Study On The Correlation Between The Glutamate Metabolism And Microcirculatory Perfusion Following Hypoxic-ischemic Brain Damage

Objective:Neonatal hypoxic-ischemic encephalopathy?HIE?is the most common cause of permanent dysneuria and death.The occurrence of HIE is a result of the interactions and influences of multiple factors that contribute to an abnormal pathological environment in the brain.The excitotoxicity caused by the massive accumulation of glutamate after hypoxia-ischemia?HI?is the central link for HIE.In this study,we measured glutamate-related metabolites in a neonatal piglet model of hypoxic-ischemic brain damage?HIBD?and analyzed the changes in EAAT2 and AMPAR subunit GluR2protein levels by histological analysis and to explore the role of glutamate in HIBD.In addition to the excitotoxicity of glutamate,hemodynamic disorders of the brain are also a risk factor for HIBD.In this study,microcirculatory perfusion was acquired by intravoxel incoherent motion?IVIM?scanning with quantitative parameters;thereafter,interactive changes and possible interaction mechanisms were further analyzed.Methods:1.Altered glutamatergic activity in a piglet model of hypoxic ischemic brain damage using proton magnetic resonance spectroscopy.Twenty-five newborn Yorkshire piglets?P3-5?were selected and then randomly assigned to the normal control group?n=5?and the model group subjected to hypoxia-ischemia?HI??n=20?.The HIBD model group was further allocated into 4 subgroups?n=5 per group?according to the following time points after HI:0-6,8-12,24-30,and 48-72 h.Spectral raw data obtained by¹H-MRS scanning were quantitatively analyzed using linear combination model software?LCModel,Version 6.3-1B,S.W.Provencher?.In this study,the absolute concentrations of Glu,Gln,Glx,NAA,Cho,and Cr in the basal ganglia were analyzed.Additionally,we measured the ratios of Glu/Cr,Gln/Cr,Glx/Cr,NAA/Cr,and Cho/Cr?namely the relative concentration?which were also be provide by LCModel software.We detected changes in protein levels of EAAT2 and GluR2 by immunohistochemical staining.Correlations between spectral data and pathological results were analyzed using Spearman rank correlation analysis.2.Early changes in glutamate metabolism and perfusion in basal gangalia following hypoxia-ischemia in neonatal piglets:a multi-sequence 3.0T MR study.Twenty-five newborn male or female Yorkshire piglets were randomly selected from the Laboratory Animal Center of Shengjing Hospital of China Medical University.Piglets were subjected to common carotid artery occlusions with concomitant drop in inhaled O₂ to 6%for 40 minutes.Before an operation,and 0–6 h,8–12 h,24–30 h and 48–60 h after HI insult,animals were subjected to a ¹H-MRS scan and then changes in glutamate metabolism in damaged basal ganglia were analyzed using LCModel software.Meanwhile,IVIM scan was performed.IVIM raw data were transferred to post-processing software?home-made Matlab script;Philips Healthcare,China?,and then post-processed with a bi-exponential model to give pseudo-color images of diffusion coefficient D,pseudo-diffusion coefficient D*and perfusion fraction f.The latter two parameters D*and f can provide information on microcirculatory perfusion.Correlations between metabolism involving the release of glutamate and changes in IVIM-derived perfusion parameters were further analyzed.Results:1.Altered glutamatergic activity in a piglet model of hypoxic ischemic brain damage using proton magnetic resonance spectroscopy.After HI,there was a sharp increase in Glu level in the basal ganglia at 0–6 h,followed by a transient decrease at 8–12 h,to a level that was still higher than that in the control group;thereafter,it increased again,demonstrating a?two-phase?change.NAA/Cr ratios gradually declined over time after HI injury,and was negatively correlated with the severity of basal ganglia damage?R_s=-0.456,P=0.022?.Cho level increased after HI and was correlated positively with the severity of basal ganglia damage?the absolute Cho concentration,R_s=0.703,P<0.001;Cho/Cr,R_s=0.638,P=0.001?.While no differences in the absolute concentrations of Gln,NAA or Cr were observed between the different groups.Immunohistochemical staining indicated that EAAT2 expression in basal ganglia transiently declined during the early stage after HI,then significantly increased at 8-12h,and thereafter declined again.Changes in EAAT2 protein level were significantly and negatively correlated with those of Glu level?including the absolute Glu concentration and Glu/Cr ratio??the absolute Glu concentration,Rs=-0.662,P<0.001;Glu/Cr,Rs=-0.664,P<0.001?.GluR2 protein level in basal ganglia significantly declined at different time points compared with the control group?P<0.05?.The GluR2 expression tended to decline over time of HI,and was negatively correlated with the pathological scores severity?Rs=-0.876,P<0.001?.The GluR2 expression also was correlated with the the absolute Glu concentration and Glu/Cr ratio?Rs=-0.797,P<0.001;Rs=-0.567,P=0.003?.2.Early changes in glutamate metabolism and perfusion in basal gangalia following hypoxia-ischemia in neonatal piglets:a multi-sequence 3.0T MR study.After HI,the perfusion fraction f,trended to recover gradually at<12 h after HI,but was still lower than that for the control group;thereafter,it further decreased and displayed an opposing trend to the changing trend in the Glu concentration.Another IVIM-derived perfusion parameter——pseudo-diffusion coefficient D*,was not statistically significant.In addition,this study also demonstrated that the diffusion coefficient D,a parameter reflecting the true diffusion motion of water molecules,was markedly decreased at 0–6 h after HI and then gradually recovered over time.Glu showed a significant negative correlation with f?Rs=-0.643,P=0.001?;Glx also showed a negative correlation with f?Rs=-0.478,P=0.016?;However,no significant correlation was observed between the Gln concentration and f.In addition,D*did not correlate with the concentration of Glu,Gln or Glx.Conclusions:In conclusion,this study confirmed that ¹H-MRS is a powerful non-invasive technique in detecting the metabolites in vivo.Our results confirmed that Glu level tended to elevated after HI,and then EAAT2 and AMPAR subunit GluR2 were activated.In addition,changes in Glu levels were inversely correlated with changes in EAAT2 and GluR2 expression after HI,indicating that EAAT2 and GluR2 may have important roles in regulating the release of Glu.Therefore,¹H-MRS can be of use in estimating the activation status of EAAT2 and GluR2 in vivo.NAA/Cr,Cho/Cr and Cho could be considered as the indicators for assessing the degree of brain injury and poor prognosis.Future studies with a larger sample size need to be conducted to validate this view.In addition,we demonstrated the dynamic changes of microcirculatory perfusion in HI brain tissues using IVIM quantitative parameters,and we found that f could be used as a biomarker to evaluate microcirculatory perfusion and reflect the hemodynamic changes of the brain after HI.Our data highlight the potential of combining changes in Glu concentration and f to explore the close relationship between cerebral dysmetabolism and microcirculatory disturbance after HI.