Ge Feng, Xu Jiefeng, Zhu Jinjiang, Cao Guangli, Wang Xuguang, Zhou Meiya, Chen Tiejiang, Zhang Mao
Department of Emergency Medicine, Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang Provincial Key Laboratory of Severe Trauma and Burn Diagnosis and Treatment, Zhejiang Provincial Research Center for Clinical Medicine of Acute and Critical Illness, Hangzhou 310009, Zhejiang, China.
Department of Emergency Medicine, Yiwu Central Hospital, Yiwu 322000, Zhejiang, China.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2025 Feb;37(2):133-139. doi: 10.3760/cma.j.cn121430-20240605-00487.
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).
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 2.5×10/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).
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].
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.
探讨人胚胎干细胞来源的间充质干细胞（hESC-MSC）减轻猪心脏骤停（CA）复苏后脑损伤的机制。
将29只健康雄性大白猪随机分为假手术组（n = 9）、心肺复苏（CPR）组（n = 10）和hESC-MSC组（n = 10）。假手术组仅完成动物准备。CPR组和hESC-MSC组通过电刺激诱导室颤10分钟并进行6分钟CPR建立CA-CPR猪模型。在成功复苏后5分钟，hESC-MSC组在1小时内通过静脉微量泵注射2.5×10/kg的hESC-MSC。在复苏前以及复苏后4、8、24、48和72小时采集静脉血样本。采用酶联免疫吸附测定（ELISA）检测神经元特异性烯醇化酶（NSE）和S100B蛋白（S100B）水平。在复苏后24、48和72小时，采用神经功能缺损评分（NDS）和脑功能分级（CPC）评估动物的神经功能。每组随机选取3只动物在复苏后24、48和72小时安乐死，迅速获取海马组织。采用免疫荧光染色检测hESC-MSC在海马中的分布。采用免疫组织化学染色检测海马中星形胶质细胞和小胶质细胞的激活以及神经元的存活情况。采用TdT介导的dUTP缺口末端标记法（TUNEL）检测凋亡程度。
复苏后24小时，CPR组和hESC-MSC组脑损伤标志物血清NSE和S100B水平显著高于假手术组，随后逐渐升高。hESC-MSC组复苏各时间点血清NSE和S100B水平均显著低于CPR组[NSE（μg/L）：4小时时20.69±3.62 vs. 28.95±3.48，72小时时27.04±5.56 vs. 48.59±9.22；S100B（μg/L）：4小时时2.29±0.39 vs. 3.60±0.73，72小时时2.38±0.15 vs. 3.92±0.50，均P < 0.05]。在神经功能方面，与假手术组相比，CPR组和hESC-MSC组在复苏后24小时NDS评分和CPC评分显著升高，随后逐渐降低。复苏后24小时，hESC-MSC组的NDS和CPC评分显著低于CPR组（NDS：111.67±20.21 vs. 170.00±21.79，CPC：2.33±0.29 vs. 3.00±0.00，均P < 0.05）。在荧光显微镜下观察到hESC-MSC组在复苏后24、48和72小时海马中hESC-MSC阳性标志物CD73、CD90和CD105的表达，表明hESC-MSC可归巢至受损海马。此外，与假手术组相比，复苏后24、48和72小时CPR组海马中星形胶质细胞、小胶质细胞比例和凋亡指数显著增加，神经元比例显著降低。与CPR组相比，复苏后24小时hESC-MSC组海马中星形胶质细胞、小胶质细胞比例和凋亡指数降低，神经元比例显著增加[星形胶质细胞比例：（14.33±1.00）% vs.（30.78±2.69）%，小胶质细胞比例：（12.00±0.88）% vs.（27.89±5.68）%，凋亡指数：（12.89±3.86）% vs.（52.33±7.77）%，神经元比例：（39.44±3.72）% vs.（28.33±1.53）%，均P < 0.05]。
复苏早期应用hESC-MSC可减轻CA猪复苏后脑损伤和神经功能障碍。其机制可能与抑制免疫细胞激活、减少细胞凋亡和促进神经元存活有关。
