Effects of mild hypothermia on the ROS and expression of caspase-3 mRNA and LC3 of hippocampus nerve cells in rats after cardiopulmonary resuscitation

doi:10.5847/wjem.j.issn.1920-8642.2014.04.010

Abstract

Abstract:

BACKGROUND: Cardiac arrest (CA) is a common and serious event in emergency medicine. Despite recent improvements in resuscitation techniques, the survival rate of patients with CA is unchanged. The present study was undertaken to observe the effect of mild hypothermia (MH) on the reactive oxygen species (ROS) and the effect of neurological function and related mechanisms.
METHODS: Sixty-five healthy male Sprague Dawley (SD) adult rats were randomly (random number) divided into 2 groups: blank control group (n=5) and CPR group (n=60). CA was induced by asphyxia. The surviving rats were randomly (random number) divided into two groups: normothermia CPR group (NT) and hypothermia CPR group (HT). Normothermia of 37 °C was maintained in the NT group after return of spontaneous circulation (ROSC), hypothermal intervention of 32 °C was carried out in the HT group for 4 hours immediately after ROSC. Both the NT and HT groups were then randomly divided into 2 subgroups 12 hours and 24 hours after ROSC (NT-12, NT-24, HT-12, HT-24 subgroups). During observation, the neurological deficit scores (NDSs) was recorded, then the bilateral hippocampi were obtained from rats' head, and monoplast suspension of fresh hippocampus tissue was made immediately to determine the level of intracellular ROS by flow cytometry. Transmission electron microscope was used to observe the ultramicro changes of cellular nucleus and mitochondria. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of caspase-3 mRNA, and western-blotting (WB) was used to determine the level of LC3 in frozen hippocampus tissue. Measured data were analyzed with paired sample t test and One-Way ANOVA.
RESULTS: Of 60 rats with CA, 44 (73%) were successfully resuscitated and 33 (55%) survived until the end of the experiment. The NDSs of rats in the NT and HT groups were more significantly reduced than those in the BC group (F=8.107, P<0.05), whereas the NDSs of rats in the HT-12 and HT-24 subgroups were significantly increased in comparison with those NDSs of rats in the NT-12 and NT-24 subgroups, respectively (t=9.692, P<0.001; t=14.374, P<0.001). The ROS in hippocampus nerve cells in the NT and HT groups significantly increased compared to the BC group (F=16.824, P<0.05), whereas the ROS in the HT-12 and HT-24 subgroups significantly reduced compared with that ROS in the NT-12 and NT-24 subgroups, respectively (t=9.836, P<0.001; t=7.499, P<0.001). The expression of caspase-3 mRNA in hippocampus nerve cells in the NT and HT groups were significantly increased compared to the BC group (F=24.527, P<0.05), whereas the expression of caspase-3 mRNA in rats of the HT-12 and HT-24 subgroups was significantly reduced compared to the NT-12 and NT-24 subgroups, respectively (t=6.935, P<0.001; t=4.317, P<0.001). The expression of LC3B-II/I in hippocampus nerve cells of rats in the NT and HT groups significantly increased compared to the BC group (F=6.584, P<0.05), whereas the expression of LC3B-II/I in rats of the HT-12 and HT-24 subgroups significantly reduced compared to the NT-12 and NT-24 subgroups, respectively (t=10.836, P<0.001; t=2.653, P=0.02). Ultrastructure damage of nucleus and mitochondria in the NT group was more evident than in the BC group, and eumorphism of nucleus and mitochondria were maintained in rats of the HT group compared with the NT group.
CONCLUSION: Mild hypothermia lessened the injury of nerve cells and improved the neurological function of rats that survived from cardiac arrest by reducing the ROS production of nerve cells and inhibiting the expression of caspase-3 mRNA and LC3, leading to cellular apoptosis and massive autophagy in rats that survived from cardiac arrest after CPR.

Key words: Mild hypothermia, Cardiopulmonary resuscitation, Reactive oxygen species, Caspase-3, LC3, Autophagy

Jian Lu, Yi Shen, Hui-yin Qian, Li-jun Liu, Bao-chun Zhou, Yan Xiao, Jin-ning Mao, Guo-yin An, Ming-zhong Rui, Tao Wang, Chang-lai Zhu. Effects of mild hypothermia on the ROS and expression of caspase-3 mRNA and LC3 of hippocampus nerve cells in rats after cardiopulmonary resuscitation[J]. World Journal of Emergency Medicine, 2014, 5(4): 298-305.

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Groups	Survival number (n)	The time from asphyxiation to SBP≤25 mmHg (s)	The time of SBP ≤25 mmHg (s)	The time of resuscitation (s)	The time from induction to 32 °C of HT group (min)
NT-12	9	212±33	241±32	81±30	—
NT-24	7	244±33	248±31	84±23	—
HT-12	8	229±47	249±35	70±8	24.5±3.1
HT-24	9	219±28	247±21	79±15	27.3±3.7

Groups	T (center,°C)	HR (beats/min)	RR (beats/min)	MAP (mmHg)
NT (n=16)
Baseline	36.6±0.7	255±42	78±4	94±21
PR-0.5 h	36.5±0.5	232±58	78±1	66±9
PR-4 h	36.6±0.5	248±61	89±11	91±28
HT (n=17)
Baseline	36.4±0.5	238±37	77±1	100±21
PR-0.5 h	32.4±0.6	260±62	77±1	78±15
PR-4 h	32.1±0.2	257±57	75±10	96±18

Groups	pH	PaO₂ (mmHg)	PaCO₂ (mmHg)	SaO₂ (%)	Lac (mg/dL)
NT (n=16)
Baseline	7.36±0.06	91±29	42±6	94±8	2.2±0.9
PR-0.5 h	7.23±0.11	84±24	56±16	92±9	6.9±2.8
PR-4 h	7.29±0.07	87±27	49±10	90±9	3.4±1.8
HT (n=17)
Baseline	7.39±0.03	85±9	41±6	96±2	2.1±0.9
PR-0.5 h	7.35±0.11	81±13	50±11	94±16	7.2±2.9
PR-4 h	7.33±0.08	71±10	44±6	92±4	2.3±1.1^*

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