Sciatic Nerve Ligation In Mice Research Articles

Probucol is anti-hyperalgesic in a mouse peripheral nerve injury model of neuropathic pain

2,6-di-tert-butylphenol (2,6-DTBP) ameliorates mechanical allodynia and thermal hyperalgesia produced by partial sciatic nerve ligation in mice, and selectively inhibits HCN1 channel gating. We hypothesized that the clinically utilized non-anesthetic dimerized congener of 2,6-DTBP, probucol (2,6-di-tert-butyl-4-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)sulfanylpropan-2-ylsulfanyl]phenol), would relieve the neuropathic phenotype that results from peripheral nerve damage, and that the anti-hyperalgesic efficacy in vivo would correlate with HCN1 channel inhibition in vitro. A single oral dose of probucol (800 mg/kg) relieved mechanical allodynia and thermal hyperalgesia in a mouse spared-nerve injury neuropathic pain model. While the low aqueous solubility of probucol precluded assessment of its possible interaction with HCN1 channels, our results, in conjunction with recent data demonstrating that probucol reduces lipopolysaccharide-induced mechanical allodynia and thermal hyperalgesia, support the testing/development of probucol as a non-opioid, oral antihyperalgesic albeit one of unknown mechanistic action.

Antinociceptive effect of ethanolic extract of Bauhinia brachycarpa Benth on neuropathic pain model induced by partial sciatic nerve ligation

Ethnopharmacological relevanceThe plant Bauhinia brachycarpa Benth (BBB) has been traditionally used for treating muscle aches such as bone pain, and neuralgia for a long time, on account of its sedative and antinociceptive activities in Yunnan province of China. However, there was no experimental evidence to confirm its traditional medicinal use. Aim of the studyThe antinociceptive effect and possible mechanism of ethanolic extract of BBB on neuropathic pain was evaluated through a model of partial sciatic nerve ligation in mice. Materials and methodsA commonly employed animal model induced by partial sciatic nerve ligation (PSNL) in mice was established in the aim of studying neuropathic pain. Ethanolic extract of BBB (1000, 500, 250 mg/kg) and pregabalin (60 mg/kg) were intragastric administrated daily for 7 days post-PSNL. Mechanical and thermal hyperalgesia were assessed throughout the experimental period.After the experiment, the levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in serum were determined by ELISA. The mRNA expressions of ionized calcium binding adaptor molecule 1 (Iba-1), CD16, CD206, arginine-1 (Arg-1), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) in the spinal cord were detected by qPCR. The protein levels of Iba-1, CD16, CD206, and p38 phosphorylation in the spinal cord were detected by immunohistochemistry and western blotting. The phytochemical analysis of BBB was performed through the colorimetric test. ResultsNeuropathic pain induced by PSNL was significantly alleviated by BBB treatment, which decreased pro-inflammatory cytokines such as TNF-α, IL-1β and iNOS, increased anti-inflammatory cytokine such as IL-10 and Arg-1, and attenuated p38 phosphorylation. BBB also reduced the number of Iba-1 and CD16 positive cells, but it enhanced the number of CD206 positive cells. n-Butanol portion that was partitioned from the ethanolic extract had the highest content of total flavonoids among all the portions, and the antinociceptive effect of n-butanol portion is better than that of other portions. ConclusionsOur findings indicate that the antinociceptive effect of BBB is mediated by inhibiting the inflammatory response and regulating the differentiation of microglia. The antinociceptive effect of BBB was related to the content of total flavonoids.

Calcitonin gene-related peptide is not involved in neuropathic pain induced by partial sciatic nerve ligation in mice

BackgroundOptimal neuropathic pain (NeP) therapy has still not been established despite great efforts to develop new strategies for NeP analgesia. One possible target might be calcitonin gene-related peptide (CGRP). This is because the expression of CGRP and its receptors in the dorsal horn of the spinal cord might be associated with the persistence of pain symptoms including symptoms of NeP. We previously developed αCGRP knockout mice, and we aimed in this study to clarify the roles of CGRP in NeP by partial sciatic nerve ligation (PSNL) using the knockout mice. MethodsPSNL was performed in αCGRP knockout mice and wild-type (WT) mice, and spontaneous pain behavior and mechanical and thermal hyperalgesia were evaluated after PSNL. CGRP immunoreactivity (IR) was also observed in the superficial dorsal horn and deep dorsal horn of L4 to L5 segments of the spinal cord in WT mice after PSNL. ResultsSpontaneous pain behavior and mechanical and thermal hyperalgesia after PSNL were not different between αCGRP knockout mice and WT mice throughout the observation period. The expression of CGRP-IR was not different between the PSNL model and the sham operation model at 1 day and 7 days after surgery. ConclusionThe results suggest that the involvement of αCGRP may differ depending on the type and site of nerve injury, and clinical indications for anti-CGRP treatment of NeP should be carefully based on various pathophysiological conditions of NeP.

Calcitonin Gene-Related Peptide Is Not Involved in Neuropathic Pain Induced by Partial Sciatic Nerve Ligation in Mice

Calcitonin Gene-Related Peptide Is Not Involved in Neuropathic Pain Induced by Partial Sciatic Nerve Ligation in Mice

Analysis of the effects of a tricyclic antidepressant on secondary sleep disturbance induced by chronic pain in a preclinical model

Chronic pain and sleep have a bidirectional relationship that promotes a vicious circle making chronic pain more difficult to treat. Therefore, pain and sleep should be treated simultaneously. In our previous study, we suggested that hyperactivation of ascending serotonergic neurons could cause secondary sleep disturbance in chronic pain. This study aimed to demonstrate the effects of a tricyclic antidepressant (amitriptyline) and a selective 5-hydroxy-tryptamine 2A (5-HT2A) antagonist (MDL 100907) that adjust serotonergic transmission, on secondary sleep disturbance induced in a preclinical chronic pain model. We produced a chronic neuropathic pain model by partial sciatic nerve ligation in mice, analyzed their electroencephalogram (EEG) and electromyogram (EMG) using the SleepSign software, and evaluated the sleep condition of the pain model mice after administration of amitriptyline or MDL 100907. Amitriptyline improved thermal hyperalgesia and the amount of sleep, especially non-REM sleep. Time change of normalized power density of δ wave in the nerve ligation group with amitriptyline administration showed a normal pattern that was similar to sham mice. In addition, MDL 100907 normalized sleep condition similar to amitriptyline, without improvement in pain threshold. In conclusion, amitriptyline could improve sleep quantity and quality impaired by chronic pain. 5-HT2A receptor antagonism could partially contribute to this sleep improvement, but is not associated with pain relief.

Design and synthesis of novel anti-hyperalgesic agents based on 6-prenylnaringenin as the T-type calcium channel blockers

Since 6-prenylnaringenin (6-PNG) was recently identified as a novel T-type calcium channel blocker with the IC50 value around 1 µM, a series of flavanone derivatives were designed, synthesized and subsequently evaluated for T-channel-blocking activity in HEK293 cells transfected with Cav3.2 T-type channels using a patch-clamp technique. As a result, several new flavanones blocked Cav3.2-dependent T-currents more potently than 6-PNG. In the synthesized compounds, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(2-hydroxyphenyl)chroman-4-one 8j, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11b, 6-(2-cyclopentylideneethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11d, and 6-(2-Cyclopentylethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 12c were more potent blocker than 6-PNG with the IC50 value of 0.39, 0.26, 0.46, and 0.50 µM, respectively. Among the above four derivatives, the compound 8j provided the best result in the in vivo experiments; i.e. systemic administration of 8j at the minimum dose completely restored neuropathic pain induced by partial sciatic nerve ligation in mice.

Effect of repeated oral administration of chondroitin sulfate on neuropathic pain induced by partial sciatic nerve ligation in mice

We examined whether chondroitin sulfate (CS), a compound used to treat osteoarthritis and joint pain, is effective against partial sciatic nerve ligation (PSNL)-induced neuropathic pain. Repeated oral administration of CS (300 mg/kg, b.i.d. for 20 days) resulted in inhibition of tactile allodynia observed 21 days after PSNL. On day 21, phosphorylation of spinal p38 mitogen-activated protein kinase (MAPK) was attenuated by CS. CS also inhibited c-Fos upregulation in ipsilateral deep dorsal horn (laminae III–IV) neurons, which receive Aβ-fiber afferent inputs. These findings suggest that CS attenuates PSNL-induced tactile allodynia by inhibiting spinal p38 MAPK phosphorylation and Aβ-fiber activation.

Peripheral nerve injury-induced rearrangement of neural circuit in the spinal dorsal horn revealed by cross-correlation analysis.

Peripheral nerve injury often induces abnormal pain states, such as hyperalgesia and allodynia. In this study, we attempted to elucidate how neurons are synaptically integrated into the neuronal circuitry in the spinal dorsal horn and how synaptic connectivity patterns among dorsal horn neurons are altered by peripheral nerve injury. Experiments were performed on 6-8-week-old ICR mice. Partial sciatic nerve ligation was performed. Transverse slices of the lumbar spinal cord were prepared. Spike activities were simultaneously recorded from multiple neurons in the superficial dorsal horn (SDH) using a multi-electrode array system, and cross-correlograms between spike trains of neuron pairs were constructed. In sham-operated control mice, except for the flat cross-correlogram, the most common pattern was a cross-correlogram suggestive of common excitatory synaptic inputs to neuronal pairs. Peripheral nerve ligation increased the incidences of cross-correlograms suggestive of common excitatory synaptic inputs and excitatory synaptic connections, and decreased that of inhibitory synaptic connections. Additionally, bath-applied capsaicin, an agonist for transient receptor potential vanilloid 1 receptor, increased the frequency of action potentials. The effects of capsaicin stimulation on the incidence of cross-correlograms with various patterns were significantly different between sham-operated control and sciatic nerve-ligated mice. The present observations seem to indicate that neurons in the SDH form excitatory and/or inhibitory synapses with nearby neurons, and that synaptic connections among neurons in the SDH may remarkably change after the development of neuropathic pain.

Adult hippocampal neurogenesis in neuropathic pain and alkyl glycerol ethers treatment

Neuropathic pain manifested by a number of sensory symptoms is often accompanied by disorders of higher nervous activity, such as memory impairment, depression, anxiety, anhedonia, etc. This emphasizes the involvement of supraspinal structures including the hippocampus in neuropathic pain pathogenesis. In the present study, we focused on the impact of chronic neuropathic pain on hippocampal neurogenesis and microglial state. In addition, we test the effect of alkyl glycerol ethers on hippocampal neuronal and microglial plasticity as well as behavioral parameters. Neuropathic pain was induced using the model of sciatic nerve chronic constriction injury. We found an impairment of working memory and locomotor activity in animals with neuropathic pain, which was prevented by alkyl glycerol ethers treatment. Sciatic nerve ligation in mice contributed to the decrease in hippocampal neurogenesis intensity. Alkyl glycerol ethers administration significantly reduced this effect. Neuropathic pain-associated neurogenesis reduction was accompanied by an increased percentage of Iba1-labeled area in the CA1 hippocampal region on the 14th and 28th days after surgery. In addition, we observed a decrease in hippocampal pro-inflammatory microglia marker CD86 immunostaining on day 28 after surgery in alkyl glycerol ethers-treated mice with sciatic nerve ligation. These results are consistent with data on pro- and anti-inflammatory cytokines expression in the hippocampus. Alkyl glycerol ethers administration increased IL-10 and decreased IL-1β hippocampal expression in animals with neuropathic pain. Taken together, these data suggest that neuropathic pain-behavior in rodents is accompanied by changes in microglia polarization, thereby contributing to neurogenesis impairment and cognitive disturbances. Alkyl glycerol ethers prevented M1 microglial activation, contributing to the maintenance of normal neurogenesis levels within the hippocampus and normalizing working memory.

Persistent Alterations in Microglial Enhancers in a Model of Chronic Pain.

Chronic pain is a common and devastating condition that induces well-characterized changes in neurons and microglia. One major unanswered question is why these changes should persist long after the precipitating injury has healed. Here, we suggest that some of the longer-lasting consequences of nerve injury may be hidden in the epigenome. Cell sorting and sequencing techniques were used to characterize the spinal cord immune response in a mouse model of chronic neuropathic pain. Infiltration of peripheral myeloid cells was found to be absent, and RNA sequencing (RNA-seq) of central microglia revealed transient gene expression changes in response to nerve ligation. Conversely, examination of microglial enhancers revealed persistent, post-injury alterations in close proximity to transcriptionally regulated genes. Enhancers are regions of open chromatin that define a cell's transcription factor binding profile. We hypothesize that changes at enhancers may constitute a mechanism by which painful experiences are recorded at a molecular level.

Involvement of mesolimbic dopaminergic network in neuropathic pain relief by treadmill exercise: A study for specific neural control with Gi-DREADD in mice.

BackgroundExercise alleviates pain and it is a central component of treatment strategy for chronic pain in clinical setting. However, little is known about mechanism of this exercise-induced hypoalgesia. The mesolimbic dopaminergic network plays a role in positive emotions to rewards including motivation and pleasure. Pain negatively modulates these emotions, but appropriate exercise is considered to activate the dopaminergic network. We investigated possible involvement of this network as a mechanism of exercise-induced hypoalgesia.MethodsIn the present study, we developed a protocol of treadmill exercise, which was able to recover pain threshold under partial sciatic nerve ligation in mice, and investigated involvement of the dopaminergic reward network in exercise-induced hypoalgesia. To temporally suppress a neural activation during exercise, a genetically modified inhibitory G-protein-coupled receptor, hM4Di, was specifically expressed on dopaminergic pathway from the ventral tegmental area to the nucleus accumbens.ResultsThe chemogenetic-specific neural suppression by Gi-DREADD system dramatically offset the effect of exercise-induced hypoalgesia in transgenic mice with hM4Di expressed on the ventral tegmental area dopamine neurons. Additionally, anti-exercise-induced hypoalgesia effect was significantly observed under the suppression of neurons projecting out of the ventral tegmental area to the nucleus accumbens as well.ConclusionOur findings suggest that the dopaminergic pathway from the ventral tegmental area to the nucleus accumbens is involved in the anti-nociception under low-intensity exercise under a neuropathic pain-like state.

Suppression of bone marrow–derived microglia in the amygdala improves anxiety-like behavior induced by chronic partial sciatic nerve ligation in mice

Chronic neuropathic pain causes abnormal sensitivities such as hyperalgesia and allodynia, and emotional abnormalities such as anxiety and depression. Although spinal cord microglia are involved in abnormal sensitivity to neuropathic pain, no previous studies have examined the mechanism of neuropathic pain–induced anxiety. Here, we examined the involvement of bone marrow (BM)–derived microglia aggregated in the amygdalae of mice with chronic neuropathic pain in the development of anxiety-like behavior. We prepared partial sciatic nerve ligations (PSNL) in mice that received bone marrow transplantation from green fluorescent protein (GFP)–Tg mice after irradiation with head protection, and examined GFP-positive microglia in the central nuclei of the amygdalae (CeA). On day 28 after PSNL, BM-derived microglia aggregated in the CeA concurrent with anxiety-like behavior. BM-derived microglia in the CeA highly expressed interleukin (IL)–1β and C-C chemokine receptor type 2 (CCR2). In addition, neurons in the CeA highly expressed monocyte chemotactic protein–1 (MCP-1), a ligand for CCR2, in PSNL-treated mice compared to sham-operated mice, suggesting that the MCP-1/CCR2 axis is involved in the recruitment of BM-derived microglia. Oral administration of a CCR2 antagonist decreased the number of BM-derived microglia in the CeA, and successfully reversed the anxiety-like behavior and hypersensitivity to mechanical stimuli in PSNL-treated mice. Microinjections of an IL-1β receptor antagonist directly into the CeA successfully reversed the anxiety-like behavior in the PSNL-treated mice even though the neuropathic pain persisted. These results suggest that the recruitment of BM-derived microglia to the CeA via the MCP-1/CCR2 axis and neuron–microglia interactions might be important in the pathogenesis of neuropathic pain–induced anxiety.

Astrocytic activation in the anterior cingulate cortex is critical for sleep disorder under neuropathic pain

Insomnia, depression, and anxiety disorder are common problems for people with neuropathic pain. In this study, mild noxious heat stimuli increased the duration and number of spontaneous pain-like behaviors in sciatic nerve-ligated mice. We used functional magnetic resonance imaging to visualize the increased blood oxygenation level-dependent signal intensity in the anterior cingulate cortex (ACC) of mice with sciatic nerve ligation under mild noxious stimuli. Such stimuli significantly increased the release of glutamate in the ACC of nerve-ligated mice. In addition, sciatic nerve ligation and mild noxious stimuli changed the morphology of astrocytes in the ACC. Treatment of cortical astrocytes with glutamate caused astrocytic activation, as detected by a stellate morphology. Furthermore, glutamate induced the translocation of GAT-3 to astrocyte cell membranes using primary cultured glial cells from the mouse cortex. Moreover, the GABA level at the synaptic cleft in the ACC of nerve-ligated mice was significantly decreased exposure to mild noxious stimuli. Finally, we investigated whether astrocytic activation in the ACC could directly mediate sleep disorder. With the optogenetic tool channel rhodopsin-2 (ChR2), we demonstrated that selective photostimulation of these astrocytes in vivo triggered sleep disturbance. Taken together, these results suggest that neuropathic pain-like stimuli activated astrocytes in the ACC and decreased the extracellular concentration of GABA via an increase in the release of glutamate. Furthermore, these findings provide novel evidence that astrocytic activation in the ACC can mimic sleep disturbance in mice.

Involvement of protein isoprenylation in neuropathic pain induced by sciatic nerve injury in mice

Isoprenylation is crucial step for activating many intracellular signaling. The present study examined whether inhibition of the protein isoprenylation could affect neuropathic pain in partial sciatic nerve-ligated mice. Intrathecal treatment with a geranylgeranyl transferase I inhibitor GGTI-2133, but not with a farnesyl transferase inhibitor FTI-277, dose-dependently blocked the thermal hyperalgesia in partial sciatic nerve-ligated mice. Intrathecal treatment with GGTI-2133 also attenuated the mechanical allodynia in partial sciatic nerve-ligated mice. Phosphorylated MARCKS expression was increased in the ipsilateral side of the spinal cord dorsal horn in partial sciatic nerve-ligated mice, and this increase was attenuated by GGTI-2133 but not by FTI-277. These results suggest that protein isoprenylation by geranylgeranyl transferase I is involved in the neuropathic pain.

Depression-related behavior and mechanical allodynia are blocked by 3-(4-fluorophenylselenyl)-2,5-diphenylselenophene in a mouse model of neuropathic pain induced by partial sciatic nerve ligation

Clinically, it is suggested that chronic pain might induce mood disorders like depression and anxiety. Based on this antidepressant drugs have emerged as a new therapy for pain. In this study, the effect of acute and subchronic treatments with 3-(4-fluorophenylselenyl)-2,5-diphenylselenophene (F-DPS) on behavioral changes induced by partial sciatic nerve ligation (PSNL) was evaluated. At the 4th week after surgery, PSNL caused a significant depression-like behavior in mice evaluated in the forced swimming test (FST) and the tail suspension test (TST), which was accompanied by increased pain sensitivity. The anxiety-like behavior assessed in the light–dark test (LDT) was not modified by PSNL. Acute treatment with F-DPS, at a dose of 1 mg/kg, intragastrically (i.g.) administered 30 min before the FST, produced a significant anti-immobility effect in PSNL mice. The antidepressant drug paroxetine showed acute antidepressant-like action at a dose 10 times higher than F-DPS. Subchronic treatment with F-DPS (0.1 mg/kg, i.g.) reversed depression-like behavior of sciatic nerve-ligated mice in the TST and FST and produced a significant anxiolytic-like action in both sham-operated and PSNL animals. Although the acute F-DPS treatment did not produce anti-allodynic effect, F-DPS subchronic treatment significantly reduced pain sensitivity in PSNL mice. These findings demonstrated that F-DPS blocked behavioral changes induced by neuropathic pain, suggesting that it might be attractive in the pharmacological approach of pain-emotion diseases.

Changes in circadian rhythm for mRNA expression of melatonin 1A and 1B receptors in the hypothalamus under a neuropathic pain-like state

Several clinical reports on neuropathic pain of various etiologies have shown that it significantly interferes with sleep. Inadequate sleep due to neuropathic pain may contribute to the stressful negative consequences of living with pain. It is generally recognized that melatonin (MT) system in the hypothalmus is crusial for circadian rhythm and sleep-wake transition. However, little, if any, is known about whether neuropathic pain could affect the MT system associated with sleep disturbance. In this study, we investigated the possible changes in circadian rhythm for the expression of MT receptors, especially MT1A and MT1B receptors, in the hypothalamus of mice with sciatic nerve ligation. The samples for real-time RT-PCR assay were prepared at 8:00, 14:00, 20:00, and 2:00 on day 7 after sciatic nerve ligation or sham operation. The mRNA expression of MT1A and MT1B receptors at 2:00 in sciatic nerve-ligated mice, which exhibited thermal hyperalgesia along with an increase in wakefulness and a decrease in nonrapid eye movement sleep, was significantly greater than those in sham-operated mice, whereas the levels of both MT1A and MT1B receptors at 8:00 in sciatic nerve-ligated mice were significantly lower than those in sham-operated mice. These findings suggest that neuropathic pain-like stimuli lead to sleep disturbance in parallel with changes in circadian rhythm for mRNA expression of MT 1A and 1B receptors in the hypothalamus of mice.

Orally active opioid μ/δ dual agonist MGM-16, a derivative of the indole alkaloid mitragynine, exhibits potent antiallodynic effect on neuropathic pain in mice.

(E)-Methyl 2-((2S,3S,7aS,12bS)-3-ethyl-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate (7-hydroxymitragynine), a main active constituent of the traditional herbal medicine Mitragyna speciosa, is an indole alkaloid that is structurally different from morphine. 7-Hydroxymitragynine induces a potent antinociceptive effect on mouse acute pain through μ-opioid receptors. In this study, we developed dual-acting μ- and δ-opioid agonists MGM-15 and MGM-16 from 7-hydroxymitragynine for the treatment of acute and chronic pain. MGM-16 showed a higher potency than that of 7-hydroxymitragynine and MGM-15 in in vitro and in vivo assays. MGM-16 exhibited a high affinity for μ- and δ-opioid receptors, with K(i) values of 2.1 and 7.0 nM, respectively. MGM-16 showed μ- and δ-opioid full agonistic effects in a guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding assay and in a functional test using electrically elicited guinea pig ileum and mouse vas deferens contractions. Systemic administration of MGM-16 produced antinociceptive effects in a mouse acute pain model and antiallodynic effects in a chronic pain model. The antinociceptive effect of MGM-16 was approximately 240 times more potent than that of morphine in a mouse tail-flick test, and its antiallodynic effect was approximately 100 times more potent than that of gabapentin in partial sciatic nerve-ligated mice, especially with oral administration. The antinociceptive effect of MGM-16 was completely and partially blocked by the μ-selective antagonist β-funaltrexamine hydrochloride (β-FNA) and by the δ-selective antagonist naltrindole, respectively, in a tail-flick test. The antiallodynic effect of MGM-16 was completely blocked by β-FNA and naltrindole in a neuropathic pain model. These findings suggest that MGM-16 could become a class of a compound with potential therapeutic utility for treating neuropathic pain.

Changes in the circadian rhythm of mRNA expression for µ‐opioid receptors in the periaqueductal gray under a neuropathic pain‐like state

Variation in the production of opioid receptors over a 24-h period is considered to contribute to circadian alterations in neuropathic pain. In this study, we investigated the possible changes in the circadian rhythm of mRNA expression for µ-opioid receptor (MOR), κ-opioid receptor (KOR), and adrenaline α2a receptor (α2a) in the periaqueductal gray, frontal cortex, thalamus, and spinal cord following sciatic nerve ligation in mice. In sham-operated mice, the latencies of hind paw-withdrawal in response to thermal stimuli at 14:00 and 20:00 were significantly greater than that at 8:00 and the latency at 2:00 was significantly less than those at 14:00 and 20:00, indicating a "rest" period-dominant circadian rhythm for thermal pain-thresholds. In sciatic nerve-ligated mice, the latencies of hind paw-withdrawal in response to thermal stimuli at 14:00 and 20:00 were significantly less than that at 8:00, and the latency at 2:00 was significantly greater than those at 14:00 and 20:00. A correlative tendency between the time-variation of pain latency and the time-variation of MOR mRNA expression was observed in the periaqueductal gray of sham-operated and sciatic nerve-ligated mice. In contrast, neither mouse showed a strong circadian rhythm for the expressions of KOR and α2a mRNAs in any region. The present data suggest that changes in MOR mRNA expression in the periaqueductal gray may be synchronized with the circadian rhythm for the pain threshold for noxious thermal stimuli. In contrast, neuropathic pain in mice exhibited a negative circadian pattern for the expression of MOR, KOR, and α2a receptors in the frontal cortex, thalamus, and spinal cord.

Multiple analyses of epigenetics modification in the spinal cord of sciatic nerve-ligated mice under neuropathic pain

Multiple analyses of epigenetics modification in the spinal cord of sciatic nerve-ligated mice under neuropathic pain

Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice

BackgroundEnkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn.ResultsTo develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP) under the control of the preproenkephalin (PPE) gene (penk1) promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the penk1 promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side) compared with the contralateral (non-ligated side). These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors.ConclusionsThese results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA), and are activated by forskolin and in conditions of nerve injury. The penk1-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and modulation of nociceptive information.