Effect Of Sevoflurane Preconditioning On Hippocampal CA1 Region Neurons Autophagy And Long-term Cognitive Function In Newborn Rat With Asphyxia

ObjectiveWe established new born rat model with asphyxia on mimicking transient hypoxia, and study the model practicability. The present study was designed to investigate effect of sevoflurane preconditioning on hippocampal CA1 region neurons autophagy and the effect of long-term cognitive function in new born rat with asphyxia, and explore the possible cerebral protection and possible mechanism.Methods1、With reference to Grojean and Jialin Yu, new born age 7 day P(7) rats were made to breathe 100% nitrogen 10 minutes to establish the model of neonatal rat with asphyxia. Neurological behavior, arterial blood gas analysis, short-term neural function, brain water content,histological examination under the microscope were adopted to verify the model.2、Two hundred and eighty-two P7 SD rats were randomly divided into control group(C group), hypoxia group(H group), sevoflurane preconditioning group(S group), Immunohistochemistry and western blot were used to detected the change of autophagy related proteins LC3-Ⅱ; HE staining and DAPI staining were applied to test pathological histology change of hippocampal CA1 region neurons. The rats brain sensorimotor functional outcome was evaluated by suspension test(P21) and thebalancing test(P23), long-term cognitive function was evaluated by the Morris Water Maze test(P28-P33).Results1、Breathing 100% nitrogen ten minutes was taken for appropriate time to prepare model. During hypoxia, the performance of neurological behaviors such as cyanosis, abnormal fidgety, limb movement disorder,unconsciousness and urinary and fecal incontinence were found in all of rats. It turned out SPO2 declined, acidosis, oxygen partial pressure reduction and high carbon dioxide partial pressure during hypoxia. The brain water content of model group was higher than normal group’s(P <0.05). The rats of model group completed Cliff avoidance test and Negative reflection test longer normal group’s(P<0.05). Through hematoxylin-eosin staining we found that the cortical and hippocampus neurons were severely damaged after 10 minutes hypoxia.2、HE staining showed C group rats hippocampus CA1 region normal neurological morphology; H group displayed obviously abnormal hippocampus CA1 region structure: disordered arrangement and large numbers of nuclear shrinkage; S group displayed lighter neurological morphology damage than H group. DAPI staining showed that there are significant differences in the number of changed neurons nucleus at hippocampal CA1 region within three groups(P <0.05); compared with C group, H and S group showed more numbers of nuclear morphological changed neurons in hippocampal CA1 region(P <0.05). S group showed less nuclear morphological changed neurons than H group(P <0.05).Hippocampal CA1 region neurons LC3-Ⅱimmunohistochemistry results: LC3-Ⅱexpression localized at the cytoplasm of neuron. There are almost not positive cells at each point in C group; S group showed a small amount of positive cells thirty minutes after one hour sevoflurane preconditioning(P <0.05); compared with C group, H and Sgroup showed more LC3-Ⅱpositive cells 6h after hypoxia, increased significantly 12 h after hypoxia, reached a peak 24 h after hypoxia, then gradually decreased with lighter color(P <0.05); S group showed less LC3-Ⅱexpression than H group at each point after hypoxia(P <0.05).LC3 results in western blot : C group LC3-Ⅱ/LC3-Ⅰratio had no obvious change in each points; LC3- Ⅱ /LC3- Ⅰ ratio increased 30 min after one hour sevoflurane preconditioning(P < 0.05); compared with C group, H and S group LC3-Ⅱ/LC3- Ⅰratio increased at 6h after hypoxia, reached a maximum at 24 h, than gradually reduced. Compared with H group, LC3-Ⅱ/LC3-Ⅰratio of S group was lower at each point after hypoxia(P < 0.05).There were no significant differences in neurological behaviors both suspension test and the balancing test among three groups. Compared with C group, performances of rats from H group and S group in Morris water-maze test from P28 to P32 showed significantly longer mean avoidance latency(P <0.05); compared with the H group, S group showed shorter avoidance latency from the third day to fifth day(P <0.05). Compared with C group, performances of rats from H group and S group in space exploration test showed significantly fewer times of crossing the spot at which platform located previously(P<0.05). Compared with the H group, S group crossed the spot more times(P<0.05).Conclusions1、By means of putting P7 neonatal rat in the environment of 100% nitrogen, we successfully set up the model of neonatal rat with asphyxia mimicking hypoxic ischemic encephalopathy.2、Probable mechanism of sevoflurane preconditioning cerebral protective effect is related to raise autophagy level of neurons first and then reduce autophagy level afterhypoxia.3、Sevoflurane preconditioning could reduce hippocampal CA1 region neurons death in newborn rat with asphyxia.4、Sevoflurane preconditioning could improve long-term cognitive function of in newborn rat with asphyxia.