Preliminary Study Of Microglia Activation Impairing Neurobehavior In Neonatal Rats With Hypoglycemia

Objective:Hypoglycemia(HG)is one of the most common metabolic disorders in neonatal intensive care units.Repeated and severe hypoglycemia can lead to central nervous system damage,resulting in neonatal hypoglycemic encephalopathy with sequelae such as cognitive impairment,cerebral palsy,and autism.Microglia play a crucial role in brain development,and some studies have found that the activation of microglia can be induced by hypoglycemic environment.To further clarify the relationship between microglia and neonatal hypoglycemic brain injury,this experiment investigated the changes of prepubertal neurobehavior in neonatal rats with repeated and severe hypoglycemic experiences with or without inhibition of microglia activation,which is important to further reveal the mechanism and treatment strategy of neonatal hypoglycemic brain injury.Methods:Both male and female SD rats aged at 10 days(P10)were recruited with the following four groups: hypoglycemic group(HG),Control group(Ctrl),hypoglycemic treated with minocycline group(HG+Mino)or vehicle group(HG+Veh).Firstly,the following four behavioral tests were performed in early adolescence:(1)The motor development of the rats was evaluated by wire hanging test at P14;(2)The voluntary locomotor ability was assessed by the open field test at P21 and the anxiety was detected by the light and dark box test at P22.(3)The spatial learning and memory ability of rats were tested by morris water maze at from P28 to P33.Brains were taken at P15 for frozen sectioning and Iba1 staining to observe the activation of microglia in the anterior cingulate cortex,ventral and dorsal hippocampal DG areas of rats in HG,Ctrl,HG+Mino and HG+Veh groups.Results:(1)Modeling of each group: During the hypoglycemia modeling for 3 consecutive days,compared with the initial blood glucose,the HG,HG+Mino and HG+Veh groups had significantly lower blood glucose at 1 h,2 h,3 h and 4 h after hypoglycemia,and significantly different with the Ctrl group.There was no significant difference in weight,brain weight and brain volume among the Ctrl,HG,HG+Mino and HG+Veh groups.(2)Wire hanging test: The experimental results of P14 and P16 showed that there was no significant difference in the suspension duration between HG group and Ctrl group,and between HG+Mino group and HG+Veh group.However,in P15,there were significant differences in the suspension duration between HG group and Ctrl group,and between HG+Veh group and HG+Mino group.(3)Open field: A pairwise comparison between the HG group and the Ctrl group,and between the HG+Mino group and the HG+Veh group showed no significant difference in the total distance of movement within 10 minutes.(4)Light/dark box: Compared with Ctrl group,HG group showed a decrease in the total time in the open box and the number of times in the open box.Compared with HG+Veh group,the total time in the open tank and the number of times entering the open tank in HG+Mino group were significantly increased.(5)Morris water maze: When comparing the HG group with the Ctrl group and the HG+Mino group with the HG+Veh group,there was no difference in the escape latency and path length of the rats during the visible platform and the escape latency during the hidden platform.However,the number of rats crossing the original position of the hidden platform was significantly reduced in the HG group during the no platform,while the number of rats crossing the original position of the hidden platform was significantly increased in the HG+Mino group.(6)Iba1 staining: Compared with Ctrl,microglia in the anterior cingulate cortex,dorsal hippocampus and ventral hippocampus DG region were significantly activated in HG group,with larger cell bodies and shorter or disappeared processes.After the intervention of microglia inhibitors,the activation of microglia in each brain area of HG+Mino group was reduced,and most of them showed highly branched microglia.Conclusions:In conclusion,this study confirmed that repeated severe hypoglycemia in neonatal period can induce the activation of microglia cells.They exhibited motor retardation,anxiety-like behavior,and spatial learning memory deficits in early adolescence.Inhibition of microglia activation improves spatial learning memory deficits and motor developmental delays caused by recurrent severe hypoglycemia,and relieves anxiety.These findings provide a new theoretical basis for the treatment of neonatal hypoglycemic brain injury.