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

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 defi cit 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 m RNA, 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 signifi cantly 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 signifi cantly 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 m RNA in hippocampus nerve cells in the NT and HT groups were signifi cantly increased compared to the BC group(F=24.527, P<0.05), whereas the expression of caspase-3 m RNA in rats of the HT-12 and HT-24 subgroups was signifi cantly 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 signifi cantlyincreased 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 m RNA and LC3, leading to cellular apoptosis and massive autophagy in rats that survived from cardiac arrest after CPR.

A partition-type tubular scaffold loaded with PDGF-releasing microspheres for spinal cord repair facilitates the directional migration and growth of cells

The best tissue-engineered spinal cord grafts not only match the structural characteristics of the spinal cord but also allow the seed cells to grow and function in situ.Platelet-derived growth factor（PDGF） has been shown to promote the migration of bone marrow stromal cells;however,cytokines need to be released at a steady rate to maintain a stable concentration in vivo.Therefore,new methods are needed to maintain an optimal concentration of cytokines over an extended period of time to effectively promote seed cell localization,proliferation and differentiation.In the present study,a partition-type tubular scaffold matching the anatomical features of the thoracic 8–10 spinal cord of the rat was fabricated using chitosan and then subsequently loaded with chitosan-encapsulated PDGF-BB microspheres（PDGF-MSs）.The PDGF-MS-containing scaffold was then examined in vitro for sustained-release capacity,biocompatibility,and its effect on neural progenitor cells differentiated in vitro from multilineage-differentiating stress-enduring cells（MUSE-NPCs）.We found that pre-freezing for 2 hours at-20°C significantly increased the yield of partition-type tubular scaffolds,and 30 μL of 25% glutaraldehyde ensured optimal crosslinking of PDGF-MSs.The resulting PDGF-MSs cumulatively released 52% of the PDGF-BB at 4 weeks in vitro without burst release.The PDGF-MS-containing tubular scaffold showed suitable biocompatibility towards MUSE-NPCs and could promote the directional migration and growth of these cells.These findings indicate that the combination of a partition-type tubular scaffold,PDGF-MSs and MUSENPCs may be a promising model for the fabrication of tissue-engineered spinal cord grafts.