Intrathecal co-administration of ketamine and morphine preventing activation of astrocytes and decreasing releases of IL-1β and IL-6 from spinal cord in rats of morphine tolerance

Objective： To investigate the effects of intrathecal administration of ketamine, a non-competitive N-methy-D-aspartate receptor antagonist, combined with morphine on the activation of astrocytes and releases of IL-1β and IL-6 from spinal cord in the rats of morphine tolerance. Methods： Twenty-seven Sprague-Dawley male rats were randomly divided into sham-operated, morphine tolerance, and morphine plus ketamine group. The subarachnoid catheterication of all the rats was prepared by the method of Jianping Yang. Morphine 20μg in 10μl was adminstrated intrathecally to induce spinal morphine tolerance once daily for 5 consecutive days. Morphine and ketamine 250μg in 10μl total volume was given in morphine plus ketamine group. Three groups all received intrathecal morphine 5μg in 10μl for morphine challenge test at 24h after last administration of the morphine. After morphine challenge test, lumbar spinal tissues were taken for measurement of glial fibrillary acidic protein （GFAP） of astrocyte in lumbar spinal horn cord by immunohistochemistry and IL-1β and IL-6 of spinal cord by ELISA. Results： The decrease of %MPE induced by chronic intrathecal morphine was inhibibed by ketamine and hyperalgesia and allodynia induced by morphine-withdrawl were alleviated. The average areas, the average absorbency （A^-）, the integral absorbency （A） of GFAP immuno-reactive cells in the dorsal horn, and IL-1β and IL-6 of spinal cord were significantly larger in morphine tolerance group than in morphine plus ketamine group. Conclusion：Co-administration of ketamine and morphine enhance antinociceptive effect of morphine and prevent the development of morphine tolerance. Ketamine might attenuate the activation of astrocytes and inhibit the release of IL-1β and IL-6 from spinal cord in repeated intrathecal morphine rats.     

Effects of morphine and electroacupuncture on substance P level in spinal cord and their relation to pain threshold in rats

Immunoreactive substance P(Ir-SP)level,and substance P likeimmunoreactivity(SP-Li)in the spinal cord were observed with radioimmunoassay andimmunohistochemistry in rats after they were given an intraperitoneal injection of mor-phine(7.5mg/kg)or electroacupunctured(3V and 3Hz)on the “Jiaji point”.It wasfound that the pain threshold(PT),Ir-SP level and SP-Li in the dorsal horn of the spi-nal cord were more significantly increased in the animals after the administration ofmorphine or electroacupuncture than in the control(P<0.05～0.01).The combined effectsof morphine and electropacupuncture were even more powerful than either of the agentswas administered singly.Naloxone could block the analgesic effect and the elevation ofIr-SP due to morphine or electroacupuncture.The findings suggest that there is a synergismbe tween morphine and electroacupuncture and the analgesic effect of the 2 depends uponthe increase of Ir-SP level of the spinal cord mediated through the opiate receptors.     

Effect of continuous spinal anesthesia with ropivacaine on the ultrastructure of spinal cord and nerve roots in rats

Effect of continuous spinal anesthesia with ropivacaine on the ultrastructure of spinal cord and nerve roots in rats@孙志华$Dept Anesthesiol,Xiangya Hosp,Zhongnan Univ,Changsha 410008     

Switching from morphine to fentanyl attenuates the decline of μ-opioid receptor expression in periaqueductal gray of rats with morphine tolerance

Background Opioid switching is a therapeutic maneuver to improve analgesic response and/or reduce adverse side effects although the underlying mechanisms remain unknown.The μ-opioid receptor （MOR） has an important role in mediating the actions of morphine and other analgesic agents.This study is aimed at exploring the changes of MOR in the periaqueductal gray （PAG） in rats when morphine is substituted for equianalgesic fentanyl.Methods Forty rats were randomly assigned to five treatment groups：7 days normal saline group （N group）,7 days fentanyl group （F group）,7 days morphine group （M group）,7 days morphine and 7 days fentanyl-switching group （MF group）,and 14 days morphine group （MM group）.Rats repeatedly received subcutaneous injections of morphine sulfate （10 mg/kg） or equianalgesic fentanyl sulfate （0.1 mg/kg） twice daily.Rats＇ antinociceptive response to thermal pain was evaluated by the tail flick latency assay.MOR mRNA and protein expression in the PAG were measured using RT-PCR and Western blotting analyses respectively.Results This study showed that after morphine was substituted with fentanyl on day 8,the tail flick latency （TFL） increased from （3.9±0.4） seconds to （11.4±0.4） seconds.The results also demonstrated that both MOR mRNA and protein expression in the PAG of rats in the MF group were less than that in the M group （P〈0.05） but more than that in MM group （P〈0.05）.Conclusions Equianalgesic fentanyl was still antinociceptive effective in rats with morphine tolerance,which may be due to the switching from morphine to fentanyl attenuating the decline of MOR expression in the PAG of rats.     

Influence of neurotrophin-3 on Bcl-2 and Bax expressions in spinal cord injury of rats

Objective： To study the protective mechanisms of neurotrophin-3 （NT-3） on the spinal cord injury. Methods：Totally 105 SD rats were randomly divided into 3 groups： control group, experimental group and sham operation group. Rats from the former 2 groups were inflicted to animal model of acute spinal cord injury according to Allen＇s （WD） by situating a thin plastic tube in the subarachnoid space below the injury level for perfusion. Rats in experimental group received 20 ul NT-3 （200 ng） from the tube at 0, 4, 8, 12, 24 h and 3, 7 d after injury, and those in control group got an equal volume of normal saline at the same time. The animals in sham operation group only received opening vertebral plate and tube was put in subarachnoid space. The rats were sacrificed at 4, 8, 12, 24 h and 3, 7, 14 d post injury （n=5）. The expression levels of Bcl-2 and Bax proteins in spinal cord of rats were detected by immunohistochemistry assay. Results： The level of Bax protein in control group significantly increased as compared with those in sham operation group, and the peak reached at 8 h after spinal cord injury. The Bcl-2 proteins were always weakly positive. The Bax proteins in NT-3 group significantly decreased but the Bcl-2 proteins obviously increased as compared with those in control group. Conclusion： NT-3 can protect spinal cord from injury in vivo. One of the mechanisms is that NT-3 can inhibit abnormal expression of Pax protein, and increase the expression of Bcl-2 protein, then inhibit apoptosis after spinal cord injury.     

Effect of neurotrophin-3 on SOD and MDA in rats after acute spinal cord injury

Objective：To investigate the effect of neurotrophin-3 on the expressions of SOD and MDA in the injured spinal cord of rats. Methods： Totally 105 SD rats were randomly divided into 3 groups （n= 35）： sham group, control group and experimental group. Animal model of acute spinal cord was inflicted with Allen＇s method by a thin plastic tube situated in subarachnoid space below the injury level for perfusion. Rats in experimental group received 20μl NT-3 （200ng） from the tube at 0, 4, 8, 12, 24 h and 3, 7 d after injury, and those in control group got the equal volume of normal saline at the same time points. The animals in sham group only received opening vertebral plate and putting tube in subarachnoid space. The rats were sacrificed at 4, 8, 12, 24 h, and 3, 7, 14 d postinjury （n=5）. And the levels of superoxide dismutase （SOD） and malondialdehyde （MDA） in blood were observed with colorimetric method. Results： The serum level of SOD reduced obviously and the level of MDA raised obviously in rats after the injury, and the activity of SOD reached the lowest on day 3 and the concentration of MDA reached peak at the 7 d. In the experimental group, the SOD level was obviously higher （P〈0. 01）, and MDA level was lower than the control （P〈0. 01）. Conclusion： NT-3 can mitigate secondary injury of spinal cord in vivo. One of mechanisms is that inhibits abnormal expression of MDA and elevates the activity of SOD, thus the injury of free radical and lipid peroxidation is attenuated.     

Immunohistochemical changes of synaptophysin in the rat spinal cord after chronic constriction injury of sciatic nerve and ketamine intervention study

Immunohistochemical changes of synaptophysin in the rat spinal cord after chronic constriction injury of sciatic nerve and ketamine intervention study@陈敏$Dept Anesthesiol,Tongji Hosp,Tongji Med Col,Huazhong Univ Sci Technol,Wuhan 430030     

Survival and migration of Schwann cells after the vascularized peripheral nerve grafted into spinal cord in rats

Objective：To study the survival and ability of inducing axonal regeneration of the Schwann cells after the peripheral nerve being grafted into spinal cord. Methods：A total of 30 adult female Wistar rats were randomly divided into the VN （vascularized peripheral nerve） and PN （peripheral nerve） groups. A 5-mm spinal cord defect of the left posterior column was made at the T1-3 vertebral level. The defect was grafted with the vascularized or isolated peripheral nerve respectively. The survival and proliferation of the Schwann cells were assessed by histological and morphometric analysis 8 weeks after the operation. Resuits：In the VN group, the peripheral nerve grew into the cord with lots of Schwann cells survived and proliferated, and had more NF and S-100 positive fibers than in the PN group. Conclusion：The vascularized peripheral nerve enhances the survival and proliferation of the Schwann cells and prompts the regener- ation of injured axon of the central nerve system to certain degree.     

Hyperbaric oxygen intervention on expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in spinal cord injury models in rats

Background Hyperbaric oxygen （HBO） intervention is a main therapeutic method and the curative effect has been certified for spinal cord injury （SCI）, but the mechanisms of the neuroprotective effect of HBO on SCI remain elusive. This study aimed to observe the change in expression of hypoxia-inducible factor-1α （HIF-1α） and vascular endothelial growth factor （VEGF） after SCI at different time points and to investigate the neuroprotective mechanism of HBO on SCI in rats.     

The effect of spinal cord injury on the expression of TGF-β and TNF-α in rat articular cartilage

Objective： To observe the expression of TGF-β and TNF-α in the spinal cord injured rat model and discuss the significance of the articular cartilage metabolism. Methods： 36 SD female rats were randomly divided into 2 groups： Rats models of spinal cord injury were implemented by Allen method. T10 laminectomy was performed in the control group. Both groups of rats were killed respectively in 1w, 3w and 6w. Hematoxylin-eosin stain was given to each slice in the model group and control group. Immunohistochemical stain was applied by using ABC method in the expression of TGF-β and TNF-α. Those expressed level were performed in image analysis and statistics process. Results： TGF-β and TNF-α were mainly distributed on the surface layer of the articular cartilage, with a weak expression in control group. The expression of TNF-α in the model group was more significant than that in the control group in the lw, and still remained an evident difference with that in control group until the 6w（P 〈 0.05）. TGF-β expression of the model group had no remarkable difference with the control group in the lw （P 〉 0.05） and prominently became stronger at 6w（P 〈 0.05）. Conclusion： The expression of TNF-α occurred early in the development of spinal cord injury, and the expression of TGF-β became stronger with the revival of spinal neural function. Both expressions were strengthened in articular cartilage in the 3rd week.     

The effect of spinal cord injury on the expression of TGF-β and TNF-α in rat articular cartilage

Objective: To observe the expression of TGF-β and TNF-α in the spinal cord injured rat model and discuss the significance of the articular cartilage metabolism. Methods: 36 SD female rats were randomly divided into 2 groups: Rats models of spinal cord injury were implemented by Allen method. T10 laminectomy was performed in the control group. Both groups of rats were killed respectively in 1w, 3w and 6w. Hematoxylin-eosin stain was given to each slice in the model group and control group. Immunohistochemical stain was applied by using ABC method in the expression of TGF-β and TNF-α. Those expressed level were performed in image analysis and statistics process. Results: TGF-β and TNF-α were mainly distributed on the surface layer of the articular cartilage, with a weak expression in control group. The expression of TNF-α in the model group was more significant than that in the control group in the 1w, and still remained an evident difference with that in control group until the 6w(P ＜ 0.05). TGF-β expression of the model group had no remarkable difference with the control group in the lw (P ＞ 0.05) and prominently became stronger at 6w(P ＜ 0.05). Conclusion: The expression of TNF-o occurred early in the development of spinal cord injury, and the expression of TGF-β became stronger with the revival of spinal neural function. Both expressions were strengthened in articular cartilage in the 3rd week.     

Expression and significance of IL-6 mRNA and its protein in liver after injury in rats

Thereisagraduallyincreasedinterestoninterle'ukin--6(II---6)anditbecomesahotspotofresearchbecauseofitsrelationshipwithothercytokines,itsparticipationinthesynthesisofacutephaseproteinsanditseffectsontheearlydamagesoforgansinsystemicinjury['j.ThisstudywasdesignedtoobservethedistributionandcelllocalizationofII---6andIL--6mRNAinliverafterburninjury,endotoxemiaorthecombinationofthetwoinrats1andtheeffectsofII---6onthedevelopmentofliverdamageswereinvestigated.MATERIALSANDMETHODSOnehundreda…     

Expression of NF-kB in Schwann apoptosis in spinal cord following cells and its effect on motor neuron sciatic nerves injury in rats

Objective：To explore the expression of nuclear factor-kappa B （NF-kB） in Schwann cells （SCs） and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in adult rats. Methods： Thirty-six adult Sprague-Dawley （SD） rats were divided randomly into normal control group （n=6）, and sciatic nerves crushing group （n= 30）, and the later was further equally randomized into 5 subgroups： 1, 3, 7, 14, and 21 d post-injury groups. The expression of NF-kB of normal and injured nerves were examined by immunohistochemistry staining, and the apoptosis of motor neurons in spinal cord of lumbar 4 to lumbar 6 （L4-L6） was investigated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling （TUNEL） assay. Both were qua.ntitated by image analysis. Results： In crushing group, except 21 d post-injury group, the expression of NF-kB was markedly higher than that in the normal control group （P〈0.05, P〈0. 01）. At 1 d after sciatic nerves crushing, the expression of NF-kB was obviously up-regulated, reached peak at 3 d, and recovered at 21 d. The same trend was observed in the time-course on motor neuron apoptosis after sciatic nerves injury. Correlation analyses revealed that motor neuron apoptosis was significantly and positively correlated with the expression of NF-kB following sciatic nerves injury （r= 0. 976 0, P〈0. 01）. Conclusion： After injury of sciatic nerves, the presence and up-regulation of NF-kB in SCs may be involved in motor neuron apoptosis in L4-L6 spinal cord.     

Expression of IL-1β and TNF-α in MCMV Myocarditis and Its Role

Autoi mmune myocarditis in Coxsackievirus B3(CB3)-infected mice is associated with infiltrationof the heart by inflammatory cells that secreteTNF-αand IL-1[1].In this study we examine theexpression of cytokines , TNF-αand IL-1βpro-duced locallyinthe heart inresponse to MCMVin-fectionin order to determine their potential role inthe development of acute myocarditis .1MATERIALS AND METHODS1 .1Reagents and AntibodiesMonoclonal goat anti-mouse IL-1β, TNF-αanti-body and SABC …     

Hyperbaric oxygen intervention on expression of hypoxia inducible factor-1α and vascular endothelial growth factor after spinal cord injury models in rats

【Abstract】Objective: To observe the change of expression of Hypoxia inducible factor-1α (HIF-1α) and Vascular endothelial growth factor (VEGF) after spinal cord injury at different time and to investigate the Neuroprotective mechanism of hyperbaric oxygen (HBO) on spinal cord injury (SCI) in rats. Methods: 160 adult Sprague-Dawley rats, weighing between 250g and 300g,were randomly assigned to 4 experimental groups (n=40 per group). SCI group: SCI was created with special NYU impactor of Allen’s by a 25gram-centimeter (GCM) impacting energy on T10 of the spinal cord. SCI HBO group: hyperbaric oxygen therapy after spinal cord injury model was established. Sham operation（SH）group: only laminectomy of T10 and no impact on the spinal cord was done. SH HBO group: hyperbaric oxygen therapy after sham operation. The hindlimb functional recovery was evaluated using Basso Beattie Bresnahan (BBB) score and the expression of HIF-1α and VEGF were observed with fluorescent quantitation PCR and Western-Blot method of six rats picked randomly from each group at different times of 1, 3, 7 and 14 days after operation. Result: Rats in the SCI group and SCI HBO group were paralyzed completely after operation with BBB 0-1 score. Rats in the SH group and SH HBO group could walk after sham operation with BBB 20-21 score. The BBB scores of rats in SCI HBO group was higher than that in SCI group at 7d and 14d time point obviously (P＜0.05);The expression of HIF-1α and VEGF in SCI group and SCI HBO group was higher than in SH group and SH HBO group at any time point obviously (P＜0.05);while the SCI HBO group presented the least expression of HIF-1α at 3d,7d and 14d time points (P＜0.05) and more expression of VEGF at 7d and 14d (P＜0.05) than that of the SCI group with significant difference. Conclusion: The experimental research showed HBO could improve the hind limb functional recovery after SCI in rats;The higher expression of HIF-1α and VEGF could promote repair of damaged spinal cord after SCI;The elevation and duration of the expression of VEGF and the expression of HIF-1α by HBO intervention maybe inversely related in the repair of damaged spinal cord and neuroprotective effect.     

The effects of ciliary neurotrophic factor on neurological function and glial activity following contusive spinal cord injury in the rats

Ciliary neurotrophic factor (CNTF) has been implicated in the pathophysiology of injury to the central nervous system. The rapid increase in CNTF production following spinal cord injury (SCI) in rats is thought to serve a role in the neuronal survival and functional recovery. In this study, 40 SD rats were divided into four groups.- sham-     

Changes in Behavior and Amino Acid Neurotransmitters in the Brain of Rats with Seizure Induced by IL-1β or IL-6

To explore the mechanism of epilepsy induced by IL-1β and 1L-6, the changes of glutamie acid (Glu) and GABA immunoreaetion in the cerebral cortex and hippoeampus of rats with seizure induced by IL-1β or IL-6 were studied. Rats were randomly divided into 3 groups: control group (intraeerebroventrieular injection (iev) of NS), IL-1β group (iev injection of IL-1β) and IL-6 group (i. e. v. injection of IL-6). 120 min after the iev injection of reagents of IL-1β or IL-6, behavioral changes were observed and Glu and GABA in the cerebral cortex and hippoeampus were examined by means of immunohistoehemistry. Our results showed that no seizure developed in the control group, while moderate seizure was observed in IL-1β group and IL-6 group. Compared with the controls, the immunoreaetion of Glu was significantly increased, while GABA was obviously decreased in IL-1β group and IL-6 group after 120 min. Our study suggested that the IL-1β and IL-6 might promote and induce epilepsy by increasing Glu and decreasing GABA in the cerebral cortex and hippoeampus.     

Role of protein tyrosine kinase in IL-1β induced activation of mitogen-activated protein kinase in fibroblast-like synoviocytes of rheumatoid arthritis

Objectives To study mitogen-activated protein kinase (MAPKs) activation in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) under the stimulation of IL-1β, and to elucidate the role of protein tyrosine kinase (PTK) in the activation of MAPKs. Methods Primary cultures of RA FLS were used. Western blot was applied to examine transient changes in protein tyrosine phosphorylation status and MAPKs activation in RA FLS stimulated with IL-1β at various doses, and over different periods. Genistein, the specific PTK inhibitor, was used to evaluate the inhibitory role in activation of MAPKs by IL-1β.Results IL-1β transiently increased protein tyrosine phosphorylation, and activated the MAPKs cascades (mainly ERK(2), JNK(2) and P(38)) in RA FLS. There was no obvious difference in MAPKs activation among different doses of IL-1β (1 IU/ml,10 IU/ml, 100 IU/ml), but the peak activation of ERK(2), JNK(2) and P(38)took place at 5 min, 15 min and 1 min, respectively, after stimulation with IL-1β. The activation of ERK(2)was inhibited by genistein, but the inhibitory role on that of JNK and P(38)was relatively weak. Conclusions During signal transduction of IL-1β in RA FLS, tyrosine phosphorylation was increased transiently, the MAPKs cascade was activated in a few minutes, and there was heterogenicity in the activation among three subfamily members. PTK had a role in the activation of ERK, but had weak effects on that of JNK and P(38).