Effect of different delayed cooling time on organ injuries in rat models of exertional heat stroke

doi:10.12122/j.issn.1673-4254.2024.10.03

Abstract

Abstract:

Methods To investigate how the timing of cooling therapy affects organ injuries in rats with exertional heat stroke (EHS) and explore the possible mechanisms. Methods A total of 60 adult male Wistar rat models of EHS were randomized into model group without active cooling after modeling, immediate cooling group with cold water bath immediately after modeling, delayed cooling groups with cold water bath at 5, 15 and 30 min after modeling, with another 12 mice without EHS as the normal control group. The changes in core body temperature of the mice were recorded and the cooling rate was calculated. After observation for 24 h, the mice were euthanized and blood samples were collected for detection of interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-10, and interferon-γ, followed by pathological examination of the vital organs. The rats that died within 24 h were immediately dissected for examination. Results The number of deaths of the model rats within 24 h increased significantly with the time of delay of cooling treatment. The delay of cooling was positively correlated (r=0.996, P=0.004) while the cooling rate negatively correlated with the mortality rate (r=-0.961, P=0.009). The inflammatory cytokine levels presented with different patterns of variations among the cooling intervention groups. All the rat models of EHS had significant organ damages characterized mainly by epithelial shedding, edema, effusion, and inflammatory cell infiltration, and brain and renal injuries reached the peak level at 24 h after EHS. Conclusion EHS causes significant nonspecific pathologies of varying severities in the vital organs of rats, and the injuries worsen progressively with the delay of cooling. There is a significant heterogeneity in changes of serum inflammatory cytokines in rats with different timing of cooling intervention following EHS.

Key words: exertional heat stroke, animal models, cooling interventions, mortality

Jinbao ZHAO, Yiqin JIA, Handing MAO, Shijiao WANG, Fan XU, Xin LI, Ye TAO, Lei XUE, Shuyuan LIU, Qing SONG, Biye ZHOU. Effect of different delayed cooling time on organ injuries in rat models of exertional heat stroke[J]. Journal of Southern Medical University, 2024, 44(10): 1858-1865.

Figures/Tables 10

Fig.1 Core temperature of a rat model measured via the rectum.

Fig.2 Experimental platform of EHS model rats.

Tab.1 Body weight, age, and core temperature （Tc） of the rats before and after modeling in different groups (n=12, Mean±SD)

Group	Weight (g)	Age (week)	Pre-modelling Tc (℃)	Post-modelling Tc (℃)
Control	251.90±7.29	7.0±0.8	37.85±0.81	-
EHS	251.75±6.23	6.9±0.8	37.97±0.85	43.62±0.68
Immediate cooling group	252.77±9.01	7.2±0.5	38.07±0.76	43.22±0.68
Delayed cooling group A	250.45±6.36	6.8±0.7	38.06±0.94	43.32±0.99
Delayed cooling group B	250.29±7.47	7.2±0.8	37.95±0.83	43.52±0.73
Delayed cooling group C	250.68±6.81	7.0±0.9	37.68±0.65	43.16±0.75

Tab.2 Effect of cooling treatment on 24-h mortality rate of the rat models of EHS (n=12)

Group	Delay time for cooling (min)	Cooling rate (℃/min)	Number of deaths within 24 h	24-h mortality rate
CON	-	-	0	0
EHS	-	0.08±0.05	7	58.3%
Immediate cooling group	0	0.36±0.11*	1	8.3%*
Delayed cooling group A	5	0.26±0.07*^#	2	16.7%*
Delayed cooling group B	15	0.20±0.05*^#	3	25.0%*
Delayed cooling group C	30	0.16±0.04*^{#^}	5	41.7%^#

Tab.3 Correlation analysis of delay time of cooling treatment， cooling rate and 24-h mortality rate of the rat models

Variables Statistical indicators		Cooling rate	Delay time for cooling	Mortality rate
Cooling rate	Pearson correlation coefficient	1	-0.912	-0.961
Cooling rate	P	-	0.088	0.009
Delay time for cooling	Pearson correlation coefficient	-0.912	1	0.996
Delay time for cooling	P	0.088	-	0.004
Mortality rate	Pearson correlation coefficient	-0.961	0.996	1
Mortality rate	P	0.009	0.004	-

Fig.3 Representative images of rat kidney sections in each group (HE staining, original magnification: ×200). A: Control group. B: EHS group. C: Immediate cooling group. D: Delayed cooling group A. E: Delayed cooling group B. F: Delayed cooling group C.

Fig. 4 Representative images of rat brain tissue sections in each group (HE staining, ×200). A: Control group. B: EHS group. C: Immediate cooling group. D: Delayed cooling group A. E: Delayed cooling group B. F: Delayed cooling group C. Black arrows indicate neurons with pyknosis accompanied by collapse and karyolysis of Nissl body.

Fig. 5 HE staining of rat lung tissue sections in each group (×200). A: Control group. B: EHS group. C: Immediate cooling group. D: Delayed cooling group A. E: Delayed cooling group B. F: Delayed cooling group C.

Fig. 6 HE staining of rat small intestine tissues in each group (×200). A: Control group. B: EHS group. C: Immediate cooling group. D: Delayed cooling group A. E: Delayed cooling group B. F: Delayed cooling group C.

Fig. 7 Comparison of inflammatory factors among the groups. *P<0.05 vs Control group; #P<0.05 vs EHS group; ^P<0.05 vs immediate cooling group; + P<0.05 vs delayed cooling group A; &P<0.05 vs delayed cooling group B.

References 34

1	全军热射病防治专家组, 全军重症医学专业委员会. 中国热射病诊断与治疗专家共识[J]. 解放军医学杂志, 2019, 44(3): 181-96.
2	Liu SY, Song JC, Mao HD, et al. Expert consensus on the diagnosis and treatment of heat stroke in China[J]. Mil Med Res, 2020, 7(1): 1.
3	Katch RK, Scarneo SE, Adams WM, et al. Top 10 research questions related to preventing sudden death in sport and physical activity[J]. Res Q Exerc Sport, 2017, 88(3): 251-68.
4	Yankelson L, Sadeh B, Gershovitz L, et al. Life-threatening events during endurance sports: is heat stroke more prevalent than arrhythmic death[J]? J Am Coll Cardiol, 2014, 64(5): 463-9.
5	McGeehin MA, Mirabelli M. The potential impacts of climate variability and change on temperature-related morbidity and mortality in the United States[J]. Environ Health Perspect, 2001, 109(): 185-9.
6	Centers for Disease Control and Prevention (CDC). Heat-related deaths: United States, 1999-2003[J]. MMWR Morb Mortal Wkly Rep, 2006, 55(29): 796-8.
7	Maule A. Heat exhaustion and heat stroke among active component members of the U.S. armed forces, 2018-2022[J]. MSMR, 2023, 30(4): 3-7.
8	Liu CC, Shih MF, Wen YS, et al. Dexamethasone improves heat stroke-induced multiorgan dysfunction and damage in rats[J]. Int J Mol Sci, 2014, 15(11): 21299-313.
9	Wen FL, Xu YJ, Xue LE, et al. Proteomics analyses of acute kidney injury biomarkers in a rat exertional heat stroke model[J]. Front Physiol, 2023, 14: 1176998.
10	毛汉丁, 王娇, 王宇曦, 等. 植入式生物遥测技术在劳力型热射病大鼠实验模型中的应用[J]. 解放军医学院学报, 2021, 42(10): 1068-73, 1088.
11	李庆华, 孙荣青, 吕宏迪, 等. 热打击后不同核心温度对大鼠血清炎性细胞因子及MODS的影响[J]. 中华危重病急救医学, 2018, 30(5): 439-43.
12	曹策, 李玲美, 訾明杰, 等. 医学研究中动物实验样本量的确定方法[J]. 中国比较医学杂志, 2023, 33(2): 99-105.
13	Lin XJ, Lin CH, Liu RX, et al. Myricetin against myocardial injury in rat heat stroke model[J]. Biomedecine Pharmacother, 2020, 127: 110194.
14	He SX, Li R, Peng YM, et al. ACSL4 contributes to ferroptosis-mediated rhabdomyolysis in exertional heat stroke[J]. J Cachexia Sarcopenia Muscle, 2022, 13(3): 1717-30.
15	Sato A, Ikawa Y, Inoue N, et al. Massive intestinal liquid retention in a case of severe heat stroke[J]. J Paediatr Child Health, 2019, 55(2): 248-9.
16	Gathiram P, Wells MT, Raidoo D, et al. Changes in lipopolysaccharide concentrations in hepatic portal and systemic arterial plasma during intestinal ischemia in monkeys[J]. Circ Shock, 1989, 27(2): 103-9.
17	Dokladny K, Moseley PL, Ma TY. Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability[J]. Am J Physiol Gastrointest Liver Physiol, 2006, 290(2): G204-12.
18	Yang WR, Li BB, Hu Y, et al. Oxidative stress mediates heat-induced changes of tight junction proteins in porcine Sertoli cells via inhibiting CaMKKβ‑AMPK pathway[J]. Theriogenology, 2020, 142: 104-13.
19	Leon LR, Helwig BG. Heat stroke: role of the systemic inflammatory response[J]. J Appl Physiol, 2010, 109(6): 1980-8.
20	刘树元, 汪茜, 邢令, 等. 劳力型热射病器官损伤机制与救治策略[J]. 武警医学, 2020, 31(5): 450-4.
21	Hu J, Kang HJ, Liu C, et al. Response of regulatory T cells to classic heat stroke in mice[J]. Exp Ther Med, 2018, 16(6): 4609-15.
22	Ganesan S, Volodina O, Pearce SC, et al. Acute heat stress activated inflammatory signaling in porcine oxidative skeletal muscle[J]. Physiol Rep, 2017, 5(16): e13397.
23	徐向迎, 宗晶, 王小洁. 重症劳力性热射病患者的胃肠道屏障破坏及对策探讨[J]. 海军医学杂志, 2023, 44(4): 414-6.
24	Lim CL. Heat sepsis precedes heat toxicity in the pathophysiology of heat stroke-a new paradigm on an ancient disease[J]. Antioxidants, 2018, 7(11): 149.
25	Bouchama A, Knochel JP. Heat stroke[J]. N Engl J Med, 2002, 346(25): 1978-88.
26	Bouchama A, Roberts G, Al Mohanna F, et al. Inflammatory, hemostatic, and clinical changes in a baboon experimental model for heatstroke[J]. J Appl Physiol, 2005, 98(2): 697-705.
27	Epstein Y, Yanovich R. Heatstroke[J]. N Engl J Med, 2019, 380(25): 2449-59.
28	Leon LR, Helwig BG. Role of endotoxin and cytokines in the systemic inflammatory response to heat injury[J]. Front Biosci, 2010, 2(3): 916-38.
29	Bouchama A, Kunzelmann C, Dehbi M, et al. Recombinant activated protein C attenuates endothelial injury and inhibits procoagulant microparticles release in baboon heatstroke[J]. Arterioscler Thromb Vasc Biol, 2008, 28(7): 1318-25.
30	Li Y, Palmer A, Lupu L, et al. Inflammatory response to the ischaemia-reperfusion insult in the liver after major tissue trauma[J]. Eur J Trauma Emerg Surg, 2022, 48(6): 4431-44.
31	Cannon JG. Inflammatory cytokines in nonpathological states[J]. News Physiol Sci, 2000, 15: 298-303.
32	Christman JW, Sadikot RT, Blackwell TS. The role of nuclear factor-kappa B in pulmonary diseases[J]. Chest, 2000, 117(5): 1482-7.
33	Schwarze J, Cieslewicz G, Joetham A, et al. Critical roles for interleukin-4 and interleukin-5 during respiratory syncytial virus infection in the development of airway hyperresponsiveness after airway sensitization[J]. Am J Respir Crit Care Med, 2000, 162(2 Pt 1): 380-6.
34	Shanley TP, Schmal H, Friedl HP, et al. Regulatory effects of intrinsic IL-10 in IgG immune complex-induced lung injury[J]. J Immunol, 1995, 154(7): 3454-60.

[1]	GE Yue, LI Jianwei, LIANG Hongkai, HOU Liusheng, ZUO Liuer, CHEN Zhen, LU Jianhai, ZHAO Xin, LIANG Jingyi, PENG Lan, BAO Jingna, DUAN Jiaxin, LIU Li, MAO Keqing, ZENG Zhenhua, HU Hongbin, CHEN Zhongqing. Construction and validation of an in-hospital mortality risk prediction model for patients receiving VA-ECMO: a retrospective multi-center case-control study [J]. Journal of Southern Medical University, 2024, 44(3): 491-498.
[2]	LUO Xiao, CHENG Yi, WU Cheng, HE Jia. An interpretable machine learning-based prediction model for risk of death for patients with ischemic stroke in intensive care unit [J]. Journal of Southern Medical University, 2023, 43(7): 1241-1247.
[3]	LI Bo, WANG Lei, ZHAO Wei, FAN Yuhan. Morphology of the esophagus of ferrets and expression profile of molecular markers [J]. Journal of Southern Medical University, 2023, 43(3): 428-435.
[4]	CHEN Na, LI Renhua, WANG E, HU Dehua, TANG Zhaohui. Outcomes of patients experiencing cardiovascular adverse events within 1 year following craniotomy for intracranial aneurysm clipping: a retrospective cohort study [J]. Journal of Southern Medical University, 2022, 42(7): 1095-1099.
[5]	. Establishment of animal models of epidermal growth factor receptor inhibitor-related rashes [J]. Journal of Southern Medical University, 2021, 41(3): 352-357.
[6]	. A new method for establishing a ventricular fibrillation model by TCEI in Tibetan miniature pig [J]. Journal of Southern Medical University, 2019, 39(11): 1370-1375.
[7]	. Improvement of a mouse model of valproic acid-induced autism [J]. Journal of Southern Medical University, 2019, 39(06): 718-.
[8]	. Establishment of New Zealand rabbit models of aplastic anemia [J]. Journal of Southern Medical University, 2017, 37(12): 1660-.
[9]	. Establishment of mouse model of bone metastasis of prostate cancer and breast cancer via femoral artery injection of tumor cells [J]. Journal of Southern Medical University, 2017, 37(07): 914-.
[10]	. Establishment of a modified rabbit model of acute lung injury induced by cardiopulmonary bypass [J]. Journal of Southern Medical University, 2017, 37(06): 797-.
[11]	. Predictive value of miRNA-29a and miRNA-10a-5p for 28-day mortality in patients with sepsis-induced acute kidney injury [J]. Journal of Southern Medical University, 2017, 37(05): 646-.
[12]	. Impact of premature rupture of membranes on neonatal complications in preterm infants with gestational age <37 weeks [J]. Journal of Southern Medical University, 2016, 36(07): 887-.
[13]	. Association of heart valve calcification with cardiovascular outcomes in patients on maintenance hemodialysis [J]. Journal of Southern Medical University, 2016, 36(07): 941-.
[14]	. Pathological and magnetic resonance imaging findings in a neonatal Tibet minipig model of hypoxic-ischemic encephalopathy [J]. Journal of Southern Medical University, 2016, 36(05): 705-.
[15]	. Proteomics study of synovial lesions in rats with Mtb-induced rheumatoid arthritis [J]. Journal of Southern Medical University, 2014, 34(09): 1272-.