Microglial Mechanisms Of Memory Impairment In Rats With Type 2 Diabetes

Background: Diabetes mellitus(DM)is a complex metabolic disease,which may have destructive effects on many organs of the body.According to the statistics,DM is the leading cause of end-stage renal disease,blindness and cardiovascular disease.It is also a common cause of neuropathy.Because of the aging of the population,it appears more and more cognitive dysfunction of diabetic patients,which seriously affect the quality of life of patients with DM.Although a lot of researches have been done,the pathogenesis of the diabetic cognition impairment remains unknown.The treatment options are very limited.Therefore,it is very important to investigate mechanisms and to search for effective therapeutic targets.Specific Aims: The present study aims to explore molecular mechanisms underlying the diabetic memory impairment in rats with type 2 diabetes mellitus(T2DM).The specific aims include the followings:(1)To determine whether there is memory impairment in type 2 diabetic rats.(2)To examine whether microglia activation in hippocampus participates in the memory impairment of rats with T2 DM.(3)To investigate roles of P2X4 receptors expression in the hippocampus and its mechanism in the development of memory impairment in type 2 diabetic rats.Methods:(1)Adult male SD rats were used as experimental animals.The rats were fed with high fat diet for a period of 3 weeks.A small dose of streptozotocin(STZ,30 mg/kg)was intraperitoneally injected one these rats at the end of the second week.The same volume of normal saline(NS)was used as controls.(2)The water maze and T-maze test were performed to detect the level of learning and memory in control rats and rats with T2 DM.The pain threshold of rat hindpaw was examined by Von Frey filament(VFF)and thermal stimulation.(3)Immunofluorescence assay was used to observe the status of hippocampal cells and the localization of P2X4 receptors(P2X4Rs)in the hippocampus of rats with T2 DM.(4)The expression of P2X3 Rs,P2X4Rs,P2X7 Rs and ATM in hippocampus and P2X4 Rs in anterior cingular cortex and basolateral amygdala of rats were detected by Western blot and Real-time PCR.(5)DNA damage of P2X4 Rs in hippocampus of rats was detected by Long Amplicon PCR(LA-PCR).(6)Minocycline(Mino)was intraperitoneally(50mg/kg)injected in rats with T2 DM from the second day before Morris water maze to the fifth day for one week.Results:(1)The body weight of rats fed with high fat food was significantly increased faster than that of control diet rats.The fasting blood glucose of rats increased to above 16mmol/L after the injection of STZ,and it at least lasts to the eleventh weeks.IPITT was performed to test the insulin tolerance in rats.Compared with control rats,the rats fed with high-fat food were not sensitive to insulin.(2)In the water maze test,the Escape Latency and Path Length of type 2 diabetic rats had no difference with control rats.But the Passing times and Time in the quadrant of type 2 diabetic rats were significantly less than control rats,besides,the Mean distance of type 2 diabetic rats was clearly more than control ones.(3)In the T-maze test,the correct choice rate of type 2 diabetic rats was significantly lower than that of control rats.And the Win-shift failure and Lose-shift failure of type 2 diabetic rats were markedly more than that of control rats.(4)The status of neurons and astrocytes in hippocampus of rats with type 2 diabetes mellitus did not differ from those of control rats,but the number of activated microglia was significantly more than that of control ones.(5)After intraperitoneal injection of minocycline,the number of activated microglia was significantly reduced.More importantly,type 2 diabetic rats showed the reduced mean distance in the Morris water maze test after intraperitoneal injection of minocycline.(6)Compared with the control rats,the expression and mRNA level of P2X4 R were significantly decreased in the hippocampus of type 2 diabetic rats.But the expression of ATM in the hippocampus of type 2 diatetec rats was obviously increased,compared to the control ones.(7)LA-PCR experiments showed that the DNA amplification of P2X4 R in the hippocampus of type 2 diabetic rats was significantly less than that of control rats.Conclusions:(1)There is memory impairment in type 2 diabetic rats.(2)The excessive activation of microglia in the hippocampus is involved in the memory impairment in type 2 diabetic rats.(3)The DNA damage of P2X4 R gene in microglia led to the down-regulation of P2X4 R protein expression in the hippocampus of type 2 diabetic rats,which is the cause of excessive activation of microglial cells and may be the molecular mechanism of memory impairment in type 2 diabetic rats.