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Association between CIRP expression and hypoxic‑ischemic brain injury in neonatal rats

  • Authors:
    • Lifang Chen
    • Qiaohuan Tian
    • Weihua Wang
  • View Affiliations

  • Published online on: July 11, 2019     https://doi.org/10.3892/etm.2019.7767
  • Pages: 1515-1520
  • Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The role of cold inducible RNA‑binding protein (CIRP) in mediating ischemic brain injury in neonatal rats under chronic hypobaric hypoxia was investigated. The neonatal rat model of chronic hypobaric hypoxia and the cell culture model of SH‑SY5Y cells exposed to hypoxia (1% O2) were constructed. The expression of CIRP and hypoxia‑inducible factor‑1α (HIF‑1α) was detected after hypoxic exposure, and the apoptosis‑related proteins were analyzed via terminal deoxynucleotidyl transferase‑mediated dUTP nick end-labeling (TUNEL) and western blot analysis to detect neuronal apoptosis. Moreover, the effects of CIRP overexpression on HIF‑1α and neuronal apoptosis were identified. Chronic hypobaric hypoxia can lead to HIF‑1α expression and neuronal apoptosis in the body. CIRP was induced at early exposure (3 d/7 d). However, the CIRP level in the hypoxic group was obviously lower than that in the control group with the prolongation of exposure time (21 d). In addition, the knockdown of HIF‑1α significantly reduced the neuronal apoptosis under hypoxic conditions, indicating that HIF‑1α may promote apoptosis during exposure. The overexpression of CIRP significantly inhibited the upregulation of HIF‑1α during hypoxia and the HIF‑1α‑mediated neuronal apoptosis. Results of the current study showed that, CIRP is involved in the ischemic brain injury induced by chronic hypoxia through downregulation of HIF‑1α expression.

Introduction

According to previous studies, severe and chronic hypoxia leads to neuronal death in the Cornu Ammonis 3 (CA3) and CA4 regions in hippocampus dentate gyrus, indicating that neuronal apoptosis in this brain region is one of the main causes of chronic hypobaric hypoxia-induced cognitive impairment (1).

Cold inducible RNA-binding protein (CIRP) was screened as the DNA damage-induced gene transcript initially, which plays a key role in controlling the cell response under various environmental stresses, such as low temperature and ultraviolet light (2,3). Previous studies have revealed that CIRP migrates from the nucleus to the cytoplasm under environmental stress, which regulates its target messenger RNA (mRNA) at the post-transcriptional level and exerts a neuroprotective effect (4,5). For example, CIRP can inhibit the neuronal apoptosis through inhibiting the mitochondrial apoptosis pathway during mild hypothermia (6). Besides, CIRP protein in cortical neuron of rats inhibits H2O2-induced neuronal apoptosis under low temperature, thereby protecting the brain. There have been reports that CIRP is up-regulated in acute mild (8% O2) or severe (1% O2) hypoxia response (7). However, the expression features of CIRP in brain tissues under chronic hypobaric hypoxia remain unclear, and whether CIRP can serve as a neuroprotective factor under chronic hypobaric hypoxia has not been confirmed (8).

As the most important transcription factor in cell hypoxia response, hypoxia-inducible factor-1α (HIF-1α) is closely related to the hypoxia-induced neuronal apoptosis (9). Under hypoxic stress, HIF-1α can inhibit its anti-apoptosis effect through increasing the anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2) (1012). Besides, HIF-1α is related to neuronal apoptosis after brain injury through regulating p53 and Bcl-2 nineteen-kilodalton interacting protein 3 (BNIP3) in apoptotic neurons (13). To weaken the hypoxia-induced neuronal apoptosis in the brain region of cognitive function, clarifying the detailed regulatory mechanism of HIF-1α under hypoxic stress and searching for protective factors are of medical significance.

Chang et al (14), found that CIRP can bind to mRNA of HIF-1α and several protein translation factors on polysomes, and increase the protein translation under cell stress. Considering that CIRP plays an important role in the stress-induced neuronal apoptosis, it is assumed as a neuroprotective factor. CIRP can be involved in HIF-1α-mediated neuronal apoptosis under chronic hypobaric hypoxia and exert a neuroprotective effect. A microRNA (miRNA) is a small and non-coding RNA, which plays a vital role in the regulation of such biological processes as cell differentiation, proliferation and apoptosis. Under hypoxic conditions, a hypoxia-sensitive miRNA family named hypoxamiRs will be induced, and these miRNAs are specifically involved in controlling various processes, such as tumorigenesis, angiogenesis and apoptosis.

To confirm the above hypothesis, dynamic changes in CIRP/HIF-1α expression and neuronal apoptosis were detected in rats exposed to chronic hypobaric hypoxia and SH-SY5Y cells exposed to hypoxia (1% O2). To investigate the potential association between CIRP change and hypoxia-induced neuronal apoptosis, the effects of CIRP overexpression on HIF-1α expression and neuronal apoptosis were detected.

Materials and methods

Materials
Main reagents

Rabbit anti-human CIRP, HIF-1α, Bax, Bcl-2, caspase-3 and β-actin polyclonal antibodies were purchased from ProteinTech Group, Inc. (Chicago, IL, USA) (1:300; cat. nos. 10209-2-AP, 20960-1-AP, 50599-2-Ig, 12789-1-AP, 19677-1-AP, 20536-1-AP, respectively), rabbit anti-human cleaved caspase-3 (1:200; cat. no. 9661, Cell Signaling Technology, Danvers, USA) and Opti-MEM Medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were also used.

The present study was approved by the Ethics Committee of Liaocheng Third People's Hospital (Liaocheng, China).

Model establishment
In vivo hypobaric hypoxia animal models

Newborn male Sprague-Dawley rats (n=40) were kept in an animal room of the Research Institute in cages with 12/12 h dark-light cycle before exposure to hypobaric hypoxia and provided with sufficient pellet feed and water at 23°C. The humidity was 60%. All rats were randomly divided into normal control group (n=6) and hypoxia group (n=6).

In vitro chronic 1% hypoxic cell models

Human neuron-like SH-SY5Y neuroblastoma cells (ATCC® CRL-2266™) were placed in the RPMI-1640 medium containing 2 mM L-glutamine supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/streptomycin. The culture was kept in a standard wet incubator with 5% CO2 at 37°C, and the original medium was replaced with fresh medium once every 2 days. When 90% cells were fused, the medium was divided as 1:4. Cells were placed in the calibration gas containing 1% O2 or 3% O2 (the concentration of CO2 was adjusted to 5% under these two conditions) and the cells were placed in a humidified microaerophilic culture system (DWS HypOxystation) to prepare the anaerobic environment. The cells were kept in an incubator at 37°C at different times. Control culture was kept for the same time under normal oxygen content.

Methods
Terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis

TUNEL was carried out to evaluate cell apoptosis according to the manufacturer protocol. The procedure was as follows: Induced apoptotic cells were fixed in 4% paraformaldehyde phosphate-buffered saline (PBS) at room temperature for 30 min, washed with PBS 3 times and then incubated on ice using 0.1% Triton X-100. After that, the treated cells were mixed with TUNEL reaction mixture, followed by reaction in the dark for 1 h at 37°C. The nuclei were labelled with Hoechst-33342. Subsequently, the cells were observed under a fluorescence microscope to count the proportion of cell apoptosis.

Western blot analysis

After exposure for the specified time, 3 rats in each group were decollated and western blot analysis was performed to determine HIF-1α, CIRP, cleaved caspase-3/caspase-3 and Bax/Bcl-2 levels in the hippocampus. The hippocampus was removed from brain tissue of rats after cervical dislocation and rapidly placed in prepared pre-cooled 0.9% NaCl solution. Resected tissue was preserved in liquid nitrogen. Samples of tissue and cells were lysed and homogenized, after which the concentration of protein obtained was determined. Western blot analysis was carried out and FluorChem FC2 imaging system (ProteinSimple, San Jose, CA, USA) based on ECLO was used to detect the immune response signal. Gray values of bands in each group were analyzed using ImageJ software. Each protein band was normalized into β-actin value and presented as the intensity ratio. Western blot analyses were performed in triplicate.

Flow cytometry for analysis of cell apoptosis

Flow cytometry was performed for further analysis of cell apoptosis. After hypoxic exposure, SH-SY5Y cells were treated with trypsin, centrifuged at 3,000 × g for 8 min at 4°C and washed twice. Then, the cells were re-suspended using binding buffer, and added with 5 ml FITC-labeled Annexin-V and 5 ml PI, followed by incubation in the dark at room temperature for 15 min. Samples were analyzed within 1 h after staining.

Plasmid construction and transfection

CIRP complementary deoxyribonucleic acid (cDNA) was cloned in pEGFP-N2 vector and control transfection was performed via pEGFP-N2 without CIRP. Overexpression of CIRP in cells was confirmed via western blot analysis using anti-CIRP antibody in accordance with the protocol. According to procedures provided by the manufacturer, SH-SY5Y cells were transfected with CIRP cDNA using Lipofectamine 2000 transfection reagents. In brief, the cells were inoculated into a 6-well plate with 3×105 cells in each well and grew overnight until 80% of cells were fused. Transfection complex composed of 2.5 µg pEGFP-N2 vector plasmid DNA or pEGFP-N2-CIRP plasmid DNA and 6 µl Lipofectamine reagent was added into the well with Opti-MEM medium. Transfection efficiency and viability of cells were analyzed at 48 h after lipid transfection.

Statistical analysis

Continuous variables were presented as mean ± standard error of mean (SEM) and Student's t test was applied for analysis. Statistical analyses were completed using GraphPad Prism v.5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Exposure to chronic hypobaric hypoxia led to the increased apoptotic rate of hippocampal neurons in rats and significant changes in CIRP expression

To investigate the effect of exposure to chronic hypoxia on hypoxia-sensitive hippocampal neurons, adult rats were placed in an animal decompression chamber under 349 mmHg. The expression of apoptosis-related proteins, caspase-3, Bcl-2 associated X protein (Bax) and Bcl-2, in hippocampal neurons were detected, and the CIRP expression during hypobaric hypoxia was also detected at day (d) 3, 7 and 21. Compared with those in control group, the cleaved caspase-3/caspase-3 and Bax/Bcl-2 ratios in hypoxia group at 7 d and 21 d were significantly increased (Fig. 1A and B). Results of western blot analysis revealed that the CIRP levels at 3 and 7 d in hypoxia group were obviously higher than those in control group (P<0.01), while the CIRP level at 21 d in hypoxia group was obviously lower than that in control group (P<0.01). CIRP was induced at the early stage of hypoxia exposure, and inhibited continuously with the prolongation of exposure time (Fig. 1C).

Exposure to chronic hypobaric hypoxia induces apoptosis of SH-SY5Y cells and significantly reduces the CIRP expression

To detect the role of CIRP in hypoxic-related neuronal apoptosis, the in vitro chronic hypoxia model was constructed. SH-SY5Y cells were cultured in an anoxic chamber with 1% O2 for 48 h to simulate the chronic hypoxic condition in tissues. Results of western blot analysis showed that compared with those in control group, the CIRP expression was significantly decreased, and cleaved caspase-3/caspase-3 and Bax/Bcl-2 ratios were significantly increased in hypoxia group (P<0.01; Fig. 2B and C). Therefore, it is speculated that exposure to 1% hypoxia for 48 h leads to the increased apoptotic rate of SH-SY5Y cells.

The CIRP expression was detected after exposure to 1% hypoxia, and results of western blot analysis manifested that the CIRP expression in hypoxia group was remarkably decreased compared with that in control group, which was consistent with in vivo results (Fig. 2D).

Overexpression of CIRP inhibits the upregulation of HIF-1α in hypoxia and inhibits hypoxia-induced neuronal apoptosis

To study the potential association between CIRP decrease and hypoxia-induced brain injury, the effects of CIRP overexpression on HIF-1α expression and hypoxia-induced apoptosis were detected. SH-SY5Y cells were transfected with p-EGFP-N2-CIRP plasmid, and apoptosis was detected after exposure to hypoxia (1% O2) for 48 h. The overexpression of CIRP in transfected cells was confirmed via western blot analysis (Fig. 3C), which obviously decreased the HIF-1α expression under 1% hypoxic conditions (P<0.01; Fig. 3B) and significantly reduced neuronal apoptosis induced by hypoxia (Fig. 3A). The above results suggest that the overexpression of CIRP can alleviate hypoxia and induce apoptosis of SH-SY5Y cells by regulating HIF-1α expression.

Discussion

The aim of the present study was to investigate the molecular mechanism of CIRP participating in apoptosis during chronic hypobaric hypoxia stress. It was found that the CIRP expression was downregulated in hippocampal neurons and SH-SY5Y cells of rats exposed to hypoxia. Moreover, the overexpression of CIRP could effectively inhibit the upregulation of HIF-1α, thus inhibiting the hypoxia-induced neuronal apoptosis.

CIRP is involved in the neuronal apoptosis induced by a variety of environmental stresses, such as low temperature, oxidative stress, inflammation and DNA damage (15,16). In cortical neurons of rats, CIRP inhibits the etoposide-induced apoptosis through regulating levels of p53 and its downstream targets (8). To the best of our knowledge, no studies are available on the role of CIRP under chronic hypobaric hypoxia. It was found in the present study that in the hippocampus of rats exposed to chronic hypobaric hypoxia, CIRP expression was increased at early exposure, decreased after 7 d and continuously inhibited. Accordingly, the neuronal apoptosis and proportion of apoptosis-related proteins in the hippocampal CA3 region were increased from 7 d after exposure. In SH-SY5Y cells exposed to 1% O2, CIRP expression was increased in the first 12 h of exposure, then decreased at 24 h after hypoxia exposure and continuously inhibited. Therefore, it can be speculated that the downregulation of CIRP may be involved in the chronic hypobaric hypoxia-induced neuronal apoptosis.

The role of HIF-1α, the most important transcription factor in cell hypoxia response, in hypoxia-induced apoptosis has been discussed widely (17). HIF-1α can initiate the hypoxia-mediated apoptosis by increasing the expression of Bcl-2 binding protein, thus inhibiting the anti-apoptotic effect of Bcl-2 (11). Chang et al found that CIRP can bind to mRNA of HIF-1α and several protein translation factors on polysomes, and increase the protein translation under cell stress (14). In the present study, the overexpression of CIRP obviously decreased the HIF-1α level and the apoptotic rate of SH-SY5Y cells exposed to 1% O2, suggesting that the overexpression of CIRP can inhibit the HIF-1α expression and alleviate the hypoxia-induced apoptosis. Recently, Luo et al (18), reported several HIF inhibitors under chronic hypoxia, and found that several kinds of genes, such as peroxiredoxin 2 (PRDX2) and PRDX4, inhibit the HIF-1α mRNA level and transcriptional activity. Therefore, it is speculated that CIRP may repress HIF-1α during chronic hypobaric hypoxia-induced neuronal apoptosis.

CIRP significantly increased HIF-1α expression under normoxia compared with that under hypoxia. Several previous studies have proved that HIF-1α accumulates under hypoxia (1921). Wang et al (21), confirmed that the accumulation of HIF-1α under normoxia is possibly related to the increased glycolysis or glutamine dissolution. It has been proved that CIRP is widely involved in cellular metabolism, so it can be inferred that the transfection of CIRP under normoxia can change the cellular metabolism, resulting in the accumulation of HIF-1α. Moreover, HIF-1α is mainly regulated by the protein stability in an oxygen-dependent way. Under normoxia, HIF-1α can be rapidly degraded by the proteasome, failing to exert its functions. In the present study, the apoptotic rate and levels of apoptosis-related proteins in cells transfected with CIRP under normoxia had no difference from those in control group. It can be observed that although CIRP transfection significantly increases the HIF-1α accumulation under normoxia, it seemingly has no effect on apoptosis of SH-SY5Y cells. The detailed mechanism of CIRP in promoting HIF-1α under normoxia remains to be clarified.

In conclusion, the present study indicates that exposure to hypobaric hypoxia leads to hypoxia injury in the hippocampus of rats and neuronal apoptosis. At the same time, hypoxia exposure to 1% O2 increases levels of HIF-1α and apoptosis-related proteins, and apoptotic rate of SH-SY5Y cells. CIRP is considered to exert a neuroprotective effect under chronic hypobaric hypoxia stress. The overexpression of CIRP can effectively inhibit the HIF-1α expression in cells, thus alleviating the hypoxia-induced apoptosis. However, the CIRP expression decreases gradually with the prolongation of exposure time.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

LC assisted with TUNEL analysis and wrote the manuscript. LC and QT were responsible for model establishment. WW performed western blot analysis. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Liaocheng Third People's Hospital (Liaocheng, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Maiti P, Singh SB, Muthuraju S, Veleri S and Ilavazhagan G: Hypobaric hypoxia damages the hippocampal pyramidal neurons in the rat brain. Brain Res. 1175:1–9. 2007. View Article : Google Scholar : PubMed/NCBI

2 

Pan F, Zarate J, Choudhury A, Rupprecht R and Bradley TM: Osmotic stress of salmon stimulates upregulation of a cold inducible RNA binding protein (CIRP) similar to that of mammals and amphibians. Biochimie. 86:451–461. 2004. View Article : Google Scholar : PubMed/NCBI

3 

Nishiyama H, Higashitsuji H, Yokoi H, Itoh K, Danno S, Matsuda T and Fujita J: Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene. Gene. 204:115–120. 1997. View Article : Google Scholar : PubMed/NCBI

4 

Tang JJ, Tang C and Nie PT: The cytoprotective mechanisms of CIRP upon stresses. Sheng Li Ke Xue Jin Zhan. 44:67–71. 2013.(In Chinese). PubMed/NCBI

5 

Al-Fageeh MB and Smales CM: Cold-inducible RNA binding protein (CIRP) expression is modulated by alternative mRNAs. RNA. 15:1164–1176. 2009. View Article : Google Scholar : PubMed/NCBI

6 

Zhang HT, Xue JH, Zhang ZW, Kong HB, Liu AJ, Li SC and Xu DG: Cold-inducible RNA-binding protein inhibits neuron apoptosis through the suppression of mitochondrial apoptosis. Brain Res. 1622:474–483. 2015. View Article : Google Scholar : PubMed/NCBI

7 

Li S, Zhang Z, Xue J, Liu A and Zhang H: Cold-inducible RNA binding protein inhibits H2O2-induced apoptosis in rat cortical neurons. Brain Res. 1441:47–52. 2012. View Article : Google Scholar : PubMed/NCBI

8 

Lee HN, Ahn SM and Jang HH: Cold-inducible RNA-binding protein, CIRP, inhibits DNA damage-induced apoptosis by regulating p53. Biochem Biophys Res Commun. 464:916–921. 2015. View Article : Google Scholar : PubMed/NCBI

9 

Zhang Q, Tang X, Lu QY, Zhang ZF, Brown J and Le AD: Resveratrol inhibits hypoxia-induced accumulation of hypoxia-inducible factor-1alpha and VEGF expression in human tongue squamous cell carcinoma and hepatoma cells. Mol Cancer Ther. 4:1465–1474. 2005. View Article : Google Scholar : PubMed/NCBI

10 

Tang N, Wang L, Esko J, Giordano FJ, Huang Y, Gerber HP, Ferrara N and Johnson RS: Loss of HIF-1alpha in endothelial cells disrupts a hypoxia-driven VEGF autocrine loop necessary for tumorigenesis. Cancer Cell. 6:485–495. 2004. View Article : Google Scholar : PubMed/NCBI

11 

Salceda S and Caro J: Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem. 272:22642–22647. 1997. View Article : Google Scholar : PubMed/NCBI

12 

Takubo K, Goda N, Yamada W, Iriuchishima H, Ikeda E, Kubota Y, Shima H, Johnson RS, Hirao A, Suematsu M, et al: Regulation of the HIF-1alpha level is essential for hematopoietic stem cells. Cell Stem Cell. 7:391–402. 2010. View Article : Google Scholar : PubMed/NCBI

13 

Zaidi AU, McDonough JS, Klocke BJ, Latham CB, Korsmeyer SJ, Flavell RA, Schmidt RE and Roth KA: Chloroquine-induced neuronal cell death is p53 and Bcl-2 family-dependent but caspase-independent. J Neuropathol Exp Neurol. 60:937–945. 2001. View Article : Google Scholar : PubMed/NCBI

14 

Chang ET, Parekh PR, Yang Q, Nguyen DM and Carrier F: Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells. Oncotarget. 7:10578–10593. 2016.PubMed/NCBI

15 

Khan MM, Yang WL, Brenner M, Bolognese AC and Wang P: Cold-inducible RNA-binding protein (CIRP) causes sepsis-associated acute lung injury via induction of endoplasmic reticulum stress. Sci Rep. 7:413632017. View Article : Google Scholar : PubMed/NCBI

16 

Qiang X, Yang WL, Wu R, Zhou M, Jacob A, Dong W, Kuncewitch M, Ji Y, Yang H, Wang H, et al: Cold-inducible RNA-binding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis. Nat Med. 19:1489–1495. 2013. View Article : Google Scholar : PubMed/NCBI

17 

Dai S, Huang ML, Hsu CY and Chao KS: Inhibition of hypoxia inducible factor 1alpha causes oxygen-independent cytotoxicity and induces p53 independent apoptosis in glioblastoma cells. Int J Radiat Oncol Biol Phys. 55:1027–1036. 2003. View Article : Google Scholar : PubMed/NCBI

18 

Luo W and Wang Y: HIF repressors under chronic hypoxia. Aging (Albany NY). 8:418–419. 2016. View Article : Google Scholar : PubMed/NCBI

19 

Doe MR, Ascano JM, Kaur M and Cole MD: Myc posttranscriptionally induces HIF1 protein and target gene expression in normal and cancer cells. Cancer Res. 72:949–957. 2012. View Article : Google Scholar : PubMed/NCBI

20 

Iida Y, Aoki K, Asakura T, Ueda K, Yanaihara N, Takakura S, Yamada K, Okamoto A, Tanaka T and Ohkawa K: Hypoxia promotes glycogen synthesis and accumulation in human ovarian clear cell carcinoma. Int J Oncol. 40:2122–2130. 2012.PubMed/NCBI

21 

Wang H, Zhao L, Zhu LT, Wang Y, Pan D, Yao J, You QD and Guo QL: Wogonin reverses hypoxia resistance of human colon cancer HCT116 cells via downregulation of HIF-1α and glycolysis, by inhibiting PI3K/Akt signaling pathway. Mol Carcinog. 53 (Suppl 1):E107–E118. 2014. View Article : Google Scholar : PubMed/NCBI

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September 2019
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APA
Chen, L., Tian, Q., & Wang, W. (2019). Association between CIRP expression and hypoxic‑ischemic brain injury in neonatal rats. Experimental and Therapeutic Medicine, 18, 1515-1520. https://doi.org/10.3892/etm.2019.7767
MLA
Chen, L., Tian, Q., Wang, W."Association between CIRP expression and hypoxic‑ischemic brain injury in neonatal rats". Experimental and Therapeutic Medicine 18.3 (2019): 1515-1520.
Chicago
Chen, L., Tian, Q., Wang, W."Association between CIRP expression and hypoxic‑ischemic brain injury in neonatal rats". Experimental and Therapeutic Medicine 18, no. 3 (2019): 1515-1520. https://doi.org/10.3892/etm.2019.7767