Objective To investigate the effect of human hepatocyte growth factor (hHGF) genetic modification on the ameliorating effects of mesenchymal stem cells (MSCs) implantation on pulmonary microvascular rarefaction in a rat model of pulmonary hypertension (PH).Methods MSCs were obtained from F344 rats and transduced with lentiviral vector modified with human HGF (hHGF-MSCs) or empty vector (EGFP-MSCs).Sixty-six 7 week old male F344 rats weighing 180-250 g were used in this study.PH was induced by left pneumonectomy and subcutaneous monocrotaline (MCT) 60 mg/kg injected at 2 weeks after operation.The animals with PH were randomly divided into 3 groups:control group (group C),EGFP-MSCs group (group E) and HGF-MSCs group (group H).Groups H and E received hHGF-MSCs or EGFP-MSCs 5 × 105 in DMEM 1 ml iv at 3 weeks after subcutaneous MCT injection,while group C received plain DMEM 1 ml.Mean pulmonary arterial pressure (mPAP) was measured and right ventricular hypertrophy and angiogenesis in the lung were assessed and the content of rat HGF (rHGF) and hHGF protein in lung tissue and pulmonary capillary density (by immuno-histochemistry) was measured at 2 weeks after MSCs implantation.The survival rates within 45 days after MCT administration were compared among the 3 groups.Results No hHGF was detected in groups C and E.Both hHGF-MSCs and EGFP-MSCs significantly reduced MPAP and right ventricular hypertrophy and increased pulmonary capillary density and survival rates in groups H and E as compared with group C and the efficacy of hHGF-MSCs was significantly greater than that of EGFP-MSCs.Barium angiography revealed that distal pulmonary vasculature was significantly increased in group H as compared with groups E and C.The survival of the rats receiving hHGF-MSCs was significantly longer in group H than that in groups E and C.Conclusion hHGF genetic modification can improve the ameliorating effects of MSCs implantation on PH-related microvascular rarefaction.

Objective To investgate the changes in the expression of hepatocyte growth factor (HGF)and c-met in the lungs in a rat model of pulmonary hypertension.Methods Eighty 7 week old male SD rats weighing 180-250 g were randomly divided into 2 groups ( n =40 each ):control group (group C) and pulmonary hypertension group (group PH).Pulmonary hypertension was induced by left pneumonectomy and subcutaneous monocrotaline (MCT) 60 mg/kg 2 weeks later.Pulmonary artery pressure and the ratio between the weight of right ventricle and left ventricle + interventricular septum ( RV/LV + S) were measured at 7,14,21 and 28 d after MCT administration.HGF and c-met protein and mRNA expression and TGF-β content in the lung tissue were determined.Results Pulmonary hypertension and right ventricular hypertrophy associated with hypertrophy of pulmonary artery tunica media and muscularization of small pulmonary arteries developed after MCT administration in PH group.In PH group HGF protein and mRNA expression in the lungs was significantly down-regulated as compared with group C.There were no significant differences in c-met protein and mRNA expression in the lungs between the 2 groups.The TGF-β content in the lungs was significantly increased in group PH as compared with group C.Conclusion Decrease in HGF production in the lungs plays an important role in the pulmonary hypertension.Increasing of pulmonary TGF-β may play an important role in the down-regulation of pulmonary HGF expression during pulmonary hypertension.

ObjectiveTo investigate the effect of ketamine on nicotine-induced current in rat superior cervical ganglion neurons.MethodsNewborn Wistar rats were used in this study.Neurons were isolated enzymatically from superior cervical ganglia of newborn rats in an aseptic condition and cultured in 90％ DMEM/F12,10％ horse serum containing penicillin 100 μg/ml for 5-7 d.Nicotine-induced current was measured and recorded using whole-cell patch clamp technique.A mixture of nicotine 50 μmol/L and different concentrations of ketamine ( 10,25,50,100 μmol/L) was added to the isolated neurons.The effect of ketamine on nicotine-induced current was evaluated.ResultsNicotine-induced peak current was inhibited by ketamine in a concentration-dependent manner.The time constant of fast and slow desensitizing phase of the nicotine acetylcholine receptor was shortened after being exposed to the mixture of nicotine 50 μmol/L + 50 or 100 μmol/L ketamine as compared to nicotine 50 μmol/L-induced current.The median effective concentration of ketamine inhibiting nicotine-induced current was less than 20 μmol/L.ConclusionKetamine can decrease nicotine-induced current in rat superior cervical ganglion neurons in a concentration-dependent manner indicating that inhibition of sympathetic activity is involved in the mechanism of decrease in BP by ketamine in specific condition.

BACKGROUND: As an important part of systemalimbica, hippocampus involves in emotion, perceiving and learning memory and can be affected by anesthesia.OBJECTIVE: With target controlled infusion of propofol, the depth of anesthesia was well controlled. And under anesthesia in various depths, cfos gene expressions in different regions of hippocampus in rabbits were detected to find the target site for central nervous inhibition by propofol.DESIGN :It was a randomized controlled study.SETTING:Department of Anesthesiology ,Shenzhen Second People's Hospital; Department of Anesthesiology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was conducted in the Neurobiological Laboratory of Tongji Medical College, Huazhong University of Science and Technology from May 2000 to June 2001. Thirty Japanese white rabbits were selected and randomly divided into control group, light anesthesia group and deep anesthesia group, with 10 rabbits in each group.METHODS:Intravenous cannulas were placed in external jugular vein (EJV) and femoral artery in all animals. According to the propofol plasma concentration, the infusion of propofol and the depths of anesthesia were well controlled. In light anesthesia group, the plasma concentration of propofol was (9.28±0.12)mg/L. In deep anesthesia group, the plasma concentration of propofol was (11.63±0.29)mg/L. Thirty minutes after being anesthetized, the animals in the two experimental groups were decapitated and the animals in control group were killed by air embolism through ear vein. Coronal sections were sliced continuously, in thickness of 7μm and 1 slice in 100 μm tissue was selected. In situ hybridization was performed to detect the c-f os mRNA in Area CA1, CA3 and dentate gyrus of the hippocampus. In each rabbit, 5 sections were selected randomly.Under a light microscope, photos were taken in 15-20 fields. And then average absorbency and average grayscale were calculated. The grayscale scores were classified as 256 scales. A lower grayscale score indicated a higher positive rate.MAIN OUTCOME MEASURES: ①Under various depths of anesthesia,in situ hybridization results of Area CA1, CA2 and dentate gyrus of the anesthesia, average grayscale scores of Area CA1, CA2 and dentate gyrus of the hippocampus in rabbits were assessed.② Under various depths of anesthesia, average grayscale scores of Area CA1, CA2 and dentate gyrus of the hippocampus in rabbits were calculated.RESULTS:Thirty rabbits entered the statistical analysis procedure.①Under various depths of anesthesia, in situ hybridization results of Area CA1 of the hippocampus in rabbits: In control group, brown, sparse or dense, light-stained or deep-stained c-fos positive cells could be observed. In light anesthesia group, dense, moderately stained c-fospositive neurons could be observed. In deep anesthesia group, cells were denser with deeper stained cytoplasma. ② Under various depths of anesthesia, in situ hybridization results of dentate gyrus of the hippocampus in rabbits: In light anesthesia group, positive cells were strongly stained in deep brown with transparent and vacuolar nuclei. In deep anesthesia group, a large number of c-fos positive cells in great dense could be observed. ③Under various depths of anesthesia, grayscale scores of different regions of the hippocampus in rabbits: Compared with control group, grayscale scores of Area CA1 and dentate gyrus of the hippocampus were significantly decreased in both light and deep anesthesia groups [(168±5), (80±7), (59±5)% ,P ＜ 0.05; (163±8),(103±15), (67±6)%,P ＜ 0.05,P ＜ 0.01]. This was more significant in deep anesthesia group than in light anesthesia group (P ＜ 0.01 ). For Area CA3, the grayscale scores in each group were similar.CONCLUSION: ①With the increasing depth of propofol anesthesia, c-fos gene expression is increased in hippocampus in rabbits. ② After anesthesia, the average grayscale score of Area CA1 and dentate gyrus of the hippocampus are significantly decreased, and this is more significant after deep anesthesia. However, there is no significant change in Area CA3. This indicates that the central inhibitory receptor sites of propofol are various in different brain regions, which supposes that the Area CA3 is not the central receptor sites of propofol.

To investigate the protective effect of genistein on endotoxin-induced acute lung injury in rats, and explore the underlying mechanisms, 32 male Sprague-Dawley rats were randomly divided into 4 experimental groups: saline control, genistein alone, lipopolysaccaride alone, and genistein pretreatment. Each treatment group consisted of eight animals. Animals were observed for 6 h after LPS challenge, and the wet/dry (W/D) weight ratio of the lung and bronchoalveolar lavage fluid(BALF) protein content were used as a measure of lung injury. Neutrophil recruitment and activation were evaluated by BALF cellularity and myeloperoxidase (MPO) activity. RT-PCR analysis was performed in lung tissue to assess gene expression of ICAM-1. The histopathological changes were also observed using the HE staining of lung tissue. Our results showed that lung injury parameters, including the wet/dry weight ratio and protein content in BALF, were significantly higher in the LPS alone group than in the saline control group (P＜0.01). In the LPS alone group, a larger number of neutrophils and greater MPO activity in cell-free BAL and lung homogenates were observed when compared with the saline control group (P＜0.01). There was a significant increase in lung ICAM-1 mRNA in response to LPS challenge (P＜ 0. 01, group L versus group S).Genistein pretreatment significantly attenuated LPS-induced changes in these indices. LPS caused extensive lung damage, which was also lessened after genistein pretreatment. All above-mentioned parameters in the genistein alone group were not significantly different from those of the saline control group. It is concluded that genistein pretreatment attenuated LPS-induced lung injury in rats.This beneficial effect of genistein may involves, in part, an inhibition of neutrophilic recruitment and activity, possibly through an inhibition of lung ICAM-1 expression.

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L1), 6 h (group L2), 12 h (group L3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L1, L2, L3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L1, L2, L3) were also significantly higher than that of control group (P<0.01). Moreover, among group L1, L2 and L3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.
Animals ; Endotoxins ; Female ; Lung ; pathology ; Male ; Matrix Metalloproteinase 8 ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Respiratory Distress Syndrome, Adult ; chemically induced ; enzymology ; etiology ; pathology

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L1), 6 h (group L2), 12 h (group L3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L1, L2, L3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L1, L2, L3) were also significantly higher than that of control group (P<0.01). Moreover, among group L1, L2 and L3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.
Endotoxins ; Lung/pathology ; Matrix Metalloproteinase 8/*metabolism ; Random Allocation ; Rats, Sprague-Dawley ; Respiratory Distress Syndrome, Adult/chemically

To assess the potential therapeutic effect of propofol in the treatment of endotoxemia, 76 rats were randomly assigned to 5 groups: control group(A), endotoxemic group(B), pre-treatment group(C), simultaneous treatment group(D) and post-treatment group(E). Five h after endotoxin injection, PO2, pH, MAP, plasma concentrations of Nitrite/nitrate (NO2-/NO3-) and mortality rates were assessed in each group. After the rats were sacrificed, lung tissue was sampled to measure myeloperoxidase (MPO) activity and tumor necrosis factor (TNF)-alpha contents. It was found that endotoxin injection produced progressive hypotension, metabolic acidosis, and a large increase in the plasma NO2-/NO3- concentrations and increased mortality rates in 5 h. Endotoxin injection significantly increased MPO activity and TNF-alpha contents in lung tissue (P < 0.01 or P < 0.05). These changes response to endotoxin were significantly attenuated in the groups B, C and D. But these beneficial effects were blunted in the group E. The results suggest that propofol administration may offer advantages in endotoxemia.
Animals ; Free Radical Scavengers ; therapeutic use ; Lipopolysaccharides ; Lung ; metabolism ; Male ; Nitric Oxide ; blood ; Peroxidase ; metabolism ; Propofol ; therapeutic use ; Random Allocation ; Rats ; Rats, Wistar ; Shock, Septic ; chemically induced ; drug therapy ; Tumor Necrosis Factor-alpha ; metabolism

AIM: To investigate the effects of rosiglitazone(ROSI),an agonist of peroxisome proliferator-activated receptor ?(PPAR?),on the lung expression of intercellular adhesion molecule-1(ICAM-1) and cytokine-induced neutrophil chemoattractant(CINC) in rats with acute lung injury. METHODS: Thirty-six male Wistar rats were randomly divided into six groups: control group,ROSI group,GW9662(a PPAR? antagonist) group,lipopolysaccharide(LPS,6 mg/kg,iv) group,ROSI-LPS group(0.3 mg/kg ROSI iv 30 min prior to LPS) and GW9662-ROSI-LPS group(0.3 mg/kg GW9662,iv,20 min before ROSI).Four hours after LPS injection,wet/dry weight(W/D) ratio,myeloperoxidase (MPO) activity,malondialdehyde(MDA) and CINC-1 concentrations were assayed in the lung tissues.Immunohistochemical analysis of ICAM-1 expression was also studied.RESULTS: Pretreatment with ROSI significantly attenuated LPS-induced increases in W/D ratio,MPO activity,MDA and CINC-1 concentrations as well as ICAM-1 expression in the lung tissues.The specific PPAR? antagonist GW9662 antagonized the effects of ROSI.CONCLUSION: Pretreatment with ROSI reduces LPS-induced lung injury in rats.The mechanism involves inhibition of the lung expression of ICAM-1 and CINC-1 by the activation of PPAR?.

Objective To investigate the effects of genistein pretreatment on endotoxin-induced acute lung injury in rats and assess the possible mechanism. Methods Thirty-two male Wistar rats weighing 240-280 g were randomly divided into 4 groups ( n = 8, each group) : group Ⅰ control; group Ⅱ genistein; group ? LPS and group Ⅳ genistein pretreatment. The jugular vein was cannulated for administration of fluid and drug. The animals were anesthetized with intraperitoneal 3% pentobarbital 40 mg? kg-1 . In group Ⅰ and Ⅱ Ⅳ normal saline (NS) 1 ml?kg-1 was Ⅳ given 30min after IP NS 1 ml?kg-1 (Ⅰ ) or genistein 50 mg?kg-1 (Ⅱ ). In group ? and Ⅳ LPS 6 ml? kg-1 was Ⅳ given 30 min after IP NS 1 ml?kg-1 (?) or genistein 50 mg? kg -1 (Ⅳ). Four hours after.LPS injection, rats were sacrificed. The lungs were removed for evaluation of histological injury and determination of wet/dry lung weight (W/D) ratio, myeloperoxidase (MPO) activity, malondialdehyde (MDA) content, expression of TNF-f55 mRNA, HO-1 mRNA, TNF-f55, and HO-1. Bronchoalveolar lavage fluid (BALF) was collected for determination of PMN count, protein content, and MPO activity.Results LPS administration induced marked lung injury and significant increases in W/D ratio, MPO activity, and MDA content in the lung tissues, and PMN count, protein content, and MPO activity in BALF. All of these changes were significantly reduced by genistein pretreatment. Genistein also markedly suppressed LPS-induced expression of TNF-a mRNA and protein, and enhanced LPS-induced expression of HO-1 mRNA and protein. Conclusion Pretreatment with genistein has protective effect against endotoxin-induced acute lung injury. The underlying mechanism is via an inhibition of neutrophilic recruitment and activity, a down-regulation in TNF-f55 production, and a up-regulation in HO-1 expression.