The distribution of Mas-related G protein-coupled receptor A in cerebrospinal fluid-contacting nucleus of normal rats and its up-regulation in neuropathic pain / 生理学报

This study was aimed to observe the distribution of Mas-related G protein-coupled receptor A (MrgA) in cerebrospinal fluid (CSF)-contacting nucleus of normal rats and its expression in neuropathic pain, and to provide morphological evidence for CSF-contacting nucleus to participate in neuropathic pain. The model of neuropathic pain with chronic constriction injury (CCI) of the sciatic nerve was made in Sprague-Dawley rats. The thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were measured. The expressions of MrgA in the CSF-contacting nucleus were examined by double labeling with immunofluorescent staining. The results showed that on the 5th, 7th, 10th and 14th days, the values of MWT and TWL in CCI group were all lower than those in sham group (P < 0.05). MrgA was found to be distributed in CSF-contacting nucleus of normal rats; and the expression was markedly up-regulated in rats at the peak of neuropathic pain. Our data suggest that CSF-contacting nucleus may participate in neuropathic pain through the MrgA-mediated signaling pathway.

GluN2B-BDNF pathway in the cerebrospinal fluid-contacting nucleus mediates nerve injury-induced neuropathic pain in rats / 生理学报

The present study was aimed to investigate the role of GluN2B-BDNF pathway in the cerebrospinal fluid-contacting nucleus (CSF-CN) in neuropathic pain. Intra-lateral ventricle injection of cholera toxin subunit B conjugated with horseradish peroxidase (CBHRP) was used to label the CSF-CN. Double-labeled immunofluorescent staining and Western blot were used to observe the expression of GluN2B and BDNF in the CSF-CN. Chronic constriction injury of sciatic nerve (CCI) rat model was used to duplicate the neuropathic pain. Pain behavior was scored to determine the analgesic effects of GluN2B antagonist Ro 25-6981 and BDNF neutralizing antibody on CCI rats. GluN2B and BDNF were expressed in the CSF-CN and their expression was up-regulated in CCI rats. Intra-lateral ventricle injection of GluN2B antagonist Ro 25-6981 or BDNF neutralizing antibody notably alleviated thermal hyperalgesia and mechanical allodynia in CCI rats. Moreover, the increased expression of BDNF protein in CCI rats was reversed by intra-lateral ventricle injection of Ro 25-6981. These results suggest that GluN2B and BDNF are expressed in the CSF-CN and alteration of GluN2B-BDNF pathway in the CSF-CN is involved in the modulation of the peripheral neuropathic pain.

The distribution of MAP kinase phosphatase-1 in the cerebrospinal fluid-contacting nucleus and its functional contribution to depressive behaviors / 生理学报

The purpose of this research is to explore the distribution and expression of MAP kinase phosphatase-1 (MKP-1) in cerebrospinal fluid (CSF)-contacting nucleus in depression, and provide experimental evidence to reveal the biological function and regulatory mechanisms of CSF-contacting nucleus in depression. Depression model was produced by chronic forced swimming stress (CFSS) in Sprague-Dawley (SD) rats. Intracerebroventricular injection of cholera toxin subunit B (CTb) labeled with horseradish peroxidase (CB-HRP) was used to specifically mark distal CSF-contacting nucleus. The rate of animal growth and behavioral tests including sucrose preference test (SPT) and open field test (OFT) were used to validate the model of depression. The expressions of MKP-1 and fos proteins in CSF-contacting nucleus were detected by immunofluorescence. Software Image-Pro Plus version 6.0 was used to count the positive neurons. The results showed that, the distributions of MKP-1 were found in the CSF-contacting nucleus. After 28 days of swimming, the rats in stress group had a lower growth rate, a less consumption of sucrose and lower scores of OFT compared to control group. The number of neurons double labeled with CB-HRP/fos or CB-HRP/MKP-1 in stress group was significantly higher than that in control group (P < 0.01). These results suggest that the CSF-contacting nucleus may be involved in the process of depression via the MKP-1.

Phosphorylation of protein kinase C in cerebrospinal fluid-contacting nucleus modulates the inflammatory pain in rats / 生理学报

The present study was aimed to investigate the role of cerebrospinal fluid-contacting nucleus (CSF-CN) neurons in modulation of inflammatory pain and underlying mechanism. The inflammatory pain model was made by subcutaneous injection of the complete Freund's adjuvant (CFA) into the left hind paw of rats. The phosphorylation level of PKC (p-PKC) was examined by Western blot. Thermal withdrawal latency (TWL) of the rats was measured to assess inflammatory pain. The results showed that, compared with the sham controls, the inflammatory pain model rats showed shortened TWL on day 1, 3, and 7 after CFA injection, as well as increased level of p-PKC in CSF-CN neurons at 24 h after CFA injection. The administration of GF109203X, a PKC inhibitor, into lateral ventricle decreased the level of p-PKC protein expression and increased TWL in the model rats. These results suggest that blocking the PKC pathway in CSF-CN neurons may be an effective way to reduce or eliminate the inflammatory pain.

Neuropathic pain enhances expression of HCN2 channel in rat cerebrospinal fluid-contacting nucleus / 生理学报

The purpose of this research is to explore the distribution and expression of hyperpolarization-activated cyclic nucleotide-gated channels subtype 2 (HCN2) in cerebrospinal fluid (CSF)-contacting nucleus in neuropathic pain, and provide experimental evidence to reveal the biological function and regulation mechanisms of CSF-contacting nucleus in neuropathic pain. Neuropathic pain model was produced by chronic constriction injury (CCI) in Sprague-Dawley (SD) rats. Intracerebroventricular injection of cholera toxin subunit B (CTb) labeled with horseradish peroxidase (CB-HRP) was used to specifically mark distal CSF-contacting nucleus. The thermal withdrawal latency and mechanical withdrawal threshold of rats were recorded to detect the change of pain threshold. The expressions HCN2 channel and c-Fos proteins in CSF-contacting nucleus were detected by immunofluorescence and Western blot. The results showed that, compared with the control group, CTb-treated rats did not show any differences in the expressions of HCN2 channel and c-Fos proteins in CSF-contacting nucleus, as well as pain threshold. At 7, 14 d after CCI operation, the model rats showed not only significantly increased expressions of HCN2 channel and c-Fos in CSF-contacting nucleus, but also decreased pain threshold. ZD7288, a HCN2 channel blocker, could reverse the above changes in neuropathic pain model rats. These results suggest that the CSF-contacting nucleus may be involved in the process of neuropathic pain via the HCN2 channel.

HCN ion channel: biological characteristics and functions in pain / 生理学报

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in vertebrate are reverse voltage-dependent, and its activation depends on the hyperpolarization of cell and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP) or other signal transduction cascades. The distribution, quantity, and activation states of HCN channels differ in tissues throughout the body. By modulating If/If current, HCN channels may influence the resting membrane potential, and thus importantly regulate neuronal excitability, dendritic integration of synaptic potentials, and synaptic transmission. Evidence exhibits that HCN channels participate in pain and other physiological and pathological process. Pharmacological treatment targeting HCN channels is of benefit to relieve pain and other related diseases.

Targeted damage of the cerebrospinal fluid-contacting nucleus contributes to the pain behavior and the expression of 5-HT and c-Fos in spinal dorsal horn of rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>The changes of pain threshold and expression of 5-hydroxytryptamine(5-HT) and c-Fos in spinal dorsal horn of rats were observed after targetedly damaged the cerebraspinal fluid-contacting nucleus (CSF-contacting nucleus) to provide experimental evidence for the mechanism of regulating pain CSF-contacting nucleus involved in.</p><p><b>METHODS</b>Male adult SD rats were divided into control, sham, choleratoxin subunit B conjugated with horse-radish peroxidase (CB-HRP)and damage groups randomly. The pain threshold using mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded and analyzed. Immunofluorescence method was used to observe the expression of 5-HT and c-Fos in spinal dorsal horn.</p><p><b>RESULTS</b>Compared with the control, sham and CB-HRP groups, the MWT and TWL of the damage group were significantly increased (P < 0.05). The results of immunofluorescence showed that 5-HT was detected in neurons of CSF-contacting nucleus. In the damage group, the number of neurons of CSF-contacting nucleus reduced gradually, and no survived neurons were observed at the 10th day. Meanwhile, both the expression of 5-HT and c-Fos in spinal dorsal horn increased gradually, and negatively correlated with the change of pain threshold.</p><p><b>CONCLUSION</b>The method of targeted damaging CSF-contacting nucleus by cholera toxin subnit B conjugated with saporin(CB-SAP) is scientific and reliable, and it results in the changes of pain threshold and expression of 5-HT and c-Fos in spinal dorsal horn of rats. This study suggests that CSF-contacting nucleus participate in the regulation of pain, moreover, 5-HT and c-Fos play important roles in this regulation.</p>

Distribution and effects of 5-HT(1A) receptors in distal cerebral spinal fluid-contacting neurons in rat brain parenchyma in neuropathic pain / 生理学报

The present study aimed to explore the effects of 5-HT(1A) receptors in the distal cerebral spinal fluid-contacting neurons (CSF-CNs) in rat brain parenchyma in neuropathic pain. The model of neuropathic pain with chronic constriction injury (CCI) of the sciatic nerve was made in Sprague-Dawley rats. The behavioral studies of animal were scored and the paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured. The distribution and expression of 5-HT(1A) receptors were observed in the distal CSF-CNs in brain parenchyma with double labeling of cholera toxin subunit B with horseradish peroxidase (CB-HRP) and 5-HT(1A) receptors with immunhistochemistry. The relationship between 5-HT(1A) receptors in distal CSF-CNs and neuropathic pain was analyzed. The results were as follows. On days 1, 3, 7, 14 of neuropathic pain, the PWL was 19.37±2.74, 12.04±1.77, 8.74±1.15 and 12.31±1.94, respectively; the PWT was 18.58±3.62, 13.05±1.81, 6.66±1.43 and 11.55±2.01, respectively. CB-HRP-labeled neurons of two clusters were always found in definite region but not in other area in brain parenchyma. The number of neurons double-labeled with CB-HRP/5-HT(1A) receptors in each group was 276.14±36.00, 161.72±28.41, 108.64±6.81, and 139.76±44.64, which was about 95%, 60%, 40% and 55% of all CB-HRP-labeled neurons in the four courses of neuropathic pain, respectively. It is suggested that the distal CSF-CNs are always located in a special region in rat brain parenchyma and most of them have 5-HT(1A) receptors. A negative correlation is found between the expression of 5-HT(1A) receptors and neuropathic pain.

Expression of drebrin in the distal cerebrospinal fluid contacting neurons of rats with chronic constriction injury of sciatic nerve / 生理学报

To observe the expression of drebrin in the distal cerebrospinal fluid contacting neurons (dCSF-CNs) of rats with chronic constriction injury (CCI) of sciatic nerve by immunofluorescence technique, male Sprague-Dawley rats were randomly divided into three groups: control group, sham surgery group and CCI group. The behavior of rats was scored. After choleratoxin subunit B-conjugated horseradish peroxidase (CB-HRP, 3 muL) was injected into the lateral cerebroventricle to trace dCSF-CNs, the expression of drebrin was observed in the dCSF-CNs through immunofluorescence double staining and laser scanning confocal microscopy technique. The results showed that only the pain threshold of CCI group was decreased. The dCSF-CNs were clearly displayed in three groups. No drebrin expression was observed in the control and sham groups. In CCI group, drebrin was markedly expressed in intracytoplasm. It is suggested that the technique displaying dCSF-CNs with immunofluorescence is successful and the dCSF-CNs are possibly involved in the transmission of nociceptive information under the neuropathic pain state.

The lesion of CSF contacting neurons in rat brain parenchyma inhibits the development of morphine dependence and withdrawal / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effect of CSF contacting neurons (CSF-CNs) lesion in rat dorsal raphe nucleus (DRN) on the scores of morphine withdrawal symptoms precipitated by naloxone and the nNOS expression in dorsal horn of spinal cord, and study the relationship between the distal CSF-CNs in rat brain parenchyma and the development of morphine dependence and withdrawal.</p><p><b>METHODS</b>Chemical lesion of neurons the injection of cholera toxin subunit B with horseradish peroxidase (CB-HRP) into one of the rats lateral ventricles, TMBST reaction, nNOS immunohistochemistry and Western blot were used in this study.</p><p><b>RESULTS</b>The withdrawal symptoms by the naloxone precipitated attenuated obviously after the lesion of CSF-CNs in rat DRN, scores of all signs were significantly decreased about 38% compared to that of withdrawal group without lesion (P < 0.05). The withdrawal symptoms scores of vehicle withdrawal group and side lesion withdrawal group were not changed significantly (P > 0.05). Neurons in the location of CSF-CNs concentrated in the rat brain slices of lesion group were damaged obviously, there were only few CB-HRP positive neurons around the lesion location. But the location and the quantity of the CB-HRP positive neurons in the brain slices of the group without lesion was stable relatively, and their appearance was very clear. After the lesion, the nNOS expression and the quantity of the nNOS positive neurons in dorsal horn of spinal cord decreased significantly compared to that of withdrawal group without lesion (P < 0.05), but it also increased significantly compared to that of normal group and dependence group (P < 0.01).</p><p><b>CONCLUSION</b>The lesion of distal CSF contacting neurons attenuated the scores of morphine withdrawal symptoms precipitated by naloxone and the nNOS expression in dorsal horn of spinal cord. The distal CSF contacting neurons in rat brain parenchyma partly participated in the development of morphine dependence and naloxone precipitated withdrawal possibly by the modulation of NO (nitric oxide).</p>

Distribution and expression of p38 MAPK in the distal cerebrospinal fluid contacting neurons in brain of rat by noise stress / 中国应用生理学杂志

<p><b>AIM</b>To observe the distribution and expression of p-p38MAPK in the distal cerebrospinal fluid contacting neurons in brain of rat by noise stress.</p><p><b>METHODS</b>By a double-labelled method combing the tracing of CB-HRP and the immunohistochemical technique p-p38MAPK, the distribution and expression of p-p38MAPK in the distal cerebrospinal fluid contacting neurons(csf cn) were observed following noise stress. Expression of p-p38MAPK and double-labelled of CB-HRP/p-p38MAPK were also observed in rat brain after noise stress.</p><p><b>RESULTS</b>Two groups of CB-HRP labeled neuron clusters consistently appeared in certain regions of the brainstem but none in other regions of the brain. Without noise stress exposure, only a few neurons were found double-labeled by CB-HRP/p-p38MAPK. After 1 day noise stress exposure, only few neurons double-labeled by CB-HRP/p-p38MAPK were observed in the above-mentioned regions. After 5 days, the number of neurons double-labeled by CB-HRP/p-p38MAPK increased significantly compared with the control group (P < 0.05). After 10 days, the number of neurons double-labeled by CB-HRP/p-p38MAPK increased significantly compared with the control group (P < 0.05). After 20 days, both of the numbers of neurons double-labeled by CB-HRP/p-p38MAPK increased significantly compared with that of the control group (P < 0.01).</p><p><b>CONCLUSION</b>Two groups of distal cerebrospinal fluid contacting neuron clusters consistently existed in certain regions of the brain parenchyma, and in these clusters only a few neurons con rained p-p38MAPK. After noise stress exposure of different durations (days 1, 5, 10, 20), the number of distal cerebrospinal fluid contacting neurons with p-p38MAPK increased significantly with increasing days. The results indicate that distal cerebrospinal fluid contacting neurons are special neurons existing consistently in brain, including distal cerebrospinal fluid contacting neurons with p-p38MAPK which may participate in the whole procedure of signal transduction or central modulation in noise stress response and play greater roles with increasing days.</p>

Activation of ERK1/2 in spinal cord contributes to the development of acute cystic pain in rabbits / 神经科学通报·英文版

Objective To investigate the role of activated extracellular signal-regulated kinase 1/2 (ERK1/2) in spinal cord in the development of cystic pain in rabbit. Methods We observed the relationship between the activation of ERK1/2 in spinal cord and nociceptive behaviors, as well as the effect of U0126, a mitogen-activated protein kinase (MEK, upstream protein of ERK1/2) inhibitor, on cystic pain in rabbits by behavioral test, immunohistochemistry and western blot analysis. Results After injecting 0.5 ml formalin into gallbladder, the behaviors such as grasping of the cheek and licking of the abdomen increased in 30 min, with a significant increase in pERK1/2 expression in the spinal cord, as well as the pERK1/2 immunoreactive cells located in laminae V-VII and X of the dorsal horn and ventral horn of T6 spinal cord. Administration of U0126 (100 - 400 mu g/kg body weight, i.v., 10 min before instillation of formalin) could attenuated nociceptive behaviors dose-dependently, but could not restrain the nociceptive behaviors completely even at the maximal efficient dose of 400 mu g/kg body weight. Conclusion Activated ERK1/2 in the spinal cord at least partly participates in the development of acute inflammatory cystic pain induced by formalin in rabbits.

cAMP response-element binding protein participates in the phosphorylated extracellular signal-regulate kinase mediated neuropathic pain / 生理学报

It has been reported that extracellular signal-regulate kinase (ERK) is involved in the modulation of nociceptive information and central sensitization produced by intense noxious stimuli and/or peripheral tissue inflammation. Few studies have explored the relationship between ERK and cAMP response-element binding protein (CREB) in neuropathic pain after nerve injury, such as chronic constriction injury (CCI) of the sciatic nerve. In the present study, CCI model was employed to investigate the activation of ERK on the expression of phosphorylated CREB (pCREB) in chronic neuropathic pain. Lumbar intrathecal catheters were chronically implanted in male Sprague-Dawley rats. The left sciatic nerve was loosely ligated proximal to the sciatica's trifurcation at around 1.0- mm intervals with 4-0 silk suture. Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphorothioate-modified antisense oligonucleotides (ODN) were intrathecally administered one day before and three consecutive days after CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal lantency (PWL) to radiant heat and von Frey filaments respectively. The expression of pCREB and Fos were assessed by both Western blot and immunohistochemical analysis. The results showed that intrathecal injection of U0126 or ERK antisense ODN attenuated significantly CCI-induced mechanical and thermal hyperalgesia. Correlating with behavior results, the injection also markedly suppressed the increase of CCI-induced pCREB and c-Fos expression. The results obtained suggest that CREB participates in the pERK-mediated neuropathic pain.

Different roles of the spinal protein kinase C alpha and gamma in morphine dependence and naloxone-precipitated withdrawal / 生理学报

Our previous studies showed that spinal neurons sensitization was involved in morphine withdrawal response. This study was to investigate the roles of spinal protein kinase C (PKC) alpha, gamma in morphine dependence and naloxone-precipitated withdrawal response. To set up morphine dependence model, rats were subcutaneously injected with morphine (twice a day, for 5 d). The dose of morphine was 10 mg/kg in the first day and was increased by 10 mg/kg each day. On day 6, 4 h after the injection of morphine (50 mg/kg), morphine withdrawal syndrome was precipitated by an injection of naloxone (4 mg/kg, i.p.). Chelerythrine chloride (CHE), a PKC inhibitor, was intrathecally injected 30 min before the administration of naloxone. The scores of morphine withdrawal symptom and morphine withdrawal-induced allodynia were observed. One hour after naloxone-precipitated withdrawal, Fos protein expression was assessed by immunohistochemical analysis and Western blot was used to detect the expression of cytosol and membrane fraction of PKC alpha and gamma in the rat spinal cord. The results showed that intrathecal administration of CHE decreased the scores of morphine withdrawal, attenuated morphine withdrawal-induced allodynia and also inhibited the increase of Fos protein expression in the spinal cord of morphine withdrawal rats. The expression of cytosol and membrane fraction of PKC alpha was significantly increased in the spinal cord of rats with morphine dependence. Naloxone-precipitated withdrawal induced PKC alpha translocation from cytosol to membrane fraction, which was prevented by intrathecal administration of CHE. During morphine dependence, but not naloxone-precipitated withdrawal, PKC gamma in the spinal cord translocated from cytosol to membrane fraction, and intrathecal administration of CHE did not change the expression of PKC gamma in the spinal cord of naloxone-precipitated withdrawal rats. It is suggested that up-regulation and translocation of PKC in the spinal cord contribute to morphine dependence and naloxone-precipitated withdrawal in rats and that PKC alpha and gamma play different roles in the above-mentioned effect.

Activation of p38 mitogen-activated protein kinase in spinal cord contributes to chronic constriction injury-induced neuropathic pain / 生理学报

The present study aimed to investigate the role of spinal p38 mitogen-activated protein kinase (p38 MAPK) activation in chronic constriction injury (CCI) of the sciatic nerve induced neuropathic pain. CCI model was produced by loosely ligating the left sciatic nerve proximal to the sciatica's trifurcation with 4-0 silk thread in male Sprague-Dawley rat. SB203580, a specific inhibitor of the p38 MAPK, was intrathecally administered on day 5 post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal lantency (PWL) to radiant heat and the paw withdrawal threshold (PWT) to von Frey filaments respectively. The protein levels of the phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated cAMP response element binding protein (pCREB) were assessed by Western blot analysis. The results showed that CCI significantly increased the expressions of cytosolic and nuclear p-p38 MAPK in the spinal cord. Intrathecal administration of SB203580 dose-dependently reversed the established mechanical allodynia and thermal hyperalgesia induced by CCI. Correlated with behavior results, SB203580 dose-dependently inhibited the CCI-induced increase of the expressions of cytosolic and nuclear p-p38 MAPK and nuclear pCREB in the spinal cord. Taken together, these findings suggest that the activation of p38 MAPK pathway contributes to the development of neuropathic pain induced by CCI, and that the function of p-p38 MAPK may partly be accomplished via the CREB-dependent gene expression.

Microinjection of L-NAME into dorsal raphe nucleus inhibits nociceptive response in sigmoid pain model of rats / 生理学报

By means of Fos immunocytochemistry, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and microinjection methods, the role of nitric oxide synthase (NOS) of dorsal raphe (DR) neurons in the modulation of rats sigmoid pain was studied. The results showed: (1) Rats exhibited aversive behavioral responses related to visceral pain after injecting formalin into the sigmoid wall. NOS neurons in DR were up-regulated, in addition, about 8% of NOS-labeled neurons were Fos positive. By contrast, there were no Fos/NOS double-labeled neurons in the control group. (2) Formalin-induced sigmoid pain scores and the expression of Fos in the spinal cord at S1 segment were decreased after microinjecting L-NAME into the DR. These findings suggest that NOS neurons are involved in the modulation of formalin-induced sigmoid pain and that NO may play an important role in the transmission of visceral nociceptive message in the midbrain.

Propofol depresses c -fos expression of NOS neurons in the spinal cord of rats with inflammatory pain / 生理学报

In formalin pain model, the effect of propofol on Fos expression in the spinal cord was examined by means of c -fos oncogene immunohistochemistry and NADPH-d histochemistry. Fos-like immunoreactive (FLI) neurons were mainly found in the ipsilateral dorsal horn after injection of formalin, some of which were FLI/NOS double-labeled neurons. Most of the FLI or FLI/NOS double-labeled neurons were observed in the medial part of lamina and the outer lamina . Before or after injection of formain, i.p. injection of propofol significantly decreased the number of FLI and FLI/NOS double-labeled neurons in all laminae (P<0.05 or P<0.01). By single i.p. injection of propofol or normal saline, few FLI neurons were found in the spinal cord. The results suggest that the antinociceptive function of propofol is possibly involved in the depression of the NOS neurons in the spinal cord.