those in the sham-operated rats. No change was observed in other brain regions. Rats injected in the ventral posterolateral thalamic nucleus (VPL) with 5IA demonstrated a significant and dose-dependent anti-allodynic effect and this effect was completely antagonized by mecamylamine, injected with 5IA, into the VPL. The blockade of nACh receptors in the VPL by mecamylamine decreased by 70% the anti-allodynic effect of 5IA, given i.c.v. Moreover, mecamylamine given intra-VPL by itself, induced significant hyperalgesia. Our findings suggest that the nACh receptors expressed in the VPL play an important role in the anti-allodynic effects produced by exogenous and endogenous agonists.

Injured nerve-derived COX2/PGE2 contributes to the maintenance of neuropathic pain in aged rats

Neuropathic pain (NeP) is a debilitating disease afflicting mostly the aged population. Inflammatory responses in injured nerves play a pivotal role in the pathogenesis of NeP. Injured nerve derived cyclooxygenase 2/prostaglandin E2 (COX2/PGE2) contributes to the genesis of NeP at the early stage in young rats. Here we show that COX2/PGE2 is involved in the maintenance of NeP at a chronic stage in aged rats. Eighteen months after partial sciatic nerve ligation (PSNL), NeP remained prominent in aged rats. COX2 expressing macrophages and PGE2 levels were increased in injured nerves. PGE2 receptors (EP1 and EP4) and pain-related ion channel transient receptor potential vanilloid-1 (TRPV1) were increased in the ipsilateral dorsal root ganglion (DRG) neurons of aged PSNL rats. Perineural injection of a selective COX2 inhibitor NS-398 relieved NeP, reversed PSNL increased expression of EP1, EP4 and TRPV1 and suppressed the levels of pain-related peptide substance P and calcitonin gene-related peptide in DRG neurons. These data suggest that injured nerve-derived PGE2 contributes to the maintenance of NeP at the chronic stage in aged rats. Chronically facilitating the synthesis of pain-related molecules in nociceptive DRG neurons is a novel mechanism underpinning the contribution of PGE2.

Levels of neuropeptides nocistatin, nociceptin/orphanin FQ and their precursor protein in a rat neuropathic pain model

Neuropeptides nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are related to pain modulation. The amounts of these peptides and their precursor protein, prepronociceptin (ppN/OFQ) in the brain, spinal cord and serum samples of rats with partial sciatic nerve ligation (PSNL) were compared with those in naïve rats using radioimmunoassay (RIA). There was a significant rise in the levels of ppN/OFQ, N/OFQ and NST in the brains of PSNL rats. Their spinal cords showed significantly increased ppN/OFQ and NST levels but no change in N/OFQ levels. The PSNL rats also had increased serum NST (statistically significant) and N/OFQ (statistically insignificant) with decreased ppN/OFQ suggesting important roles of these peptides in neuropathic pain mechanism.

Intrathecal injection of cAMP response element binding protein (CREB) antisense oligonucleotide attenuates tactile allodynia caused by partial sciatic nerve ligation

The transcription factor cAMP responsive element binding protein (CREB) is important in regulating immediate-early genes and some late-effector genes involved in neuroplasticity in response to peripheral injury and stressful insults. Partial nerve injury elicited neuropathic pain is accompanied by increased phosphorylation of CREB in the ipsilateral spinal cord dorsal horn (Ma and Quirion, Pain 93 (2001) 295; Miletic et al., Pain 99 (2002) 493). The aim of this study is to determine whether increased phosphorylation of CREB in the dorsal horn contributes to the pathogenesis of neuropathic pain. Three weeks following partial sciatic nerve ligation (PSNL), daily intrathecal injection of antisense CREB oligodeoxynucleotide (ODN, 20 μg/day) for 5 days significantly attenuated tactile allodynia. The attenuation lasted for more than 4 days. Total CREB and phosphorylated CREB in both ipsilateral and contralateral dorsal horn neurons were dramatically reduced in antisense ODN injected PSNL rats 1 week after injection. The extent of reduction of total CREB and phosphorylated CREB containing cells in the dorsal horn ipsilateral to injury was greater than in the contralateral dorsal horn. These data suggest that phosphorylation of CREB is an important contributing event in the central plasticity of nerve injury and in the pathogenesis of neuropathic pain.

Intraplantar injection of a cyclooxygenase inhibitor ketorolac reduces immunoreactivities of substance p, calcitonin gene-related peptide, and dynorphin in the dorsal horn of rats with nerve injury or inflammation

We previously reported that partial sciatic nerve ligation (PSNL) dramatically up-regulates cyclooxygenase 2 (COX2) in injured sciatic nerve, and local injection of the COX inhibitor, ketorolac, reverses tactile allodynia and suppresses increased phosphorylation of the transcription factor cAMP responsive element binding protein [Eur J Neurosci 15 (2002) 1037]. These findings suggest that peripheral prostaglandins (PGs) are over-produced and contribute to the central plasticity and the maintenance of neuropathic pain after nerve injury. PGs, particularly PGE2, are well known to facilitate the release of the pro-nociceptive neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP) from primary sensory afferents. Thus, suppressing peripheral PG over-production may inhibit the release of these two neuropeptides from primary afferents and thereby increase the content of these neuropeptides remaining in afferent terminals in the dorsal horn. In this study we tested this hypothesis by examining the immunoreactivities of SP and CGRP in the dorsal horn of PSNL rats intraplantarly injected with saline and ketorolac. Four weeks after PSNL, SP- and CGRP-immunoreactivities (IR) in the ipsilateral dorsal horn were not significantly different from the contralateral side. Five days following intraplantar injection of ketorolac, CGRP- and SP-IR in the ipsilateral and contralateral dorsal horn were dramatically reduced compared with saline-injected PSNL rats. Local ketorolac also suppressed PSNL-induced increase in dynorphin-IR in dorsal horn neurons. Since abundant production of PGs during inflammation is well documented, we further examined the effect of intraplantar ketorolac on neuropeptide expression in the dorsal horn following carrageenan inflammation. We observed that co-administration of ketorolac with carrageenan in the hindpaw also reduced SP- and dynorphin-IR in the ipsilateral and contralateral dorsal horn. These findings are in contrast to our hypothesis, suggesting that peripherally over-produced PGs following nerve injury and inflammation possibly contribute to the production of SP and CGRP in primary sensory neurons, to the up-regulation of dynorphin in the dorsal horn neurons, and finally to the mechanisms of neuropathic and inflammation pain.

Four PGE2 EP receptors are up-regulated in injured nerve following partial sciatic nerve ligation

We previously reported that cyclooxygenase 2 (COX2) is up-regulated in macrophages in injured nerve of rats with partial sciatic nerve ligation (PSNL) and that local injection of the COX inhibitor ketorolac reversed tactile allodynia (Eur. J. Neurosci. 15: 1037–1047, 2002). These findings suggest that prostaglandins (PGs) are overproduced in injured nerve and are involved in the pathogenesis of neuropathic pain. In this study, we examined whether overproduced PGs alter the expression of PGE2 receptors, EP1–EP4, in injured nerve of PSNL rats. We found that cell profiles immunoreactive (IR) for four EP receptors, EP1, EP2, EP3, and EP4, are dramatically increased in injured nerve 2 and 4 weeks after PSNL. EP4-IR cells were the most abundant among these receptor-expressing cells. Immunoreactivities of all four EP receptors were localized to the cell nucleus. These EP-IR cells were never found in uninjured nerve. More than 80% EP1- and about 30% EP4-IR cells were identified as infiltrating macrophages since they coexpressed ED1. Only 3% EP2- and 6% EP3-IR cells coexpressed ED1. These findings suggest that majority of EP2-, EP3-, and EP4-IR cells are other types of inflammatory cells than macrophages. About 48% of macrophages expressed EP1 and 45% expressed EP4. Only 3 and 6% of macrophages, respectively, expressed EP2 and EP3. Perineural injection of ketorolac reversed tactile allodynia and suppressed the up-regulation of EP1 and EP4, but not the recruitment of ED1-IR marcrophages, in injured nerve. Our data suggest that following PSNL, PGE2 is one of the possible PGs overproduced in injured nerve and PG overproduction is involved in the up-regulation of EP receptors in injured nerve.

Intrathecal lidocaine reverses tactile allodynia caused by nerve injuries and potentiates the antiallodynic effect of the COX inhibitor ketorolac.

Systemic lidocaine and other local anesthetics reduce hypersensitivity states induced by both acute inflammation and peripheral nerve injury in animals and produce analgesia in some patients with chronic pain. The mechanisms underlying the antiallodynic effect of systemic lidocaine are unclear, although most focus is on peripheral mechanisms. Central mechanisms, particularly at the spinal dorsal horn level, are less known. In this study, the authors aimed to determine whether intrathecal lidocaine has an antiallodynic effect on established mechanical allodynia in two well-characterized neuropathic pain rat models: partial sciatic nerve ligation (PSNL) and spinal nerve ligation (SNL). Lidocaine (100-300 micro g) was intrathecally injected in PSNL and SNL rats. The withdrawal threshold of both hind paws in response to mechanical stimulation was measured using a series of calibrated von Frey filaments. This single injection reduced ongoing tactile allodynia in PSNL and SNL rats. The antiallodynic effect of intrathecal lidocaine lasted longer in PSNL (> 3 days) than in SNL rats (< 3 days). Intraperitoneal lidocaine (300 micro g) had no effect on tactile allodynia in PSNL rats. In SNL rats, prior intrathecal lidocaine (200 and 300 micro g) potentiated the antiallodynic effect of intrathecal ketorolac, a nonselective cyclooxygenase inhibitor. Intrathecal ketorolac alone had no antiallodynic effect on SNL rats. However, prior intrathecal lidocaine (100 micro g) failed to potentiate the antiallodynic effect of intrathecal ketorolac. The authors' data suggest that intrathecal lidocaine possibly suppressed the hyperexcitability of the dorsal horn neurons and likely interacted with eicosanoid systems in the spinal dorsal horn.

Substance P release in the spinal dorsal horn following peripheral nerve injury

Spinal microdialysis was used to study the potassium induced in vivo release of substance P (SP) in the rat dorsal horn at different time points (3, 14, and 60 days) following partial sciatic nerve ligation (PNL) or sciatic nerve axotomy. The withdrawal threshold to innocuous mechanical stimuli was investigated with von Frey filaments in the PNL rats prior to microdialysis. The release of SP was significantly elevated at 60 days following PNL but not following complete nerve injury. However, the PNL rats in all time groups displayed mechanical hypersensitivity, which implies that this late change in SP release seems to be unrelated to the development of neuropathy. The present results indicate that there is an increase of the releasable pool of SP in the dorsal horn at late post-operative times after PNL. This change in SP release may reflect an altered sensory processing or may instead relate to adaptive responses to promote recovery.

Morphological and pharmacological evidence for the role of peripheral prostaglandins in the pathogenesis of neuropathic pain.

Inflammatory mediators produced in the injured nerve have been proposed as contributing factors in the development of neuropathic pain. Prostaglandins (PGs) are probably included in these important inflammatory mediators. In the present study, 2 and 4 weeks following partial sciatic nerve ligation (PSNL), we observed a dramatic increase in the prostaglandin synthesizing enzyme cyclooxygenase (COX)2-immunoreactive (IR) cell profiles in the injury site and adjacent region. Some of these COX2-IR cells were identified as macrophages because they coexpressed ED1. None of these COX2-IR cell profiles coexpressed the Schwann cell marker S100. In the contralateral sciatic nerve and sciatic nerve from normal rats, we failed to observe any of these COX-IR cell profiles. We also observed COX1-IR cell profiles (presumably Langerhans cells) in the epidermis of the footpad of both normal and PSNL rats. Interestingly, a greater number of COX1-IR cell profiles were observed in the epidermis of the ipsilateral footpad of PSNL rats. Local injection of ketorolac, a nonselective COX inhibitor, into the ipsilateral plantar side or into the injury site of the sciatic nerve, effectively reversed the tactile allodynia induced by PSNL for > 5 days. Intraperitoneal or intramuscular injection of ketorolac had a similar but shorter antiallodynic effect. Intraplantar or peri-neural injection of ketorolac dramatically suppressed the PSNL-induced increase in the phosphorylation of a transcription factor cAMP response element binding protein (CREB) in the ipsilateral dorsal horn of L4 and L5 spinal cord of PSNL rats. Intraplantar or peri-neural injection of ketorolac at the time of lesion did not prevent mechanical hypersensitivity but reduced it with a slow onset 3 weeks after lesion. Our data suggest that PSNL induces over-production of PGs in peripheral tissues and that PGs probably sensitize nociceptors and are involved in central plasticity and sensitization at the spinal cord level, thus contributing to the maintenance of tactile allodynia.