Objective To investigate the effect of lentivirus-mediated RNA interference(RNAi) on the expression of PKCγ mRNA and protein in rat neurons.Methods Primary cultured cortical neurons from SD rat (16 days of pregnency) embryos were randomly divided into 3 groups with 6 wells in each group:control group (group C),negative group(group NC)and RNAi group.Group C received no treatment.Each well in group NC was given negative lentivirus 3 × 105.Each well in group RNAi was given the recombinant lentiviral vector containing PKCγ shRNA(LV-PKC7 shRNA).The expression of PKCT mRNA and protein in rat neurons was detected by RT-PCR and Western blot 5 days later.The interference efficiency was ealculated.Results Compared with group NC,the expression of PKCγ mRNA and protein was down-regulatedin group RNAi(P<0.05),but no significant change Was found in group NC(P>0.05).The interference efficiency of gene and protein were 99.3%and 85.2%respectively.Conclusion Lentivirus-mediated RNAi can down restate the expression of PKC7 gene and protein in rat neurons.

Objective To evaluate the effect of neurotrophin-3 (NT-3) on the expression of serine/threonine protein kinase (Akt) during ropivacaine-induced neurotoxicity to the spinal cord of rats.Methods Healthy adult male Sprague-Dawley rats,aged 1-2 months,weighing 280-320 g,were used in the study.A catheter was inserted at L5,6 interspace into the epidural space of rats.A total of 108 rats,in which intrathecal catheters were successfully implanted,were randomly divided into 3 groups (n =36each):control group (group C),1％ ropivacaine group (group R),and 1％ ropivacaine + NT-3 group (group NT).The equal volume of normal saline was given in group C,1％ ropivacaine 0.12 ml/kg was injected via the intrathecal catheter once every 1.5 h for 8 times in total in R and NT groups.In addition,NT-3 0.1 mg/kg was simultanenously injected via the intrathecal catheter in group NT.On days 1,3,5,7,14 and 28 after the end of administration (T1-6),6 rats were sacrificed in each group.Their lumbar enlargements were removed for determination of neuronal apoptosis (using TUNEL) and Akt expression (by immuno-histochemistry).The apoptotic rate was calculated.Results Compared with group C,the apoptotic rate was significantly increased at T1-4,and Akt expression was significantly up-regulated at T1-3 in group R,and the apoptotic rate were significantly increased,and Akt expression was significantly up-regulated at T1-3 in group NT (P＜0.05).Compared with group R,the apoptotic rate was significantly decreased at T3,4,and Akt expression was significantly down-regulated at T2.3 in group NT (P＜0.05).Conclusion The mechanism by which NT-3 reduces ropivacaine-induced neurotoxicity to the spinal cord may be related to down-regulation of the expression of Akt in rats.

Objective To investigate the effect of intrathecal injection of lentiviral vector-mediated up-regulation of glial cell line-derived neurotrophicfactor (GDNF) on neuropathic pain of chronic constriction injury (CCI) rats.Methods The CCI model was prepared by ligating the sciatic nerve of Sprague-Dawley (SD) rats.Seven days after CCI modeling,a single intrathecal injection of lentiviral vectors (LV)-GDNF was given.Before CCI and 3,5,7,14,and 21 days after CCI modeling,the mechanical pain threshold was tested in rats,and 21 days after surgery,Western blot was used to detect the expression of GDNF protein.Results On 21 days after CCI modeling,GDNF expression was reduced compared to sham group.After intrathecal injection of LV-GDNF,GDNF expression was up-regulated in the spinal cord,and CCI-induced mechanical hyperalgesia in rats was alleviated.Conclusions Intrathecal injection LV-GDNF can up-regulate the expression of GDNF and alleviate neuropathic pain in CCI rats.

ObjectiveTo evaluate the changes in the expression of purinergic P2X4 receptor (P2X4R) in spinal cord dorsal horn and dorsal root ganglion in a rat model of acute morphine tolerance or inflammatory pain.MethodsThirty male SD rats were randomly divided into 3 groups:normal saline group (group NS,n =5);acute morphine tolerance group (group M,n =5) and inflammatory pain group (group F,n =20).Inflammatory pain was induced by subcutaneous injection of 4％ formalin 50 μl into the plantar surface of left hindpaw in group F.The animals received intrathecal morphine 10 μg ( 10 μl) once every 2 h for 6 times (T1-6) in group M,while the equal volume of normal saline was given instead in group NS.The rats were then sacrificed 2 h after the last time administration.Paw withdrawal latency (PWL) to a thermal nociceptive stimulus was measured at T1-6 in groups M and NS,or on day 4,7,10 and 14 after establishing the model of inflammatory pain in group F,The rats were sacrificed after measurement of PWL and spinal cord dorsal horn and dorsal root ganglion were removed to detect the expression of P2X4 R by immuno-histochemisty.ResultsCompared with the baseline value,PWL was significantly decreased on day 4-14 after intlammatory pain in group F,and PWL was significantly increased at T1-5,while no significant change was found at T6 in group M ( P ＞ 0.05).Compared with group NS,the expression of P2X4 R was up-regulated in group M,and the expression of P2X4 R was up-regulated on day 4 after inflammatory pain,peaked on day 7 after inflammatory pain,and then was down-regulated gradually on day 10 and 14 after inflammatory pain in group F ( P ＜ 0.01).ConclusionThe expression of purinergic P2Y4 R is up-regulated in spinal cord dorsal horn and dorsal root ganglion in rats with acute morphine tolerance or inflammatory pain,and the change may be related to the development of acute morphine tolerance or inflammatory pain.

Objective To construct a lentiviral vector of RNA interference (RNAi) of PKCγ gene. Methods The effective sequence of siRNA targeting PKCγ gene was confirmed in our previous study. The complementary DNA containing both sense and antisense oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFP vector, which contained U_6 promoter and green fluorescent protein (GFP) . The resulting lentiviral vector containing PKCγshRNA was named lentivinis RNAi vector of PKCγ, and it was confirmed by realtime PCR and sequencing. 293T cells were cotransfected with lentiviral vector pGCSIL-CTP, pHelper 1.0 and pHelper 2.0. All virus stocks were produced by calcium phosphate-mediated transfection. The titer of virus was tested according to the expression level of GFP. Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of PKCγ producing PKCγshRNA was constructed successfully. The titer of concentrated virus was 1 ×10~9 TU/ml. Conclusion The lentivinis RNAi vector of PKCy was constructed successfully.

Objective To study the effect of ivabradine on hyperpolarization activated cation current in canine pulmonary vein(PY) sleeve cardiomyocytes with atrial fibrillation.Methods Dissociation of PVs yielded single cardiomyocytes from a Landengorff column without or with pacemaker activity from long-term rapidly atrial pacing (RAP) canines.If current was measured with the whole-cell patch-clamp technique.Results Compared with the control group,the rapidly atrial pacing canine PV cardiomyocytes had spontaneous diastolic depolarization and had larger If densities.Ivabradine (Iva,1 μM),a selective inhibitor of the If current,markedly reduced If currents in the RAP from -2.66±0.4 pA/pF to -1.58±0.1 pA/pF at the test potential of-120 mV (P＜0.01,n=12).Inhibition effect of Iva of If current showed concentration-dependent range from 0.1 to 10.0μM,with IC50 of 2.2 μ M ( 1.8-2.9 μM,95% CL).Furthermore,V1/ of steady-state activated curve was shifted from -84.3±4.9 mV to -106.9±3.4 mV and k value of steady-state activated curve was changed from 12.1+2.6 mV to 9.9±3.4 mV by the application of.1.0 μM Iva ( P＜0.01,n=12).Conclusions Our study revealed that Ivarbadine may significantly decrease If of rapidly atrial pacing pulmonary vein sleeve ceUs with atrial fibdllation.(J Geriatr Cardiol 2008;5:39-42)

OBJECTIVE: To investigate the incidence of awareness during general anesthesia and analyze the risk factors in anesthetic practice and patient populations. METHODS: A total of 2 300 patients who underwent general anesthesia were included. Perioperative data and anesthetic drugs were collected prospectively. Patients were interviewed twice postoperatively with the same structured questionnaire. Each patient was classified into categories as no awareness, possible awareness, and awareness. RESULTS: Twenty-one patients (0.91%) definitely reported awareness, and another 205 (8.91%) reported possible awareness. Few of the patients with awareness required psychological intervention. ASA physical status III-IV and propofol maintenance were associated risk factors of awareness. CONCLUSION The incidence of intraoperative awareness is high in the clinical practice in major medical centers.
Adult ; Anesthesia, General ; adverse effects ; Anesthetics, Intravenous ; adverse effects ; Awareness ; China ; epidemiology ; Female ; Humans ; Incidence ; Intraoperative Complications ; epidemiology ; Male ; Middle Aged ; Propofol ; adverse effects ; Risk Factors

OBJECTIVE: To investigate the effect of pretreatment with neurotrophin-3 (NT-3) on intrathecal ropivacaine in rats. METHODS: A total of 144 male Sprague Dawley rats weighing 280-320 g were successfully implanted with microspinal cather following the improved methods of Yaksh. The rats were randomly divided into 4 groups and given saline (Group NS, n=36), 0.5% ropivacaine (Group M, n=36), 1% ropivacaine (Group R, n=36), and ropivacaine+NT-3 (Group T, n=36). The rats received 0.12 mL/ kg body weight of ropivacaine at 0.5% or 1%, or normal saline only, via an implanted intrathecal catheter at 90-min interval for 12 h in Group NS, M, R and T. In the meantime the rats also received NT-3 0.1 mg/kg in group T. On days 1, 3, 5, 7, 14 and 28, we assessed the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL), behavioural change and histopathological damage score changed for possible neuronal injury within the spinal cord. RESULTS: Compared with Group NS and Group M, the PWMT and PWTL were significantly higher on 1, 3, 5 d and the histopathological damage score was significantly higher on 1, 3, 5, 7, 14 d in Group R (P<0.05). Compared with Group T, the PWMT and PWTL in Group R were significantly higher on 1, 3, 5 d and histopathological damage score was significantly higher on 5, 7, 14 d (P<0.05). CONCLUSION NT-3 pretreatment in mice has obvious protective effect against repeated intrathecal injection of 1% ropivacaine in the spinal nerve.
Amides ; adverse effects ; Animals ; Injections, Spinal ; Male ; Neurotrophin 3 ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ropivacaine ; Spinal Cord ; drug effects

This research focuses on the application of multiple interactive modes in online teaching, combined with the actual teaching cases of the anesthesia equipment course of Xiangya Anesthesiology Specialty of Central South University, showing in detail the preparations for interactive teaching before anesthesia equipment learning, the interaction in online classrooms, the extension of interactive teaching outside the classroom, and the evaluation of interactive teaching feedback mechanism throughout the implementation process. By establishing a "host-guest-viewer" mode, the effect of online live broadcasting is maximized. Through the 360-degree materialized explanation with students as the main body, we will make opening in the pain points and blocking points of online teaching in which students do not go to class and students have no thinking, and promote the improvement of online teaching quality and efficiency. In the following practice, we must continue to work on issues such as the improvement of teacher talent quality, the building of an efficient talent team, and the construction of practical application value evaluation systems for teaching.

Objective:The activation of astrocytes is an important process in the formation of chronic pain.This study aims to observe the activation of A1 reactive astrocytes in the medullary dorsal horn in the rat model of trigeminal neuralgia,and to explore the mechanism of central sensitization caused by A1 reactive astrocyte. Methods:The adult male rats were randomly divided into a sham group and a chronic constriction injury of infraorbital nerve(ION-CCI)group.The facial mechanical pain threshold and thermal withdrawal latency were measured before the operation and on the 1st,3rd,7th,10th,and 14th day after the operation.After pain behavior observation,the expression of glial fibrillary acidic protein(GFAP)in the medullary dorsal horn was observed by immunohistochemistry and immunofluorescence colocalization of GFAP,complement 3(C3)/S100A10,and 4',6-diamidino-2-phenylindole(DAPI)was analyzed.Primary astrocytes were cultured and randomly divided into a naive group and a DHK group.The DHK group was treated with 1 mmol/L of astrocyte activation inhibitor dihydrokainic acid(DHK).Fura-2/AM was used to stain the astrocytes and the calcium wave of the 2 groups under the stimulation of high potassium was recorded and compared.The expression of C3 was detected by Western blotting. Results:The facial mechanical pain threshold and thermal withdrawal latency of the ION-CCI group were significantly lower than those of the sham group(both P<0.05).There were a large number of GFAP positive astrocytes in the medullary dorsal horn of the ION-CCI group.The fluorescence intensity of GFAP in the ION-CCI group was higher than that in the sham group(P<0.05).GFAP and C3/S100A10 were co-expressed in astrocytes.Compared with the sham group,the fluorescence intensity of C3 and the protein expression of C3 in the ION-CCI group were increased(both P<0.05).The expression of C3 in ION-CCI group was significantly increased(P<0.05).Compared with the naive group,the C3 protein expression was significantly decreased in the DHK group(P<0.05).The intensity of calcium fluorescence was increased after high potassium stimulation in both groups.Furthermore,the peak and increase amplitude of calcium fluorescence in the naive group were much higher than those in the DHK group(both P<0.05). Conclusion:A1 reactive astrocytes in the medullary dorsal horn of trigeminal neuralgia model rats are increased significantly,which may participate in central sensitization of trigeminal neuralgia by impacting astrocyte calcium wave.