Experimental Study Of Intermittent Normobaric Hypoxia Preconditioning In The Prevention And Treatment Of Diabetes With Ischemic Stroke

Background:Ischemic stroke(IS)is the main cause of disability and death worldwide.Due to the aging population and unhealthy lifestyle,the incidence rate of cerebrovascular diseases is very high.Type 2 diabetes is a common complication of IS.DM increases the risk and severity of IS,and affects the recovery of neurological function after IS.Therefore,in addition to timely and effective reperfusion therapy for IS,active control of these risk factors is crucial for reducing the incidence and recurrence rate of IS.Evidence from animal and human studies suggests that hypoxia conditioning(HC)can improve common vascular risk factors in IS.In addition,HC plays a neuroprotective role by increasing resistance to acute ischemia and hypoxic stress and promotes post injury repair and regeneration processes to alleviate brain injury.Intermittent hypoxia conditioning(IHC)refers to alternating exposure to normal and low oxygen for a short time every day,lasting for several days or weeks.For IS patients and high-risk groups,IHC is expected to become an auxiliary clinical treatment strategy for the prevention and treatment of IS.However,whether IHC has beneficial effects depends not only on the hypoxia paradigm,including dose,exposure time,equipment requirements,but also on the differences among subjects,including gender,age,complications,circadian rhythm,and other factors.Therefore,the optimal treatment mode and applicable conditions for IHC need to be further explored and studied.Purpose:(1)Part 1: Whether IH preconditioning and IH postconditioning can activate the body to produce endogenous protection against ischemic-hypoxic brain injury and promote neurological recovery through non-invasive stimulation during the prevention and treatment phase of IS.(2)Part 2: To explore the effects of IHC on body weight,blood glucose,cognitive memory function,and cerebral microvascular structure in db/db diabetic mice during active phase(ZT15)and inactive phase(ZT3).(3)Part3 : The effect of active phase(ZT15)and inactive phase(ZT3)IH preconditioning on the prognosis of acute ischemic brain injury in db/db diabetic mice.Methods:(1)Part 1: Male C57BL/6 mice were divided into 5 groups: sham operaction group(sham),d MCAO group(d MCAO),IH preconditioning before d MCAO(IH preconditioning),IH postconditioning was perfomed 24 hours after d MCAO(acute phase IH postconditioning),IH postconditioning was perfomed 72 hours after d MCAO(subacute phase IH postconditioning).IHC was given for 7 days,and the animal behavioral tests were made by adhesive removal test,grid walking test and novel object recognition test to assess neurological functions such as sensory,motor,cognitive and memory functions of mice.(2)Part 2: Male db/db mice were divided into three groups: nomorxia group(normoxia),inactive phase IH group(IH ZT3),and active phase IH group(IH ZT15).IHC was given for 2 weeks,and the mice were monitored for changes in body weight,fasting blood glucose,and random blood glucose.The neurological functions of mice such as sensory,motor,cognitive and memory functions were assessed by adhesive removal test,grid walking test,novel object recognition test.The cerebral microvascular structures were observed by Lectin immunofluorescence staining.(3)Part 3:Male db/db mice were divided into four groups: sham operation group(sham),normoxia + d MCAO group,active phase IH preconditioning(IH ZT15)+ d MCAO group,inactive phase IH preconditioning(IH ZT3)+ d MCAO group.IHC was given for 2 weeks,and the mice were evaluated for sensory,motor,cognitive and memory neurological functions through animal behavioral tests such as adhesive removal test,grid walking test,novel object recognition test.The cortical cerebral blood perfusion of mice was measured by laser speckle blood flow imaging system.The infarct volume was measured by MAP2 immunofluorescence staining.The cerebral microvascular structures were observed by Lectin immunofluorescence staining.Results:(1)Part 1: Compared with the sham group,the C57BL/6 mice d MCAO model can cause an increase in sticky paper contact time and remove time,and more grid walking foot fault rate(P<0.001).Compared with the d MCAO group,there was a statistically significant difference in the shortening of sticky paper contact time and remove time after ischemic brain injury in C57BL/6 mice in the IH preconditioning group(P<0.001).In contrast,there was no improvement in sticky paper contact time and remove time,or grid walking foot fault rate in the acute and subacute IH postconditioning groups.Compared with baseline,cognitive memory function for recognition of old and new objects was not affected in all groups of C57BL/6 mice.(2)Part 2: Compared with the normoxia group,the body weights of db/db mice in both the active phase(ZT15)IH group and the inactive phase(ZT3)IH group decreased to a different extent during 14 days of IHC,with a statistically significant difference in weights on day 7 of IH(P<0.05).IHC had no significant effect on blood glucose in db/db mice.Compared with the normoxia group and the active phase(ZT15)IH group,the inactive phase(ZT3)IH group resulted in a statistically significant difference in the impaired recognition of old and new objects in db/db mice(P<0.05).Compared with the normoxia group and the inactive phase(ZT3)IH group,the cerebral cortical microvasculature of db/db mice in the active phase(ZT15)IH group were enriched,and the cerebral microvessel areas,lengths,and diameters increased,with statistically significant differences(P<0.05).(3)Part 3: On the third day after dMCAO,80% mortality was observed in the normoxia group of db/db mice,no mortality in the IH ZT15 group,and 60% mortality in the IH ZT3 group.Compared with the sham and normoxia groups,fasting and random blood glucose of db/db mice in the IH ZT15 group on day 3 after d MCAO did not change significantly,whereas fasting and random blood glucose were significantly decreased in the IH ZT3 group(P<0.01).In all d MCAO model groups,weight loss on the third day after d MCAO was significant compared with preoperative baseline level(P<0.0001),whereas weight loss was mild in the IH ZT15 d MCAO group compared with the sham group.In comparison with the sham group,the d MCAO caused impaired sensory and motor neurological functions in db/db mice(P<0.001).Compared with the normoxia group,the IH ZT15 group showed a statistically significant reduction in sticky paper contact time and remove time,and grid walking foot fault rate(P<0.05).Compared with the normoxia group and the IH ZT3 group,the IH ZT15 group had less impaired cognitive function on day 3 after d MCAO(P<0.05).Compared with the normoxia group and the IH ZT3 group,cortical collateral blood flow perfusion in the ischemic region of the cortex was significantly improved in the IH ZT15 group 3 days after d MCAO(P<0.0001).Compared with the normoxia group and the IH ZT3 group,the infarct volume caused by d MCAO in db/db mice in the IH ZT15 group was small and statistically different(P<0.05).Compared with the normoxia and IH ZT3 group,the IH ZT15 group had an abundance of cortical microvasculature in the peri-infarct cortex and an increase in cerebral microvessel areas,lengths,and diameters,which were statistically different(P<0.05).Conclusions:(1)The dMCAO model in C57BL/6 mice can cause sensory and motor neurological impairments,but not cognitive and memory impairments.IH preconditioning has a neuroprotective effect on C57BL/6 mice and can attenuate sensory and motor neurological impairments caused by d MCAO.IH postconditioning given during the acute and subacute phases of ischemic brain injury did not significantly improve sensory and motor neurological impairments in C57BL/6 mice.(2)IHC had a weight-reducing effect on db/db mice,with no significant effect on blood glucose.Inactive phase(ZT3)IH resulted in impaired cognitive and memory function in db/db mice,and active phase(ZT15)IH did not affect cognitive function.Active phase IHC can remodel the cerebral microvascular structure of db/db mice.(3)Active phase(ZT15)IH preconditioning has neuroprotective effects against ischemic brain injury in db/db mice,which reduces mortality after d MCAO, attenuates neurological impairments such as sensory,motor,cognitive and memory in mice,improves cortical collateral blood flow perfusion to the ischemic area,reduces core infarct volume,and increases cortical microvessel density in peri-infarct areas.Active-phase IHC increase the brain’s resistance to ischemic injury by remodeling the cerebrovascular network and increasing collateral blood perfusion to ischemic brain areas.