title: Effects of mild hypothermia on the ROS and expression of caspase-3 mRNA and LC3 of hippocampus nerve cells in rats after cardiopulmonary resuscitation.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Emergency Medicine, Suzhou Municipal Hospital, Suzhou 215002, China ; Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Lu
authors.affiliation: Department of Emergency Medicine, Suzhou Municipal Hospital, Suzhou 215002, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Hui-Yin
authors.initials: HY
authors.lastname: Qian
authors.affiliation: Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Liu
authors.affiliation: Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Bao-Chun
authors.initials: BC
authors.lastname: Zhou
authors.affiliation: Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Jin-Ning
authors.initials: JN
authors.lastname: Mao
authors.affiliation: Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Guo-Yin
authors.initials: GY
authors.lastname: An
authors.affiliation: Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Ming-Zhong
authors.initials: MZ
authors.lastname: Rui
authors.affiliation: Department of Emergency and Critical Care Medicine, Second Affiliated Hospital of Soochow University, Suzhou 215007, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: Electron Microscope Laboratory, Medical College of Nantong University, Nantong 226200, China.
authors.forename: Chang-Lai
authors.initials: CL
authors.lastname: Zhu
keywords: Autophagy; Cardiopulmonary resuscitation; Caspase-3; LC3; Mild hypothermia; Reactive oxygen species