Persistent Nociception Facilitates the Extinction of Morphine-Induced Conditioned Place Preference.

BACKGROUND: As opioid abuse and addiction have developed into a major national health crisis, prescription of opioids for pain management has become more controversial. However, opioids do help some patients by providing pain relief and improving the quality of life. To better understand the addictive properties of opioids under chronic pain conditions, we used a conditioned place preference (CPP) paradigm to examine the rewarding properties of morphine in rats with persistent nociception. METHODS: Spared nerve injury (SNI) model was used to induce persistent nociception in rats. Nociceptive behavior was assessed by von Frey test. CPP test was used to examine the rewarding properties of morphine. RESULTS: Our findings are as follows: (1) SNI rats did not show a difference compared with sham rats in magnitude of morphine-induced CPP 1 day after last morphine injection (2-way analysis of variance; for SNI versus sham, F[1,42] = 0.014, P = .91; and 95% confidence intervals for difference of means, -5.9 [-58 to 46], 0.76 [-51 to 53], and 0.90 [-51 to 53] for 2.5, 5, and 10 mg/kg, respectively); (2) increasing morphine dosage (2.5, 5, and 10 mg/kg) did not further increase the magnitude of CPP in both sham and SNI rats (for dosage: F[2,42] = 0.94, P = .40); and (3) morphine-induced CPP persisted in sham rats but extinguished in SNI rats when tested at 8 days after last morphine injection (for sham versus SNI: Bonferroni correction, P < .006 for both 5 and 10 mg/kg doses; and 95% confidence intervals for difference of means, 80.3 [19.7-141] and 87.0 [26.3-148] for 5 and 10 mg/kg, respectively). CONCLUSIONS: Our data provide new evidence supporting the notion that the brain's reward circuitry changes in the context of persistent pain. This observational study suggests that future investigation into the neurobiology of opioid reward requires consideration of the circumstances in which opioid analgesics are administered.

Abstinence to chronic methamphetamine switches connectivity between striatal, hippocampal and sensorimotor regions and increases cerebral blood volume response.

Methamphetamine (meth), and other psychostimulants such as cocaine, present a persistent problem for society with chronic users being highly prone to relapse. We show, in a chronic methamphetamine administration model, that discontinuation of drug for more than a week produces much larger changes in overall meth-induced brain connectivity and cerebral blood volume (CBV) response than changes that occur immediately following meth administration. Areas showing the largest changes were hippocampal, limbic striatum and sensorimotor cortical regions as well as brain stem areas including the pedunculopontine tegmentum (PPTg) and pontine nuclei - regions known to be important in mediating reinstatement of drug-taking after abstinence. These changes occur concomitantly with behavioral sensitization and appear to be mediated through increases in dopamine D1 and D3 and decreases in D2 receptor protein and mRNA expression. We further identify a novel region of dorsal caudate/putamen, with a low density of calbindin neurons, that has an opposite hemodynamic response to meth than the rest of the caudate/putamen and accumbens and shows very strong correlation with dorsal CA1 and CA3 hippocampus. This correlation switches following meth abstinence from CA1/CA3 to strong connections with ventral hippocampus (ventral subiculum) and nucleus accumbens. These data provide novel evidence for temporal alterations in brain connectivity where chronic meth can subvert hippocampal - striatal interactions from cognitive control regions to regions that mediate drug reinstatement. Our results also demonstrate that the signs and magnitudes of the induced CBV changes following challenge with meth or a D3-preferring agonist are a complementary read out of the relative changes that occur in D1, D2 and D3 receptors using protein or mRNA levels.

Different effects of dexmedetomidine and midazolam on the expression of NR2B and GABAA-α1 following peripheral nerve injury in rats.

Neuropathic pain is a complex, chronic pain condition and the treatment is a major clinical challenge. Recent studies have shown that two FDA approved drugs dexmedetomidine (DEX) and midazolam (MZL), may be useful in treating neuropathic pain, but the mechanism is not fully dementated. Here, we investigated the effects and mechanisms of DEX and MZL treatment in the peripheral nerve injury model. Intramuscular injection with DEX and MZL attenuated the development of mechanical allodynia and thermal hyperalgesia in rats with chronic constriction injury (CCI). Concurrently, the expression of NMDA receptor subunit 2B (NR2B), GABA (A) receptor subunit alpha1 (GABAA-α1), and Sonic Hedgehog (SHH) displayed different temporal patterns in the thalamus and the ipsilateral dorsal horn of the spinal cord after CCI. Such that (1) NR2B expression was decreased on day 1 and 14, whereas GABAA-α1 expression was increased on day 1 in the thalamus, and NR2B expression was decreased on day 1, whereas GABAA-α1 expression was increased on day 1 and day 30 in the ipsilateral spinal cord dorsal horn after DEX treatment. (2) NR2B expression was increased on day 1, then decreased on day 14 and returned to baseline on day30, whereas GABAA-α1 expression was no significant changes on day 1, 14, 30 in the thalamus, and NR2B expression was decreased on day 14 and 30, whereas GABAA-α1 expression was no changes on day 1 and 14 but increased on day 30 after MZL treatment. Furthermore, the mechanical allodynia was significantly attenuated after PUR administration. Meanwhile the expression of NR2B was significantly decreased, and the expression of GABAA-α1 was significantly increased, in the thalamus and in the ipsilateral spinal cord dorsal horn when detected on postoperative day 1, 7, and 14. Our findings indicate that DEX and MZL have different mechanisms in CCI rats, suggesting different strategies could be considered in managing neuropathic pain in different individuals. © 2018 IUBMB Life, 70(2):143-152, 2018.

Ultrasmall Superparamagnetic Iron Oxide Imaging Identifies Tissue and Nerve Inflammation in Pain Conditions.

Objective: Correlation between radiologic structural abnormalities and clinical symptoms in low back pain patients is poor. There is an unmet clinical need to image inflammation in pain conditions to aid diagnosis and guide treatment. Ferumoxytol, an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle, is clinically used to treat iron deficiency anemia and showed promise in imaging tissue inflammation in human. We explored whether ferumoxytol can be used to identify tissue and nerve inflammation in pain conditions in animals and humans. Methods: Complete Freud's adjuvant (CFA) or saline was injected into mice hind paws to establish an inflammatory pain model. Ferumoxytol (20 mg/kg) was injected intravenously. Magnetic resonance imaging (MRI) was performed prior to injection and 72 hours postinjection. The changes in the transverse relaxation time (T2) before and after ferumoxytol injection were compared between mice that received CFA vs saline injection. In the human study, we administered ferumoxytol (4 mg/kg) to a human subject with clinical symptoms of lumbar radiculopathy and compared the patient with a healthy subject. Results: Mice that received CFA exhibited tissue inflammation and pain behaviors. The changes in T2 before and after ferumoxytol injection were significantly higher in mice that received CFA vs saline (20.8 ± 3.6 vs 2.2 ± 2.5, P = 0.005). In the human study, ferumoxytol-enhanced MRI identified the nerve root corresponding to the patient's symptoms, but the nerve root was not impinged by structural abnormalities, suggesting the potential superiority of this approach over conventional structural imaging techniques. Conclusions: Ferumoxytol-enhanced MRI can identify tissue and nerve inflammation and may provide a promising diagnostic tool in assessing pain conditions in humans.

Nortriptyline Enhances Morphine-Conditioned Place Preference in Neuropathic Rats: Role of the Central Noradrenergic System.

BACKGROUND: Combination drug therapy is commonly used to treat chronic pain conditions such as neuropathic pain, and antidepressant is often used together with opioid analgesics. While rewarding is an intrinsic property of opioid analgesics, it is unknown whether the use of an antidepressant would influence opioid reward, which may contribute to opioid addiction. In this study, we examined whether nortriptyline (a tricyclic antidepressant and a first-line medication for neuropathic pain) would enhance the morphine rewarding property in both naive and chronic constriction sciatic nerve injury (CCI) rats. METHODS: The rewarding effect of these drugs was assessed using conditioned place preference (CPP). The real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay analysis were used to investigate the function of central noradrenergic system. RESULTS: In naive rats, coadministration of nortriptyline with morphine did not change the acquisition of morphine-induced CPP. However, nortriptyline enhanced the acquisition, delayed the extinction, and augmented the reinstatement of morphine-induced CPP in CCI rats. In CCI rats treated with both nortriptyline and morphine, the expression of α2A-adrenergic receptors, norepinephrine transporter, and tyrosine hydroxylase was markedly decreased in the locus coeruleus, whereas the norepinephrine concentration in the nucleus accumbens was remarkably increased. CONCLUSIONS: These results demonstrate that nortriptyline enhanced morphine reward when both drugs were used to treat neuropathic pain in rats and that this behavioral phenotype is likely to be mediated by upregulation of the central noradrenergic system. These findings may have implications in opioid therapy commonly used for chronic pain management.

A leptin-mediated central mechanism in analgesia-enhanced opioid reward in rats.

Opioid analgesics are commonly used in chronic pain management despite a potential risk of rewarding. However, it remains unclear whether opioid analgesia would enhance the opioid rewarding effect thereby contributing to opioid rewarding. Utilizing a rat paradigm of conditioned place preference (CPP) combined with ankle monoarthritis as a condition of persistent nociception, we showed that analgesia induced by either morphine or the nonsteroid anti-inflammatory drug ibuprofen increased CPP scores in arthritic rats, suggesting that analgesia itself had a rewarding effect. However, arthritic rats exhibited a significantly higher CPP score in response to morphine than ibuprofen. Thus, the rewarding effect of morphine was enhanced in the presence of persistent nociception, producing a phenomenon of analgesia-enhanced opioid reward. At the cellular level, administration of morphine activated a cascade of leptin expression, glial activation, and dopamine receptor upregulation in the nucleus accumbens (NAc), while administration of ibuprofen decreased glial activation with no effect on leptin expression in the NAc. Furthermore, the morphine rewarding effect was blocked in leptin deficient ob/ob mice or by neutralizing leptin or interleukin-1ß in the NAc without diminishing morphine analgesia. The data indicate that systemic opioid can activate a leptin-mediated central mechanism in the NAc that led to the enhanced opioid rewarding effect. These findings provide evidence for an interaction between opioid analgesia and opioid rewarding, which may have implications in clinical opioid dose escalation in chronic pain management.

Loss of MEF2C function by enhancer mutation leads to neuronal mitochondria dysfunction and motor deficits in mice.

Genetic changes and epigenetic modifications are associated with neuronal dysfunction in the pathogenesis of neurodegenerative disorders. However, the mechanism behind genetic mutations in the non-coding region of genes that affect epigenetic modifications remains unclear. Here, we identified an ALS-associated SNP located in the intronic region of MEF2C (rs304152), residing in a putative enhancer element, using convolutional neural network. The enhancer mutation of MEF2C reduces own gene expression and consequently impairs mitochondrial function in motor neurons. MEF2C localizes and binds to the mitochondria DNA, and directly modulates mitochondria-encoded gene expression. CRISPR/Cas-9-induced mutation of the MEF2C enhancer decreases expression of mitochondria-encoded genes. Moreover, MEF2C mutant cells show reduction of mitochondrial membrane potential, ATP level but elevation of oxidative stress. MEF2C deficiency in the upper and lower motor neurons of mice impairs mitochondria-encoded genes, and leads to mitochondrial metabolic disruption and progressive motor behavioral deficits. Together, MEF2C dysregulation by the enhancer mutation leads to mitochondrial dysfunction and oxidative stress, which are prevalent features in motor neuronal damage and ALS pathogenesis. This genetic and epigenetic crosstalk mechanism provides insights for advancing our understanding of motor neuron disease and developing effective treatments.

Spinal leptin contributes to the pathogenesis of neuropathic pain in rodents.

Pain after nerve injury, a phenomenon referred to as neuropathic pain, is a debilitating clinical condition, but the underlying mechanisms remain unclear. As leptin, an adipocytokine produced mainly by nonneuronal tissue, has been implicated in the regulation of neuronal functions, we examined the role of leptin in neuropathic pain using a rat model of the condition chronic constriction sciatic nerve injury (CCI). We report that leptin critically contributed to pain behaviors following CCI. Specifically, spinal administration of a leptin antagonist prevented and reversed neuropathic pain behaviors in rats. Further examination revealed that levels of both leptin and the long form of the leptin receptor (Ob-Rb) were substantially increased within the ipsilateral spinal cord dorsal horn after peripheral nerve injury. Mechanistic studies showed that leptin upregulated the expression of both the spinal NMDA receptor and IL-1beta through the JAK/STAT pathway. Furthermore, these CCI-induced behavioral and cellular responses were diminished in leptin-deficient mice and mimicked by spinal administration of exogenous leptin in naive rats. Our findings reveal a critical role for spinal leptin in the pathogenesis of neuropathic pain and suggest what we believe to be a novel form of nonneuronal and neuronal interactions in the mechanisms of pathological pain.

A functional relationship between trigeminal astroglial activation and NR1 expression in a rat model of temporomandibular joint inflammation.

OBJECTIVE: To examine the hypothesis that glial activation would regulate the expression of the N-methyl-D-aspartate receptor subunit 1 (NR1) in the trigeminal subnucleus caudalis (Sp5C) after temporomandibular joint (TMJ) inflammation. METHODS: Inflammation of TMJ was produced in rats by injecting 50 µL complete Freund's adjuvant (CFA) into unilateral TMJ space. Sham control rats received incomplete Freund's adjuvant injection. Mechanical nociception in the affected and non-affected TMJ site was tested by using a digital algometer. Fractalkine, fluorocitrate, and/or MK801 were intracisternally administrated to examine the relationship between astroglial activation and NR1 upregulation. RESULTS: CFA TMJ injection resulted in persistent ipsilateral mechanical hyperalgesia 1, 3, and 5 days after CFA injection. The inflammation also induced significant upregulation of CX3C chemokine receptor 1 and glial fibrillary acidic protein (GFAP) beginning on day 1 and of NR1 beginning on day 3 within the ipsilateral Sp5C. Intracisternal administration of fluorocitrate for 5 days blocked the development of mechanical hyperalgesia as well as the upregulation of GFAP and NR1 in the Sp5C. Conversely, intracisternal injection of fractalkine for 5 days exacerbated the expression of NR1 in Sp5C and mechanical hyperalgesia induced by TMJ inflammation. Moreover, once daily intracisternal fractalkine administration for 5 days in naïve rats induced the upregulation of NR1 and mechanical hyperalgesia. CONCLUSIONS: These results suggest that astroglial activation contributes to the mechanism of TMJ pain through the regulation of NR1 expression in Sp5C.

Role of 5-HT1A-mediated upregulation of brain indoleamine 2,3 dioxygenase 1 in the reduced antidepressant and antihyperalgesic effects of fluoxetine during maintenance treatment.

The reduced antidepressant and antihyperalgesic effects of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine during maintenance treatment has been reported, but little is known about the molecular mechanism of this phenomenon. In three comorbid pain and depression animal models (genetic predisposition, chronic social stress, arthritis), we showed that the fluoxetine's antidepressant and antihyperalgesic effects were reduced during the maintenance treatment. Fluoxetine exposure induced upregulation of the 5-hydroxytryptamine 1A (5-HT1A) auto-receptor and indoleamine 2,3 dioxygenase 1 (IDO1, a rate-limiting enzyme of tryptophan metabolism) in the brainstem dorsal raphe nucleus (DRN), which shifted the tryptophan metabolism away from the 5-HT biosynthesis. Mechanistically, IDO1 upregulation was downstream to fluoxetine-induced 5-HT1A receptor expression because 1) antagonism of the 5-HT1A receptor with WAY100635 or 5-HT1A receptor knockout blocked the IDO1 upregulation, and 2) inhibition of IDO1 activity did not block the 5-HT1A receptor upregulation following fluoxetine exposure. Importantly, inhibition of either the 5-HT1A receptor or IDO1 activity sustained the fluoxetine's antidepressant and antihyperalgesic effects, indicating that 5-HT1A-mediated IDO1 upregulation in the brainstem DRN contributed to the reduced antidepressant and antihyperalgesic effects of fluoxetine. These results suggest a new strategy to improving the therapeutic efficacy of SSRI during maintenance treatment.

A rat model of radicular pain induced by chronic compression of lumbar dorsal root ganglion with SURGIFLO.

BACKGROUND: Radicular pain is a common and debilitating clinical pain condition. To date, the mechanisms of radicular pain remain unclear, partly because of the lack of suitable preclinical models. The authors report a modified rat model of radicular pain that could mimic a subset of clinical radicular pain conditions induced by the soft tissue compression on dorsal root ganglion. METHODS: A rat model of radicular pain was produced by infiltrating the L5 intervertebral foramen with 60 microl of a hemostatic matrix (SURGIFLO; Johnson & Johnson, Somerville, NJ) resulting in chronic compression of lumbar dorsal root ganglion. Thermal hyperalgesia and mechanical allodynia were measured with or without epidural treatment with triamcinolone. Western blot was used to assess the expression of the NR1 subunit of the N-methyl-D-aspartate receptor and inhibitory factor kappabeta-alpha, an inflammatory marker, within the affected L5 dorsal root ganglion and spinal cord dorsal horn. RESULTS: Chronic compression of lumbar dorsal root ganglion resulted in: (1) persistent mechanical allodynia and thermal hyperalgesia up to 4 or 5 postoperative weeks and (2) up-regulation of the N-methyl-D-aspartate receptor and inhibitory factor kappabeta-alpha within the ipsilateral L5 dorsal root ganglion and spinal cord dorsal horn. Epidural administration of triamcinolone (6.25-100 microg) on postoperative day 3 dose-dependently attenuated both thermal hyperalgesia and mechanical allodynia in rats with chronic compression of lumbar dorsal root ganglion. CONCLUSION: The data suggest that this modified rat model of chronic compression of lumbar dorsal root ganglion may be a useful tool to explore the mechanisms as well as new therapeutic options of radicular pain.

Exacerbated mechanical allodynia in rats with depression-like behavior.

Although a clinical connection between pain and depression has long been recognized, how these two conditions interact remains unclear. Here we report that both mechanical allodynia and depression-like behavior were significantly exacerbated after peripheral nerve injury in Wistar-Kyoto (WKY) rats, a genetic variation of Wistar rats with demonstrable depression-like behavior. Administration of melatonin into the anterior cingular cortex contralateral to peripheral nerve injury prevented the exacerbation of mechanical allodynia with a concurrent improvement of depression-like behavior in WKY rats. Moreover, there was a lower plasma melatonin concentration and a lower melatonin receptor expression in the anterior cingular cortex in WKY rats than in Wistar rats. These results suggest that there exists a reciprocal relationship between mechanical allodynia and depression-like behavior and the melatoninergic system in the anterior cingular cortex might play an important role in the interaction between pain and depression.

Cognitive impairment in a rat model of neuropathic pain: role of hippocampal microtubule stability.

Clinical evidence indicates that cognitive impairment is a common comorbid condition of chronic pain. However, the cellular basis for chronic pain-mediated cognitive impairment remains unclear. We report here that rats exhibited memory deficits after spared nerve injury (SNI). We found that levels of stable microtubule (MT) were increased in the hippocampus of the rats with memory deficits. This increase in stable MT is marked by α-tubulin hyperacetylation. Paclitaxel, a pharmacological MT stabilizer, increased the level of stable MT in the hippocampus and induced learning and memory deficits in normal rats. Furthermore, paclitaxel reduced long-term potentiation in hippocampal slices and increased stable MT (evidenced by α-tubulin hyperacetylation) levels in hippocampal neuronal cells. Intracerebroventricular infusion of nocodazole, an MT destabilizer, ameliorated memory deficits in rats with SNI-induced nociceptive behavior. Expression of HDAC6, an α-tubulin deacetylase, was reduced in the hippocampus in rats with cognitive impairment. These findings indicate that peripheral nerve injury (eg, SNI) affects the MT dynamic equilibrium, which is critical to neuronal structure and synaptic plasticity.

Increased HCN Channel Activity in the Gasserian Ganglion Contributes to Trigeminal Neuropathic Pain.

Orofacial neuropathic pain caused by trigeminal nerve injury is a debilitating condition with limited therapeutic options. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability and are involved in the development and maintenance of chronic pain. However, the effect of HCN channel activity in the Gasserian ganglion on trigeminal neuropathic pain has not been examined. We evaluated nociceptive behaviors after microinjection of the HCN channel blockers ZD7288 or ivabradine into the Gasserian ganglion in rats with trigeminal nerve injury. Both blockers dose-dependently ameliorated evoked and spontaneous nociceptive behavior in rats with trigeminal neuropathic pain. Moreover, the clinically available HCN channel blocker ivabradine showed a prolonged antinociceptive effect. In the Gasserian ganglion, HCN1 and HCN2 are major HCN isoforms. After trigeminal nerve injury, the counts of HCN1 as well as HCN2 immuno-positive punctae were increased in the ipsilateral Gasserian ganglions. These results indicate that the increased HCN channel activity in the Gasserian ganglion directly contributes to neuropathic pain resulting from trigeminal nerve injury. PERSPECTIVE: Trigeminal nerve damage-induced orofacial pain is severe and more resistant to standard pharmacological treatment than other types of neuropathic pain. Our study suggests that targeting HCN channel activities in the Gasserian ganglion may provide an alternative treatment of trigeminal neuropathy including trigeminal neuralgia.

Time-dependent effect of epidural steroid on pain behavior induced by chronic compression of dorsal root ganglion in rats.

Although epidural steroid injection has been commonly used to treat radicular pain, its clinical efficacy remains controversial. In a rat model of radicular pain induced by chronic compression of lumbar dorsal root ganglion (CCD), we examined the effect of epidural steroid on CCD-induced pain behavior. Triamcinolone [a glucocorticoid receptor (GR) agonist] or RU38486 (a GR antagonist) was given epidurally once either on day 3 (early treatment) or day 10 (late treatment) after CCD. The results showed that 1) early treatment with triamcinolone and RU38486 alone, respectively, reduced and exacerbated mechanical allodynia and thermal hyperalgesia, 2) late treatment with triamcinolone alone failed to improve mechanical allodynia and only transiently attenuated thermal hyperalgesia, and 3) late treatment with RU38486 alone improved mechanical allodynia and thermal hyperalgesia in CCD rats. Moreover, a second dose of triamcinolone given on day 10 paradoxically exacerbated pain behavior in CCD rats that received a first dose of triamcinolone on day 3. These results indicate that the effect of epidural steroid on radicular pain may be time-dependent. Clinical implications for epidural steroid treatment are discussed in light of these preclinical findings.

An Improved Rodent Model of Trigeminal Neuropathic Pain by Unilateral Chronic Constriction Injury of Distal Infraorbital Nerve.

The number of studies on trigeminal nerve injury using animal models remains limited. A rodent model of trigeminal neuropathic pain was first developed in 1994, in which chronic constriction injury (CCI) is induced by ligation of the infraorbital nerve (IoN). This animal model has served as a major tool to study trigeminal neuropathic pain. Unfortunately, the surgical procedure in this model is complicated and far more difficult than ligation of peripheral nerves (eg, sciatic nerve). The aim of this study was to improve on the current surgical procedure of IoN ligation to induce trigeminal neuropathic pain in rats. We show that the IoN can be readily accessed through a small facial incision. CCI can be induced by ligation of a segment at the distal IoN (dIoN). This dIoN-CCI procedure is simple, minimally invasive, and time-saving. Our data show that the dIoN-CCI procedure consistently induced acute as well as chronic nociceptive behaviors in rats. Daily gabapentin treatment attenuated mechanical allodynia and reduced face-grooming episodes in dIoN-CCI rats. PERSPECTIVE: The orofacial pain caused by trigeminal nerve damage is severe and perhaps more debilitating than other types of neuropathic pain. However, studies on trigeminal neuropathic pain remain limited. This is largely because of the lack of proper animal models because of the complexity of the existing surgical procedures required to induce trigeminal nerve injury. Our improved dIoN-CCI model is likely to make it more accessible to study the cellular and molecular mechanisms of neuropathic pain caused by trigeminal nerve damage.

Gut microbiota is critical for the induction of chemotherapy-induced pain.

Chemotherapy-induced pain is a dose-limiting condition that affects 30% of patients undergoing chemotherapy. We found that gut microbiota promotes the development of chemotherapy-induced mechanical hyperalgesia. Oxaliplatin-induced mechanical hyperalgesia was reduced in germ-free mice and in mice pretreated with antibiotics. Restoring the microbiota of germ-free mice abrogated this protection. These effects appear to be mediated, in part, by TLR4 expressed on hematopoietic cells, including macrophages.

Central glucocorticoid receptors modulate the expression and function of spinal NMDA receptors after peripheral nerve injury.

Central glucocorticoid receptors (GRs) and NMDA receptors (NMDARs) have been shown to play a significant role in the mechanisms of neuropathic pain after peripheral nerve injury; however, how central GRs and NMDARs interact in this process remains unknown. Here we show that the expression and function of spinal NMDARs after peripheral nerve injury were modulated by central GRs. Chronic constriction nerve injury (CCI) in rats induced a time-dependent upregulation of NR1 and NR2 subunits of the NMDAR within the spinal cord dorsal horn ipsilateral to CCI. The upregulation of NMDARs was significantly diminished by intrathecal administration (twice daily for postoperative days 1-6) of either the GR antagonist RU38486 or an antisense oligonucleotide against GRs. Moreover, this CCI-induced expression of NMDARs was significantly attenuated in rats receiving intrathecal treatment with an interleukin-6 (IL-6) antiserum and in mice with protein kinase Cgamma (PKCgamma) knock-out. Because IL-6 and PKCgamma mediated the upregulation of central GRs after CCI as demonstrated previously, the results suggest that IL-6 and PKCgamma served as cellular mediators contributing to the GR-mediated expression of NMDARs after CCI. Functionally, nociceptive behaviors induced by NMDAR activation and CCI were reversed by a single intrathecal administration of the GR antagonist RU38486. Conversely, a single intrathecal injection with the noncompetitive NMDAR antagonist MK-801 reversed neuropathic pain behaviors exacerbated by the GR agonist dexamethasone in CCI rats. These data suggest that interactions between central GRs and NMDARs through genomic and nongenomic regulation may be an important mechanism critical to neuropathic pain behaviors in rats.

Expression of spinal NMDA receptor and PKCgamma after chronic morphine is regulated by spinal glucocorticoid receptor.

Spinal NMDA receptor (NMDAR), protein kinase C (PKC), and glucocorticoid receptor (GR) have all been implicated in the mechanisms of morphine tolerance; however, how these cellular elements interact after chronic morphine exposure remains unclear. Here we show that the expression of spinal NMDAR and PKCgamma after chronic morphine is regulated by spinal GR through a cAMP response element-binding protein (CREB)-dependent pathway. Chronic morphine (10 microg, i.t.; twice daily for 6 d) induced a time-dependent upregulation of GR, the NR1 subunit of NMDAR, and PKCgamma within the rat's spinal cord dorsal horn. This NR1 and PKCgamma upregulation was significantly diminished by intrathecal coadministration of morphine with the GR antagonist RU38486 or a GR antisense oligodeoxynucleotide. Intrathecal coadministration of morphine with an adenylyl cyclase inhibitor (2',5'-dideoxyadenosine) or a protein kinase A inhibitor (H89) also significantly attenuated morphine-induced NR1 and PKCgamma expression, whereas intrathecal treatment with an adenylyl cyclase activator (forskolin) alone mimicked morphine-induced expression of GR, NR1, and PKCgamma. Moreover, the expression of phosphorylated CREB was upregulated within the spinal cord dorsal horn after chronic morphine, and a CREB antisense oligodeoxynucleotide coadministered intrathecally with morphine prevented the upregulation of GR, NR1, and PKCgamma. These results indicate that spinal GR through the cAMP-CREB pathway played a significant role in NMDAR and PKCgamma expression after chronic morphine exposure. The data suggest that genomic interaction among spinal GR, NMDAR, and PKCgamma may be an important mechanism that contributes to the development of morphine tolerance.

Downregulation of spinal glutamate transporter EAAC1 following nerve injury is regulated by central glucocorticoid receptors in rats.

Previous studies have shown that glucocorticoid receptors (GR) were upregulated, whereas glutamate transporters were downregulated, within the spinal cord dorsal horn after peripheral nerve injury. However, the relationship between the expression of spinal GR and glutamate transporter after nerve injury remains unknown. In the present study, we examined the hypothesis that central GR would regulate the expression of spinal glutamate transporter EAAC1 following chronic constriction nerve injury (CCI) in rats. CCI induced a significant downregulation of EAAC1 expression primarily within the ipsilateral spinal cord dorsal horn when examined on postoperative day 7 using both Western blot and immunohistochemistry. The downregulation of EAAC1 was significantly diminished after either the GR antagonist RU38486 (4 > 2 = 0.5 microg = vehicle) or a GR antisense oligonucleotide was administered intrathecally twice daily for postoperative day 1-6. Moreover, CCI induced a significant downregulation of nuclear factor kappaB (NF-kappaB) within the ipsilateral spinal cord dorsal horn, which also was attenuated by either RU38486 (4 > 2 = 0.5 microg = vehicle) or a GR antisense oligonucleotide. The immunohistochemical data indicated a pattern of colocalization between GR and EAAC1 as well as GR and NF-kappaB within the spinal cord dorsal horn. Since, NF-kappaB has been shown to regulate the expression of those cellular elements linked to inflammation and tissue injury and its activity can be negatively regulated by GR activation, these results suggest that spinal GR through NF-kappaB may play a significant role in the regulation of EAAC1 expression after peripheral nerve injury, a cellular pathway that may contribute to the development of neuropathic pain behaviors in rats.