ObjectiveTo investigate the expression of caspase-3 and Toll-like receptor 4 (TLR4) in the incised rat skin healing process and its relationship with the wound time and to provide an experimental evidence for the prediction of injury time. MethodsAfter the rat incised wound model was established, hematoxylin-eosin dyeing technology and immunohistochemical staining technique were used to observe the expression of caspase-3 and TLR4. Then Image Pro Plus Image analysis software and SPSS statistical analysis software were used to deal with the experimental results. ResultsCaspase-3- and TLR4-positive cells were detected in epidermis, hair follicle and sebaceous gland cells in the control skin. The expression of caspase-3 and TLR4 of the ante mortem groups were significantly different compared with the control group except the 0 h group (P<0.05). Caspase-3- and TLR4-positive cells were detected in neutrophils around the hair follicle half an hour later. Caspase-3- and TLR4-positive cell rate increased with the infiltration of inflammatory cells. Caspase-3- and TLR4-positive cell rate reached the maximum on the 3 rd day, and then it began to decrease, and they were mainly expressed in fibroblasts and mononuclear macrophages. Caspase-3- and TLR4-positive cells were mainly expressed in fibroblasts on the 10th day. There was no significant differences between the postmortem injury groups and the normal control groups (P>0.05). ConclusionCaspase-3- and TLR4-positive cell rate is time dependent and stable in 25℃ temperature environment which makes it possible to determine the time of injury.
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
ObjectiveTo investigate the establishment of rat models with chronic obstructive pulmonary disease (COPD) combined with type 2 diabetes mellitus (DM). MethodsEighty Sprague-Dawley (SD) male rats were randomly divided into four groups:COPD group (n=20), DM group (n=20), COPD combined with DM group (n=20) and normal group (n=20). COPD rats were established by cigarette smoke. Type 2 diabetes rats were modeled by streptozotocin injection. COPD combined with DM rats were modeled by cigarette smoking and streptozotocin injection at the same time. Pathological examination and blood glucose were tested after three months. ResultsBronchial epithelium was seriously shedding in COPD+DM group, with alveolar structure damaged and some alveolar fused into bullae. The blood glucose level in COPD+DM group was (27.1±1.1) mmol/L, which was statistically different from other groups (P<0.05). ConclusionRat model of COPD combined with type 2 DM could be established by cigarette smoking and streptozotocin injection, which can provide an animal model for further medical research.
Objective Dexamethasone (DXM) can regulate the balance of neutrophil and cytokine and enhance the ischemia-reperfusion tolerance of the skin flap; amlodipine besylate (AB) can selectively expand the peripheral blood vesselsand rel ieve the vascular smooth muscle spasm. To investigate the percutaneous penetration abil ity of DXM/AB compound gel and evaluate its effect on survival of ischemic skin flap. Methods Sodium carboxymethylcellulose was used to make blank gel, which was mixed in DXM, AB, azone (AZ), and progylene glycol (PG) respectively to make the compound gel containing 0.3%DXM/0.5%AB only (group D), the compound gel containing 3%AZ/2%PG, 3%AZ, and 2%PG (groups A, B, and C), the 0.3%DXM gel containing 3%AZ/2%PG (group E), the 0.5%AB gel containing 3%AZ/2%PG (group F). The accumulative penetration of DXM and AB in compound gel, 0.3%DXM gel, 0.5%AB gel through excised rat skin and its penetration within flap tissue were investigated by ultraviolet spectrophotometry. Fifty SD rats were selected to make 100 mm × 10 mm random flap at the back, and were randomly divided into 5 groups according to different gels which were used to treat flaps (n=10): compound gel group (group A1), 0.3%DXM gel group (group B1), 0.5%AB gel group (group C1), blank gel group (group D1), and peritoneal injection of DXM (5 mg/kg) and AB (2 mg/kg) (group E1). The survival area of ischemic random skin flap was measured on the 7th day by planimetry. Twenty-four SD rats were selected to make 100 mm × 10 mm random flap at the back, and were randomly divided into 2 groups (n=12). The accumulative penetration of DXM and AB within skin flap were also detected at 2 and 6 hours after appl ication of 2 g of compound gel containing 3%AZ/2%PG (group A2) and peritoneal injection AB (2 mg/kg) / DXM (5 mg/kg) (group B2). Results The accumulative penetration of DXM and AB in compound gel were increased in time-dependent manner (P lt; 0.05), and it was the highest in group A, and was significantly higher than that in group B and group C (P lt; 0.01), but there was no significant difference when compared with group E or group F (P gt; 0.05). The accumulative penetration of DXM and AB in groups A, B, and C were significant higher than that in group D (P lt; 0.05). After 7 days, the survival area of flaps in groups A1, B1, C1, D1, and E1 were (695.0 ± 4.6), (439.3 ± 7.1), (477.5 ± 14.5), (215.2 ± 3.8), and (569.4 ± 9.7) mm2, respectively; group A1 was significantly higher than other groups (P lt; 0.05). After 2 and 6 hours, the quantities of DXM and AB in skin flap of group A2 were significantly higher than that of group B2 (P lt; 0.05). Conclusion In 0.3%DXM/0.5%AB compound gel, DXM and AB might penetrate into skin tissue, which could significantly increase the survivalarea of ischemic skin flap.
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.
ObjectiveTo characterize the dynamic expression of Robo3 in the rat model of temporal lobe epilepsy(TLE), and assess the potential contribution of Robo3 to epileptogenesis. MethodsMale Sprague-Dawley (SD) rats were randomly divided into the control group (n=6) and the experimental groups (n=30, 6 per group). The experimental groups were injected intraperitoneally (i.p.) with an aqueous solution of lithium-pilocarpine, and sacrificed at different time points (1, 7, 14, 30 and 60 days) following the seizure. The control group was i.p. with 0.9% sodium chloride instead of pilocarpine. Quantitative real-time PCR were used to detected the mRNA expression of Robo3 and Western bolt were used to detected the protein expression of Robo3. ResultsQuantitative real-time PCR showed that the expression of Robo3 were significantly lower in the rat temporal lobe tissues of the latent and the chronic period group as compared with the controls(P < 0.05), but no significant differences were identified between the acute period group and the controls(P > 0.05). Western blot showed that the protein expression of Robo3 were significantly lower in the rat temporal lobe tissues of the latent and the chronic period group as compared with the controls(P < 0.05), no significant differences were identified between the acute period group and the controls(P > 0.05). ConclusionsRobo3 may be involved in the pathogenesis of temporal lobe epilepsy.
ObjectiveTo observe the dynamic changes of neuroglobin (NGB) expression in hippocampus after status epilepticus(SE) in rats, and to explore the role of NGB in epileptic seizures.Methods40 healthy male Sprague Dawley rats were randomly divided into two group according to random number table method:control group (n=5) and epilepsy model group(n=35).Epilepsy model group according to observation time was divided into:0h, 1h, 3h, 12h, 24h, 10d and 30d.Intraperitoneal injection Lithium-pilocarpine (20 mg/kg~127 mg/kg, Li-PC) to establish the rat model of SE.Observe the behavioral changes in rats with epilepsy.Nissl staining was used to detect the neuronal damage in hippocampus. Streptavidin-biotin-peroxidase complex immunohistochemical method was used to detect the expression level of NGB in hippocampus;ResultsAfter SE, the neurons in hippocampus were severely damaged with the progress of epileptic seizures, the number of surviving neurons in CA1, CA3 regions showed a near linear decline.Among them, the number of surviving neurons in (12h, 24h, 10d, 30d)CA1, (0h, 12h, 24h, 10d, 30d)CA3 and(12h, 24h, 10d, 30d) DG area were significantly lower than that of the control group (P < 0.05).The expression level of NGB in CA1, CA3 and DG region of hippocampus were increased after SE, and both of CA1 and DG were reached peak in 24h after SE, but was still higher than the control group.And the CA3 area showed a continue rising trend.Among them, CA1(24h, 10d, 30d), CA3(24h, 10d, 30d) and DG(12h, 24h, 10d, 30d) were higher than that of control group significantly (P < 0.05).In addition, it was found that there was a positive correlation between the number of surviving neurons in CA3 area and the expression level of NGB (R=0.306, P=0.011).ConclusionUp-regulation of NGB expression in hippocampus after status epilepticus, and was positively correlated with the number of neurons in the CA3 area, suggesting that up regulation of NGB expression may be a compensatory protective mechanism of ischemic injury induced by seizures, and participate in the protection of epilepsy related neuronal damage.
ObjectiveTo investigate the role of PI3K/AKT/mTOR signaling pathway in skeletal muscle atrophy in rats with chronic obstructive pulmonary diseases(COPD). MethodsPassive cigarette smoking was used to establish COPD model.The protein expression of PI3K, total mTOR, phosphorylated-mTOR, total GSK-3β, phosphorylated-GSK-3β, total 4E-BP1, phosphorylated-4E-BP1, total p70S6K1 and phosphorylated-p70S6K1 in extensor digitorum longus of rats were measured by Western blot. ResultsThe protein expression of PI3K was not significantly different between two groups(P > 0.05).Compared with the control group, the protein expression of total mTOR, phosphorylated-mTOR, total GSK-3β, and phosphorylated-GSK-3βincreased significantly in the COPD group(P < 0.05).The protein expression of total 4E-BP1 and total p70S6K1 were not significantly different between two groups(P > 0.05).While the protein expression of phosphorylated-4E-BP1 and phosphorylated-p70S6K1 significantly increased in the COPD group(P < 0.05). ConclusionThe protein expressions of PI3K/AKT/mTOR signaling pathway in extensor digitorum longus increased significantly in COPD rats, suggesting that the activity of PI3K/AKT/mTOR signaling pathway increased, which may be one of the compensatory mechanism of skeletal muscle atrophy in COPD.
ObjectiveTo investigate the effects of hippocampal long-term potentiation (LTP) on cognitive dysfunction in immature epileptic rats. MethodsImmature epileptic rats were established by intraperitoneal injection of lithium chloride-pilocarpine (li-pilo). Racine classification standard modified by Becker was used to evaluate behavior of epileptic seizure, and the survival rats within RacineⅣmagnitude were selected in the experiment. The function of learning and memory of epileptic rats when they were adult was assessed using Morris water maze experiment, and their independent exploratory behavior was evaluated by the open-field test. Field potential was recorded by electrophysiological technology to detecte whether hippocampal LTP was essential of cognitive dysfunction. ResultsThe function of learning and memory was significantly impaired when compared with controls(n=8, t=10.86, P < 0.05;n=8, t=9.98, P < 0.05). In addition, independent exploratory behavior was significantly reduced when compared with controls(n=8, t=12.89, P < 0.05). Besides, CA1 hippocampal LTP induced by high-frequency stimulation presented the significant inhibition in epileptic rats with cognitive dysfunction when compared with controls(Slope:n=8, t=13.32, P < 0.05;Amplitude:n=8, t=20.02, P < 0.05). ConclusionInhibition of CA1 hippocampal LTP may be implicated in cognitive dysfunction of epileptic rats.