[Mechanism of human embryonic stem cell-derived mesenchymal stem cells on alleviating brain injury after cardiopulmonary resuscitation in swine with cardiac arrest].

Abstract

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.