Single-neuron projectome-guided analysis reveals the neural circuit mechanism underlying endogenous opioid antinociception.

Endogenous opioid antinociception is a self-regulatory mechanism that reduces chronic pain, but its underlying circuit mechanism remains largely unknown. Here, we showed that endogenous opioid antinociception required the activation of mu-opioid receptors (MORs) in GABAergic neurons of the central amygdala nucleus (CEA) in a persistent-hyperalgesia mouse model. Pharmacogenetic suppression of these CEAMOR neurons, which mimics the effect of MOR activation, alleviated the persistent hyperalgesia. Furthermore, single-neuron projection analysis revealed multiple projectome-based subtypes of CEAMOR neurons, each innervating distinct target brain regions. We found that the suppression of axon branches projecting to the parabrachial nucleus (PB) of one subtype of CEAMOR neurons alleviated persistent hyperalgesia, indicating a subtype- and axonal-branch-specific mechanism of action. Further electrophysiological analysis revealed that suppression of a distinct CEA-PB disinhibitory circuit controlled endogenous opioid antinociception. Thus, this study identified the central neural circuit that underlies endogenous opioid antinociception, providing new insight into the endogenous pain modulatory mechanisms.

p300 exerts an epigenetic role in chronic neuropathic pain through its acetyltransferase activity in rats following chronic constriction injury (CCI).

BACKGROUND: Neuropathic pain is detrimental to human health; however, its pathogenesis still remains largely unknown. Overexpression of pain-associated genes and increased nociceptive somato-sensitivity are well observed in neuropathic pain. The importance of epigenetic mechanisms in regulating the expression of pro- or anti-nociceptive genes has been revealed by studies recently, and we hypothesize that the transcriptional coactivator and the histone acetyltransferase E1A binding protein p300 (p300), as a part of the epigenetic mechanisms of gene regulation, may be involved in the pathogenesis of neuropathic pain induced by chronic constriction injury (CCI). To test this hypothesis, two different approaches were used in this study: (I) down-regulating p300 with specific small hairpin RNA (shRNA) and (II) chemical inhibition of p300 acetyltransferase activity by a small molecule inhibitor, C646. RESULTS: Using the CCI rat model, we found that the p300 expression was increased in the lumbar spinal cord on day 14 after CCI. The treatment with intrathecal p300 shRNA reversed CCI-induced mechanical allodynia and thermal hyperalgesia, and suppressed the expression of cyclooxygenase-2 (COX-2), a neuropathic pain-associated factor. Furthermore, C646, an inhibitor of p300 acetyltransferase, also attenuated mechanical allodynia and thermal hyperalgesia, accompanied by a suppressed COX-2 expression, in the spinal cord. CONCLUSIONS: The results suggest that, through its acetyltransferase activity in the spinal cord after CCI, p300 epigenetically plays an important role in neuropathic pain. Inhibiting p300, using interfering RNA or C646, may be a promising approach to the development of new neuropathic pain therapies.

Inhibition of Spinal Interleukin-33 Attenuates Peripheral Inflammation and Hyperalgesia in Experimental Arthritis.

Accumulating evidence indicates that the continuous and intense nociceptive from inflamed tissue may increase the excitability of spinal dorsal horn neurons, which can signal back and modulate peripheral inflammation. Previous studies have demonstrated that spinal interleukin (IL)-33 contributes to the hyperexcitability of spinal dorsal horn neurons. This study was undertaken to investigate whether spinal IL-33 can also influence a peripheral inflammatory response in a rat model of arthritis. Lentivirus-delivered short hairpin RNA targeting IL-33 (LV-shIL-33) was constructed for gene silencing. Rats with adjuvant-induced arthritis (AIA) were injected intrathecally with LV-shIL-33 3 days before the complete Freund's adjuvant (CFA) injection. During an observation period of 21 days, pain-related behavior and inflammation were assessed. In addition, the expression of spinal proinflammatory cytokines and the activation of spinal extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) pathways were evaluated on 9 days after CFA treatment. The existence of tissue injury or inflammation in rats with AIA resulted in the upregulation of spinal IL-33, which is predominantly expressed in neurons, astrocytes, and oligodendrocytes. Intrathecal administration of LV-shIL-33 significantly alleviated hyperalgesia, paw swelling, and joint destruction, and attenuated the expression of proinflammatory cytokines [IL-6, IL-1ß, and tumor necrosis factor-α (TNF-α)], as well as the activation of ERK and NF-κB/p65 in the spinal cord. Our data suggest that spinal IL-33 contributes to the development of both peripheral inflammation and hyperalgesia. Thus, interference with IL-33 at the spinal level might represent a novel therapeutic target for painful inflammatory disorders.

[Analgesic effect of intrathecal injection of Herpes simplex virus type I amplicon vector-mediated human preproenkephalin gene on chronic neuropathic pain: experiment with rats].

OBJECTIVE: To investigate the antinociceptive effect of intrathecal (IT) injection of Herpes simplex virus type I (HSV-1) amplicon vector-mediated HPPE on chronic neuropathic pain. METHODS: 45 Sprague-Dawley rats underwent chronic constriction. Injury (CCI) of unilateral sciatic nerve and then were randomly divided into 3 equal groups: CCI + normal saline group, undergoing insertion of microspinal catheter into the subarachnoid space at the lumber region and intrathecal delivery of NS, CCI + pHSVIRES-LacZ (SHPZ) group undergoing intrathecal delivery of and recombinant HSV-I amplicon vector pHSVIRES-HPPE-LacZ containing human pre-proenkephalin (HPPE) gene, and CCI + blank vector (SHZ) group receiving pHSV-HPPE-LacZ. Another 15 rats underwent sham operation to be used as control group. One week after IT administration 9 rats from each group were killed with their lumber segments of spinal cord removed to detect the expression of LacZ by X-gal staining, HPPE mRNA expression by RT-PCR, and L-enkephalin (L-EK) content by radioimmunoassay. Paw mechanical withdrawal threshold (PMWT) and paw withdrawal thermal latency (PWTL) were measured before CCI (baseline) and 3 days after CCI and then once a week for 5 weeks after IT administration. RESULTS: After IT administration of SHPZ expression of HPPE mRNA was detected in the spinal cord. One week after the IT injection the L-EK level of the SHPZ group was (748 +/- 185 ng/L), significantly higher than those of the Sham operation, NS, and SHZ groups [(452 +/- 89), (453 +/- 92), and (451 +/- 99) ng/L respectively, all P < 0.05]. The PWMT and PWTL levels of the SHPZ group were significantly increased since 1 week after the IT administration in comparison with the baseline values and those of the other 3 groups (all P < 0.05), and these effects peaked in the third week and then lasted to the fifth week. However, the threshold to mechanical and thermal stimuli was not affected by intrathecal delivery of vehicle or SHZ compared with the threshold before intrathecal delivery. CONCLUSIONS: Intrathecal administration SHPZ can produce significant analgesic effects on chronic neuropathic pain in rats.

[Periaqueductal gray administration of HSV-I amplicon vector-mediated HPPE gene therapy of nocicepion in rats with formalin-induced pain].

OBJECTIVE: To investigate the antinociceptive effect of periaqueductal gray (PAG) administration of herpes simplex virus type-1(HSV-I) amplicon vector-mediated human preproenkephalin gene (HPPE). METHODS: Sprague-Dawley rats weighting 260 to approximately 320 g were randomly divided into pHSVIRES-HPPE-LacZ (SHPZ) group, pHSVIRES-LacZ (SHZ) group, and saline (NS) group which included 3 d,1 week,2 week,3 week,4 week,5 week, and 6 week groups (n=51). The rats were anesthetized with intraperitoneal chloral hydrate (300 to approximately 350) mg/kg. Rats were PAG delivered with recombinant HSV-I amplicon vector SHPZ, SHZ or NS. One week after PAG administration 9 rats in each group were sacrificed and lumber segment of the spinal cord was removed for determination of expression of LacZ by X-gal staining and HPPE mRNA expression by reverse transcription-polymerase chain reaction and L-enkephalin content by radioimmunoassay in PAG. Formalin 50 microL (5%) was injected into the left hindpaw, and pain intensity scoring (PIS) was used to assess the antinociceptive effect. RESULTS: After in vivo transferring, neurocyte demonstrated strong positive signals with X-gal immunohistochemical staining. The expression of HPPE mRNA was detected in PAG after administration of SHPZ. PAG delivery of SHPZ showed antinociceptive effect on formalin-induced pain for 6 weeks compared with SHZ group. CONCLUSION: This amplicon virus can transfer HPPE into rat PAG neural cells and make it express efficiently. PAG administration of SHPZ can produce significant analgesic effect on formalin-induced pain in rats for 5 weeks.

[Three subanaesthetic dose ketamines mixed with butorphanol in the postoperative continuous intravenous analgesia].

OBJECTIVE: To determine an optimal clinical dose of ketamine after comparing the efficacy and security of 3 low dose ketamines mixed with butorphanol in the postoperative continuous intravenous analgesia. METHODS: Eighty ASA (American Society of Anesthesiologists) I-II patients scheduled for elective gynecological surgery under general anesthesia were divided randomly into 4 groups (n=20): Group B received butorphanol 3 microg/(kg x h);Group BK1 received butorphanol 2 microg/(kg x h) mixed with ketamine 60 microg/(kg x h); Group BK2 received butorphanol 2 microg/(kg x h) mixed with ketamine 90 microg/(kg.h); and Group BK3 received butorphanol 2 microg/(kg x h) mixed with ketamine 120 microg/(kg x h). Continuous intravenous infusion pump was used when the patients had obvious pain (visual analgesia scale of five), and the bolus infusion (4 mL) was given before the operation, and continuous infusion at 2 mL/h. In the postoperative period, pain was assessed using visual analogue scale (VAS) at 2,6,12,24, and 48 h.At the same time, Ramsay scores and adverse effects were recorded. RESULTS: There was no significant difference in the adverse effects and the postoperative mean arterial pressure, heart rate, respiratory rate values, and pulse oxygen among the 4 groups. Postoperative VAS values in Group BK3 was the lowest, followed by Group BK2. There was no significant difference between Group BK1 and Group B. The incidence of somnolence in Group B was higher than that in Group BK1, BK2 and BK3(P<0.05). CONCLUSION: Ketamine in subanaesthetic dose added to butorphanol for postoperative continuous intravenous infusion has a better postoperative analgesic effect and sedation. It can effectively spare butorphanol consumption without increasing adverse effects. The optimal combined dose is 90-120 microg/(kg x h).

[Effect of intrathecal pumping tramadol on the immune function in rats with formalin pain].

OBJECTIVE: To evaluate the effect of intrathecal pumping tramadol on cell-mediated immunity in rats with formalin inflammatory pain. METHODS: Thirty-two Sprague-Dawley adult male rats weighting 250 approximately 300 g were randomly divided into 4 groups (n=8 in each group):Saline group (NS) and 3 tramadol groups (T1,T2,and T3). The rats were anesthetized with intraperitoneal chloral hydrate (300 approximately 350)mg/kg. Microspinal catheter was inserted into the subarachnoid space at the lumber region according to modified Yaksh techniques. In the tramadol groups,after 5 days tramadol was continuously infused through the spinal catheter at 50 (T1),25 (T2), and 12.5 microg/h (T3) for 7 days. In the NS group normal saline was continuously infused instead of tramadol. On Day 7 formalin (5%, 50 microL) was injected into the plantar surface of the left hindpaw. The number of flinches, lickings and total time of licking was recorded for 60 min.Pain intensity scoring(PIS)(0 approximately 3;0= no pain, 3=severe pain) was used to assess the antinociceptive effect of intrathecal tramadol. The rats were killed after the evaluation of pain intensity. Body weight and spleen weight were measured and spleen index (spleen weight/body weight) was calculated. T-lymphocyte function was evaluated based on Concanavalin-A(ConA) induced splenocyte proliferation. A modified lactic acid dehydrogenase(LDH) release assay was done to assess the NK cell activity. Phenotypic expressions of cell surface markers of T lymphocyte subsets (CD3+, CD3+ CD4+, CD3+ CD8+, and CD4+/ CD8+) and NK cell(CD161+) in the spleen were analyzed by flow cytometry. RESULTS: The PIS scores were significantly lower in the T1,T2,and T3 groups than those in the NS group. The spleen index and splenocyte proliferation induced by ConA were significantly suppressed in the T1 group,and the phenotypes of T lymphocyte subsets were significantly changed,but no significant difference was found in the T2 and T3 groups compared with the NS group. There were no differences in NK cell activity in the 3 tramadol groups from the control group. CONCLUSION: Intrathecal pumping tramadol has significantly antinociceptive effect. Intrathecal pumping higher dosage tramadol (50microg/h) suppresses T lymphocyte proliferation and alteres T lymphocyte subset phenotype but does not affect NK cell activity. General analgesic dosage tramadol (25 and 12.5 microg/h) has no effect on the immune function.

[Intrathecal injection of ketamine and clonidine for chronic neuropathic pain model in rats].

OBJECTIVE: To observe the effect of intrathecal injection of ketamine and clonidine for chronic constriction injury (CCI) in rats. METHODS: Thirty-two SD male rats weighing 220-280 g were anesthetized with intraperitoneal chloral hydrate 300 mg/kg. A catheter was implanted in the subarachnoid space at the lumbal region and CCI rat models were made successfully. On the 4th day after the surgery, the rats were randomly divided into 4 group: a control group,injecting 0.9%NS 20 microL intrathecally; a ketamine group, injecting ketamine 1 mg/kg(20 microL) intrathecally; a clonidine group (CL), injecting clonidine 20 microg/kg (20 microL) intrathecally; a combined ketamine and clonidine group, injecting ketamine 0.5mg/kg and clonidine 10 g/kg (20 microL) intrathecally, once a day for 1 week. BME-410A Plantar Analgesia Tester was used to measured pain threshold before the administration and 30 min after the administration. The rats were killed after the test was finished. And then we detected the nitric oxide synthase (NOS) activity and the NO production in the spinal cord. RESULTS: The combined injection of ketamine (0.5mg/kg)and clonidine(10 g/kg) produced significantly more potent analgesia than the injection of ketamine (1 mg/ kg) or clonidine (20 microg/ kg)alone. The NOS activity and the production of NO in the combined injection group were significantly lower than those of the single injection group (P<0.05). The weight of rats post-administration increased obviously in the 4 groups (P<0.05). CONCLUSION: The combined injection of ketamine and clonidine can produce synergistic ab-irritation without obvious side effects.

[Effect of intrathecal katemine on the expression of nNOS in spinal dorsal horn in rats with formalin pain].

OBJECTIVE: To investigate the expression of neuronal nitric oxide synthase (nNOS) in spinal dorsal horn and the effect of intrathecal katemine on the expression of nNOS in the rats with formalin-induced pain. METHODS: Thirty-two Sprague-Dawley rats were randomly divided into 4 groups (n=8 in each group): a control group (C), an intrathecal saline group (NS), a katemine 50 microg group (K1), and a katemine 100 microg group (K2). The rats that were anesthetized with 10% chlroral hydrate 300 - 350 mg/kg by abdominal injection were intrathecally inserted a microspinal catheter into the lumbar region according to the method of modified Yaksh. After 5 days ,the rats in Group NS, K1 and K2 were intrathecal 20 microL saline or 10 microL katemine (50, 100 microg) followed by 10 microL saline, respectively. Thirty minutes later, 5% formalin 50 microL was subcutaneously injected into the left hindpaw. Pain intensity scoring (PIS) was used to assess antinociceptive behavior within 1 h after the formalin injection. The expression of nNOS in the spinal dorsal horn of L5 segment was assayed using immunohistochemistry 24 h later. RESULTS: Compared with Group NS, PIS of Group K1 and K2 was decreased obviously (P<0.01) in the second phase of formalin pain. The number of immunoreactive soma and immunohistochemistic dying grade of nNOS in the spinal dorsal horn of L5 segment was higher in Group NS than that in Group C (P<0.01), but Group K1 and K2 were lower than Group NS (P<0.01). CONCLUSION: There was significant antinociception of intrathecal katemine in rats with formalin pain. Intrathecal katemine significantly inhibited the increase of nNOS expression in the spinal dorsal horn of formalin-induced pain, suggesting nNOS plays an important role in nociceptive transmission and modulation of the spinal cord.

[Spinal anesthesia with low dose sufentanil-bupivacaine in transurethral resection of the prostate].

OBJECTIVE: To explore the clinical efficacy of intrathecally administered low dose sufentanil-bupivacaine in transurethral resection of the prostate (TURP). METHODS. Ninety patient (ASA I - III) undergoing TURP were randomly divided into 3 groups (n = 30); Group A, B and C. Group A received 7.5 mg bupivacaine + 5 microg sufentanil + 10% glucose; Group B received 7.5 mg bupivacaine + 7.5 microg sufentanil + 10% glucose; Group C received 15 mg bupivacaine + 10% glucose. The volume was 3 mL in every group. SP, DP, HR, SpO2, the degree of motor and sensory blockade and the side effect were observed. RESULTS: SP/DP was significantly decreased in Group C than that in Group and Group B (p<0.05), HR and SpO2 in group B were decreased to different degrees 15 min after the injection (p<0.05). The complete recovery time of motor nerve blockade and the regression time of sensory blockade were obviously prolonged in Group C (p<0.05). There were no significant differences in analgesic effect among the three groups during the operation, but the incidence of pruritus was higher in both group A and Group B than that in Group C during the first 24 hours after the injection. CONCLUSION: Spinal anesthesia with low dose sufentanil-bupivacaine possesses relatively steady hemodynamics. The blockade degree of motor and sensory blockade in this spinal anesthesia is lower than that in standard spinal bupivacaine in TURP.

[Intrathecal administration of HSV-I amplicon vector-mediated HPPE gene therapy of nocicepion in rats with formalin pain].

OBJECTIVE: To investigate the antinociceptive effect of intrathecal administration of HSV-I amplicon vector-mediated HPPE. METHODS: Sprague Dawley rats (290+/-30) g were randomly divided into pHSVIRES-HPPE-LacZ (SHPZ) group, pHSVIRES-LacZ (SHZ) group, and saline group (NS), and 3 d, 1 week, 2 weeks, 3 weeks, 4 weeks, and 5 weeks group,which were anesthetized with 10% chlroral hydrate 300- 350 mg/kg. A microspinal catheter was inserted into the lumbar subarachnoid space. Rats were intrathecally delivered with recombinant HSV-I amplicon vector SHPZ, SHZ or NS. The HPPE expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and radioimmune assay. Formalin 50 microL (5%) was injected into the left hindpaw, pain intensity scoring (PIS) was used to assess the antinociceptive effect. RESULTS: After in vivo transferring,neurocyte demonstrated strong positive signals with X-gal immunohistochemical staining. RT-PCR and L-enkephalin radioimmune assay found that the neural cells transferred foreign gene (HPPE) had effective expression. Intrathecal delivery of SHPZ showed antinociceptive effects on formalin induced pain for 5 weeks compared with SHZ. CONCLUSION: This amplicon virus can transfer HPPE into rat central nerve system neural cells and express efficiently, suggesting SHPZ is satisfactory treatment for gene therapy for chronic pain. Intrathecal delivery SHPZ demonstrated antinociceptive effects on formalin induced pain.

[Effect of intrathecal pumping morphine on immunological function in rats with formalin pain].

OBJECTIVE: To evaluate the immunological function in rats with formalin inflammatory pain through intrathecal pumping different dosages of morphine. METHODS: Thirty-two Sprague-Dawley rats were randomly divided into 4 groups (n = 8 in each group): saline group (NS) and morphine group included M1 group (10 microg/h) , M2 group (5 microg/h), and M3 group (2.5 microg/h). Chronic intrathecal catheterization was performed under anesthesia with 10% chloral hydrate (300-350) mg/kg according to M2 group (5 microg/h) and M3 group (2. 5 microg/h). Chronic intrathecal catheterization was modified Yaksh's. After 7 days, pain intensity scoring (PIS) was utilized to assess antinociceptive effect of morphine. And spleens were aseptically removed to obtain splenic cells. T lymphocyte function was evaluated based on Concanavalin-A induced splenocyte proliferation. A modified lactic acid dehydrogenase release assay was used to assess NK cell activity. Phenotypic expression of cell surface markers of T lymphocyte subsets (CD3+, CD3+ CD4+, CD3+ CD8+, and CD4+ / CD8+ ) and NK cell ( CD161+) in the spleen were analyzed by flow cytometry. RESULTS: Compared with the NS group, PIS of morphine group decreased obviously (P < 0.01) and was dose-dependent in the early and late phase of formalin pain, but there were no significant differences among morphine groups. Spleen index, splenocyte proliferation and NK cell activity were significantly suppressed by intrathecal pumping morphine. Phenotypic expression of T lymphocyte subsets and NK cell assessed by flow cytometry were different from the control group in all morphine groups. CONCLUSION: There was significant antinociception of intrathecal pumping morphine. After intrathecal pumping different dosages of morphine (10 microg/h,5 microg/h, and 2.5 microg/h), the function of cellular immunity was suppressed and was dose-dependent.

[Clinical study on bispectral index in monitoring the depth of desflurane anesthesia in the elderly].

OBJECTIVE: To evaluate the values of bispectral index (BIS) in mornitoring the depth of desflurane anesthesia in the elderly by observing the changes of BIS at various end-tidal desflurane concentrations. METHODS: BIS was used to monitor the depth of desflurane anesthesia in the elderly without surgery stimulation. Forty ASA physical status I approximately III patients undergoing general anesthesia were divided into 2 groups with 20 in each group:Group Elderly ( > or =65 years) and Group Youth ( 18 approximately 55 years). Anesthesia was induced with propofol 2. 0 mg/kg and vecuronium 0. 1 mg/kg including the endotracheal topical anesthesia with 2% lidocaine. After the desflurane in oxygen, each concentration of desflurane was maintained for 20 minutes. The changes of mean arterial blood pressure (MAP), heart rate (HR) and BIS were recorded simultaneously. The timepoints setting for observation were: preanesthesia, 2 minutes after the anesthesia, endotracheal intubation, 2 minutes after the intubation, and end-tidal concentration of desflurane at 0. 6 MAC, 1.0 MAC and 1. 3 MAC. RESULTS: During anesthesia of desflurane, MAP and HR did not change significantly in the 2 groups with increasing end-tidal desflurane concentration from 0. 6 MAC to 1. 3 MAC (P > 0.05). BIS decreased significantly than that at preanesthesia in the 2 groups during anesthesia of desflurane ( P < 0.05 ). The changes of BIS were different in the 2 groups during the whole anesthesia (P < 0.05). As the concentration of desflurane increased, BIS decreased gradually but there were no significant changes on BIS in the 2 groups ( P > 0. 05 ). In general, BIS highly correlated with the end-tidal desflurane concentration. The coefficient of product-moment correlation (r) between BIS and the end-tidal desflurane concentration was -0. 996 and -0. 946 in Group Elderly and Youth (P < 0. 05 ). CONCLUSION: BIS highly correlates with the end-tidal desflurane concentration which is used to evaluate the depth of desflurane anesthesia in the elderly and youth. There is different depth of anesthesia by BIS in the elderly or youth at the same end-tidal desflurane concentration.

[Behaviorial observation of ropivacaine in continuous spinal anesthesia and Ca2+ content of spinal cord in rats].

OBJECTIVE: To observe the behaviour and Ca2+ content of spinal cord in rats after continuous spinal anesthesia administration of different densities and doses of ropivacaine in SD rats. METHODS: Twenty-four male SD rats weighing 220 approximately 280 g were anesthetized. A polyurethane microspinal catheter was inserted into the lumbar subarachnoid space 8 cm according to the method of Yaksh's. The animals were randomly divided into 4 groups of 6 each: in group N the rats were given normal saline 40 microl intrathecally every one and half hours for 3 times; in group R1, 0.5% ropivacaine was given; in group R2 0.75% ropivacaine and in group R3 1% ropivacaine was given. The activity of rats was observed. After 6 hours rats were perfused with 4% formamint through the ascending aorta. The rats were sacrificed and L1 approximately 2 segment of spinal cord was immediately removed for Ca2+ content examination. RESULTS: A total hind limbs paralysis was seen at 30 seconds and intramuscular strain gradually came back from 30 to 90 minutes after the intrathecal administration of ropivacaine in all rats. The recovery of motor black was remarkably different in group R1, R2, and R3 (P < 0.05). The Ca2+ content of spinal cord was significantly higher in group R3 than that in group R1 and R2 (P < 0.05 ). CONCLUSION: There is no significant change of motor black time and it is related to drug dose for 0.5% , 0.75% and 1% ropivacaine in continuous spinal anesthesia. 1% ropivacaine may increase Ca2+ content in spinal cord.

[Clinical anesthetic effects of epidural ropivacaine with tramadol].

OBJECTIVE: To observe the anesthesic effects of epidural ropivacaine with tramadol during lower limbs surgery. METHODS: Thirty patients (ASA I - II) scheduled for the lower limbs surgery were randomly divided into 2 groups with 15 patients in each group: group ropivacaine (R) and group ropivacaine with tramadol (T). The puncture was performed at the interspace of L2-3. Each patient was given 2% lidocaine 3 ml with 0.75% ropivacaine 10 ml which included NS 1 ml in Group R or tramadol 50 mg in Group T. The potency of analgesia, the time of sensation block to T12 and T10, the time to the highest plane of analgesia, the lasting time of analgesia, the degree of sedation, the degree of motor block, and the side effects were recorded and analyzed during anesthesia after the first dose. RESULTS: The time of sensation block which reached T12 and T10 and the time to the highest plane of analgesia decreased significantly in Group T than that in Group R (P < 0.05). The lasting time of analgesia in Group T was longer than that in Group R (P < 0.05). There was no significant difference in the potency of analgesia, the degree of sedation and motor block, and the side effects (P > 0.05). CONCLUSION: The epidural ropivacaine with tramadol enhanced the anesthetic effects of ropivacaine.

[Effect of ulinastatin on human blood coagulation and platelet aggregation in orthopaedic surgery].

OBJECTIVE: To investigate the effect of ulinastatin (UTI) on human blood coagulation and platelet aggregation in orthopaedic surgery. METHODS: Thirty ASA I-II patients without blood dyscrasia and blood coagulation obstacle were randomly divided into two groups: Group I (UTI group, n=15) in which patients received UTI 5000 U/kg, and Group II (control group, n=15) in which patients received NS 100 ml. PT, TT, APTT, IB, INR and PAG1, PAG5, and PAGM were measured at 3 points: pre-infusion (T0), 1 hour after the infusion (T1), and 2 hours after the infusion (T2). RESULTS: Compared with the saline group, APTT and PT of UTI group were prolonged significantly than the baseline (before infusion). In Group I, after the infusion, APT, TT and PT were prolonged significantly than before the infusion. CONCLUSION: UTI 5000 U/kg can ameliorate orthopaedic patients and blood coagulation status,which may reduce microthrombus syndrome in the operation and prevent venous thrombosis after the operation.

Suppressing SNAP-25 and reversing glial glutamate transporters relieves neuropathic pain in rats by ameliorating imbalanced neurotransmission.

BACKGROUND: Neuropathic pain results from a lesion or disease affecting the somatosensory system at either the peripheral or central level. The transmission of nociception within the central nervous system is subject to modulation by release and reuptake of neurotransmitters, which maintain a dynamic balance through the assembly and disassembly of the SNARE complex as well as a series of neurotransmitter transporters (inhibitory GABA transporters GAT and excitatory glutamate transporters GT). Neuronal hyper-excitability or defected inhibition involved in neuropathic pain is one of the outcomes caused by imbalanced neurotransmission. SNAP-25, which is one of the SNARE complexes, can modulate the release of neurotransmitters. Glia glutamate transporter (GLT) is one of the two glutamate transporters which account for most synaptic glutamate uptake in the CNS. The role of SNAP-25 and GLT as well as GAT is not clearly understood. METHODS: We used the rat chronic constriction injury (CCI) model for research, and degraded SNAP-25 by a single intrathecal administration of BoNT/A. The mechanical (MWT) and thermal withdrawal latency (TWL) were tested. The level of SNAP-25, GLT, and GAT-1 were assayed using RT-PCR and Western blotting. RESULTS: SNAP-25 was suppressed by a single intrathecal administration of 0.01U BoNT/A and the reduction of SNAP- 25 was correlated with the relief of nociceptive responses in CCI rats. MWT and TWL returned to normal from the 5th to 14th day (P < 0.05) after the administration. On the 14th day after surgery, compared to the sham group, the upregulation of SNAP-25 in CCI rats was reversed after BoNT/A treatment (P < 0.05). The decreased GLT was reversed after BoNT/A treatment but increased GAT-1 was not influenced by BoNT/A treatment. CONCLUSIONS: SNAP-25 and GLT play important roles in the development of neuropathic pain, and the mechanism may involve the imbalance of neurotransmission after peripheral nerve injury. Intrathecal administration of BoNT/A reversed the upregulation of SNAP-25 and downregulation of GLT after CCI, but had no significant effect on the expression of GAT-1.

Effects of intrathecal epigallocatechin gallate, an inhibitor of Toll-like receptor 4, on chronic neuropathic pain in rats.

Numerous studies revealed that spinal inflammation and immune response play an important role in neuropathic pain. In this study, we investigated the effects of intrathecal injection of a Toll-like receptor (TLR4) inhibitor epigallocatechin gallate (EGCG) on neuropathic pain induced by chronic constriction injury of the sciatic nerve (CCI). A total of 120 rats were randomly assigned into 4 groups: sham-operated group, CCI group, CCI plus normal saline group and CCI plus EGCG group. CCI and sham surgeries were performed and both thermal hyperalgesia and mechanical allodynia were tested. Lumbar spinal cord was sampled and the mRNA and protein expressions of TLR4 and High Mobility Group 1 protein (HMGB1) were detected, the contents of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and interleukin-10 (IL-10) were measured by ELISA, and immunohistochemistry for nuclear factor kappa B (NF-κB) was also carried out. When compared with the sham group, both mechanical and heat pain thresholds were significantly decreased, and the mRNA and protein expressions of TLR4 and HMGB1, the contents of TNF-α, IL-1ß and IL-10 in the spinal cords and NF-κB expression in the spinal dorsal horn were markedly increased in CCI rats (P<0.05). After intrathecal injection of EGCG (1mg/kg) once daily from 1day before to 3days after CCI surgery, the expressions of TLR4, NF-κB, HMGB1, TNF-α and IL-1ß were markedly decreased while the content of IL-10 in the spinal cord increased significantly accompanied by dramatical improvement of pain behaviors in CCI rats (P<0.05). These results show that the TLR4 signaling pathway plays an important role in the occurrence and development of neuropathic pain, and the therapy targeting TLR4 might be a novel strategy in the treatment of neuropathic pain.

[Effect of intrathecal ketamine injection on protein kinase C expression in the spinal dorsal horn of rats with formalin-induced pain].

OBJECTIVE: To investigate the expression of protein kinase C (PKC) in the spinal dorsal horn of rats with formalin-induced pain and the effect of intrathecal ketamine on PKC expression. METHODS: Thirty-two SD rats were randomly divided into 4 equal groups, namely the control group, intrathecal saline group (NS), 50 µg ketamine group (K1) and 100 µg ketamine group (K2). The rats were anesthetized with 10% chloral hydrate, and a microspinal catheter was inserted intrathecally into the lumbar region. Five days later, the rats in groups, K1 and K2 were subjected to intrathecal administration of 50 and 100 µg ketamine (10 µl), respectively, followed by 10 µl saline, and those in NS group received 20 µl saline only. Thirty minutes later, 5% formalin (50 µl) was subcutaneously injected into the left hindpaw. The pain intensity score (PIS) was utilized to assess antinociceptive behavior within 1 h after formalin injection. Twenty-four hours later, the left hindpaw thickness was measured and the expression of PKC in the spinal dorsal horn in the L5 segment was assayed using immunohistochemistry. RESULTS: Compared to group NS, groups K1 and K2 showed significantly decreased PIS (P<0.01) in the second phase of formalin-induced pain; 24 h later, the left hindpaw thickness of group NS increased obviously in comparison with that in the control group (P<0.01), whereas the thickness was significantly reduced in group K1 and K2 as compared to that in group NS (P<0.05). The number of immunoreactive cells and the immunohistochemical score of PKC in the spinal dorsal horn were significantly higher in group NS than in group C (P<0.01), but significantly lower in groups K1 and K2 than in group NS (P<0.05). CONCLUSION: Intrathecal ketamine produces obvious antinociception against formalin-induced pain in rats and inhibits the enhanced PKC expression in the spinal dorsal horn in response to formalin-induced pain, suggesting the important role of PKC in nociceptive signal transmission and modulation in the spinal cord.

Intrathecal 5-azacytidine inhibits global DNA methylation and methyl- CpG-binding protein 2 expression and alleviates neuropathic pain in rats following chronic constriction injury.

The pathogenesis of neuropathic pain remains largely unknown. Epigenetic mechanisms may play a major role in regulating expression of pro- or antinociceptive genes. DNA methylation is a major epigenetic mechanism in vertebrates, and methyl- CpG-binding protein 2 (MeCP2) is directly involved in methylation-mediated gene silencing. To determine how changes in global DNA methylation and MeCP2 expression occur following chronic constriction injury (CCI) and how repression of DNA methylation affects these changes and attenuates neuropathic pain, we used intrathecal 5-azacytidine, a DNA methyltransferase inhibitor, in CCI rats. Rats received 0.9% saline or 5-azacytidine (10µmol·d(-1)) via spinal injection once daily from day 3 to day 14 after CCI surgery. Global DNA methylation and MeCP2 expression increased in the spinal cord in CCI rats on day 14 after CCI surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI were attenuated by intrathecal 5-azacytidine from day 5 to day 14 after CCI surgery. The increases in global DNA methylation and MeCP2 expression in the spinal cord in CCI rats were also significantly inhibited by intrathecal 5-azacytidine. These results demonstrate that increased global DNA methylation and MeCP2 expression in the spinal cord after nerve damage may play an important role in neuropathic pain. 5-azacytidine shows potential for treating neuropathic pain.