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Objective To investigate the effect of estrogen-related receptor alpha(ERRα)on lipopolysaccharide-induced inflammatory response in rat pulmonary microvascular endothelial cells (PMVECs) and its mechanism.Methods PMVECs were cultured in vitro.When the cells were in the logarithmic growth phase,the cell were ransfected with lentivirus,and a stable low-expression ERRα cell line was constructed.The cells were divided into four groups:Ctr group (normal control group),Ctr+LPS group (normal celI+LPS treatment group),shERRα1 (shERRα1 gene knockdown group),and shERRα1+LPS group (shERRα1 gene knockdown +LPS treatment group).After 20 μg/mL LPS stimulated cells in the control group and shERRal group for 6,12 and 24 h,cell counting kit-8 (cck-8) was used to detect the cell proliferation ability of each group,and enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of tumor necrosis factor alpha (TNF-α) and Interleukin-1β (IL-1β) in cell culture fluid.After 12 h LPS stimulation,the expression levels of ERRα and NF-κB related proteins (p-p65,p65,P-IKBα,IKBα) were measured by Western blot.Pairwise comparisons were performed with SNK-q test (two-tailed),and multiple-group comparisons were performed with one-way ANOVA.The non-parametric test of rank transformation was used when homogeneity of variance were not met.P value＜0.05 was considered significantly different.Results Compared with the control group,ERRα expression in the shERRα group was significantly decreased (0.09±0.01 vs 0.15±0.01).At 6,12 and 24 h after LPS stimulation,compared with the control group,the cell proliferation ability (％) of the shERRαl+LPS group was significantly reduced (99.68±4.53 vs 48.62±1.60) and the concentration of TNF-α (ng/mL) (15.76±3.38 vs 5 498.91±367.95) and IL-1β (ng/mL) (14.41±3.86 vs 6 014.92±277.33) in the cell culture supematant were significantly increased.The change was most obvious after 12 h stimulation.Meanwhile the expression of p-p65 (0.30±0.50 vs 1.05±0.07) and p-IKBα (0.27±0.04 vs 0.77±0.06) were increased significantly,while the expression of IKBα (0.96±0.07 vs 0.14±0.04) was decreased significantly in the shERRαl+LPS group (all P＜0.05).Conclusion ERRα gene attenuates LPS-induced inflammatory response in rat pulmonary microvascular endothelial cells by inhibiting NF-κB signaling pathway activation.
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Objective To investigate the effects of multidrug resistance-associated protein 4 (MRP4) on the cytoskeleton and cellular permeability of rat pulmonary microvascular endothelial cells (PMVECs) induced by lipopolysaccharide (LPS).Methods PMVECs were cultured for 3 to 6 generations were randomly divided into 4 groups:control group,LPS group,Ad-shMRP4 group (adenoviral expression of a short-hairpin RNA directed against MRP4),Ad-shRNA group.The infection rate of cells was detected by fluorescence microscope observation.The level of MRP4 was assayed by Western botting.Monolayer permeability was determined by the Transwell assay.The morphological characteristic and distribution of Factin was measured by laser confocal fluorescence microscope.Results Compared with control group,the expression of MRP4 protein was up-regulated (P ＜ 0.05) and the significant increase in the permeability of endothelial cells (2 h,6 h,12 h and 24 h respectively:0.28 ±0.02 vs.0.41 ±0.04,0.32 ±0.02,0.30 ±0.01 vs.0.53±0.04,0.39±0.03,0.33 ±0.04 vs.1.12±0.17,0.70 ±0.07,0.32±0.03 vs.0.79 ± 0.02,0.57 ± 0.05,P ＜ 0.05),the F-actin was remodeled,and the stress fibers were formed in LPS group and Ad-shMRP4 group.However,compared with LPS group,the expression of MRP4 protein was down-regulated (P ＜ 0.05) and the markedly decrease in the permeability of endothelial ceils (2 h,6 h,12 h and 24 h respectively:0.41 ± 0.04 vs.0.32 ± 0.02,0.53 ± 0.04 vs.0.39 ± 0.03,1.12 ± 0.17 vs.0.70 ± 0.07,0.79 ± 0.02 vs.0.57 ± 0.05,P ＜ 0.05) was found,and the remodeling of F-actin,and the formation of stress fibers were observed in Ad-shMRP4 group.Conclusions Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton,thus playing an important role in the protection of endothelial cell barrier.
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Objective To investigate the effects of multidrug resistance-associated protein 4 (MRP4) on the cytoskeleton and cellular permeability of rat pulmonary microvascular endothelial cells (PMVECs) induced by lipopolysaccharide (LPS).Methods PMVECs were cultured for 3 to 6 generations were randomly divided into 4 groups:control group,LPS group,Ad-shMRP4 group (adenoviral expression of a short-hairpin RNA directed against MRP4),Ad-shRNA group.The infection rate of cells was detected by fluorescence microscope observation.The level of MRP4 was assayed by Western botting.Monolayer permeability was determined by the Transwell assay.The morphological characteristic and distribution of Factin was measured by laser confocal fluorescence microscope.Results Compared with control group,the expression of MRP4 protein was up-regulated (P ＜ 0.05) and the significant increase in the permeability of endothelial cells (2 h,6 h,12 h and 24 h respectively:0.28 ±0.02 vs.0.41 ±0.04,0.32 ±0.02,0.30 ±0.01 vs.0.53±0.04,0.39±0.03,0.33 ±0.04 vs.1.12±0.17,0.70 ±0.07,0.32±0.03 vs.0.79 ± 0.02,0.57 ± 0.05,P ＜ 0.05),the F-actin was remodeled,and the stress fibers were formed in LPS group and Ad-shMRP4 group.However,compared with LPS group,the expression of MRP4 protein was down-regulated (P ＜ 0.05) and the markedly decrease in the permeability of endothelial ceils (2 h,6 h,12 h and 24 h respectively:0.41 ± 0.04 vs.0.32 ± 0.02,0.53 ± 0.04 vs.0.39 ± 0.03,1.12 ± 0.17 vs.0.70 ± 0.07,0.79 ± 0.02 vs.0.57 ± 0.05,P ＜ 0.05) was found,and the remodeling of F-actin,and the formation of stress fibers were observed in Ad-shMRP4 group.Conclusions Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton,thus playing an important role in the protection of endothelial cell barrier.
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Objective To introduce a new modified percutaneous dilational tracheostomy and compare the application effect of percutaneous dilational tracheostomy with modified percutaneous dilational tracheostomy in the treatment of critically ill patients. Methods A total of 60 critically ill patients undergoing tracheotomy were selected , and they were randomly divided into two groups according to the methods of tracheotomy. Sex, age, weight, body mass index, acute physiology and chronic health evaluation, operation time, incision size, intraoperative blood loss, incision healing time, incidences of complications after operation were compared between the two groups. Results There were not statistically significant differences of in sex, age, weight, body mass index, and acute physiology and chronic health evaluation between percutaneous dilational tracheostomy group and modified percutaneous dilational tracheostomy group (P>0.05). Operation time, incision size and intraoperative blood loss of modified percutaneous dilational tracheostomy group was statistically significantly shorter than that of percutaneous dilational tracheostomy group [(5.80 ± 1.19) min vs. (7.65 ± 1.05) min, (8.33 ± 3.30) ml vs. (11.33 ± 4.34) ml, (1.08 ± 2.96) cm vs. (1.27 ± 2.54) cm] (P0.05). Conclusions The modified percutaneous dilational tracheostomy can save operation time, and reduce intraoperative blood loss, so it can be widely used.
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Objective To investigate the effect of multidrug resistance protein 4 (MRP4) overexpression on lipopolysaccharide (LPS)-induced vascular endothelial hyperpermeability of rat pulmonary micro-vascular endothelial cells (PMVECs) and its molecule mechanism. Methods Three to six passages of PMVECs were cultured in vitro, and they were divided into three groups: the cells in LPS group were only challenged by LPS 10 μg/mL after being cultured in serum-free medium for 24 hours; the cells in Ad-shRNA and Ad-MRP4 groups were infected with the empty virus control or recombinant adenovirus expressing MRP4 for 2 hours, and then were cultured in serum-free medium for 24 hours followed by stimulation of LPS 10 μg/mL. Endothelial permeability was assayed by the Transwell chamber models at 2, 6, 12, and 24 hours after LPS stimulation. Intracellular cyclic adenosine monophosphate (cAMP) levels were detected by enzyme-linked immunosorbent assay (ELISA). The morphological characteristics and distribution of F-actin was determined by laser confocal fluorescence microscope. The protein expressions of MRP4,β-catenin, vascular endothelium-cadherin (VE-cad) and ZO-1 were measured by Western Blot. Results ① After LPS stimulation, endothelium permeability and intracellular cAMP levels in PMVECs were significantly increased, peaked at 12 hours, and then decreased after 24 hours. Compared with LPS group and Ad-shRNA group, PMVECs of Ad-MRP4 group were exhibited a significant increase in endothelial permeability [12-hour permeability (A value):1.88±0.06 vs. 1.12±0.17, 1.10±0.18] and a significant decrease in intracellular cAMP level [12-hour cAMP (μg/L):2.39±0.02 vs. 2.97±0.01, 3.00±0.02, all P 0.05).② Under laser confocal fluorescence microscope, after LPS stimulation, the stress fiber formation was induced in three groups. But there were pronounced irregular aggregation of fiber in PMVECs of Ad-MRP4 group. ③ Furthermore, compared with LPS group and Ad-shRNA group, protein expression of MRP4 in Ad-MRP4 group was dramatically increased (gray value: 0.76±0.03 vs. 0.44±0.02, 0.43±0.02, both P 0.05). Conclusion MRP4 overexpression can decrease intracellular cAMP levels, reduce intercellular junction protein expression, and then exaggerate LPS-induced vascular endothelial hyperpermeability.
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Objective To evaluate whether there is a correlation among plasma levels of procalcitonin (PCT),N-terminal pro-brain natriuretic peptide (NT-pro-BNP)and cardiac troponin T (cTnT)in patients with sepsis,as well as significance to prognosis of patients.Methods 48 patients with sepsis who were admitted to the intensive care unit of a hospital between September 2014 and March 2015 were chosen for study,patients were divided into severe and mild sepsis groups according to the disease condition,plasma levels of PCT,NT-pro-BNP,and cTnT were de-tected,mortality of patients were analyzed statistically,relation between plasma levels of PCT,NT-pro-BNP,cT-nT and patients’death were compared.Results The plasma levels of PCT,NT-pro-BNP and cTnT in severe sepsis group were significantly higher than those in mild sepsis group (all P <0.05);mortality of mild sepsis group was significantly lower than that of severe sepsis group (10.53% vs 41.38%,P <0.05);Levels of PCT,NT-pro-BNP and cTnT levels in died patients were all higher than surviving patients (all P <0.05);levels of PCT and NT-pro-BNP,NT-pro-BNP and cTnT were positively correlated respectively (rs = 0.337,P <0.05;rs =0.456,P =0.001, respectively ),while PCT was not significantly correlated with cTnT.Plasma levels of PCT,NT-pro-BNP,and cTnT were all correlated with the prognosis of patients (P <0.05),and is helpful for judging the prognosis of pa-tients,combination of three indexes had better prognostic value for the prognosis.Conclusion Combination detec-tion of plasmid levels of PCT,NP-pro-BNP,and cTnT can assess the severity of infection in patients with sepsis, and preliminarily judge the prognosis of patients with sepsis.
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Objective To investigate the protective effect of multidrug resistant associated protein 4 (MRP4) inhibitor on rats with sepsis-induced acute lung injury (ALI). Methods Sixty Sprague-Dawley (SD) rats were randomly divided into sham group, sepsis group and MRP4 inhibitor MK571 treatment group, with 20 rats in each group. Sepsis model was reproduced by cecal ligation and puncture operation (CLP), and the rats in sham group were only received celiotomy without ligation and puncture. Rats in MK571 treatment group were intraperitoneally injected with MRP4 inhibitor MK571 (20 mg/kg) 30 minutes before model reproduction, while rates in sham group and sepsis group were given the same amount of normal saline. Twenty-four hours later, the femoral artery blood of mice was collected, and arterial blood gas analysis was measured. Serum tumor necrosis-α (TNF-α) was determined by enzyme-linked immunosorbent assay (ELISA). The lung tissues were collected, and the wet/dry weight ratio (W/D) was calculated. The expression of MRP4 protein in lung tissue was determined by Western Blot. Results Compared with sham group, arterial blood pH value and arterial partial pressure of oxygen (PaO2) were significantly lowered [pH value: 7.18±0.03 vs. 7.40±0.03; PaO2 (mmHg, 1 mmHg = 0.133 kPa): 63.15±6.24 vs. 98.05±2.58], while arterial partial pressure of carbon dioxide (PaCO2) was dramatically higher in the sepsis group (mmHg: 56.60±8.30 vs. 37.85±3.18), serum TNF-α level in the sepsis group was significantly increased (ng/L: 146.24±19.99 vs. 25.77±9.83), the W/D ratio of lung tissue was significantly increased (7.75±0.47 vs. 4.09±0.58), and the expression of MRP4 protein was up-regulated in the sepsis group (gray value: 0.153±0.006 vs. 0.087±0.005, all P < 0.05). Compared with the sepsis group, arterial blood pH value (7.30±0.02 vs. 7.18±0.03) and PaO2 (mmHg: 80.30±5.34 vs. 63.15±6.24) were significantly elevated in the MK571 treatment group, while PaCO2 was dramatically decreased (mmHg: 29.25±3.24 vs. 56.60±8.30), the serum level of TNF-α was significantly decreased (ng/L: 97.96±16.72 vs. 146.24±19.99), the W/D ratio of lung tissue was significantly reduced (5.89±0.51 vs. 7.75±0.47), and MRP4 protein expression was significantly down-regulated (gray value: 0.124±0.006 vs. 0.153±0.006, all P < 0.05). Conclusion MRP4 inhibitor may improve lung function in rats with sepsis-induced ALI by down-regulating MRP4 protein expression and reducing levels of inflammatory cytokines, which exerts protective effect on ALI.
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Anti-neutrophil cytoplasm antibody-associated vasculitis (ANCA) is an autoimmune disease with multi organ involvement characterized by vascular wall inflammation and fibrinoid necrosis, including microscopic polyangitis (MPA), granuloma polyangitis (GPA), and eosinophilic granuloma polyangitis (EGPA). Because its clinical manifestations are complicated and non-speciifc, it is dififcult to make early diagnose. In recent years, some new progress has been made in diagnosis and treatment of this disease. The article will review the related information.
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Objective To explore the effects of multidrug resistance-associated protein 4 (MRP4) inhibition on pulmonary vascular endothelial barrier dysfunction in septic rats.Methods Sixty Sprague Dawley rats were randomly (random number) divided into three groups:sham-operated group,sepsis group,and sepsis plus MRP4 inhibitor treatment group,with 20 rats in each group.Sepsis was induced by cecal ligation and puncture.MRP4 inhibitor MK571 (20 mg/kg) was administrated by intraperitoneal injection 30 minutes before induction of sepsis.Twenty-four later,serum interlukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels were measured by enzyme-linked immunosorbent assay.Lung injury was assessed by histopathological examination.Lung vascular permeability was evaluated by quantitation of Evans blue dye extravasation from vascular space to lung parenchyma.Results Compared with sham group,serum IL-6 and TNF-α levels were significantly higher in sepsis group.In addition,lung injury and lung vascular permeability were elevated in sepsis group compared to sham group.Importantly,MRP4 inhibitor treatment significantly decreased serum IL-6 and TNF-α levels,improved lung injury and reduced lung vascular permeability in septic rats.Conclusions Inhibition of MRP4 protects against pulmonary vascular endothelial barrier dysfunction in septic rats.
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Objective To observe the influence of intensive insulin therapy on inflammatory fac-tors and prognosis in severe multiple trauma patients.Methods A total of 53 cases of severe multiple trauma were randomly divided into the treatment group(n =27)and the control group(n =26).Besides basic treatment,patients in the treatment group received additional intensive insulin therapy by micro-pump.The level of blood glucose in the control group was controlled under 11.1 mmol/L.Levels of TNF-α,IL-1β,IL-6,and CRP were tested before and after treatment.Multiple organ dysfunction syndrome,noso-comial infection rate,and mortality rate were also observed.Results The levels of TNF-α,IL-1β,IL-6, and CRP in the treatment group were significantly lower than those of the control group(P <0.05 or P <0.01).The incidence of multiple organ dysfunction syndrome,nosocomial infection,and mortality rate in the treatment group was lower(P <0.05).Conclusion Intensive insulin therapy can effectively decrease the expressions of inflammatory factors in patients with severe multiple trauma,improve the prognosis,re-duce the incidence of nosocomial infection and mortality.
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<p><b>BACKGROUND</b>Surfactant protein A (SP-A) contributes to the regulation of sepsis-induced acute lung injury. In a previous study, we demonstrated the expression and localization of SP-A in the kidneys. The present study evaluated the effect of SP-A on lipopolysaccharide (LPS)-induced tumor necrosis factor-a (TNF-α) expression and its underlying mechanisms in the human renal tubular epithelial (HK-2) cells.</p><p><b>METHODS</b>Indirect immunofluorescence assay was used to detect SP-A distribution and expression in HK-2 cells. HK-2 cells were treated with various concentrations of LPS (0, 0.1, 1, 2, 5, and 10 mg/L) for 8 hours and with 5 mg/L LPS for different times (0, 2, 4, 8, 16, and 24 hours) to determine the effects of LPS on SP-A and TNF-α expression. Then, HK-2 cells were transfected with SP-A siRNA to analyze nuclear factor κB (NF-κB) P65 and TNF-α expression of HK-2 cells after LPS-treatment.</p><p><b>RESULTS</b>Indirect immunofluorescence assay revealed that SP-A is localized to the membrane and cytoplasm of HK-2 cells. Interestingly, SP-A1/SP-A2 and TNF-a expression were found to be significantly increased in HK-2 cells upon LPS treatment. Transfection of LPS-treated HK-2 cells with SP-A siRNA resulted in significant increases in the levels of NF-κB P65 protein and TNF-α mRNA and protein compared to those in non-transfected LPS-treated HK-2 cells.</p><p><b>CONCLUSION</b>SP-A plays an important role in protecting cells against sepsis-induced acute kidney injury by inhibiting NF-κB activity to modulate LPS-induced increase in TNF-α expression.</p>
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Humans , Cell Line , Epithelial Cells , Cell Biology , Metabolism , Fluorescent Antibody Technique, Indirect , Kidney Tubules, Proximal , Cell Biology , Lipopolysaccharides , Pharmacology , Pulmonary Surfactant-Associated Protein A , Metabolism , Pharmacology , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
Objective To observe the pathomorphological changes and expressions of TNF-α, SOD1 and SOD2 in cardiac tissues of rats with abdominal aorta champing(AAC)and LPS attack.Methods Forty Wistar rats were randomly divided into four groups:sham operated group (Group S),ischemia reperfusion with AAC group(Group I /R),intraperitoneal injection of endotoxin group(Group LPS)and AAC +LPS group(Group I /R +LPS).Animals were sacrificed after 20 minutes of AAC and 8 hours of reperfusion.Cardiac tissues were observed with HE staining for pathmmorphological changes,and detection of TNF-α,SOD1 and SOD2 expressions were tested by immunohistochemistry.Results The mortality rate of Group I /R +LPS was highest.HE staining of the cardiac tissues revealed that the pathological changes of myocardial tissue were not obvious in Group S;myocardial cells were disordered and slightly swollen and red blood cell and inflammatory cells were observed in the intramuscular space of the Group LPS;these injuries were more serious in the Group I /R;the injuries of cardiac tissues were most serious in Group I /R +LPS.The results of immunohistochemical staining revealed that TNF-αexpression level of cardiac tissue increased as followings:Group S,Group LPS,Group I /R and Group I /R +LPS.Significant differences were observed among groups,except for the difference between Group LPS and Group I /R.The expression level of SOD1 decreased as followings:Group S,Group LPS,Group I /R and Group I /R +LPS. Significant differences were observed among groups,except for the difference between Group I /R and Group I /R +LPS.The expression level of SOD2 decreased as followings:Group S,Group LPS,Group I /R +LPS and Group I /R.Significant differences were observed among groups,except for the differences between Group LPS and I /R and Group I /R +LPS.Conclusion The mortality in rats with sepsis after abdominal aorta clamping is high.Obvious myocardial morphological changes and injuries exist either in ischemia reperfusion or sepsis state.At the same time,the expression of TNF-αis up-regulated while SOD1 and SOD2 are down-regulated.
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Objective To compare the myocardial damage in patients with severe valvular heart disease undergoing open heart surgery under propofol and sevoflurane combined anesthesia.Methods Thirty-two patients with severe heart valvular disease undergoing open heart surgery were randomized into 3 groups:midazolam group (group M,n =8),propofol group (group P,n =12) and sevoflurane group (group S,n =12).Midazolam 1-5 mg,vecuronium 0.15 mg/kg and fentanyl 10-20 μg/kg were injected intravenously in group M.Propofol 1-2 mg/kg,vecuronium 0.15 mg/kg and fentanyl 10 μg/kg were injected intravenously in group P.In group S,the patients inhaled sevoflurane until the eyelash reflex disappeared,the end-tidal concentration of sevoflurane was 0.5 ％-2.0％,and vecuronium 0.15 mg/kg and fentanyl 10μg/kg were injected intravenously.The patients were mechanically ventilated after tracheal intubation.Anesthesia was maintained with iv infusion of midazolam 0.1 mg· kg-1 · h-1,fentanyl 0.2 μg· kg-1 · min-1,and vecuronium 0.12 mg· kg-1 · h-1 in group M,with iv infusion of propofol 150 μg· kg-1 · min-1 and fentanyl μg· kg-1 · min-1 in group P,or with inhalation of 0.5％-2.0％sevoflurane in group S.CPB was established routinely.The concentration of sevoflurane was 0.5 ％-1.0％ during CPB.Venous blood samples were collected before anesthesia (T1),at 20 min and 2 h after aortic unclamping (T2,3),and at 24 h after operation (T4) for determination of the levels of plasma lactic dehydrogenase (LDH),creatine kinase (CK),creatine kinase MB (CK-MB),cardiac troponin Ⅰ (cTnⅠ),superoxide dismutase (SOD)and tumor necrosis factor (TNF)-α.Myocardial tissues were taken at T2 for determination of heme oxygenase-1 (HO-1) expression and for examination of the myocardial ultrastructure.Results Compared with group M,the levels of plasma LDH,CK-MB,and CK were significantly decreased at T2-4,the levels of plasma SOD and cTnⅠ were significantly increased at T2,3,and the expression of HO-1 was up-regulated at T2 in groups P and S,and the levels of plasma TNF-α were significantly decreased at T2-4 in group P and at T2,3 in group S (P ＜ 0.05).The pathologic changes induced by I/R were less severe in groups P and S than in group M.Conclusion Both propofol and sevoflurane can attenuate the myocardial damage in patients with severe valvular heart disease undergoing open heart surgery and the effects are comparable.
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Objective To study the role of changes of oropharyngeal microflora in pathogenesis of post-traumatic ventilator-associated pneumonia (VAP). Methods Forty-five patients with post-traumatic VAP treated with intubation and mechanical ventilation were involved in the study.Microbiologic cultures and drug-sensitivity test of oropharyngeal secretions,subglottic secretions,sputum from deep airway and gastric fluid samples were performed at days 1,3,7 and 14 after mechanical ventilation.The stool samples were collected to detect the coccus and bacillus ratio and the fungus.The concordance rate of microflora among subglottic secretions,sputum,gastric fluid and oropharyngeal secretions were compared. Results The main pathogens for VAP patients were gram-negative bacilli.The study showed an increase in aspects of the positive rate of etiology cultures of subglottic secretions,sputum and gastric fluid samples,the concordance rate of microflora among subglottic secretions,sputum,gastric fluid and oropharyngeal secretions and the number of ESBL + and multi-resistant bacteria,along with the prolonged mechanical ventilation. Conclusions The changes of oropharyngeal microflora are closely associated with the development of VAP.The enterobacteria in the gastric cavity always reversely colonize in the oropharynx and the retrograde stomach-pharynx-lower respiratory tract infection is a major infection route of VAP.
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BACKGROUND: Ketamine is a kind of frequently used general venous anesthesia drug in clinic, and the medication in vein or epidural cavum has analgesic effect. It is N-methyl-D-aspartic acid (NMDA) receptor noncompetitive antagonist, which can inhibit toxic effect of excitatory amino acids.OBJECTIVE: To observe effect of ketamine on apoptosis of dorsal horn astrocytes of spinal cord of rats induced by NMDA receptor over activation and explore its possible mechanism of action.DESIGN: Randomized controlled observation.SETTING: Department of Anesthesiology, Taihe Hospital Affiliated to Yunyang Medical College.MATERIALS: The experiment was conducted at Cell Biology Laboratory,Institute of Basic Medical Sciences, Yunyang Medical College between September 2003 and January 2005. Neonatal Wistar rats of two or three days were provided by Animal Experimental Center of Wuhan University. METHODS: Primary astrocytes in dorsal horn of T11-L6 spinal cord of Wistar rats were purified and cultured. Astrocytes were used in the experiment when its purity coefficient reached 98% assessed by gial fibrillary acidic protein. The cultured cells in 24-well plates were divided randomly into 6 groups (9 portions in each group): ①50 μL Hanks liquor was added into the control group. ②Amount of 100μmol/L was added into the NMDA group. ③Amount of 1 mmol/L was added into the ketamine group. ④100μmol/L NMDA + 0.1 mmol/L ketamine group. ⑤100 μmol/L NMDA + 0.5 mmol/L ketamine group. ⑥100μmol/L NMDA + 1 mmol/L ke tamine group. 1 mmol/L ketamine was clinical antalgic dosage. Activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) were examined after 24-hour culture. Content of Bcl-2 protein and change of morphology were observed with immunocytochemistry. Apoptosis of astrocytes was measured with flow cytometry. MAIN OUTCOME MEASURES: ① Counterstain cell staining and changes of morphology of Bcl-2 protein with immunohistochemical method and hematoxylin-esoin staining (HE). ②Apoptosis of astrocytes was detected with flow cytometry. ③Content of MDA and activity of SOD.RESULTS: ①Mean absorbance (A) of Bcl-2 as expression of Bcl-2 protein measured semiquantitatively: It was lower in the 100μmoL/L NMDA group than the control group, which had significant difference [0.054±0.021,0.108±0.039, respectively, P＜0.01]. It was higher in the 100 μmol/L NMDA + 1 mmol/L ketamine group than the 100 μmol/L NMDA group,which had significant difference [0.148±0.045, 0.054±0.021, respectively,P ＜ 0.01]. ②Apoptosis of astrocytes detected with flow cytometry: It was higher in the 100μmol/L NMDA group than the control group, which had significant difference [(25.26±6.13)%, (5.66±2.24)%, respectively, P＜0.01].It was lower in the 100μmol/L NMDA + 1 mmol/L ketamine group than in the 100μmol/L NMDA group, which had significant difference[(24.41±4.82)%, (25.26±6.13)%, respectively, P＜0.01]. ③Content of MDA and activity of SOD: 100 μmol/L NMDA made the content of MDA in astrocytes obviously increase , while the activity of SOD markedly decrease. 1 mmol/L ketamine remarkably decreased the content of MDA, distinctly increased the activity of SOD. This effectiveness had evidently dosage-effect relationship in clinical antalgic dosage, which had obviously difference as compared with that of the NMDA group (P ＜ 0.01 ). The differences between the 1 mmol/L ketamine group and the control group as well as between the 100 μmol/L NMDA + 0.1 mmol/L ketamine group and the NMDA group had insignificant difference.CONCLUSION: NMDA receptor over activation can induce apoptosis of a great number of astrocytes in spinal dorsal horn of rats. Suitable ketamine dramatically inhibits apoptosis, and its mechanism can enhance the expressionof Bcl-2 protein of astrocytes, at the same time inhibit the production of free radical and reinforce the activity of SOD.
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BACKGROUND: It has been reported that the ischemia preconditioning (IPC) had credible protective efficiency on ischemic injury of the spinal cord during aorta operation, but the mechanism of the protective efficiency of IPC had not been clarified.OBJECTIVE: To study the protective effects of repetitive IPC on ischemic injury of spinal cord and its mechanism in rabbits.DESIGN: A completely randomized controlled study based on the experimental animals.SETTING: Department of anesthesiology in a university hospital.MATERIALS: The experiment was completed in the Department of Anesthesiology, Renmin Hospital, Wuhan University during September and December 2002. Twenty-four Japanese rabbits were randomly and double-blindly divided into sham-operation group, ischemia-reperfusion group and IPC group with 8 rabbits in each group.INTERVENTIONS: In sham-operation group, abdominal aorta was not clamped. Spinal cord ischemia was induced by infra-renal aortic cross-clamp for 45 minutes in ischemia-reperfusion group. Before the 45 minutes ischemia, the rabbits in the IPC group underwent four cycles of ischemia preconditioning, i.e. clamping abdominal aorta for 5 minutes then reperfusion for 5 minutes.MAIN OUTCOME MEASURES: The concentrations of calcium, magnesium, copper and zincum in spinal cord were measured in the 7th day after operation. Postoperative neurological function, EMG of rear limb, and spinal cord histopathological changes were assessed in all groups after operation.RESULTS: The concentrations of calcium and copper in spinal cord in ischemia-reperfusion group were significantly higher than those in sham-operation group( P ＜ 0. 05 or 0. 01 ), but magnesium and zincum significantly lower( P ＜ 0. 05). Compared with IPC group, calcium in ischemia-reperfusion group was significantly higher( P ＜ 0.01 ), but zincum significantly lower( P ＜ 0.01 ) . The neurological function and histopatholohical changes in ischemia-reperfusion group were much lower than those in sham-operation group and IPC group ( P ＜ 0.05 or P ＜ 0.01) . And there was significantly worse change of EMG in ischemia-repeffusion group than that in IPC group(P ＜ 0.01).CONCLUSION: Repetitive ischemic preconditioning can protect rabbit spinal cord from ischemia reperfusion injury quickly, and one possible reason for its protective effect is to maintain the balance of calcium, magnesium,copper and zincum in ischemic region.
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Objective To investigate the effects of different concentrations of ketamine on the apoptosis of cultured spinal dorsal horn neurons and astrocytes induced by glutamate. Methods Newborn Wistar rats (1-3 days old) weighing 10-15 g were used in this study. The animals were anesthetized and the dorsal hom of T11-L5 segment of the spinal cord was separated under sterile condition. The neurons and astrocytes were obtained by trypsin digestion of the tissue and mixed and cultured for 2 weeks. The cells were then randomly divided into 6 groups ( n = 8 each) : control group (C) in which Hank solution 50 ?l was added; glutamate group (G) glutamate was added (the final concentration was 100 ?mol?L-1); ketamine group (K) ketamine was added (final concentration = 1 mmol?L-1) and glutamate-ketamine groups (GK1 , GK2, GK3) glutamate was added first (final concentration was 100 ?mol?L-1) and 30 minutes later ketamine was added (the final concentration was 0.1, 1 or 10 mmol?L-1). After being incubated for 48 h the supernatant was harvested for determination of IL-1? and TNF-? concentrations. The morphological changes of the cells were examined by Wright staining. The apoptotic neurons and astrocytes were detected by flow cytometry.Results The number of apoptotic cells was significantly greater and the concentrations of IL-1? and TNF-? were significantly higher in group G and GK1 than in group C (P
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Objective To investigate the effect of propofol on expression of cyclinD1 and apoptosis in spinal cord neurons induced by ischemia-reperfusion (I/R) .Methods Sixty male Wistar rats weighing 200-250 g were randomly divided into 2 groups ( n = 30 each): group A I/R and group B propofol + I/R. The animals were anesthetized with 7% chloral hydrate 6 ml?kg-1. The abdomen was opened and the abdominal aorta was clamped distal to the left renal artery for 20 min. The aortic cross clamp was then released to allow reperfusion. In propofol group propofol 100 mg?kg-1 was administered intraperitoneally (IP) during the operation. Neurologic function was assessed using Taylor scale (0 = unable to move hind limbs, 4 = normal function) at 6 h after operation and on postoperative day 1, 2, 3, 7 (n = 6 at each time point) . The animals were killed after neurologic function evaluation and the spinal cord was removed for microscopic examination, detection of apoptosis in the spinal cord neurons ( TUNEL) and determination of cyclinD1 expression (immuno-histochemistry) . Results The histo-pathological damage to the neurons was significantly lighter, the neurological function better and the apoptotic index and the cyclinD1 expression were significantly lower in propofol group than in I/R group. Conclusion Propofol protects spinal cord against I/R injury by reducing neuronal apoptosis through down-regulation of cyclinD1 expression.