Expression Of MMP-9 And Changes In Synaptic Ultrastructure In Different Brain Regions Of Rats In Chronic Intermittent Alcohol Exposure Model

Objective:In the present study,we aimed to identify the role of MMP-9 in the rat medial prefrontal cortex,hippocampus and amygdala during chronic intermittent alcohol exposure process and the changes of synaptic ultrastructure during this process.Using both real-time polymerase chain reaction(PCR)and western blot analyses,we examined the expression of MMP-9 in the rat model of chronic intermittent ethanol(CIE)exposure.Meanwhile,using electron microscopy measure,we examined ultrastructure changes of synapses in these rat brain regions.And Liquid chromatography tandem mass spectrometry(LC-MS/MS)to reveal other MMP-9-associated proteins that may function during the process.Methods:Seven-week-old male rats were randomly allocated into three groups:(1)Ethanol(EtOH): Animals were administered 25 % EtOH solution(4 g/kg)via gavage;(2)Maltose: Instead of EtOH gavaging,animals were treated with maltose solution(7 g/kg)at a dose that was isocaloric to that used in the EtOH group;and(3)Chow: Animals were allowed free access to food and water without gavaging.The gavaging pattern was as follows: Each set of two consecutive EtOH exposure days was followed by a 2-consecutive-day temporary withdrawal period,resulting in a total of seven on/off cycles between Day 0 and Day 28.The same schedule was used for Maltose group animals.Animals were sacrificed at Day 0,Day 2,Day 4,Day 14,Day 16,Day 26,and Day 28 to collect tissues from hippocampus,mPFC,amgydala and serum.Samples were used to detect mRNA and protein expression level,blood EtOH concentration and IP-MS.(1)Blood ethanol concentration(BEC): Blood samples were collected from rats at the tip of the tail(0.2 ml)and then centrifuged to separate serum in the EtOH group.The ethanol concentrations were immediately measured using gas chromatography.(2)Real-time polymerase chain reaction(RT-PCR): We used RT-PCR to detect the relative expression level of MMP-9 mRNA in the rat medial prefrontal cortex,hippocampus and amygdala at different stages during chronic intermittent alcohol exposure process.(3)Western blot experiment: We used western blot to detect the relative expression level of MMP-9 protein in the rat medial prefrontal cortex,hippocampus and amygdala at different stages during chronic intermittent alcohol exposure process.(4)Immunofluorescence: We used immunofluorescence to detect the level and the location of MMP-9 in the rat hippocampus,cortex and amygdala after chronic alcohol intermittent exposure.(5)Electron microscopy: We used electron microscopy to examine ultrastructure changes in synapses in rat brain.(6)IP-MS Assay: We used immunoprecipitation to reveal other MMP-9-associated proteins that may function during chronic intermittent alcohol exposure process.Results:(1)To characterize our CIE model,BECs were measured immediately following each treatment(i.e.,after the initial gavage(Day 2),middle period(Day 14),and final period(Day 26).BECs were significantly higher in treated rats than in untreated rats.Our analysis revealed a significant main effect of ethanol treatment(P<0.01).Throughout the treatment period,all animals gained weight,and there was no significant effect of CIE treatment on animal weight.(2)The level of MMP-9 mRNA expression at different stage,compared with the control group,the expression of MMP-9 mRNA increased in the hippocampal at Day 2(P<0.01)?Day 4(P<0.01)?Day 14(P<0.01)?Day 16(P<0.05)?Day 26(P<0.01)?Day 28(P<0.01).In the prefrontal cortex,the expression level of MMP-9 mRNA also appeared similarly from Day 2 to Day 28,in Day 2(P<0.05),Day 4(P<0.05),Day 14(P<0.05),Day 16(P<0.05),Day 26(P<0.05),Day 28(P<0.05).In the amygdala,the expression of MMP-9 mRNA was not significantly different from the control group.There was no significant difference in the expression levels of MMP-9 mRNA in the maltose group at different time points in each brain region.(3)Western blot showed that,in the hippocampus,MMP-9 protein expression,including total MMP-9 and active MMP-9,was significantly increased compared with Day 0(P<0.05).The gradual increase continued until Day 28(P<0.01).In the medial prefrontal cortex,the protein expression of total MMP-9 was significantly increased(P<0.05)at Day 14,Day 16,Day 26 and Day 28.The protein expression of active-MMP9 was significantly increased(P<0.05)at Day 14,Day 26,and Day 28.In the amygdala,the expression of MMP-9 protein was not significantly different from the control group.There was no significant difference in the expression levels of MMP-9 protein in the maltose group at different time points in rat brain regions.(4)Electron microscopy showed that,compared with the Day 0,in the late stage of chronic intermittent ethanol exposure to alcohol,the thickness of the postsynaptic membrane was increased in the three brain regions of the hippocampus,cortex and amygdala.The curvature of the interface increases,and the gap between the membranes before and after the synapse increases slightly.(6)Immunoprecipitation analyses revealed the MMP-9-associated proteins,including Itgb1,Src,Eef1a2,Tubulin,Actin,and Histone H2 B during intermittent ethanol exposure process.Conclusion:(1)The expression of MMP-9 was significantly increased in the rat hippocampus and medial prefrontal cortex during intermittent ethanol exposure process,while in amygdala there was no significant differences.(2)Intermittent ethanol exposure increased synaptic interface curvature,and the thickness of postsynaptic density(PSD)and the width of the synaptic cleft was also increased.(3)Several MMP-9-related proteins during intermittent ethanol exposure process were involved,which providing an experimental basis for further mechanistic studies.