慢性吗啡耐受大鼠脊髓背角神经元磷酸化突触素Ⅰ表达的变化

目的 观察慢性吗啡耐受大鼠脊髓背角神经元磷酸化突触素Ⅰ(p-Synapsin Ⅰ)表达的变化.方法 雄性SD大鼠45只,体重150～180 g,月龄1～2月,随机分为5组(n=9):假手术组(S组)、生理盐水组(NS组)、吗啡组(M组)、氯胺酮组(K组)和吗啡+氯胺酮组(M+K组).除S组外,所有大鼠均行鞘内置管,恢复3 d后鞘内给药,NS组给予生理盐水40 μl,M组给予吗啡20 μg,K组给予氯胺酮30μg,M+K组分别给予吗啡20μg及氯胺酮30 μg,2次/d,连续7 d.于给药前(T_0,基础状态)、给药后1、3、5、7 d及停药后1d(T_(1～5))时测定机械缩爪阈值(PWT)与热缩爪潜伏期(PWL),最后一次测定痛阈后处死大鼠,取L3～6脊髓背角,测定p-Synapsin Ⅰ(Ser603)的表达.结果 与基础值比较,M组T_(1,2)时PWT升高,T_(4,5)时PWT降低,T1～3时PWL延长,T_5时PWL缩短,M+K组T_(1～5),时PWT升高,PWL延长(P＜0.05).与S组和NS组比较,M组T_(1,2)时PWT升高,T_(4,5)时PWT降低,T1～3时PWL延长,T_5时PWL缩短,M+K组T_(1～5),时PWT升高,PWL延长(P＜0.05),K组PWT和PWL差异无统计学意义(P＞0.05).与M组比较,M+K组T_(2～5)时PWT升高,T_(3～5)时PWL延长(P＜0.05).与S组和NS组比较,M组和M+K组p-Synapsin Ⅰ(Ser603)表达上调(P＜0.05),K组p-Synapsin Ⅰ(Ser603)表达差异无统计学意义(P＞0.05);与M组比较,M+K组p-Synapsin Ⅰ(Ser603)表达下调(P＜0.05).结论 脊髓背角神经元Synapsin Ⅰ的磷酸化参与了大鼠慢性吗啡耐受的形成,吗啡促进Synapsin Ⅰ磷酸化的部分机制与激活N-甲基-D-天冬氨酸受体有关.     

米诺环素对骨癌痛-吗啡耐受大鼠脊髓CX3C趋化因子受体1mRNA表达的影响

目的 探讨米诺环素对骨癌痛-吗啡耐受大鼠脊髓CX3C趋化因子受体1(CX3CR1)mRNA表达的影响.方法 清洁级雌性SD大鼠,体重180～200 g,月龄3月,经L3,4间隙行鞘内置管,取鞘内置管成功的大鼠60只,采用随机数字表法,将大鼠随机分为4组:正常对照组(C组,n=10)、米诺环素对照组(M组,n=10)、骨癌痛-吗啡耐受组(BM组,n=20)和米诺环素治疗组(BM+M组,n=20).C组不作任何处理;BM组和BM+M组右侧胫骨上段骨髓腔注入Walker256细胞10 μl(400个/μl)制备骨癌痛模型,术后第10天开始鞘内注射吗啡20 μg/kg(100 μl),2次/d,连续7 d,制备骨癌痛-吗啡耐受模型,注射吗啡第8天分别经鞘内注射20μl生理盐水或米诺环素0.25 mg/kg(20 μl),1次/d,连续3 d;M组不行手术,于BM组注射吗啡第8天时鞘内注射米诺环素0.25 mg/kg,1次/d,连续3 d.于术前、术后3、6、9 d、鞘内注射吗啡4、7、10、12 d(T0～7)时测定机械缩足阈值(MWT)和机械缩足持续时间(MWD).C组和M组于T7时,BM组和BM+M组于T6,7时取L4～6脊髓节段,采用免疫组化法检测小胶质细胞标记物——OX-42表达,采用实时PCR法检测CX3CR1 mRNA表达水平.结果 与C组和M组比较,BM组T2,3.5～7时、BM+M组T2,3,5,时MWT降低,MWD延长,CX3CR1 mRNA和OX-42表达上调(P＜0.01).与BM组比较,BM+M组L6,7时MWT升高,MWD缩短,CX3CR1 mRNA和OX-42表达下调(P＜0.01).C组和M组上述指标差异无统计学意义(P＞0.05).结论 米诺环素可抑制骨癌痛-吗啡耐受大鼠脊髓CX3CR1 mRNA的表达,可能是其拮抗吗啡耐受的机制之一.

Abstract：

Objective To investigate the effect of minocycline on spinal CX3 C chemokine receptor 1(CX3 CR1)mRNA expression in morphine-tolerant rats with bone cancer pain.Methods Sixty female SD rats weighing 180-200 g in which intrathecal(IT)catheter was successfully placed at L3,4 interspace without complications were randomly divided into 4 groups:control group(group C,n=10);minocycline group(group M,n=10);bone cancer pain + morphine tolerance group(group BM,n=20)and bone cancer pain+morphine tolerance+ minocycline group(group BM+M,n=20).Bone cancer pain was induced by injection of breast cancer cells(Walker256)10μl(400/μl)into upper segment of bone marrow of right tibia.Morphine tolerance was induced by IT injection of morphine 20 μg/kg twice a day for 7 consecutive days starting from the 10th day after intratibia injection in BM and BM + M groups. Minocycline 0.25 mg/kg was injected IT once a day for 3 consecutive days in group M and after the model of bone cancer pain and morphine tolerance was established in group BM + M. Mechanical withdrawal threshold (MWT) and mechanical withdrawal duration (MWD) were determined before (T0, baseline) and at3, 6 and 9 days after operation (T1-3) and at 4, 7, 10 and 12 days after IT morphine injection was started (T4-7).The animals were sacrificed at T6 and T7 respectively in BM and BM + M groups and at T7 in C and M groups.The lumbar segment of the spinal cord (L4-6) was removed for determination of CX3 CR1 mRNA (by RT-PCR) and OX-42 expression (by immuno-histochemistry) .Results There was no significant difference in MWT and MWD at all time points between group C and group M. MWT was significantly decreased while MWD prolonged in morphine tolerant rats with cancer pain in group BM as compared with C and M groups. The hyperalgesia was significantly attenuated by IT minocycline in group BM + M. Spinal CX3 CR1 mRNA and OX-42 expression was significantly increased in group BM than in C and M groups. IT minocycline attenuated the increase in spinal CX3 CR, mRNA and OX-42 expression induced by bone cancer. Conclusion IT minocycline can inhibit spinal CX3CR1 mRNA expression, thereby antagonizing morphine tolerance in morphine-tolerant rats with bone cancer pain.     

鞘内注射布托啡诺混合氯胺酮对炎性痛大鼠脊髓背角cAMP-PKA-CREB信号转导通路的影响

Objective To investigate the effect of intrathecal administration of a mixture of butorphanol and ketamine on cAMP-PKA-CREB signal transductian pathway in the spinal dorsal ham of the rats with inflammatory pain. Methods Twenty-four male SD rats, weighing 240-280 g,in which intrathecal catheters were successfully placed, were divided into 4 groups randomly (n = 6 each): inflammatory pain group (group IP), butorphanol group (group B), ketamine group (group K), and butorphanol + ketamine group (group BK). The inflammatory pain was induced by injection of 5% formalin 50 μl into the plantar surface of left hind paw. Normal saline 10 μl, butorphannl 12.5 μg, ketamine 50 μg, and a mixture of butorphanol 12.5 μg and ketamine 50 μg was injected intrathecally 30 min before subcutaneous injection of formalin in group IP, B, K and BK respectively.Pain intensity score (PIS) was used to assess pain behavior every 5 min within an hour after subcutaneous injection of formalin. The animals were killed at 2 h after subcutaneous injection of formalin, and the L5 segment of the spinal cord was removed for determination of protein kinase A (PKA) and phosphorylated cAMP response element binding protein (p-CREB) expression using immunohistochemistry. Results Fonnahn administration induced pain behaviour expressed as two phases. PIS scores, PKA and p-CBEB expression, and staining scores were significantly lower during the fast and second phases in group BK than in group IP (P < 0.05 or 0.01), while no significant differences were found in the indices mentioned above between group B and IP and between group K and IP (P>0.05). Conclusion lntrathecal injection of a mixture of butorphanol and ketamine can reduce inflammatory pain in rats, and the mechanism may be related to the cAMP-PKA-CREB signal transduction pathway.     

异丙酚对氯胺酮诱发新生大鼠脑损伤的影响

目的 探讨异丙酚对氯胺酮诱发新生大鼠脑损伤的影响.方法 新生SD大鼠80只,日龄7 d,雌雄不拘,体重12～20 g,采用随机数字表法,将大鼠随机分为4组(n=20):生理盐水对照组(NS组)腹腔注射生理盐水1 ml;氯胺酮致脑损伤组(K组)、异丙酚对照组(P组)和异丙酚+氯胺酮组(PK组)分别腹腔注射氯胺酮70mg/kg、异丙酚70mg/kg、异丙酚70mg/kg+氯胺酮70mg/kg,每隔2 h注射1次,共3次.于苏醒后24 h时各组随机取10只大鼠,处死后取海马组织,采用TUNEL法检测海马CA1区神经元凋亡情况,计算凋亡率,采用免疫组化法检测Bcl-2和Bax的蛋白表达,腹腔注射后21d时各组余大鼠采用Morris水迷宫实验测定学习记忆功能(逃避潜伏期和穿越平台次数).结果 与NS组相比,K组神经元凋亡率升高,P组和PK组Bcl-2蛋白表达上调,其余各组Bax蛋白表达上调,逃避潜伏期延长,穿越平台次数减少(P＜0.05或0.01);与K组相比,PK组神经元凋亡率降低,P组Bax蛋白表达下调,P组和PK组Bcl-2蛋白表达上调,逃避潜伏期缩短,穿越平台次数增加(P＜0.05).结论 异丙酚可减轻氯胺酮诱发新生大鼠的脑损伤,可能与其调节Bcl-2和Bax蛋白表达从而抑制海马神经元凋亡有关.

Abstract：

Objective To investigate the effect of propofol on the cerebral injury induced by ketamine in neonatal rats. Methods Eighty 7-day-old SD rats of both sexes, weighing 12-20 g, were randomly divided into 4 groups (n = 20 each): normal saline (NS) group, ketamine-induced cerebral injury group (group K), propofol group (group P) and propofol combined with ketamine group (group PK). Group NS received intraperitoneal NS 1 ml. In groups K, P and PK, ketamine 70 mg/kg, propofol 70 mg/kg and propofol 70 mg/kg + ketamine 70 mg/kg were injected intraperitoneally once every 2 h for 3 times respectively. Ten rats in each group were selected and sacrificed at 24 h after emergence from anesthesia and the hippocampi obtained to determine the neuronal apoptosis (by TUNEL) and Bcl-2 and Bax protein expression(by immunohitochemistry). The apoptosis rate was calculated.The other 10 rats in each group were selected at 21 days after the intraperitoneal injection and the learning and memory functions (escape latency and frequency of crossing the original platform) were evaluated using Morris water maze. Results Compared with group NS, the apoptosis rate was significantly increased in group K, Bcl-2 protein expression was up-regulated in groups P and PK, and Bax protein expression was up-regulated, the escape latency was significantly prolonged and the frequency of crossing the original platform was significantly decreased in the other groups (P ＜ 0.05 .or 0.01 ). Compared with group K, the apoptosis rate was significantly decreased in group PK, Bax protein expression was down-regulated in group P, and Bcl-2 protein expression was up-regulated,the escape latency was significantly shortened and the frequency of crossing the original platform was significantlyincreased in groups P and PK ( P ＜ 0.05). Conclusion Propofol can reduce the cerebral injury induced by ketamine in neonatal rats, and the regulation of the Bcl-2 and Bax protein expression and inhibition of the neuronal apoptosis in hippocampus may be involved in the mechanism.     

远位触液神经元5-HT1A受体在大鼠神经病理性痛中的作用

目的 评价远位触液神经元5-羟色胺1A(5-HT1A)受体在大鼠神经病理性痛中的作用.方法 雄性SD大鼠40只,体重230～270 g,采用随机数字表法,将其随机分为4组(n=10):假手术组(S组)、神经病理性痛组(NP组)、二甲基亚砜组(DMSO组)和8-羟基-2-(双-正丙胺基)-四氢萘满组(8-OH-DPAT组).采用坐骨神经慢性压迫法(CCI制备大鼠神经病理性痛模型,S组仅暴露坐骨神经,但不结扎.CCI后第7天,8-OH-DPAT组和DMSO组向远位触液神经元分别缓慢注射5-HT1A受体特异性激动剂8-OH-DPAT或DMSO 1 μl,5 min内注射完毕.分别于CCI前(T0)、CCI后第7天(T1)和给药后3、6 h(T2,3)时,测定缩足潜伏期(PWL)和缩足阈值(PWT).于给药后6 h时处死大鼠,取脑组织,采用免疫荧光标记法检测远位触液核神经元5-HT1A受体的表达.结果 与S组比较,NP组、DMSO组和8-OH-DPAT组T1时PWL缩短,PWT降低(P＜0.01);与DMSO组比较,8-OH-DPAT组T2和T3时PWL延长,PWT升高(P＜0.01).与S组比较,NP组和DMSO组5-HT1A受体表达下调(P＜0.01);与NP组和DMSO组比较,8-OH-DPAT组5-HT1A.受体表达上调(P＜0.01);NP组和DMSO组间5-HT1A受体表达比较差异无统计学意义(P＞0.05).结论 远位触液神经元5-HT1A受体参与了大鼠神经病理性痛的调控.

Abstract：

Objective To evaluate the role of 5-HT1A receptors in distal cerebrospinal fluid (CSF)-contacting neurons in neuropathic pain (NP) in rats. Methods Forty male SD rats weighing 230-270 g were randomly divided into 4 groups (n = 8 each): sham operation group (group S); NP group; dimethyl sulfoxide (DMSO) group and 8-OH-DPAT (a specific 5-HT1A receptor agonist) group. NP was induced by chronic constrictive injury (CCI) in groups NP, DMSO and 8-OH-DPAT. Four silk ligatures were placed on the sciatic nerve at 1 mm intervals . In group S, the sciatic nerve was exposed but not ligated. 8-OH-DPAT and DMSO 1 μl were injected into the region where most of CSF-contacting neurons are present over 5 min on 7th day after CCI in groups 8-OH-DPAT and DMSO respectively. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured before CCI, on 7th day after CCI, and at 3 and 6 h after administration. The rats were sacrificed 6 h after administration, and the brain tissues removed for determination of the expression of 5-HT1A receptors in the distal CSF-contacting neurons by immunofluorescence. Results Compared with group S, PWL was significantly shorten and PWT decreased at T, in groups NP, DMSO and 8-OH- DPAT (P ＜ 0.01) . Compared with group DMSO, PWL was significantly prolonged and PWT increased at T2 and T3 in group 8-OH-DPAT ( P ＜ 0.01). The 5-HT1A receptor expression was significantly down-regulated in groups NP and DMSO compared with group S, while up-regulated in group 8-OH-DPAT compared with groups NP and DMSO ( P ＜ 0.01). There was no significant difference in 5-HT1A receptor expression between groups NP and DMSO ( P ＞ 0.05). Conclusion 5-HT1A receptors in distal CSF-contacting neurons are involved in the regulation of NP in rats.     

鞘内注射吗啡加可乐定对切口痛大鼠脊髓背角蛋白激酶A催化亚单位表达的影响

Objective To observe the effect of intrathecal clonidine plus morphine on expression of protein kinase A (PKA) catalytic subunit in the spinal dorsal horn in a rat model of incisional pain. Methods Eighty male Sprague-Dawley rats were divided randomly into five groups: sham group, control group, pre-incisional morphine 2.5 μg group, pro-incisional clonidine 5 μg group and preincisional morphine 2.5 μg plus clonidine 5 lag group (n=16). lntrathecal catheter and the model of incisional pain were pro-duced according to Yaksh and Brennan's described method respectively. Changes of pain behavior were assessed by mechanical with-drawal threshold (MWT) and thermal withdrawal latency(TWL). The expressions of PKA catalytic subunit in the spinal dorsal horn were assessed by immunohistochemical method and western blotting analysis. Results Compared with sham group, MWT and TWL in control group were decreased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were increased significantly in control group (P<0.01). Compared with control group, MWT and TWL in pre-incision morphine 2 μg plus clonidine 5 lag group were increased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were decreased significantly in pre-incision morphine 2 μg plus clonidine 5 μg group (P<0.01). However, MWT, TWL and the number of positive cells and pro-tein expression of PKA catalytic subunit in the spinal dorsal horn changed with no statistical significance in pre-incisional morphine 2.5 μg group and pre-incisional clonidine 5 μg group compared with control group. Conclusion lntrathecal clonidine significantly enhances the antinociceptive effect of intrathecal morphine in a rat model of incisional pain, which might be associated with inhibi-tion of the increased expression of PKA catalytic subunit in spinal cord.     

鞘内注射吗啡加可乐定对切口痛大鼠脊髓背角蛋白激酶A催化亚单位表达的影响

Objective To observe the effect of intrathecal clonidine plus morphine on expression of protein kinase A (PKA) catalytic subunit in the spinal dorsal horn in a rat model of incisional pain. Methods Eighty male Sprague-Dawley rats were divided randomly into five groups: sham group, control group, pre-incisional morphine 2.5 μg group, pro-incisional clonidine 5 μg group and preincisional morphine 2.5 μg plus clonidine 5 lag group (n=16). lntrathecal catheter and the model of incisional pain were pro-duced according to Yaksh and Brennan's described method respectively. Changes of pain behavior were assessed by mechanical with-drawal threshold (MWT) and thermal withdrawal latency(TWL). The expressions of PKA catalytic subunit in the spinal dorsal horn were assessed by immunohistochemical method and western blotting analysis. Results Compared with sham group, MWT and TWL in control group were decreased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were increased significantly in control group (P<0.01). Compared with control group, MWT and TWL in pre-incision morphine 2 μg plus clonidine 5 lag group were increased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were decreased significantly in pre-incision morphine 2 μg plus clonidine 5 μg group (P<0.01). However, MWT, TWL and the number of positive cells and pro-tein expression of PKA catalytic subunit in the spinal dorsal horn changed with no statistical significance in pre-incisional morphine 2.5 μg group and pre-incisional clonidine 5 μg group compared with control group. Conclusion lntrathecal clonidine significantly enhances the antinociceptive effect of intrathecal morphine in a rat model of incisional pain, which might be associated with inhibi-tion of the increased expression of PKA catalytic subunit in spinal cord.     

鞘内注射吗啡加可乐定对切口痛大鼠脊髓背角蛋白激酶A催化亚单位表达的影响

Objective To observe the effect of intrathecal clonidine plus morphine on expression of protein kinase A (PKA) catalytic subunit in the spinal dorsal horn in a rat model of incisional pain. Methods Eighty male Sprague-Dawley rats were divided randomly into five groups: sham group, control group, pre-incisional morphine 2.5 μg group, pro-incisional clonidine 5 μg group and preincisional morphine 2.5 μg plus clonidine 5 lag group (n=16). lntrathecal catheter and the model of incisional pain were pro-duced according to Yaksh and Brennan's described method respectively. Changes of pain behavior were assessed by mechanical with-drawal threshold (MWT) and thermal withdrawal latency(TWL). The expressions of PKA catalytic subunit in the spinal dorsal horn were assessed by immunohistochemical method and western blotting analysis. Results Compared with sham group, MWT and TWL in control group were decreased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were increased significantly in control group (P<0.01). Compared with control group, MWT and TWL in pre-incision morphine 2 μg plus clonidine 5 lag group were increased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were decreased significantly in pre-incision morphine 2 μg plus clonidine 5 μg group (P<0.01). However, MWT, TWL and the number of positive cells and pro-tein expression of PKA catalytic subunit in the spinal dorsal horn changed with no statistical significance in pre-incisional morphine 2.5 μg group and pre-incisional clonidine 5 μg group compared with control group. Conclusion lntrathecal clonidine significantly enhances the antinociceptive effect of intrathecal morphine in a rat model of incisional pain, which might be associated with inhibi-tion of the increased expression of PKA catalytic subunit in spinal cord.     

鞘内注射吗啡加可乐定对切口痛大鼠脊髓背角蛋白激酶A催化亚单位表达的影响

Objective To observe the effect of intrathecal clonidine plus morphine on expression of protein kinase A (PKA) catalytic subunit in the spinal dorsal horn in a rat model of incisional pain. Methods Eighty male Sprague-Dawley rats were divided randomly into five groups: sham group, control group, pre-incisional morphine 2.5 μg group, pro-incisional clonidine 5 μg group and preincisional morphine 2.5 μg plus clonidine 5 lag group (n=16). lntrathecal catheter and the model of incisional pain were pro-duced according to Yaksh and Brennan's described method respectively. Changes of pain behavior were assessed by mechanical with-drawal threshold (MWT) and thermal withdrawal latency(TWL). The expressions of PKA catalytic subunit in the spinal dorsal horn were assessed by immunohistochemical method and western blotting analysis. Results Compared with sham group, MWT and TWL in control group were decreased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were increased significantly in control group (P<0.01). Compared with control group, MWT and TWL in pre-incision morphine 2 μg plus clonidine 5 lag group were increased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were decreased significantly in pre-incision morphine 2 μg plus clonidine 5 μg group (P<0.01). However, MWT, TWL and the number of positive cells and pro-tein expression of PKA catalytic subunit in the spinal dorsal horn changed with no statistical significance in pre-incisional morphine 2.5 μg group and pre-incisional clonidine 5 μg group compared with control group. Conclusion lntrathecal clonidine significantly enhances the antinociceptive effect of intrathecal morphine in a rat model of incisional pain, which might be associated with inhibi-tion of the increased expression of PKA catalytic subunit in spinal cord.     

鞘内注射吗啡加可乐定对切口痛大鼠脊髓背角蛋白激酶A催化亚单位表达的影响

Objective To observe the effect of intrathecal clonidine plus morphine on expression of protein kinase A (PKA) catalytic subunit in the spinal dorsal horn in a rat model of incisional pain. Methods Eighty male Sprague-Dawley rats were divided randomly into five groups: sham group, control group, pre-incisional morphine 2.5 μg group, pro-incisional clonidine 5 μg group and preincisional morphine 2.5 μg plus clonidine 5 lag group (n=16). lntrathecal catheter and the model of incisional pain were pro-duced according to Yaksh and Brennan's described method respectively. Changes of pain behavior were assessed by mechanical with-drawal threshold (MWT) and thermal withdrawal latency(TWL). The expressions of PKA catalytic subunit in the spinal dorsal horn were assessed by immunohistochemical method and western blotting analysis. Results Compared with sham group, MWT and TWL in control group were decreased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were increased significantly in control group (P<0.01). Compared with control group, MWT and TWL in pre-incision morphine 2 μg plus clonidine 5 lag group were increased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were decreased significantly in pre-incision morphine 2 μg plus clonidine 5 μg group (P<0.01). However, MWT, TWL and the number of positive cells and pro-tein expression of PKA catalytic subunit in the spinal dorsal horn changed with no statistical significance in pre-incisional morphine 2.5 μg group and pre-incisional clonidine 5 μg group compared with control group. Conclusion lntrathecal clonidine significantly enhances the antinociceptive effect of intrathecal morphine in a rat model of incisional pain, which might be associated with inhibi-tion of the increased expression of PKA catalytic subunit in spinal cord.     

慢性吗啡耐受大鼠脊髓神经元兴奋性氨基酸转运体3表达的变化

目的 观察慢性吗啡耐受大鼠脊髓神经元兴奋性氨基酸转运体3(EAAT3)表达的变化.方法 成年雄性SD大鼠45只,随机分为5组(n=9),除假手术组(S组)外,生理盐水组(NS组)、吗啡组(M组)、氯胺酮组(K组)和吗啡+氯胺酮组(M+K组)均进行鞘内置管,鞘内置管后3 d进行鞘内给药,Ns组鞘内注射生理盐水40 μl,M组给予吗啡20μg,K组给予氯胺酮30μg,M+K组给予吗啡20μg+氯胺酮30μg,2次/d,连续7 d.分别在给药前、给药1、3、5、7 d及停药后1 d时测定50%缩爪阈值(PWT)与辐射热缩爪潜伏期(PWL),最后一次测定痛阈后处死大鼠,分别采用免疫印迹分析和免疫组化法检测脊髓EAAT3的表达水平.结果 与S组比较,M组给药1、3 d时PWT升高,给药7 d及停药后1 d时PWT降低,给药1、3、5 d时PWL延长,停药后1 d时PWL缩短;M+K组给药1、3、5、7 d及停药后1 d时PWT升高,PWL延长,M组和M+K组脊髓EAAT3表达下调(P<0.05);与M组比较,M+K组给药3、5、7 d及停药后1 d时PWT升高,给药5、7 d时及停药后1 d时PWL延长.脊髓EAAT3表达上调(P<0.05).EAAT3主要分布于脊髓背角Ⅰ-Ⅱ层的感觉神经元.结论 脊髓背角神经元EAAT3表达下调参与了大鼠慢性吗啡耐受的形成,吗啡下调EAAT3表达的部分机制与激活N-甲基-D天冬氨酸受体有关.     

脊髓蛋白激酶C表达在代谢型谷氨酸受体5参与大鼠吗啡耐受形成中的作用

目的 评价脊髓蛋白激酶C(PKC)表达在代谢型谷氨酸受体5(mGluR5)参与大鼠吗啡耐受形成中的作用.方法 鞘内置管成功的雄性SD大鼠32只,体重180～240 g,随机分为4组(n=8):对照组(C组)、吗啡耐受组(M组)、反义链组(ANT组)和错义链组(MIS组).M组鞘内注射0.9%生理盐水5μl、ANT组和MIC组分别鞘内注射30 nmol反义、错义寡聚脱氧核苷酸(溶于5μl0.9%生理盐水),2次/d,连续8 d,于第6～8天鞘内注射吗啡15μg,2次/d,C组注射等容量生理盐水.于鞘内注射寡聚脱氧核苷酸6、7、8 d(T1～3)时测定大鼠的热痛阈和机械痛阈,第9天(T4)时处死大鼠取L4.5脊髓,采用RT-PCR法测定mGluR5、PKCα、PKCγ的mRNA表达,采用Western blot法测定PKCα、PKCγ的表达.结果 与C组比较,M组和MIS组T1.2时、ANT组T1～3时大鼠热痛阈和机械痛阈升高,T4时M组和MIS组脊髓mGluR5 mRNA、PKCα、PKCγ的表达上调,ANT组脊髓mGluR5 mRNA表达下调(P＜0.05);与M组比较,ANT组T2.3时大鼠热痛阈和机械痛阈升高,脊髓mGluR5 mRNA、PKCα、PKCγ的表达下调(P＜0.05),MIS组上述指标差异无统计学意义(P＞0.05).结论 脊髓PKC表达在mGluR5参与大鼠吗啡耐受形成中起重要作用.     

糖皮质激素受体在慢性吗啡耐受大鼠脊髓背角神经元凋亡中的作用

目的 评价糖皮质激素受体在慢性吗啡耐受大鼠脊髓背角神经元凋亡中的作用.方法 鞘内置管成功的健康雄性SD大鼠20只,体重300～350 g,随机分为4组(n=5):对照组(C组)、慢性吗啡耐受组(M组)、吗啡+糖皮质激素受体拮抗剂组(MR组)和吗啡+糖皮质激素受体激动剂组(MD组)分别于8:00和20:00鞘内注射生理盐水10μl、吗啡10μg、吗啡10μg+RU38486 2μg、吗啡10μg+地塞米松4μg,连续6 d.于每天8:00给药后30 min行甩尾实验,给药第7天处死大鼠,取L3～L5脊髓行TUNEL染色,光镜下观察脊髓背角神经元的凋亡情况,计算凋亡率.结果 地塞米松、RU38486分别对慢性吗啡耐受的形成起促进、抑制作用.与C组比较,M组和MD组脊髓背角神经元凋亡率升高(P＜0.05);与M组比较,MR组脊髓背角神经元凋亡率降低,MD组脊髓背角神经元凋亡率升高(P＜0.05).结论 糖皮质激素受体参与了慢性吗啡耐受形成中大鼠脊髓背角神经元凋亡的过程.     

人参皂甙Rg1对炎性痛大鼠吗啡耐受时脊髓背角细胞凋亡的影响

目的 探讨人参皂甙Rg1对炎性痛大鼠吗啡耐受时脊髓背角细胞凋亡的影响.方法 雄性SD大鼠,体重280～320 g,采用右踝关节腔内注射完全弗氏佐剂的方法制备炎性痛模型.取模型制备成功的24只大鼠,采用随机数字表法,将其随机分为4组(n=6):生理盐水对照组(C组)、吗啡耐受组(M组)、人参皂甙Rg1组(G组)及吗啡复合人参皂甙Rg1组(MG组).致炎后3d时M组、G组及MG组分别鞘内注射吗啡10 μg、人参皂甙Rg1 100μg及吗啡10μg+人参皂甙Rg1 100 μg,C组给予等容量生理盐水,吗啡2次/d,人参皂甙Rg1 1次/d,连续7d.分别于致炎前(T1)、鞘内给药前1d(T2)、给药1、3、5、7 d(T3-6)时测定机械缩足阈值(PWT).最后一次测定痛阈后处死大鼠,取L3-5节段脊髓组织,采用TUNEL染色法测定细胞凋亡情况,计算脊髓背角细胞凋亡率.结果 与T1时比较,4组T2-6时PWT降低(P＜0.01);与T2时比较,M组T3.4时PWT升高,G组和MG组T3～6时PWT升高(P＜0.05);与C组比较,M组和MG组PWT和脊髓背角细胞凋亡率升高(P＜0.05),G组上述指标差异无统计学意义(P＞0.05);与M组比较,MG组PWT升高,脊髓背角细胞凋亡率降低(P＜0.05).结论 人参皂甙Rg1预防吗啡耐受形成的机制与降低炎性痛大鼠脊髓背角细胞凋亡有关.     

乳铁蛋白对神经病理性痛大鼠脊髓背角PKG活性的影响

目的 探讨乳铁蛋白对神经病理性痛大鼠脊髓背角cGMP依赖性蛋白激酶(PKG)活性的影响.方法 雄性SD大鼠32只,体重200～250 g,随机分为4组(n=8):假手术组仅分离坐骨神经,不结扎,鞘内注射生理盐水10μl+50%二甲基亚砜(DMSO)10μl;余3组采用结扎坐骨神经的方法制备大鼠神经病理性痛模型,神经病理性痛组鞘内注射生理盐水10μl+50%DMSO10μl;乳铁蛋白组鞘内注射乳铁蛋白100μg+50%DMS010μl;PKG抑制剂KT5823组鞘内注射乳铁蛋白100μg+KT582310μl.给药后180 min内每隔30 min以热刺激法测定大鼠缩爪潜伏期,随后处死大鼠取脊髓背角,采用免疫荧光法检测PKG活性,并行定量分析.结果 与神经病理性痛组和KT5823组相比,乳铁蛋白组缩爪潜伏期延长,乳铁蛋白组脊髓背角PKG活性升高(P＜0.05);神经病理性痛组与KT5823组上述指标比较差异无统计学意义(P＞0.05).结论 乳铁蛋白可通过抑制脊髓背角PKG活性减轻大鼠神经病理性痛.     

炎性痛-吗啡耐受大鼠背根神经节辣椒素受体磷酸化水平的变化

目的 探讨炎性痛-吗啡耐受大鼠背根神经节辣椒素受体(VR1)磷酸化水平的变化.方法 鞘内置管成功的成年雄性SD大鼠20只,体重230～250 g,2～3月龄,采用左后足踝关节腔注射完全弗氏佐剂(CFA)50μl的方法制备炎性痛模型.大鼠随机分为4组(n=5),炎性痛+生理盐水组(NS组):注射剂CFA后3 d鞘内注射生理盐水10μl,2次/d,连续7 d;单纯吗啡组(M0组):不建立炎性痛模型,鞘内注射吗啡10μl/kg,2次/d,连续7 d;炎性痛+单次吗啡组(M1组):注射CFA后3 d鞘内注射吗啡10μl/kg;炎性痛+连续吗啡组(M2组):注射CFA后3 d鞘内注射吗啡10 μg/kg,2次/d,连续7 d.于鞘内置管后、注射CFA后3 d鞘内给药前、给药1～7 d测定机械缩足反射阈值和缩足潜伏期,最后一次测定痛阈后处死大鼠,采用Western blot法测定背根神经节磷酸化VR1(p-VR1)的表达水平.结果 NS组和M1组未发生吗啡耐受,M0组和M2组发生吗啡耐受.4组中,M2组背根神经节p-VR1表达水平最高(P＜0.05).结论炎性痛-吗啡耐受大鼠背根神经节VR1磷酸化水平升高,该变化可能是吗啡耐受形成的机制.     

μ受体在抗神经生长因子抗体减轻大鼠骨癌痛中的作用

目的 评价μ受体在抗神经生长因子抗体(anti-NGF)减轻大鼠骨癌痛中的作用.方法 实验一健康雌性SD大鼠60只,体重200～220 g,随机分为4组(n=15):假手术组(S组)、假手术+anti-NGF组(SN组)、骨癌痛组(P组)和骨癌痛+anti-NGF组(PN组).P组和PN组于左侧胫骨上段骨髓腔内注射10μl Walker256乳腺癌细胞(1×105个)制备骨癌痛模型;S组和SN组于左侧胫骨上段注射PBS 10μl.于肿瘤细胞接种后13 d时,进行鞘内置管.鞘内置管成功后3 d,SN组和PN组鞘内注射anti-NGF 10μg(用生理盐水稀释至10μl),S组和P组鞘内注射生理盐水10μl,2次/d,连续5 d.于肿瘤细胞接种前、肿瘤细胞接种后13、16、18、21 d时测定自发缩足次数(NSF)、热缩足潜伏期(PWL)和机械性痛阈(PWT).肿瘤细胞接种后21 d时,处死大鼠,取L4.5段脊髓背角和背根神经节,测定μ受体及其mRNA的表达.实验二健康雌性SD大鼠30只,体重200～220 g,随机分为2组(n=15):骨癌痛+anti-NGF组(PN组)和骨癌痛+纳洛酮+anti-NGF组(PNN组).于左侧胫骨上段骨髓腔内注射10μlWalker256乳腺癌细胞(1×105个)制备骨癌痛模型.于肿瘤细胞接种后13 d时,进行鞘内置管.鞘内置管成功后3 d,PN组鞘内注射鞘内注入anti-NGF 10μg(生理盐水稀释至25μl);PNN组鞘内注射纳洛酮10μg(生理盐水稀释至25μl),0.5 h后,鞘内注射anti-NGF 10μg(生理盐水稀释至25 μl),2次/d,连续5 d.于肿瘤细胞接种前、肿瘤细胞接种后13、16、18、21 d时测定大鼠NSF、PWL和PWT.结果 实验一与S组比较,SN组NSF、PWL和PWT差异无统计学意义,SN组和PN组μ受体及其mRNA表达差异无统计学意义(P＞0.05),P组和PN组瘤细胞接种后13～21 d时NSF增加,PWL缩短,PWT降低,P组μ受体及其mRNA表达下调(P＜0.05或0.01);与P组比较,PN组肿瘤细胞接种后18～21 d时NSF减少,PWL延长,PWT升高,μ受体及其mRNA表达上调(P＜0.05或0.01).实验二与PN组比较,PNN组肿瘤细胞接种后18～21 d时NSF增加,PWL缩短,PWT降低(P＜0.05或0.01).结论 anti-NGF减轻大鼠骨癌痛与μ受体的激活有关.     

慢性吗啡耐受大鼠脊髓谷氨酸转运体及谷氨酸的变化

目的：利用慢性关节炎吗啡耐受大鼠模型,观察脊髓背角谷氨酸转运体（GT）水平以及谷氨酸浓度的变化,阐述阿片耐受的机制。方法：24只SD大鼠随机分为四组（n=6）。吗啡组（M组）鞘内注入20μg吗啡,连续7 d;盐水对照组（S组）鞘内注入20μL生理盐水连续7 d;吗啡纳络酮组（MN组）鞘内注入20μg吗啡、10μg纳络酮连续7 d;纳络酮组（N组）鞘内注入10μg纳络酮连续7 d。于注药后第7 d观察大鼠的行为学变化,并取脊髓腰4~5节段,应用免疫组化法和计算机图像分析技术观察大鼠脊髓背角谷氨酸转运体（EAAT1,EAAT3）表达变化,采用毛细管电泳法检测谷氨酸浓度变化。结果：与S组、MN组、N组相比,M组大鼠脊髓背角谷氨酸转运体表达下降（P〈0.05）;缩脚潜伏期减少（P〈0.05）,谷氨酸浓度升高（P〉0.05）。上述变化在S组、MN组、N组之间没有差异（P〉0.05）。结论：形成关节炎吗啡耐受的大鼠脊髓背角EAAT1、EAAT3表达减少,谷氨酸浓度升高。     

鞘内注射右美托咪啶对骨癌痛大鼠脊髓背角磷酸化cAMP反应元件结合蛋白表达的影响

目的 探讨右美托咪啶对骨癌痛大鼠脊髓背角磷酸化cAMP反应元件结合蛋白(p-CREB)表达的影响.方法清洁级健康成年雌性Wistar大鼠64只,体重200～240 g,采用随机数字表法,将其随机分为4组(n=16):假手术组(S组)、骨癌痛组(BP组)、生理盐水组(NS组)和右美托眯啶组(D组).BP组、Ns组和D组采用胫骨骨髓腔注射10μl含Walker 256乳癌细胞2×106个缓冲液的方法制备大鼠骨癌痛模型,S组和BP组不作鞘内注射,NS组和D组于模型制备成功后7 d分别鞘内注射生理盐水10μl、右美托咪啶5μg/kg.分别于鞘内注射前1 d(T0)、注射前即刻(T1)、注射后1、6、12、24 h(T2-5,)时各组随机取10只大鼠,采用von Frey丝测定机械痛阈,各组余6只大鼠T4时处死取脊髓,采用免疫组化法测定脊髓背角p-CREB的表达.结果 与S组比较,BP组、NS组和D组T2～5时机械痛阈降低,T4时脊髓背角p-CREB表达上调(P＜0.05);与BP组比较,D组T2～5时机械痛阈升高,T4时脊髓背角p-CREB表达下调(P＜0.05),NS组机械痛阈和脊髓背角p-CREB表达差异无统计学意义(P＞0.05).结论 鞘内注射右美托咪啶可通过抑制大鼠脊髓背角CREB磷酸化减轻骨癌痛.

Abstract：

Objective To investigate the effect of intrathecal (IT) dexmedetomidine on the expression of cAMP response element-binding protein phosphorylation (p-CREB) in spinal dorsal horn in a rat model of bone cancer pain. Methods Sixty-four adult female Wistar rats weighing 200-240 g were randomly divided into 4 groups (n = 16 each): sham operation group (group S); bone cancer pain group (group BP); normal saline group ( group NS) ; dexmedetomidine group (group D) . Bone cancer pain was induced by injecting Walker 2S6 mammary gland carcinoma cell suspension (2 ×106 cells/ml) 10μl into the medullary cavity of the tibia in BP, NS and D groups. Groups S and BP received no IT injection. Croups NS and D received IT injection of NS 10 μl and dexme detomidine 5 μg/kg respectively 7 days after successful establishment of the model. Ten animals were selected from each group at 1 day before IT administration (T0), immediately before IT administration (T1 ) and at 1, 6, 12 and 24 h after IT administration (T2-5 ) and paw withdrawal threshold (PWT) to mechanical stimuli was measured with von Frey filaments. The other 6 rats in each group were sacrificed at T4 and the spinal cord was removed for determination of p-CREB expression in the spinal dorsal horn.Results PWT was significantly decreased at T1-5 and pCREB expression up-regulated at T4 in BP, NS and D groups compared with group S ( P ＜ 0.05) . Compared with group BP, PWT was significantly decreased at T2-5 and p-CREB expression down-regulated at T4 in group D ( P ＜0.03), while no significant change in PWT and p-CREB expression was found in group NS (P ＞ 0.05) .Conclusion IT dexmedetomidine can reduce the bone cancer pain through inhibiting the phosphorylation of CREB in rat spinal dorsal horn.