The Repair Effect And Mechanism Of Modafinil On Memory Impairment Induced By Isoflurane Anesthesia

Objective: Memory is an ability to store and extract information and is the most fundamental support for learning and survival in nature.However,its neural mechanisms are so far unclear.General anesthetics can be an important tool to explore the neural mechanisms associated with memory.Memory amnesia is an important role of general anesthetics to reduce surgical trauma,among others.However,studies have shown that this effect exists during the anesthesia awakening period and even lasts for a long time,which will seriously affect the patient’s brain function and rapid postoperative recovery.The dopamine system in the brain is extensively involved in cognition and learning memory.Studies have shown that modafinil,a dopamine agonist,is effective in improving memory performance.However,it is unclear whether modafinil accelerates recovery from memory impairment caused by isoflurane anesthesia and the mechanism of action.This study focused on the effects of modafinil on memory impairment after isoflurane anesthesia and its mechanism of action.Methods:Step 1: To investigate the effect of isoflurane anesthesia on memory excluding the interference caused by its effect on motor ability.Twenty-five male SPF-grade C57BL/6mice were randomly divided into five groups(n = 5): control group(Con group),0.5 h(0.5h group),1 h(1 h group),1.5 h(1.5 h group)and 2 h(2 h group)after 2 h RORR of isoflurane anesthesia.In the control group,the mice were subjected to the open field test after 50% O2 inhalation alone;in the experimental group,the mice were subjected to the open-field test at 0.5 h,1 h,1.5 h and 2 h after the recovery of the righting reflex under 1.5% isoflurane anesthesia for 2 h.Step 2: To explore the effect of isoflurane anesthesia on memory.Sixteen male SPF-grade C57BL/6 mice were randomly divided into two groups(n = 8): the control group(Con group)and the 1.5% isoflurane anesthesia for 2 h(ISO group).mice in the Con group were anesthetized by 50% O2 inhalation alone and in the ISO group by 1.5% isoflurane inhalation for 2 h.Novel object recognition experiment and conditioned fear test were performed in each group,respectively.Step 3: To explore the effect of modafinil on memory impairment after isoflurane anesthesia.Twenty-five male SPF-grade C57BL/6 mice were randomly divided into two groups(n = 7,5,5,4 and 4): isoflurane anesthesia + vehicle group(ISO+Veh group)and isoflurane anesthesia + modafinil group(ISO+MOD group),in which modafinil was set at four different concentration gradients of 10,30,60,and 90 mg/kg,respectively.The ISO+MOD and ISO+Veh groups were anesthetized with 1.5% isoflurane and injected intraperitoneally with MOD 10,30,60 and 90 mg/kg or an equivalent volume of Vehicle(saline containing 20% DMSO),respectively,and each group of mice was subjected to novel object recognition test and conditioned fear test,respectively.Step 4: To explore the mechanism of memory impairment after modafinil improvement of isoflurane anesthesia,immunofluorescence was used to observe the expression of c-Fos,a memory-related nucleus in the brain.Eighteen male SPF-grade C57BL/6 mice were randomly divided into three groups(n=6): control group(Con group),isoflurane anesthesia group(ISO group),and isoflurane anesthesia + modafinil group(MOD group).the Con group was simply inhaled with 50% O2.the ISO and MOD groups were both anesthetized with 1.5% isoflurane for 2 h.In the MOD group,10 mg/kg MOD was injected intraperitoneally after isoflurane anesthesia.After 1.5 h,the mice in the three groups were anesthetized separately and the brains were severed for immunofluorescence.Step 5: To investigate whether modafinil ameliorates memory impairment caused by isoflurane anesthesia by acting on hippocampal D1 receptor positive neurons,chemical genetic modulation of hippocampal D1 receptor positive neurons was observed for behavioral manifestations.Sixty male SPF-grade C57BL/6 mice,7-8 weeks old.A random number table method was used to divide them into two major groups(n = 30): activated h M3 Dq and inhibited h M4 Di,each group was further randomized into two subgroups(n =15): including the SAL and CNO groups.Three weeks after virus injection,saline(SAL group)or CNO(CNO group)was injected intraperitoneally after 1.5% isoflurane anesthesia to perform novel object recognition test and conditioned fear test,respectively.CNO specifically activated successfully infected h M3 Dq and h M4 Di viruses thereby enabling them to exert positive or negative regulatory effects.To verify whether hippocampal D2 receptor positive neurons are involved in modulating modafinil to improve memory deficits after isoflurane anesthesia,chemical genetic modulation of hippocampal D2 receptor positive neurons was observed for behavioral performance.Twenty-four male SPF-grade C57BL/6 mice were divided into two major groups(n = 12)using the random number table method: activated M3 Dq and inhibited M4 Di,and each group was further randomized into two subgroups(n = 6): including the SAL and CNO groups.The experimental protocol was the same as above.Results:1.Compared with the Con group,mice had significantly more motor trajectories about1.5 h after isoflurane anesthesia,with a total distance moved of 5150.0 ± 286.7 cm in 5 min and a mean speed of 16.9 ± 1.1 cm/s.The difference between the two groups was not statistically significant(P > 0.05,n = 5).In the novel object recognition experiment,the novel object recognition index was reduced in the ISO group mice compared to the Con group(P = 0.035,n = 8).In the conditioned fear experiment,the percentage of freezing time was significantly lower in the ISO group mice for the contextual memory test(P = 0.002,n= 8);the percentage of freezing time was also lower in the cue-tone memory test(P = 0.022,n = 8).The results indicated that the motor ability of mice was fully recovered around 1.5 h after 1.5% isoflurane anesthesia,but novel object recognition memory and conditioned fear memory were still not recovered.2.In the novel object recognition experiment,the novel object recognition index increased in mice in the ISO+MOD 10 mg/kg group compared with the ISO+Veh group(P= 0.026).In the conditioned fear experiment,the percentage of freezing time was increased in the ISO+MOD 10 mg/kg group compared with the ISO+Veh group in the contextual memory test(P = 0.030);the percentage of freezing time was also increased in the ISO+MOD 10 mg/kg group in the cue-tone memory test(P = 0.050).The results indicated that modafinil had an ameliorative effect on the memory for novel object recognition and conditioned fear memory impairment in mice during the awakening period caused by isoflurane anesthesia,and the effect was most obvious at 10 mg/kg,and the memory improvement effect diminished or even disappeared with increasing concentration.3.Immunofluorescence results showed that c-Fos expression in the prefrontal cortex Pr L,ACC in the anterior cingulate cortex and CA1 region of hippocampus was significantly reduced in the ISO group compared with the Con group(P < 0.001);while c-Fos expression in the Pr L,ACC and CA1 region of hippocampus was significantly increased in the MOD group compared with the ISO group(P < 0.01).The results suggest that modafinil may exert memory improvement effects by acting on memory-related brain regions such as Pr L,ACC and hippocampus.4.Chemical genetic transfection of negative h M4 Di virus results showed that in the novel object recognition experiment,CNO inhibited novel object recognition index in hippocampal D1 receptor positive neurons mice compared to the SAL group(P = 0.025,n= 15).In the conditioned fear experiment,contextual memory test,the percentage of freezing time was reduced in the CNO group of mice compared to the SAL group(P = 0.055,n = 12).In the cue-tone memory test,the percentage of freezing time decreased in the CNO group mice(P = 0.033,n = 12).Positive h M3 Dq viral results showed an increase in the index of novel object recognition in CNO activated hippocampal D1 receptor positive neurons mice compared to the SAL group in the novel object recognition experiment(P =0.037,n = 15).In the conditioned fear experiment,the percentage of freezing time was increased but not statistically different in the CNO group of mice in the contextual memory test(P = 0.231,n = 10).In the cue-tone memory test,mice in the CNO group showed an increase in percent freeze time compared to the SAL group(P = 0.042,n = 12).The results suggest a possible involvement of hippocampal D1 receptor positive neurons in modafinil’s improvement of novel object recognition and conditioned fear memory deficits in mice after isoflurane anesthesia.5.Chemical genetic transfection of negative h M4 Di virus results showed a decreased but not statistically significant percentage of freezing time in mice with CNO inhibition of hippocampal D2 receptor positive neurons compared to the SAL group in the conditioned fear experiment,contextual memory test(P = 0.241,n = 6).In the cue-tone memory test,the percentage of freezing time was reduced but not statistically significant in the CNO group of mice(P = 0.321,n = 6).Positive h M3 Dq viral results showed that in the conditioned fear experiment,the percentage of freezing time was reduced in the CNO group compared to the SAL group in the contextual memory test(P = 0.086,n = 6).In the cuetone memory test,the percentage of freeze time increased in the CNO group compared to the SAL group(P = 0.044,n = 6).The results suggest a complex role for hippocampal D2 receptor positive neurons in modulating the memory improvement effects of modafinil.Conclusion:Modafinil accelerated the recovery of memory function in mice during the wake period after isoflurane general anesthesia,and this memory improvement effect was related to the activation of hippocampal D1 receptor positive neurons.Objective: Sevoflurane exposure during infancy and early childhood increases the risk of cognitive dysfunction in adolescence.Alterations in microglia function caused by unpredictable stressful events are widely associated with deficits in synaptic maturation and impaired brain connectivity,and lead to a range of psychiatric disorders.Previous studies have found microglia activation and release of inflammatory mediators and increased microglia phagocytosis of synaptic proteins after early sevoflurane exposure,but the molecular mechanisms underlying microglia phagocytosis are still unclear.This study was conducted to investigate the effects of early sevoflurane exposure on cognitive function in adolescence and its molecular mechanisms,and to provide a new direction to address the exact pathogenesis of sevoflurane neurotoxicity and find effective therapeutic targets.Methods: Behavioral including Morris Water Maze and conditioned fear test were used to observe the effects of sevoflurane on the cognitive function of P45 in mice after early exposure(P6,P7,P8).Immunofluorescence,3D reconstruction,Western blot and Golgi staining were used to observe the activation status of microglia,the release of inflammatory mediators and the phagocytic function of microglia.Results: In Morris Water Maze and conditioned test,the Morris Water Maze escape latency was significantly longer and the number of platform traversals was significantly lower in the sevoflurane exposed group of mice compared to the control group.The fear memory test also showed that the fear extraction time was significantly shorter in the sevoflurane exposed group.Immunofluorescence showed increased microglia activity and inflammatory mediators in the hippocampus immediately after sevoflurane exposure,and Western blot and Golgi staining showed reduced expression of synapse-associated protein PSD95 and Vglut1 in P45.Conclusion: Multiple sevoflurane exposures can cause cognitive dysfunction in mice during adolescence,possibly through microglia phagocytosis of synapses.