Genome-Wide Comparative Analysis Revealed the Protective Mechanism of Mild Hypothermia on Brain Injury in Rats After Cardiopulmonary Resuscitation

With the development of Cardiopulmonary Resuscitation (CPR)
technology and the popularity of public CPR...


Introduction
With the development of Cardiopulmonary Resuscitation (CPR) technology and the popularity of public CPR, the success rate of Cardiac Arrest (CA) patients with Return of Spontaneous Circulation (ROSC) has improved significantly, but less than 10% of patients are discharged with good neurologic outcome. Most patients die of hypoxic ischemic brain damage after CPR [1]. Mild hypothermia treatment can improve discharge survival rate and improve neurological function in Out of Hospital Cardiac Arrest(OHCA) patients [2,3]. At present, the brain protection mechanism of TTM is still unclear.Biochip is a high-tech developed in the field of life sciences. The gene chip can detect the transcriptional changes of a large number of genes under different conditions, and can display gene expression levels reflecting characteristic tissue types, developmental stages, environmental condition responses, and genetic alterations, and become the preferred tool for highthroughput systems to investigate gene expression information of biological samples [4]. The effect of mild hypothermia on brain injury after ROSC is a complex and dynamic process, the traditional analytical methods cannot meet the need to analyze the expression of the entire genome in a single response. Therefore, in the current study, we plan to use the whole gene chip to analyze the effect of hypothermia on the gene expression in cortex of rat after ROSC, screen out differentially expressed genes, and elucidate its function, signaling pathway and network characteristics.  [5].
The procedures of induction of CA and CPR were done as our previously described [6,7] in CPRT1 and CPRT2 group but was not in NT1 and NT2 group. The rats in NT1 and CPRT1 group were kept in an incubator chamber to keep their esophageal temperature at 36.5-38.5℃. The rats in NT2 and CPRT2 group were placed ice cubes around the body to induce hypothermia after ROSC [8]. The esophageal temperatures were taken every 5 minutes and ice cubes were added or taken away to maintain their temperature between 33-35℃ for 12 hours and then gradually re-warmer at 0.5℃per hour.

Tissue Collection and RNA Extraction
Three of the rat's cerebral cortex was harvested at 2 hours, 4 hours, and 8 hours after ROSC in CPRT1 and CPRT2 group, the brains were harvested. The cerebral cortices were snap-frozen in liquid nitrogen; after being complete frozen, they were stored in an airtight container at -80 0 C.

Neurological Deficit Score and TUNEL Staining
The other 12 rats in CPRT1 and CPRT2 group were induced VF and CPR, then were observed for 72 h after ROSC and their Neurological Deficit Score (NDS) were assessed from 0 (no observed neurological deficit) to 500 (death or brain death) [9]

Statistical Analysis
The Statistical Program for Social Sciences (SPSS) 13.0 software (SPSS, Chicago, IL, USA) was used to perform all the statistical analyses. All data were expressed as the means ± SD or proportions where appropriate. For comparisons, unpaired t-tests were performed where appropriate between two groups. The correlation was calculated using Spearman's correlation coefficient, which is the Pearson's correlation coefficient of the indexed ranks of two data sets. P values of 0.05 (two-tailed) were considered statistically significant.

DEMs Identified Between Groups
There were no differences of body weight, Temperature between CPRT1 and CPRT2 group at baseline. There were 13/18 rats in CPRT1 group and 12/18 rats in CPRT2 group gained the ROSC. The ROSC rate of rats, epinephrine dose, defibrillation times and base life support time were no difference between CPRT1 and CPRT2 group (supplementary Table 2). The temperature was controlled according to the protocol (supplementary Figure 1). Three rats in each time point at ROSC2h, 4h and 8h between two groups were scarified and the brain were harvested for further experiment.
A total of 41012 mRNAs were detected in 4 groups. Hierarchical cluster analysis was performed to identify DEMs between groups.

DEM Expression Tendencies following ROSC
Hypothermia inhibits the expression of P21, SFN and GADD45A mRNA in P53 pathwaythe expression of P21 and SFN in ROSC 2h and 4h in CPRT2 group was significantly lower than that in CPRT1 group ( Figure 2A& 2B), However, the effect on the expression of GADD45A was slightly later, the expression of GADD45A at ROSC at 4h and 8h in CPRT2 group was significantly lower than that in CPRT1 group ( Figure 2C).Hypothermia promoted the expression of BDNF, HSPA2 at 2h, 4h, 8h after ROSC ( Figure 2D& 2E) Figure 3A). The protein levels of BDNF (2h, 4h and 8h) and HSPA2 (2h, 4h and 8h) in CPRT2 group were significantly higher than those in CPRT1 group, while the expression of c-Fos (2h, 4h and 8h), c-JUN (2h and 4h) and NUR77 (2hand 4h) was lower than that of CPRT1 group in the MAPK pathway ( Figure 3B). The protein levels of IL4 (2h and 4h) and TSLP (2h, 4h and 8h) in CPRT2 group were significantly higher than those in CPRT1 group, while the expression of CXCL2 (2h and 4h) and CCL3 (2hand 4h) was lower than that in CPRT1 group in inflammatory factor receptor pathway ( Figure 3C).

Figure 2:
The mRNA expressions tendency of DEMs after ROSC between CPRT1 and CPRT2 group.
D-G. The MHT increased the mRNA levels of BDNF and HSP72 at 2h, 4h and 8h after ROSC, but decreased the mRNA levels of c-Fos (ROSC 2h, 4h and 8h) and c-JUN (ROSC 2h, 4h) after ROSC.
H-L. The MHT increased the mRNA levels of IL-4 (ROSC 2h, 4h) and TSLP (2h, 4h and 8h), decreased the mRNA levels of CXCL2 and CCL3 at 2h and 4h after ROSC, but no effects on NUR77 expression. b) The MHT increased the protein levels of BDNF and HSP72 at 2h, 4h and 8h after ROSC, but decreased the protein levels of c-Fos (ROSC 2h, 4h and 8h) and c-JUN (ROSC 2h, 4h) after ROSC.

Hypothermia Treatment Improved Neurologic Deficit Scores and Decreased Neuronal Apoptosis following ROSC
The induction of Ventricular Fibrillation (VF) caused serious injury to the brain in rats, resulting in the death of 6/12 rats within 72 h in the CPRT1 group, whereas 8/12 rats survived to 72 h in the CPRT2 group. The ROSC rate of rats, epinephrine dose, defibrillation times and base life support time were no difference between CPRT1 and CPRT2 group (Supplementary Table 6). The temperature was controlled according to the protocol (Supplementary Figure 2).The NDS in the CPRT1 group were 425 ± 87, which were significantly higher than those (288 ± 48) in the CPRT2 group (P=0.0338) ( Figure 4A). TUNEL-positive nuclei were observed in the cortex of animals in both CPRT1 and CPRT2 groups. The number of apoptotic neurons was 9.89 ± 0.9 in the CPRT1 group, higher than the 6.   These genes are involved in inflammatory responses, cell cycle arrest and apoptosis [12,13], and MHT can inhibit the expression of these genes.The MAPK family is involved in cerebral ischemia.
It is well documented that ERK1/2 modulates neuronal survival and apoptotic cell death. Activation of this complex results in phosphorylation of many cytoplasm and membrane proteins [14].
Previous research has also reported that activation of JNK and p38 MAPK is mediated in neuronal apoptosis, infracted volume, and neurological deficits in ischemic stroke [15]. The present study shows that MAPK signaling pathways were regulated differently by MHT, which increases expressions of BDNF and HSP72, but inhibits the expression of c-fos, c-JUN and Nur77. BDNF is a member of the family of neurotrophies in the central neural system. As an attractive target gene of CREB [16], the mature BDNF plays a vital role in antiinflammation, anti-neurotoxicity, promoting neuronal survival and regeneration following ischemic brain injury [17]. HSP72, the major inducible member of the heat shock protein 70 family, has been found protecting cells from certain apoptotic stimuli such as oxidative stress, hypoxia and inflammation [18], could decreased the activation of JNK3, c-Jun and caspase-3 induced by cerebral I/R [19].
The signals of c-fos, c-jun, and nur77 were induced with different degree of intensity by hypoxia and were reduced significantly by naloxone have protections on PC12 cells survival after hypoxia [20].
Expression of c-Jun and caspase-2 is associated with neuronal cell apoptosis in the retinal ganglion cell layer [21]. The up-regulation of Nur77 mediated neuron apoptosis and mitochondrial injury via aberrant mitochondrial fragmentation in a manner dependent on the Wnt/β-catenin/INF2 pathway, while ablation of Nur77 resulted in a reduction in the infarction area, decreased neuronal apoptosis and attenuated mitochondrial injury [22]. MHT selectively acts on different components of the MAPK pathway, increasing the expression of components beneficial to neuronal survival, reducing the expression of components involved in apoptosis. The mechanism of differential effects of MHT on MAPK is not clear, it may be related to the spatial discrepancy expression of these gene [23].Inflammatory processes play a fundamental role into brain ischemia-reperfusion injury [24]. in the present study, we found that the DEMs of inflammatory factor receptor pathway are CXCL2, CCL3, IL4 and TSLP, the MHT increases the expression of IL4 and TSLP but decreases the expression of CXCL2 and CCL3. TSLP is also expressed in the Central Nervous System (CNS) where it is produced by choroid plexus epithelial cells and astrocytes in the spinal cord [25].
Study showed that TSLP involved in the pathogenesis of ischemic stroke [26] contributed to angiogenesis which is a key neuro-restorative event in response to ischemia. The cytokine IL-4 improves long-term neurological outcomes after stroke, perhaps through M2 phenotype induction in microglia/macrophages, immunomodulation with IL-4 is a promising approach to promote long-term functional recovery after stroke [27]. The expression of CXCR2 increased in the ischemic brain correlated with increased leukocyte accumulation in the ischemic brain after focal stroke.
Cytokine-induced neutrophil chemoattractant-1 was the major chemokine involved in neutrophil recruitment to the brain [28].
CCL2, CCL3 and CCL5 recruit monocytes and T cells via the chemokine receptors CCR1, CCR3, and CCR5. Increased levels expression and production of CCL3 have been described in experimental brain stroke [29].There are some limitations in the current study, the first one is the only one section of the genomewide analysis was observed, which could not fully reflect the effect of MHT on cerebral cortex gene changes after ROSC. However, when DEMs were verified at 2 hours, 4h and 8h after ROSC, its tendency was consistent with the genome-wide analysis anticipated. The second one is that the components of cerebral cortex cells are complex, and the selected DEMs can only reflect the overall changes in the entire cerebral cortex. Whether these changes are caused by neuronal cells, glial cells, and infiltrating inflammatory cells required further study to confirm, but it gave hints for future research.

Conclusion
MAPK-associated inflammatory pathway, the P53 apoptotic pathway, and the cytokine receptor pathway are associated with brain damage after cardiopulmonary resuscitation. MHT exerts brain protection by affecting the inflammatory response, apoptosis, and cytokine receptor-mediated damage pathway after ROSC.