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Objective To investigate the effect of intrathecal morphine preconditioning (ITMP) on the excitability of substantia gelatinosa (SG) neurons in the dorsal horn of the spinal cord in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty-six adult male Sprague-Dawley rats,weighing 200-300 g,in which intrathecal catheters were successfully placed without complications,were randomly divided into 3 groups (n =12 each) using a random number table:sham operation group (group S),group I/R,and group ITMP.Myocardial I/R injury was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion.In group ITMP,the rats received intrathecal morphine 3 μg/kg (10 μl) by three cycles of 5 min infusions interspersed with 5 min infusion-free periods starting from 30 min before ischemia,and the equal volume of normal saline was given instead of morphine in group I/R.At 10 min of reperfusion,6 rats randomly selected in each group were sacrificed,and the T2-6 segments of the spinal cords were acutely isolated to prepare spinal cord slices.The resting potential,threshold of action potential (APT),peak of action potential (APP) and action potential duration in SG neurons in the dorsal horn of spinal cord slices were determined using the whole-cell patch-clamp technique,and the number of action potentials evoked by currents of 40,60,80 and 100 pA was recorded.At 120 min of reperfusion,6 rats randomly selected in each group were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size (IS) and area at risk (AAR),and IS/AAR ratio was calculated.The expression of c-fos in the T2-5 dorsal horns of the spinal cords was detected by Western blot.Results Compared with group S,the IS/AAR ratio was significantly increased,the expression of c-fos was up-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was increased,APT was decreased,and APP was increased in group I/R (P＜0.05).Compared with group I/R,the IS/AAR ratio was significantly decreased,the expression of c-fos was down-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was decreased,APT was increased,and APP was decreased in group ITMP (P＜0.05).Conclusion The mechanism by which ITMP attenuates myocardial I/R injury is related to decrease in the excitability of SG neurons in the dorsal horn of the spinal cord and reduction of responses to nociceptive stimuli in rats.
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Objective To investigate the effect of intrathecal morphine preconditioning on the expression of nerve growth factor (NGF) in the dorsal root ganglia (DRG) in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty healthy adult male Sprague-Dawley rats in which intrathecal catheters were successfully placed without complications,weighing 250-350 g,were randomly divided into 5 groups (n =6 each) using a random number table:sham operation group (S group),I/R group,intrathecal morphine preconditioning group (ITMP group),μ receptor antagonist CTOP + intrathecal morphine preconditioning group (CTOP + ITMP group),and CTOP control group (CTOP group).Myocardial ischemia was induced by 30 min of occlusion of the anterior descending branch of the left coronary artery followed by 120 min of reperfusion in all the groups except S group.Intrathecal morphine preconditioning was produced by 3 cycles of 5 min intrathecal injection of morphine 3 μg/kg (10 μl) at 5 min intervals within 30 min before ischemia in ITMP group.In CTOP+ITMP and CTOP groups,1 μg/μ1 CTOP 10 μl was injected intrathecally at 10 min before morphine preconditioning and 40 min before ischemia,respectively.At 120 min of reperfusion,the rats were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size,and DRGs were removed for determination of the expression of NGF by using immunohistochemistry and Western blot.Results Compared with S group,the myocardial infarct size was significantly increased,and the expression of NGF in DRGs was significantly up-regulated in I/R group (P＜0.05).Compared with I/R group,the myocardial infarct size was significantly decreased,and the expression of NGF in DRGs was significantly down-regulated in ITMP group (P＜ 0.05),and no significant change was found in the parameters mentioned above in CTOP group (P＞0.05).Compared with ITMP group,the myocardial infarct size was significantly increased,and the expression of NGF in DRGs was significantly up-regulated in CTOP+ITMP and CTOP groups (P＜0.05).Conclusion The mechanism by which intrathecal morphine preconditioning reduces myocardial I/R injury is related to activation of spinal μ receptors,inhibition of NGF expression in DRGs,and reduction of responses to noxious stimulation in the rats.
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Objective To investigate the role of mitochondrial apoptotic pathway in intrathecal doxepin?induced apoptosis in rat neurons. Methods Twenty?four adult male Wistar rats, weighing 250-300 g, in which intrathecal catheters were successfully implanted without complications, were randomly divided into 4 groups ( n=6 each) using a random number table: control group ( group C) and different concentrations of doxepin groups (D5, D10 and D20 groups). In D5, D10 and D20 groups, 5, 10, and 20 mmol∕L doxepin 0. 2 μl∕g were injected intrathecally, respectively. In group C, the equal volume of normal saline was given instead. At 6 h after intrathecal administration, the animals were sacrificed, and the lumbar segments of the spinal cords were obtained for detection of the cell apoptosis ( by TUNEL assay) , expression of Bax and Bcl?2 ( by immunohistochemistry) , release of cytochrome c ( Cyt c) , and expression of caspase?3, caspase?8 and caspase?9 mRNA ( using real?time reverse transcriptase polymerase chain reaction) . Apoptosis rate and Bcl?2∕Bax ratio were calculated. Results Compared with group C, the expression of Bax was significantly up?regulated, the expression of Bcl?2 was down?regulated, Bcl?2∕Bax ratio was decreased, the release of Cyt c and apoptosis rate were increased, and the expression of caspase?3 and caspase?9 mRNA was up?regulated in D10 and D20 groups, the expression of Bcl?2 was down?regulated in group D20 ( P0.05). The expression of Bax was significantly up?regulated, Bcl?2∕Bax ratio was decreased, the release of Cyt c and apoptosis rate were increased, and the expression of caspase?3 and caspase?9 mRNA was up?regulated in D10 and D20 groups, and the expression of Bcl?2 was down?regulated in group D20 as compared with group D5 ( P0.05) . Conclusion Mitochondrial apoptotic pathways, but not death receptor pathway, is involved in intrathecal doxepin?induced apoptosis in rat neurons.
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Objective To evaluate the effect of intrathecal ropivacaine on spinal histone deacetylase 1 (HDAC1) and HDAC2 expression in the rats with neuropathic pain (NP).Methods Thirty adult male Sprague-Dawley rats, weighing 220-250 g, in which intrathecal catheters were successfully placed, were randomly divided into 3 groups (n=10 each) using a random number table: sham operation group (group S), group NP, and NP + ropivacaine group (group R).NP was induced by chronic constriction injury (CCI) in anesthetized rats.Sciatic nerve was exposed and 4 loose ligatures were placed on the left sciatic nerve at 1 mm intervals with 4-0 chromic catgut.Starting from 7th day after CCI, 0.25％ ropivacaine 20 μl was injected intrathecally in group R, while the equal volume of normal saline was given instead of ropivacaine in S and CCI groups once a day for 7 consecutive days.At 1 day before CCI (T0) ,and 3, 7, 10, 14, 17 and 21 days after CCI (T1-6) , the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.After the pain threshold was measured at T4,3 rats from each group were sacrificed, and their lumbar enlargements were harvested for determination of the expression of HDAC1 and HDAC2 by Western blot.Results Compared with group S, the MWT was significantly decreased, and the TWL was shortened at T2-6, and the expression of HDAC 1 and HDAC2 was up-regulated at T4 in NP and R groups (P＜0.05).Compared with group NP, the MWT was significantly increased at T4, and the TWL was prolonged at T3.4, and the expression of HDAC1 and HDAC2 was downregulated at T4 in group R (P＜0.05).Conclusion The mechanism by which intrathecal ropivacaine alleviates NP may be related to inhibited up-regulation of spinal HDACI and HDAC2 expression in rats.
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Objective To investigate the effect of intrathecal ketamine on the intracellular calcium ion concentration [Ca2+ ]1 in the spinal dorsal root ganglion (DRG) neurons in a rat model of chronic neuropathic pain produced by chronic constrictive injury (CCI) .Methods Thirty-six male SD rats weighing 160-180 g were randomly divided into 3 groups ( n = 12 each) : Ⅰ sham-operated group; Ⅱ CCI group and Ⅲ ketamine + CCI group. The animals were anesthetized with intraperitoneal pentobarbital 40 mg?kg-1 . The right sciatic nerve was exposed and 4 loose ligatures were placed on the trunk of the nerve at 1-2 mm interval. In sham-operated group (Ⅰ) the sciatic nerve was exposed but not ligated. Intrathecal catheter was implanted at L4,5 and correct placement was confirmed by aspiration of cerebro-spinal fluid. In group Ⅲ ketamine 1 mg ? kg-1 was administered intrathecally. 30 min before and on the 1st, 2nd, 3rd, 5th, 7th, 9th and 11th day after operation. In group Ⅰ and Ⅱ normal saline (NS) was given intrathecally instead of ketamine. Thermal and mechanical hyperalgesia were measured with ice-cold water and von-Frey filaments on the 7th and 14th day after operation. The animals were killed by cervical dislocation on the 7th (n = 6) and 14th ( n = 6) day. Bilateral DRG of L4-6 spinal nerve were removed and homogenized and centrifuged at 5 000 r/min. DRG neurons were isolated and [ Ca2+ ] i was measured by flow cytometry.Results In group Ⅱ (CCI) pain threshold to von-Frey hair stimulation decreased by 80.3% (on the 7th day) and 84.8% (14th day) while pain threshold to noxious thermal stimulation increased by 309.4% (the 7th day) and 336.2% (14th day) (P