| Objective Neurological complications seriously affect survival rate and quality of life in patients with extracorporeal cardiopulmonary resuscitation（ECPR）undergoing cardiac arrest（CA）.This study proposed selective hypothermic cerebral perfusion（SHCP）as a novel approach to protect the brains of these patients.This study aimed to determine the neuroprotective effect of this novel method and explore its possible mechanism.Methods SPF male Sprague-Dawley（SD）rats,that aged 6-8 weeks and weighing250-280 g,were randomly allocated to Sham,ECPR,and SHCP combined ECPR（CP-ECPR）groups.Under condition of deep anesthesia,asphyxial CA was induced by respiratory inhibition in rats.In the ECPR group,circulatory resuscitation was performed at 6 min after asphyxial CA by venous-arterial extracorporeal membrane oxygenation（VA-ECMO）（jugular vein/femoral artery cannulated）.The vital signs were monitored and kept stable[mean arterial pressure（MAP）≥60 mm Hg].Both body temperature and brain temperature were maintained at 36±0.5℃during operation.After 3 h of VA-ECMO,the animals were weaned off and sacrificed under deep anesthesia.Both their brain tissues and blood samples were collected.The CP-ECPR rats were rescued with the same method.Meanwhile,the right carotid artery catheterization serving as the cerebral perfusion was connected to the ECMO device through a three-way cock to achieve selective brain cooling,so that the body core temperature and brain temperature in the CP-ECPR rats were maintained at 36±0.5℃and 27±1℃,respectively.The left femoral artery catheterization was performed for hemodynamic monitoring in the Sham group without CA and VA-ECMO process.Their right femoral artery and right jugular vein were ligated.Measurement indicators:（1）The restoration of spontaneous circulation（ROSC）time,nasopharyngeal temperature,rectal temperature,and MAP were recorded during operation.（2）Enzyme Linked immunosorbent assay（ELISA）was used to detect the levels of biochemical serum markers of brain injury for each group.（3）The pathomorphological changes of the hippocampal CA1 and CA3 regions were evaluated by hematoxylin-eosin（HE）staining and Nissl staining.The number of normal neurons in the hippocampal CA1 area were counted.（4）Three biological replicates further received RNA sequencing（RNA-seq）in the ECPR and CP-ECPR groups to identify differentially expressed genes（DEGs）.KEGG pathway analysis was used to determine the functional changes between the two groups and speculated the possible mechanism of SHCP.（5）Using quantitative real-time polymerase chain reaction（q RT-PCR）to verify the RNA-seq results.（6）Ionized calcium binding adaptor molecule 1（Iba1）immunohistochemical（IHC）staining was used to evaluate the microglia activation for the hippocampal CA1 and CA3 regions in each group.（7）The concentrations of interleukin-1β（IL-1β）,interleukin-6（IL-6）,interleukin-8（IL-8）,and tumor necrosis factor-α（TNF-α）in brain tissues and serum were measured by ELISA.Results（1）There was no significant difference in brain temperature and body temperature between the Sham group and the ECPR group（P>0.05）.Compared with the ECPR group,the application of SHCP could quickly reduce the brain-targeted temperature from 36.37±0.24℃to 26.74±0.47℃（P<0.05）within 30 min after the beginning of resuscitation in the CP-ECPR group.（2）ELISA results showed that S-100β protein（S100β）,neuron-specific enolase（NSE）,and ubiquitin C-terminal hydrolase-L1（Uch-L1）serum concentration in the ECPR rats were significantly higher than those in the sham group（P<0.05）.In the CP-ECPR group,the biomarkers were significantly decreased compared with those of the ECPR group（P<0.05）,indicating that SHCP could alleviate brain injury in rats with ECPR.（3）HE staining results found that pyramidal cells in the hippocampal CA1 and CA3regions of the Sham group had complete morphology and clear structure,while the other two groups had different degrees of the pathological injury,which exhibited irregular arrangement,fuzzy cell outline,and some pyknotic nuclei in triangular or irregular shapes.Compared to the ECPR group,the CP-ECPR group had the lower pathological scores in the hippocampal CA1 region（P<0.05）,which indicated that the nerve injury was alleviated in the CP-ECPR rats.（4）For Nissl staining,there were a large number of pyramidal neurons in the hippocampal CA1 and CA3 regions of the Sham group,with abundant Nissl bodies.Compared to the Sham group,staining was lighter in the ECPR group.The number of surviving pyramidal neurons robustly reduced（P<0.05）.The CP-ECPR group had more normal neurons in the hippocampal CA1 region（P<0.05）with deeper staining than those in the ECPR group,indicating that SHCP has a protective effect on neurons during ECPR.（5）There were 594 DEGs in brain tissues between the ECPR and CP-ECPR groups,of which 523 DEGs were down-regulated in the CP-ECPR group.The KEGG pathway analysis showed that more DEGs were involved in“TNF signaling pathway”,“Cytokine-cytokine receptor interaction”,and“NF-kappa B signaling pathway”,suggesting that the role of SHCP might be correlated to downregulating inflammation response during ECPR.（6）The expression levels of Il6,Ccl2,Cxcl1,and Icam1 m RNA in the CP-ECPR group were significantly lower than those in the ECPR group（P<0.05）,which confirmed the RNA-seq results.（7）IHC results revealed that in the hippocampus of the Sham rats,microglia were sparse with many cell processes.The Iba1+microglia in the ECPR group were denser,the number of activated microglia markedly increased（P<0.05）,and the cell processes became fewer and shorter.After the support of SHCP,the numbers of Iba1 positive microglia in the hippocampus CA1 region significantly decreased in the CP-ECPR group（P<0.05）,which demonstrated that SHCP could inhibit microglia activation in ECPR rats.（8）The levels of IL-1β,IL-6,IL-8,and TNF-αin both brain tissues and serum of the ECPR and CP-ECPR groups were higher than those of the Sham group（P<0.05）.The use of SHCP reduced the release of these pro-inflammatory mediators when compared with those in the ECPR group（P<0.05）,suggesting that SHCP could alleviate inflammatory response in rats receiving ECPR.Conclusions In this study,ECPR models for asphyxial CA rats were successfully established.Selective brain cooling was achieved by SHCP technique as a novel hypothermia therapy.The preliminary data indicates that the combined application of SHCP could attenuate brain injury via downregulation of neuro-inflammation in the ECPR rats.This study provides a potential therapy and theoretical basis for improving the neurological prognosis in patients with ECPR.The feasibility and safety of this treatment need to be verified in the future. |
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