Objective To study the relationship between the expression ratio of induced nitric oxide synthase (iNOS) over glial fibrillary acidic protein (GFAP) and the time of injury after brain concussion in rat, in order to acquire a new visual angle for determining injury time of cerebral concussion. Methods Eighty-five healthy Sprague-Dawley rats were divided into three groups randomly: model group (n=25), experimental group (n=55), and control group (n=5). The rats in the model group were used to confirm the attack hight to make the model of brain concussion; according to the time of execution, rats in the experimental group were then subdivided into 11 groups with 5 rats in each subgroup, and their execution time was respectively hour 0.5, 1, 3, 6, 12, 24, 48, 96, 168, 240, and 336; the rats in the control group were executed after fed for 24 hours. After the model of cerebral concussion was established through freefalling dart method, hematoxylin-eosin staining and immunohistochemistry staining of iNOS and GFAP were conducted for the brain of the rats. All related experimental results were studied by using microscope with image analytical system and homologous statistics. Results The ratio of positive expression of iNOS over that of GFAP increased gradually during hour 0.5- 3 after injury in brain (from 5.03 to 10.47). At the same time, the positive expression of iNOS increased significantly (from 14.61% to 37.45%). However, the increase of the positive expression of GFAP was not obvious. Between hour 3 and 12, the ratio began to decline to 4.98, which was still at a high level, and during the same time period, the positive expressions of iNOS and GFAP also experienced the same change pattern. Later, the ratio began to decline between hour 12 and 336 after injury (from 4.98 to 0.95). All ratios at this time were lower than those between hour 0.5 and 12. The positive expression of iNOS and GFAP both increased to a climax before declining. Conclusions The ratio of positive expression of iNOS over GFAP and the respective change pattern of iNOS and GFAP can be used as the evidence of estimating the injury time of cerebral concussion. We can use the ratio of two or more markers to provide a new visual angle for concluding the concussion injury time.
ObjectiveTo explore the antioxidant effects of reduced glutathione on rat pulmonary fibrosis compared with traditional corticosteroid. MethodsOne-hundred and eight healthy SD rats were randomly divided into 6 groups,ie. a control group,a model group,a dexamethasone group,a low-dose glutathione group,a middle-dose glutathione group,and a high-dose glutathione group,with 18 rats in each group. The pulmonary fibrosis model was established by intratrachially instillation of bleomycin in all rats except the control group. The severity of lung fibrosis was evaluated by HE staining and Masson staining of collagen,and measurement of glutathione,hydroxyproline,superoxide dismutase (SOD),glutathion peroxidase (GSH-Px)in lung tissue homogenate by photocolorimetric method. ResultsOn 7th day and 14th day after bleomycin instillation, the severity of alveolitis in the model group,the dexamethasone group,and three glutathione intervention groups was significantly reduced compared with the control group (P<0.05). On 28 day, the severity of lung fibrosis was significantly reduced in the dexamethasone group and three glutathione intervention groups compared with the model group (P<0.05). On 7th day,lung glutathione content was significantly lower in the model group compared with the control group (P<0.05), significantly higher in the dexamethasone group and three glutathione intervention groups compared with the model group (P<0.05), significantly lower in the dexamethasone group and the low-dose glutathione group compared with the control group (P<0.05), and significantly higher in the high-dose glutathione group compared with the dexamethasone group (P<0.05). On 7th,14th,and 28th day,the hydroxyproline content in the dexamethasone group and three glutathione intervention groups decreased significantly compared with the model group (P<0.05). On 14th day,the hydroxyproline content in the middle-dose and high-dose glutathione groups was significantly lower than that in the dexamethasone group (P<0.05). SOD and GSH-Px were significantly reduced in the model group compared with the control group on all time points (P<0.05),but significantly increased after intervention by different doses of glutathione (P<0.05). ConclusionReduced glutathione can significantly reduce the degree of pulmonary fibrosis in rats,but has no obvious advantage over dexamethasone.
ObjectiveTo explore the mechanism of lung injury in Sprague-Dawley (SD) rats induced by acute organic phosphorus pesticides (AOPP) by observing the changes of the blood serum nuclear factor (NF)-κB consistence, NF-κB level of lung tissue and lung coefficient. MethodNinety-six healthy male SD rats (six weeks old) were randomly divided into group A (control, n=48) and group B (poison, n=48). The rats of group B were given omethoate by gavage (45 mg/kg), and the rats of group A accepted normal saline. Then the rats were killed at designated observing points (30 minutes; 3, 6, 12, 24, and 48 hours), and the lung coefficient, blood serum NF-κB consistence and NF-κB level of lung tissue were measured. At the same time, we observed the pathological changes of the rats' lung tissue. ResultsCompared with group A, blood serum NF-κB consistence, NF-κB level of lung tissue and the level of lung coefficient in group B were significantly higher (P<0.01). The lung tissues of group A were normal at each time point, but in group B, the lung pathological changes gradually appeared 30 minutes later with pulmonary interstitial engorging, alveolar septum widening and some alveolus being full of red blood cells, and this situation reached its peak at hour 12. Then it gradually mitigated from 24 to 48 hours. ConclusionThere are significant increases in blood serum NF-κB consistence and NF-κB level in lung tissues in rats with lung injury induced by omethoate poisoning. The NF-κB may play a role in the process of lung injury induced by organophosphorus pesticide.
Objective To investigate the possibility of constructing eukaryotic expression vector for human glial derived neurotrophic factor （hGDNF）, transfecting it to spinal cord tissue of rats so as to treat acute spinal cord injury. Methods The eukaryotic expression vector pcDNA3-hGDNF was constructed by recombinant DNA technique, transfected into glial cell and neuron of spinal cord by liposome DOTAP as experimental group. In control group, mixture of empty vector and liposome was injected. The mRNA and protein expressions of hGNDF were detected by RT-PCR and Western blot. Results After the recombinant eukaryotic expression vector for hGDNF was digested with Hind III and XbaⅠ, electrophoresis revealed 400 bp fragment for hGDNF gene and 5 400 bp fragment for pcDNA3 vector. In the transfected spinal cord tissue, the mRNA and protein expressions of hGDNF gene were detected with RT-PCR and Western blot. Conclusion The constructed eukaryotic expression vector pcDNA3hGDNF could be expressed in the transfected spinal cord tissue of rat, so it provide basis for gene therapy of acute spinal cord injury.
Objective To investigate the expression pattern of hypoxia-inducible factor 1α (HIF-1α) in experimental secondary spinal cord injury (SSCI) in rats and its potential effects on SSCI. Methods A total of 66 SD rats (female or male) with weight (250 ± 20) g were randomly divided into 3 groups: normal control group (group A, n=6), pseudo injury group (group B, n=6), and spinal cord injury (SCI) group (group C, n=54). In group A, no treatment was given as normal control. In groupB, only laminectomy was appl ied. In group C, laminectomy was appl ied and static compression model of SCI was built at T10 level. The expression of HIF-1α was measured with HE and immunohistochemical staining in groups A, B (1 hour after pseudo injury), and C (1, 3, 6, 12 hours and 1, 2, 3, 7, 14 days after SCI). Results All rats survived to the end of the experiment. HE staining showed that the spinal tissue of groups A and B were dense and the nucleus were round and big with l ight staining and clear nucleolus. The injured neuron at 1-12 hours after SCI of group C presented pyknosis and deep eosin staining. The swelling axon with bubbles and the disintegrated and disorganized medullary sheath in white matter appeared at 1-3 days after SCI. The hyperplasia of gl ial cells were obvious and gray matter cells were broken and apoptosis with cavities in injured spinal segment was observed at 7 and 14 days after SCI. Immunohistochemical staining showed that HIF-1α was poorly expressed in group A and increased a l ittle in group B. The positive expression in group C increased at 3 hours after SCI, which was found in spinal cord anterior horn neurons and a small amount of gangl ion cells. It reached peak at 1 day, maintained at a high level during 1-3 days and then decl ined. At 14 days, it appeared only in a small amount of gangl ion cells of white matter. There was no significant difference in the number of HIF-1α positive cells between groups A and B (t=1.325, P=0.137). The number of HIF-1α positive cells at each time point in group C was more than those in groups A and B (P lt; 0.05), and there were significant differences between all time points in group C (P lt; 0.05). Conclusion The expression of HIF-1α increases after SCI, it is related to the ischemia hypoxia after SSCI, and the expression pattern was correlated with the injury time.
ObjectiveTo explore the effect of exogenous ghrelin on early recovery of rats after subtotal gastrectomy. MethodsTwelve rats undergoing subtotal gastrectomy (B-Ⅰtype) were randomly divided into two groups, and saline or ghrelin was intraperitoneally injected in two groups, respectively. The body weight and daily food intake were measured before operation and on 1-7 d after operation. Rats were killed on day 7 after operation and the expressions of ghrelin mRNA in the fundus of stomach and anastomotic stoma was determined by realtime fluorescent quantitative PCR assay. The anastomotic bursting pressure and hydroxyproline content of anastomotic stoma tissues were also detected. ResultsThere was no significant difference (P＞0.05) in pre and postoperative body weight between two groups. Gradual decrease in postoperative body weight among the rats of saline group was observed which was significantly lower than that before operation (Plt;0.01). Body weight reached it’s lowest on day 1 after operation (Plt;0.01), after which it gradually increased but was still lower than that before operation (Plt;0.01). The postoperative body weight of rats in ghrelin group gradually decreased too, and was also significantly lower than preoperative body weight (Plt;0.01), except for the day 1 after operation (P=0.693). It reached the lowest on day 4 after operation (Plt;0.01), then it gradually increased but was still lower than that before operation (Plt;0.05 or Plt;0.01). The cumulative food intake of rats in ghrelin group was （52.50±6.77） g, which was significantly higher than that in saline group 〔(45.67±7.47) g〕, Plt;0.05. On day 7 after operation, relative expression of ghrelin mRNA in the fundus of stomach of rats in ghrelin group was 0.08±0.04, which was significantly lower than that in saline group (0.22±0.07), Plt;0.01. Compared with saline group, ghrelin-treated rats displayed significantly higher bursting pressure 〔（155.83±6.62） mm Hg vs. （172.33±10.44） mm Hg, Plt;0.05〕 higher hydroxyproline content 〔 (0.43±0.05) μg/mg wet tissue vs. （0.50±0.29） μg/mg wet tissue, Plt;0.01〕 at the anastomotic stoma. ConclusionGhrelin may effectively promote the early recovery of rats after subtotal gastrectomy.
Objective To investigate the effects of granulocyto-colony stimulating factor (G-CSF) on the mobil ization of endothel ial progenitor cells (EPCs) in the rats with myocardial infarction (MI), to observe the density of neovascularization and the mRNA expressions of vascular endothel ial growth factor (VEGF) and its receptor (Flk-1) in the border area of MI. Methods Thirty-six adult male rats (weighing 250-280 g) were divided randomly into control group, MI group, and G-CSF group. In MI group and G-CSF group, the models of MI were establ ished by left anterior descenting coronary artery l igation and were treated with intraperitoneal injection of sal ine (0.3 mL/d) or G-CSF [30 μg/(kg?d)] for 5 days. In control group, after open chest operation, chest was closed without treatment. The level of EPCs was surveyed and the plasma concentrations of VEGF and C-reaction protein (CRP) were measured at 7 days. The mRNA expressions of VEGFand its receptor Flk-1 in the border area of infarct myocardium were determined through RT-PCR. Results Compared withcontrol group, the number of circulating white blood cell (WBC) and EPCs levels, and the serum concentrations of VEGF and CRP were all significantly increased in MI group and G-CSF group (P lt; 0.05); when compared with MI group, the number of circulating WBC and EPCs levels, and the serum concentrations of VEGF were increased and the concentration of CRP was decreased in G-CSF group (P lt; 0.05). Compared with control group, the mRNA expressions of VEGF and Flk-1, and the density of neovascularization in the border area of infarct myocardium were increased in MI group and G-CSF group, whereas those in G-CSF group were significantly augmented compared with MI group (P lt; 0.05). Conclusion In the rats with MI, G-CSF could promote EPCs mobil ization, increase the mRNA expressions of VEGF and Flk-1, and augment the density of neovascularization in the border area of infarct myocardium.
Objective Human acellular amniotic membrane (HAAM) contains collagens, glucoproteins, proteinpolysaccharide,integrin, and lamellar, which can supply rich nutrition to cell prol iferation and differentiation. To explore the possibil ity of HAAM with adi pose-derived stem cells (ADSCs) as a good engineered skin substitute for repairing skin defect. Methods Primary ADSCs were obtained from inguinal fat of 30 healthy 4-month-old SD rats, male or female, weighing 250-300 g, and cultured in vitro and purified. The 3rd passage ADSCs were used to detect CD44, CD49d and CD34 by immunocytochemistry staining. After physical and trypsin preparation, the HAAM was observed by HE staining and scanning electron microscope(SEM) respectively. ADSCs were seeded on epithel ial side of HAAM at the density of 2 × 105/cm2, cocultured, and observed by SEM at different time. MTT test was used to detect viabil ity of cells that seeded on HAAM, the group without HAAM was used as control. Thirty SD rats were made models of full-thickness skin wound and randomly divided into three groups (A, B, and C). Wound was repaired with HAAM/ADSCs composites in group A, with HAAM in group B, and with gauze as control in group C. The rats underwent postoperative assessment of wound heal ing rate and histological observation at the 1st, 2nd, and 4th weeks. Results HE staining showed that the 3rd passage ADSCs was spindle-shaped with an ovoid nucleus which located in the middle of cell; the immunocytochemistry staining showed positive result for CD44 and CD49d and negative result for CD34. There were no residues of cells in the HAAM by HE staining. SEM showed that there were different structures at the two sides of HAAM；one side had compact reticular structure and the other side had fibrous structure. After 3 days of co-culture, ADSCs showed good growth on HAAM; the cells were closely packed onto the HAAM, attached firmly and prol iferated to confluence on the stromal surface of HAAM. MTT test showed that the cells on the HAAM grew well and had b prol iferation vital ity. There was no significant difference between ADSCs cultured in the HAAM and control group (P gt; 0.05). One, 2, 4 weeks after graft, there were significant differences in wound heal ing rate between group A and groups B, C (P lt; 0.05), between group B and group C (P lt; 0.05). HE staining showed that wound healed faster in group A than in groups B, C. Cytokeratin 19 (CK19) immunohistochemical statining showed that there were more CK19 positive cells in group A than in groups B, C. Conclusion The graft of HAAM with ADSCs plays an effective role in promoting the repair of full-thickness skin wound
Objective To observe barrier function changes of gut mucosa in rats with acute respiratory distress syndrome(ARDS)．Methods Forty SD rats were randomized to an experiment group (n：30)and a control group(n=10)．Oleic acid was injected via vena femoralis to establish ARDS ratmode1．Subgroups in the experiment group were randomly divided by time 30 min，2 h，4 h interval after injection(n=10 in each subgroup)．Concentration of D-lactate and endotoxin and activity of diamineoxidase in blood plasma were measured．Histopathological changes of small intestine were observed under light microscope．Results Compared with the control group，the activation of diamine oxidase in the experiment group was higher after 30 min of injection(Plt;0.01)．Concentration of D-lactate，the activity ofdiamine oxidase and endotoxin level in the experiment group were all elevated after 2 hours of injection(all Plt;0.05)，and further elevated after 4 hours．In the rats’villous interstitial after 2 hours of the injection，there were edema，hyperemia，and infiltration of neutrophils，eosinophils and lymphocytes．After 4 hours ofthe injection，the villous epithelium showed desquamation，necrosis，denaturalization and erosion，associated with infihration of lymphocytes and neutrophils in the mucosa．Conclusion In oleic acid-induced ARDS．permeability of gut mueosa increases and gut barrier is dysfunctional．
ObjectiveTo investigate the differentiation of rat adipose-derived stem cells (ADSCs) into neuronlike cells by indirect co-culture with Schwann cells (SCs) in vitro so as to look for the ideal seed cells for tissue engineering. MethodsSCs were isolated from sciatic nerves of 1-2 days old Sprague-Dawley rats with enzymatic digestion method. Immunofluorescence staining was used to identify SCs with the marker S-100. ADSCs were isolated from the epididymal fat pads of adult male Sprague-Dawley rats by means of differential attachment. And the cell phenotypes (CD29, CD34, CD45, CD73, CD90, and CD105) of ADSCs at passage 3 were determined by flow cytometry analysis. Primary SCs and ADSCs at passage 3 were co-cultured at a ratio of 2:1 in Transwell culture dishes (experimental group), and ADSCs cultured alone served as control group. Immunofluorescence and flow cytometry were adopted to investigate the neural differentiation of ADSCs at 14 days. The expression differences for neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), neuronal nuclei protein (NeuN), and glial fibrillary acidic protein (GFAP) were detected, and the percentage of positive cells was calculated. ResultsADSCs were successfully extracted and can passage in a considerable large amount. Flow cytometry analysis showed that ADSCs at passage 3 were positive for CD29, CD90, CD73, and CD105 expression, but negative for CD34 and CD45 expression. The ADSCs of the experimental group showed contraction of nucleus, increasing of soma refraction, and several long and thick protrusions of cell body. The cell shape had no obvious change in the control group. Both immunofluorescence and flow cytometry analysis results showed the expressions of MAP2, NSE, NeuN, and GFAP at 14 days after co-cultured with SCs, and the positive cell ratios were significantly higher than those in the control group (P<0.01). ConclusionCo-culture with SCs not only can promote the survival regeneration of ADSCs, but also can induce the differentiation of ADSCs into neuron-like cells.