C-fos Expression In Spinal Neurons Research Articles

Kappa-opioid receptor-mediated thermal analgesia evoked by the intrathecal administration of the chemokine CCL1 in mice.

The chemokine CC motif ligand 1 (CCL1) participates in immune cell recruitment and, as other chemokines, is also involved in nociceptive processing. In contrast with previous reports indicating its participation in allodynia and cold hypernociception when spinally administered, its ability to evoke heat thermal analgesia, mediated by circulating leukocytes and endocannabinoids, after systemic administration has recently been reported. Aiming to explore the role played by CCL1 on spinal nociception, we study here the effect of its intrathecal administration on thermal nociception in mice. Behavioral nociceptive assays, immunohistochemical experiments, white cell blood depletion procedures and qRT-PCR experiments were performed. The intrathecal administration of CCL1 (0.3-30 ng) produced analgesia as measured by the unilateral hot plate test. This effect peaked 1 h after injection, was prevented by the CCR8 antagonist R243 and was accompanied by a reduction of c-Fos expression in spinal neurons. Whereas blood leukocyte depletion did not modify it, analgesia was abolished by the microglial inhibitor minocycline, but not the astroglial inhibitor aminoadipate. Furthermore, antinociception remained unmodified by the coadministration of cannabinoid type 1 or 2 receptors antagonists. However, it was reversed by naloxone but not by selective blockade of mu- or delta-opioid receptors. The inhibitory effect induced by the selective kappa-opioid receptor antagonist, nor-binaltorphimine, and by an anti-dynorphin A 1-17 antibody indicates that analgesia evoked by spinal CCL1 is mediated by endogenous dynorphins acting on kappa-opioid receptors. Endogenous dynorphin and microglia behave as key players in heat thermal analgesia evoked by spinal CCL1 in mice.

Withania somnifera (L.) Dunal root extract alleviates formalin-induced nociception in mice: involvement of the opioidergic system.

Withania somnifera (L.) Dunal extracts (WSEs) may possess therapeutic perspectives in the treatment of inflammation and pain. We aimed to evaluate the antinociceptive property of a WSE in the formalin test and to investigate the involvement of several neurotransmitter systems in this effect. The time spent licking the formalin-injected paw was recorded in CD1 mice after pretreatment with increasing doses of WSE. Also, c-Fos spinal cord expression and the effects of different compounds were investigated under these experimental conditions. Finally, the efficacy of WSE was analyzed following an injection of glutamate. WSE reduced the antinociceptive response during the tonic but not the acute phase of the formalin test and decreased formalin-induced c-Fos expression in spinal neurons. These effects were antagonized by the opioid antagonist naltrexone, whereas GABA, cannabinoid, δ-opioid, and nitric oxide compounds were ineffective. The administration of WSE also reduced nociception and c-Fos expression induced by glutamate injection. These results showed that WSE is effective in assays of chemical-induced nociception, indicating that this plant has potential valuable properties for the treatment of specific painful conditions. The antinocicetive effects of WSE in the formalin test appeared to be specifically mediated by the opioidergic system, although the involvement of the glutamatergic system cannot be excluded.

7-Nitroindazole enhances c-Fos expression in spinal neurons in rats realizing operant movements

The expression of c-Fos and NADPH-diaphorase reactivity (NADPH-dr) in the cervical spinal cord was studied in adult male Wistar rats that realized operant reflexes after inhibition of neuronal nitric oxide synthase. Fos-immunoreactive neurons were visualized immunohistochemically in the C6/C7 spinal segments in the control, realized operant movements animals, and/or 7-nitroindazole (7-NI) injected rats. The mean numbers of immunoreactive interneurons and motoneurons (per section) were significantly greater in the Nucleus proprius (+240%) and motor nuclei (+600%) in rats of the 7-NI-pretreated and operant reflex realized group than in the isolated operant reflex realized group. Our study showed intensive staining of NADPH-dr axon terminals on the somata and initial parts of dendrites of motoneurons in experimental rats when the disodium salt of malic acid was added to the staining solution. Suppression of NO release is associated with potentiation of neuronal activation induced by descending supraspinal and proprioceptive signaling within the spinal cord.

Effect of Long-Term Prazosin and Yohimbine Administration on c-Fos Expression in Spinal Neurons: Inhibitory Effect of Alpha-1 and Alpha-2 Adrenoceptors on Afferents from the Lower Urinary Tract

Introduction: To investigate the effects of long-term administration of the α₁-adrenoceptor antagonist prazosin and the α₂-adrenoceptor antagonist yohimbine on afferent inputs from the lower urinary tract by evaluating c-Fos expression in the spinal cord. Materials and Methods: Prazosin or yohimbine was administered for 4 weeks in rats using an osmotic pump. Effects of these agents on urodynamic parameters were determined by continuous cystometry in conscious rats. After cystometry, c-Fos expression in the dorsal horn of the L6 spinal cord was measured by immunohistochemistry. Results: The administration of prazosin (0.12 mg/kg/day) or yohimbine (0.10 mg/kg/day) significantly increased micturition interval and bladder capacity, but did not affect micturition pressure and residual urine volume. The numbers of c-Fos-positive neurons in the dorsal horn were significantly lower in rats that received prazosin than in controls. Yohimbine reduced the number of c-Fos-positive neurons in part of the dorsal horn. Conclusions: Long-term administration of prazosin and yohimbine at clinically recommended doses can exert inhibitory effects on afferent pathways from the lower urinary tract during the storage phase. These reductions of the afferent input result in the increased bladder capacity and increased micturition interval.

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1

Prostaglandins (PG) are known to induce pain perception indirectly by sensitizing nociceptors. Accordingly, the analgesic action of nonsteroidal anti-inflammatory drugs (NSAIDs) results from inhibition of cyclooxygenases and blockade of PG biosynthesis. Cyclopentenone PGs, 15-d-PGJ(2), PGA(2), and PGA(1), formed by dehydration of their respective parent PGs, PGD(2), PGE(2), and PGE(1), possess a highly reactive alpha,beta-unsaturated carbonyl group that has been proposed to gate the irritant transient receptor potential A1 (TRPA1) channel. Here, by using TRPA1 wild-type (TRPA1(+/+)) or deficient (TRPA1(-/-)) mice, we show that cyclopentenone PGs produce pain by direct stimulation of nociceptors via TRPA1 activation. Cyclopentenone PGs caused a robust calcium response in dorsal root ganglion (DRG) neurons of TRPA1(+/+), but not of TRPA1(-/-) mice, and a calcium-dependent release of sensory neuropeptides from the rat dorsal spinal cord. Intraplantar injection of cyclopentenone PGs stimulated c-fos expression in spinal neurons of the dorsal horn and evoked an instantaneous, robust, and transient nociceptive response in TRPA1(+/+) but not in TRPA1(-/-) mice. The classical proalgesic PG, PGE(2), caused a slight calcium response in DRG neurons, increased c-fos expression in spinal neurons, and induced a delayed and sustained nociceptive response in both TRPA1(+/+) and TRPA1(-/-) mice. These results expand the mechanism of NSAID analgesia from blockade of indirect nociceptor sensitization by classical PGs to inhibition of direct TRPA1-dependent nociceptor activation by cyclopentenone PGs. Thus, TRPA1 antagonism may contribute to suppress pain evoked by PG metabolites without the adverse effects of inhibiting cyclooxygenases.

Effect of Long-Term Oxybutynin Administration on c-Fos Expression in Spinal Neurons: Inhibition of Antimuscarinics on Bladder Afferents in Conscious Rats

To investigate the effect of long-term administration of oxybutynin on afferent input from the bladder by evaluating c-Fos expression in the spinal cord. Using an osmotic pump, long-term administration of oxybutynin (4 weeks) was performed in the rat. The effects of oxybutynin (2 doses) on the urodynamic parameters were determined by continuous cystometry in conscious rats. After cystometry, c-Fos expression in the spinal cord was measured by immunohistochemistry. The long-term administration of low-dose oxybutynin (0.36 mg/kg/d) significantly increased the micturition interval and bladder capacity, but it did not affect micturition pressure. However, administration of high-dose oxybutynin (3.6 mg/kg/d) significantly decreased the micturition pressure and increased the residual volume. In the rats that received low-dose oxybutynin, the number of c-Fos-positive neurons in the spinal cord was significantly lower than that in controls. Administration of low-dose oxybutynin decreased the c-Fos expression induced by continuous infusion of saline into the bladder. This result suggests that the antimuscarinic drug oxybutynin at clinically recommended doses can exert an inhibitory effect on afferent input from the bladder during the storage phase without affecting detrusor contractions.

C-fos Expression in Bladder-Specific Spinal Neurons after Spinal Cord Injury Using Pseudorabies Virus

Purposec-fos expression in spinal neurons that are activated by lower urinary tract stimulation are not organ specific. In this experiment, we demonstrated changes of c-fos expression in bladder-specific preganglionic neurons (PGNs) and interneurons using pseudorabies virus (PRV).Materials and MethodsForty Sprague-Dawley rats were used. We identified the neuronal pathway associated with the bladder by injecting PRV into the detrusor. An immunohistochemical method was used to stain Fos-protein encoded by the c-fos gene. Immunofluorescent staining for PRV was performed to evaluate changes in bladder-specific spinal neurons.ResultsImmunofluorescent staining with choline acetyltransferase (ChAT) revealed that the sacral parasympathetic nucleus (SPN) regions contained 9.8 PGNs/section. In rats with chronic spinal cord injury by intravesical saline instillation, 82.4 ± 10.3% of PGNs in SPN exhibited Fos-immunoreactive (IR). Two and a half days after PRV infection, PRV-IR PGNs were observed at 5.4 PGNs/ section, and 2.7 ± 1.6% of them exhibited Fos-IR. Unlike ChAT-IR PGNs, PRV-IR PGNs are bladder-specific neurons and PRV-IR and Fos-IR cells found in the back of PRV-IR PGNs are bladder-specific interneurons. Three days after PRV infection, we observed many PRV-IR and Fos-IR cells in the dorsal commissure. These neurons are interneurons distributed in the bladder.ConclusionWe confirmed that in chronic spinal cord injury, the patterns of c-fos expression in bladder-specific spinal neurons were similar to those in voiding-reflex related spinal neurons, which had already been demonstrated earlier. We believe that our methodology can be applied to study interactions between voiding and other organs as well, such as the urethra and prostate.

Transient receptor potential vanilloid 1, calcitonin gene-related peptide, and substance P mediate nociception in acute pancreatitis

The mechanism of pancreatitis-induced pain is unknown. In other tissues, inflammation activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to liberate CGRP and substance P (SP) in peripheral tissues and the dorsal horn to cause neurogenic inflammation and pain, respectively. We evaluated the contribution of TRPV1, CGRP, and SP to pancreatic pain in rats. TRPV1, CGRP, and SP were coexpressed in nerve fibers of the pancreas. Injection of the TRPV1 agonist capsaicin into the pancreatic duct induced endocytosis of the neurokinin 1 receptor in spinal neurons in the dorsal horn (T10), indicative of SP release upon stimulation of pancreatic sensory nerves. Induction of necrotizing pancreatitis by treatment with L-arginine caused a 12-fold increase in the number of spinal neurons expressing the proto-oncogene c-fos in laminae I and II of L1, suggesting activation of nociceptive pathways. L-arginine also caused a threefold increase in spontaneous abdominal contractions detected by electromyography, suggestive of referred pain. Systemic administration of the TRPV1 antagonist capsazepine inhibited c-fos expression by 2.5-fold and abdominal contractions by 4-fold. Intrathecal, but not systemic, administration of antagonists of CGRP (CGRP(8-37)) and SP (SR140333) receptors attenuated c-fos expression in spinal neurons by twofold. Thus necrotizing pancreatitis activates TRPV1 on pancreatic sensory nerves to release SP and CGRP in the dorsal horn, resulting in nociception. Antagonism of TRPV1, SP, and CGRP receptors may suppress pancreatitis pain.

The induction and potentiation of c-Jun and c-Fos expression in spinal neurons mediated by NK1 and NK2 receptors

Specific antagonists were used to determine NK1 and NK2 receptor mediation of the expression of c-Jun and c-Fos inducible transcription factors. (ITFs) in dorsal horn neurons. The induction of c-Jun by C-fiber stimulation was strongly reduced in the superficial laminae by NK1 and NK2 antagonists, but only weakly in the deep laminae by NK2 antagonism. c-Fos induction was reduced in all laminae by both NK1 and NK2 antagonism but less than with c-Jun. The potentiation of c-Jun expression, caused by a preceding stimulus, was abolished in all laminae by NK1 and NK2 antagonists, whereas that of c-Fos was reduced in all laminae but again less than that of c-Jun. Thus, the expressions of ITFs mediated by NK1 and NK2 receptors are complex, depending upon the neurons and stimulus considered, and they are not a measure of the neurons’ electrophysiological responsiveness.

Potent anti-inflammatory/analgesic effects of lornoxicam in comparison to other nsaids: A c-fos study in the rat

This study evaluates the anti-inflammatory/analgesic effects of lornoxicam, a new non-steroidal anti-inflammatory drug, using the method of c-Fos protein immunoreactivity in the carrageenan model of inflammatory nociception in the rat. The immunohistochemical revelation of inflammatory/nociceptive stimulation evoked c-Fos expression in spinal neurons was used as an indirect marker of neurons involved in spinal nociceptive transmission. Lornoxicam (0.1, 0.3, 1, 3 and 9 mg/kg; n=10 rats for each group) was preadministered intravenously 25 min before an intraplantar injection of carrageenan (6 mg/150 ml of saline). Three hours after carrageenan, the peripheral oedema (paw and ankle diameters) and the number of c-Fos-protein-like immunoreactive (c-Fos-LI) neurons in the lumbar spinal cord, were assessed. Preadministered lornoxicam dose relatedly reduced the total number of c-Fos-LI neurons (regression coefficient r=0.79; p<0.001) with the strongest effect corresponding to the 75+/-2% reduction (p<0.001) for the highest dose of 9 mg/kg, and the 45+/-3% reduction (p<0.001) for the low dose of 0.3 mg/kg. Lornoxicam (0.1, 0.3, 1, 3 and 9 mg/kg iv) significantly reduced the number of c-Fos-LI neurons in both superficial (24+/-6, 33+/-5, 53+/-4, 54+/-4, and 63+/-4% reduction, respectively, p<0.001 for all doses) and deep (28+/-4, 48+/-4, 62+/-2, 69+/-3 and 79+/-2% reduction, respectively, p<0.001 for all doses) laminae of the dorsal horn of the spinal cord. These reducing effects were dose related in both superficial and deep laminae (regression coefficient r=0.66 and r=0.08, respectively; p<0.001 for both). The lowes dose of lornoxicam (0.1 mg/kg iv) had a similar effect in both superficial and deep laminae, whereas the four higher doses (0.3, 1, 3 and 9 mg/kg iv) had a significantly stronger effect on the number of c-Fos-LI neurons in deep laminae as compared to that in superficial laminae. Lornoxicam (0.1, 0.3, 1, 3 and 9 mg/kg iv) dose relatedly reduced the carrageenan induced oedema at both the paw and ankle levels (regression coefficient r=0.63 and r=0.53, respectively, p<0.001 for both), with a stronger effect on the ankle diameter (34+/-8, 61+/-9, 66+/-8, 80+/-6 and 83+/-5% reduction, respectively p<0.001 for all doses). Furthermore reductions of the carrageenan evoked peripheral oedema and spinal c-Fos expression were positively correlated (correlation coefficient r=0.74 and r=0.57 for the paw and ankle diameter respectively, p<0.001 for both). These correlations suggest a predominant peripheral site, without excluding central site of action of lornoxicam in the carrageenan-induced inflammation. Our results provide clear evidence for a potent anti-inflammatory/analgesic effects of low doses of lornoxicam which have a reduced risk of side effects. Taken together, the results of the present study revealed the effects of lornoxicam in the same range as those of other previously studied NSAIDs, more precisely, closely comparable to the effects of ketoprofen.

The massive expression of c-fos protein in spinal dorsal horn neurons is not followed by long-term changes in spinal nociception.

It has been suggested that the expression of c-fos and other immediate early genes in spinal dorsal horn neurons would trigger changes in the phenotype of nociceptive neurons which may lead to long-term changes in spinal nociception. To test this hypothesis, we have used a minimally invasive intrathecal stimulation and injection technique which can be applied to adult Sprague-Dawley rats under brief ether anesthesia to induce massive c-fos expression in spinal neurons without affecting peripheral nociceptors. Electrical intrathecal stimulation (0.5 ms pulses, 15 V, 3 Hz for 15 min) or intrathecal injection of N-methyl-D-aspartate (25 nmol) produced massive c-Fos immunoreactivity in neurons throughout the sacral spinal cord and the dorsal horn of the lumber spinal cord. Immunoreactivity declined to control values at mid-thoracic levels. To assess effects of these intrathecal stimuli on nociception, hot-plate and tail-flick latencies and mechanical thresholds of hindlimb withdrawal reflexes were measured once every day for 14 days before and up to 14 days after conditioning stimulation. Spontaneous locomotion of each animal was video-taped daily for 5 min and analysed off-line. On the day of the intrathecal stimulation the tests were performed 1 h before and also 6 h after conditioning stimulation. Thermal and mechanical nociceptive thresholds were temporarily enhanced 6 h after intrathecal stimulation but they were not different from controls one to 14 days later. Thus, the massive expression of c-fos in spinal neurons is not, as previously suggested, a sufficient condition for the induction of long-term changes in spinal nociception.

Induction of c-fos expression in spinal neurons by nociceptive and nonnociceptive stimulation of LUT

Expression of c-fos gene in spinal neurons was detected with an immunocytochemical technique to study the spinal processing of nociceptive and nonnociceptive input from the lower urinary tract (LUT) of the urethan-anesthetized rat. Two preparations were used to activate afferent pathways in the LUT: 1) the urinary bladder was exposed through an abdominal incision, and saline or 1% acetic acid solution was infused directly into the bladder lumen and expelled through the urethra; and 2) the bladder was catheterized through the urethra, and the urethral outlet was ligated to allow distension and reflex contractions to occur under isovolumetric conditions. The first preparation mimicked the physiological changes occurring during normal voiding, whereas the second preparation generated high (presumably nociceptive) pressures when the bladder contracted against a closed outlet. The results indicate that distension-induced voiding increased c-fos expression largely in the region of the sacral parasympathetic nucleus (52% of the total number of cells/L6 section) and the dorsal commissure (25% of the total number of cells), whereas nociceptive stimuli markedly increased the number of c-fos-positive cells in the dorsal commissure (3.5 x increase above the number induced by distension, representing 50% of the total number of cells/L6 section). Bladder contractions against a closed outlet elicited a distribution of c-fos-positive cells similar to that induced by chemical irritation. Drugs that suppressed bladder reflexes did not reduce c-fos induced by distension, indicating that voiding reflexes do not contribute to c-fos expression.

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

This study utilized neuronal c-fos expression to examine the spinal pathways involved in processing nociceptive and non-nociceptive afferent input from the lower urinary tract (LUT) of the urethane-anesthetized rat. C-fos protein was detected immunocytochemically in only a small number of cells (< 2 cells/L6 section) in control animals. However, chemical irritation with 1% acetic acid or mechanical stimulation of the LUT markedly increased the number of c-fos-positive neurons (56-180 cells/L6 section) in four regions of the caudal lumbosacral (L6-S1) spinal cord: medial dorsal horn (MDH), lateral dorsal horn, dorsal commissure (DCM), and sacral parasympathetic nucleus (SPN). Only small numbers of c-fos-positive cells were detected in rostral lumbar segments, a region that is thought to receive nociceptive input from the LUT via afferent pathways in sympathetic nerves. The distribution of c-fos-positive cells in the L6 spinal cord varied according to the stimulus (i.e., urethral catheter, bladder distension, or chemical irritation). Distension of the urinary bladder increased the number of c-fos-positive cells mainly in DCM and SPN regions of the cord. In contrast, irritation of the LUT increased c-fos expression largely in DCM and MDH areas. Spinal cord transection (T8 level) did not alter the c-fos expression induced by a catheter or chemical irritation, indicating that gene expression was mediated by spinal pathways. Denervation experiments showed that c-fos expression was induced by activation of afferent pathways in the pelvic and pudendal nerves. These results suggest that neurons in several regions of the spinal cord are involved in processing afferent input from different parts of the LUT. Neurons in the DCM appear to have an important role since they respond to both nociceptive and non-nociceptive inputs and to visceral (pelvic nerve) and somatic (pudendal nerve) afferent pathways. Thus, these neurons may be involved in the mechanisms of visceral-somatic referred pain.