OBJECTIVE: To investigate the mechanism of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSC) in alleviating brain injury after resuscitation in swine with cardiac arrest (CA). METHODS: Twenty-nine healthy male large white swine were randomly divided into Sham group (n = 9), cardiopulmonary resuscitation (CPR) group (n = 10) and hESC-MSC group (n = 10). The Sham group only completed animal preparation. In CPR group and hESC-MSC group, the swine model of CA-CPR was established by inducing ventricular fibrillation for 10 minutes with electrical stimulation and CPR for 6 minutes. At 5 minutes after successful resuscitation, hESC-MSC 2.5×10⁶/kg was injected via intravenous micropump within 1 hour in hESC-MSC group. Venous blood samples were collected before resuscitation and at 4, 8, 24, 48 and 72 hours of resuscitation. The levels of neuron specific enolase (NSE) and S100B protein (S100B) were detected by enzyme linked immunosorbent assay (ELISA). At 24, 48 and 72 hours of resuscitation, neurological deficit score (NDS) and cerebral performance category (CPC) were used to evaluate the neurological function of the animals. Three animals from each group were randomly selected and euthanized at 24, 48, and 72 hours of resuscitation, and the hippocampus tissues were quickly obtained. Immunofluorescence staining was used to detect the distribution of hESC-MSC in hippocampus. Immunohistochemical staining was used to detect the activation of astrocytes and microglia and the survival of neurons in the hippocampus. The degree of apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: The serum NSE and S100B levels of brain injury markers in CPR group and hESC-MSC group were significantly higher than those in Sham group at 24 hours of resuscitation, and then gradually increased. The levels of NSE and S100B in serum at each time of resuscitation in hESC-MSC group were significantly lower than those in CPR group [NSE (μg/L): 20.69±3.62 vs. 28.95±3.48 at 4 hours, 27.04±5.56 vs. 48.59±9.22 at 72 hours; S100B (μg/L): 2.29±0.39 vs. 3.60±0.73 at 4 hours, 2.38±0.15 vs. 3.92±0.50 at 72 hours, all P < 0.05]. In terms of neurological function, compared with the Sham group, the NDS score and CPC score in the CPR group and hESC-MSC group increased significantly at 24 hours of resuscitation, and then gradually decreased. The NDS and CPC scores of hESC-MSC group were significantly lower than those of CPR group at 24 hours of resuscitation (NDS: 111.67±20.21 vs. 170.00±21.79, CPC: 2.33±0.29 vs. 3.00±0.00, both P < 0.05). The expression of hESC-MSC positive markers CD73, CD90 and CD105 in the hippocampus of hESC-MSC group at 24, 48 and 72 hours of resuscitation was observed under fluorescence microscope, indicating that hESC-MSC could homing to the damaged hippocampus. In addition, compared with Sham group, the proportion of astrocytes, microglia and apoptotic index in hippocampus of CPR group were significantly increased, and the proportion of neurons was significantly decreased at 24, 48 and 72 hours of resuscitation. Compared with CPR group, the proportion of astrocytes, microglia and apoptotic index in hippocampus of hESC-MSC group decreased and the proportion of neurons increased significantly at 24 hours of resuscitation [proportion of astrocytes: (14.33±1.00)% vs. (30.78±2.69)%, proportion of microglia: (12.00±0.88)% vs. (27.89±5.68)%, apoptotic index: (12.89±3.86)% vs. (52.33±7.77)%, proportion of neurons: (39.44±3.72)% vs. (28.33±1.53)%, all P < 0.05]. CONCLUSIONS Application of hESC-MSC at the early stage of resuscitation can reduce the brain injury and neurological dysfunction after resuscitation in swine with CA. The mechanism may be related to the inhibition of immune cell activation, reduction of cell apoptosis and promotion of neuronal survival.
Animals ; Heart Arrest/therapy* ; Cardiopulmonary Resuscitation ; Swine ; Humans ; Male ; Human Embryonic Stem Cells/cytology* ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/cytology* ; Phosphopyruvate Hydratase/blood* ; Brain Injuries/therapy* ; S100 Calcium Binding Protein beta Subunit ; Apoptosis ; Disease Models, Animal

Objective:To explore the protective efficacy of sulforaphane (SFN) in alleviating intestinal mucosal injury after resuscitation in pigs and its possible mechanism.Methods:This experiment was performed in the laboratory animal center, Zhejiang university. Using a random number table, twenty-four domestic healthy male white pigs were randomly divided into the Sham group, cardiopulmonary resuscitation (CPR) group, and SFN group, in which the Sham group had 6 pigs, and the other two groups had 9 pigs, respectively. The experimental parameters of 10 min of cardiac arrest and 6 min of CPR were chosen to establish the porcine model of CPR in the CPR and SFN groups. At 5 min after resuscitation, a dose of 2 mg/kg of SFN was infused via the femoral vein within 10 min in the SFN group. At 1 h, 2 h, 4 h, and 24 h after resuscitation, vein samples were collected, and then the levels of intestinal fatty acid binding protein (IFABP) and diamine oxidase (DAO) in serum were measured by ELISA. Subsequently, 6 pigs were chosen to be euthanized in each group, and then tissue samples were harvested from distal ileum to measure the level of cell apoptosis by TUNEL, the activities of superoxide dismutase (SOD) and catalase (CAT) and the contents of glutathione (GSH) and malondialdehyde (MDA) by biochemical method, the contents of 4-hydroxy-2-nonenal (4-HNE) by ELISA, the fluorescence intensity of reactive oxygen species (ROS) by immunofluorescence staining, and the expression levels of zonula occluden-1 (ZO-1), occludin, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) by Western blot. Continuous variables were compared with one way analysis of variance among the three groups, and Bonferroni test was used for further pairwise comparison.Results:During the observation period after resuscitation, the serum levels of biomarkers of intestinal mucosal injury including IFABP and DAO were significantly higher in the CPR and SFN groups than in the Sham group (all P<0.05). However, the serum levels of IFABP at 2 h, 4 h, and 24 h after resuscitation and the serum levels of DAO at 1 h, 2 h, 4 h, and 24 h after resuscitation were significantly lower in the SFN group than in the CPR group (all P<0.05). At 24 h after resuscitation, apoptotic index was significantly increased, SOD and CAT activities and GSH contents were significantly decreased, MDA and 4-HNE contents and ROS production were significantly increased, ZO-1 and occludin expression were significantly down-regulated, and Nrf2 and HO-1 expression were significantly up-regulated in the CPR and SFN groups when compared with the Sham group (all P<0.05). However, apoptotic index was significantly decreased, SOD and CAT activities and GSH contents were significantly increased, MDA and 4-HNE contents and ROS production were significantly decreased, and ZO-1, occludin, Nrf2, and HO-1 expression were significantly up-regulated in the SFN group when compared to the CPR group (all P<0.05). Conclusion:SFN could effectively protect against intestinal mucosal injury after resuscitation in pigs, and its mechanism was possibly related to the inhibition of oxidative stress and cell apoptosis via the activation of Nrf2/HO-1 pathway.

Objective:To establish pig cardiac arrest resuscitation model, and explore the effect of automatic compression synchronous ventilation (ACSV) on cardiopulmonary resuscitation in pigs.Methods:Twelve male white pigs with body weight of (38±3) kg were divided into ACSV group and intermittent positive pressure ventilation (IPPV) group with 6 pigs in each group by random number table method. A porcine cardiac arrest and resuscitation model was prepared with ventricular fibrillation induced by alternating current release via right ventricular electrode for 6 min and compression for 8 min. Mechanical chest external compression depth 5 cm, frequency 100 times/min. The tidal volume of ACSV group was 3 mL/kg and the frequency was 100 times/min. In the IPPV group, the tidal volume was 7 mL/kg and the frequency was 10 times/min. Arterial blood was drawn before resuscitation and at 1, 4 and 7min during resuscitation for blood gas analysis. Coronary perfusion pressure (CPP), end-respiratory carbon dioxide (ETCO 2) and carotid blood flow (CBF) were monitored during resuscitation. Stroke volume (SV) and global ejection fraction (GEF) were recorded by pressure monitoring catheter before and 1, 2 and 4 h after resuscitation. Venous blood samples were collected at each time point and 24 h after resuscitation to detect cardiac troponin I (cTnI), neuron specific enolase (NSE), alamine aminotransferase (ALT), creatinine (Cr), and intestinal fatty acid binding protein (IFABP). Results:(1) During resuscitation, CPP, ETCO 2 and CBF in ACSV group were slightly higher than those in IPPV group, but the differences between groups were not statistically significant. (2) There was no significant difference in pH, PaCO 2, HCO 3- and lactic acid between the two groups during resuscitation. The PaO 2 in ACSV group was higher than that in IPPV group, and the difference was statistically significant at 4 and 7 min. (3) The success rate of resuscitation in both groups was 83.3%, and there was no significant difference in SV and GEF before and after resuscitation. (4) After resuscitation, cTnI, NSE, ALT, Cr, iFABP and other indexes in ACSV group were lower than those in IPPV group, and there were statistically significant differences in cTnI at 24 h after resuscitation, ALT at 2 h and 24 h after resuscitation, and IFABP at 4 h and 24 h after resuscitation (all P<0.05). Conclusions:This study preliminarily suggested that the novel ACSV could significantly improve the oxygen supply level during cardiopulmonary resuscitation in pigs, while keeping the compression efficiency unchanged, avoiding hyperventilation, and reducing multiple organ damage after resuscitation, which is worthy of further study.

Objective:To evaluate the safety and efficacy of a novel domestic extracorporeal membrane oxygenation (ECMO) mainframe in a porcine model, and to provide the basis for further clinical application.Methods:Five domestic healthy male white pigs, weighing (51±4) kg, were selected. The ECMO system was established by using a novel ECMO mainframe with imported membrane oxygenator and pipeline, and continued to run for 72 hours. ECMO parameters are as follows: veno-arterial ECMO, centrifugal pump speed 3 000-3 500 r/min, continuous infusion of heparin anticoagulation to maintain the activate clotting time (ACT) of 140-200 s. Real-time monitoring of speed, flow, pressure before pump, pressure after pump, pressure after membrane and other equipment parameters, and the equipment performance was scored. The changes of hemodynamics, blood lactic acid and blood routine were monitored dynamically. Repeated measures analysis of variance was used to compare different time points within the group. At the end of the experiment, the thrombosis in the pump head and oxygenator was observed. The animals were sacrificed to obtain the tissue samples of the main organs for gross observation and pathological injury evaluation.Results:All animals successfully ran the ECMO system for 72 hours. (1) The centrifugal pump speed should be maintained at 3 029-3 483 r/min, the flow rate was maintained at 2.24-2.60 L/min, The pressure before the pump between minus 107.57 and minus 31.86 mmHg, the pressure after the pump was 197.50-282.43 mmHg, and the pressure after the membrane was 178.71-261.5 mmHg, all were in the normal range, and there was no significant difference between different time points (all P>0.05). The performance scores of the mainframe were all 4 points or above, indicating that the use requirements were met. (2) The heart rate of the animals was 50-80 beats /min, the mean arterial pressure was 85-115 mmHg, and the lactic acid was 0.996-2.25 mmol/L, all within the normal range, and there was no significant difference between different time points (all P>0.05). The free hemoglobin was 8.98-16.39 mg/L, and the hemoglobin was 6.58-7.52 g/L, both within a reasonable range, and there was no significant difference between different time points (all P>0.05). The platelet count was 69.6-231.6×10 9/L, and showed a continuous downward trend ( P<0.05). ACT was maintained at 135-169 s, which was within the target range, and there was no significant difference between different time points ( P<0.05). (3) At the end of the experiment, there was no obvious thrombosis in the pump head and oxygenator, no obvious thrombosis or infarction in the heart, brain, liver, lung and kidney, and no obvious hemorrhage or necrosis under the microscope. Conclusions:The ECMO established by the novel domestic ECMO mainframe combined with imported membrane oxygenator and pipeline ran smoothly for 72 hours, achieving the target of effect and safety.

Objective:To explore the clinical effects of autologous skin paste in repairing medium-thickness skin donor site wounds.Methods:The prospective randomized controlled research method was applied. From October 2018 to December 2019, 18 patients with flame burn or hydrothermal scald, conforming to the inclusion criteria were admitted to Jinhua Hospital Affiliated to Zhejiang University School of Medicine, including 15 males and 3 females, aged (45±6) years. The wounds were repaired with medium-thickness skin grafts from thigh, and the wound area was (121±33) cm 2 after medium-thickness skin grafting. The medium-thickness skin donor site wound in each patient was divided into 2 wounds in equal area and allocated into autologous skin paste group and conventional treatment group by flipping a coin, with 18 wounds in each group. The wounds in autologous skin paste group were repaired with skin paste prepared with remaining skin fragments after autologous medium-thickness skin grafting, and the wounds in conventional treatment group were covered with petroleum jelly gauze and fixed with sterile gauze. On 3, 7, 14, and 21 d after operation, the wound healing in 2 groups was observed, and the wound healing rate was calculated. The wound healing time in 2 groups was recorded. Occurrences of wound subcutaneous effusion and infection on 3, 7, 14, and 21 d after operation and wound ulceration in 3 months after operation were observed. In 6 months after operation, the Vancouver Scar Scale (VSS) was used to evaluate the scar formation of wounds in 2 groups. Data were statistically analyzed with analysis of variance for repeated measurement, chi-square test, and group t test. Results:The wounds in 2 groups did not heal on 3 and 7 d after operation. The wound healing rate in autologous skin paste group was (29.8±2.5)% and (95.6±4.7)% on 14 and 21 d after operation, which were significantly higher than (25.8±2.9)% and (82.6±8.9)% in conventional treatment group ( t=4.3, 5.6, P<0.01). The wound healing time in autologous skin paste group was (21.8±1.6) d, which was significantly shorter than (25.6±2.0) d in conventional treatment group ( t=6.24, P<0.01). On 3, 7, 14, and 21 d after operation, there were no complications such as subcutaneous effusion or infection in wounds of 2 groups. In 3 months after operation, ulceration occurred in wounds of 2 patients in autologous skin paste group, which was significantly less than 12 patients in conventional treatment group ( χ2=11.688, P<0.01). The ulcerated wounds healed after dressing changes. In 6 months after operation, the VSS score of wounds in autologous skin paste group was (9.1±1.1) points, which was significantly lower than (11.3±1.2) points in conventional treatment group ( t=-5.75, P<0.01). Conclusions:The remaining skin fragments after autologous medium-thickness skin grafting prepared into skin paste to repair medium-thickness skin donor site wounds can shorten wound healing time, improve wound healing quality, and reduce degree of scar hyperplasia, with a good clinical effect.

Objective To study the effect of type 2 diabetes mellitus (T2DM) on the surgical treatment of proximal humerus fractures in senile patients .Methods A respective analysis was made on the 83 senile patients with proximal humerus fractures (NEERⅡorⅢ), who received surgical treatment in our department from January , 2010 to January, 2015.The patients were divided into the diabetic group ( type 2 diabetes mellitus ) and the control group .Statistical analyses were made on the differences incurred by gender , the incidence of osteoporosis , the duration of perioperative period and Constant-Murley scores 1 year after surgery .Results Postoperative complications of the diabetes group were higher than those of the control group , bone mineral density was lower than that of the control group , the time taken for fracture healing was obviously prolonged , and the function scores were lower , when comparisons were made between the 2 groups.Conclusion The development of a reasonable T2DM treatment protocol was of great significance to the shortening of perioperative period , the prevention of complications , the achievement of good prognosis , and the reduction of medical costs in senile patients with proximal humerus fractures .