| Ischemic stroke has been a major disease that gravely threatens human health.Ischemic penumbra is the key area in acute stroke salvage.However,methods for accurately detection of penumbra through metabolic changes have not been developed.CZ-7,an anti-stroke drug candidate,is a derivative of Claulansine F,whose ADME process,in vivo metabolites and mechanism in brain sub-structures still remain unclear.Mass spectrometry imaging(MSI)is a novel technology for pharmacometabolomic analysis.When MSI is used for the detection of highly heterogeneous tissues,like ischemic brain,metabolomic changes of ischemic injury are always interfered by brain anatomical heterogeneity,making it difficult to find out metabolic signatures for stroke mechanism study and drug efficacy monitoring.Therefore,it is of great significance to develop data processing methods for heterogeneous tissue MSI data,which may help to discriminate regions of interest that are functional-related,and further discriminate,annotate,and interpret of metabolic signatures.In this research,we develop an AFADESI-MSI data-driven method for precisely discrimination,localization and annotation of metabolomic signatures in potentially functional-related regions.The method is applied to ischemic stroke brain,discriminating regions of ischemic core,penumbra,and normal tissue,which is further confirmed by histological staining and transmission electron microscopy analysis.In addition,metabolomics analysis is used for spatial localization and annotation of metabolic signatures in ischemic core,penumbra and healthy tissues.Colocalization of metabolic biomarkers in samples with different ischemic time gives information for dynamic changes of region location.The method is further used in ischemic brain treatment with CZ-7,annotating metabolic features in different regions,whose location and intensity changes can help for drug mechanism discovery.In addition,in vivo metabolites of CZ-7 were analyzed and identified by LC-MS-IDA-MS/MS combined with quality loss filtration method.AFADESI-MSI is applied to rat brain and wholebody tissue sections for in situ drug distribution and pharmacokinetics,which gives a new insight into drug effect evaluation.This dissertation includes three sections.The main contents are as follows:1.Method for spatial discrimination of metabolic signatures in brainMetabolomic signatures of brain injured areas are always interfered by brain anatomical heterogeneity,which hinders accuracy of unsupervised pattern recognition.In this study,method for MSI data driven based localization of metabolic signature was built through pre-selection of metabolites in clustered and targeted pathways,which ignored metabolic difference of anatomical structures and highlighted that of injury discrimination.The method is successfully used in ischemic stroke brain to localize regions of ischemic core,penumbra and healthy tissue regions.The unique metabolic profile of penumbra is further confirmed by supervised multivariate statistical analysis.In addition,DHA is selected as a biomarker to quantify ischemic injury with cutoff values of 29.9 pg/mm2 for 64.8 pg/mm2 for penumbra and healthy tissue respectively.Comparted to traditional TTC staining method,the proposed method takes advantages in high sensitivity and accuracy,with only 15 μm section used.We highlighted our method as a new approach for stroke model research,injury assessment and drug candidate evaluation.2.Spatial localization and annotation of metabolic signatures in ischemic brainSpatial localization and annotation of metabolomics in ischemic core,penumbra and healthy tissue was done by MSI metabolomics based on the proposed method to discriminate injured regions.The result shows alanine,aspirate and glutamate metabolism,sphingolipid metabolism,arginine and proline metabolism,glycerophospholipid metabolism,taurine and hypotaurine metabolism,valine,leucine and isoleucine degradation,citrate cycle(TCA cycle)and cysteine and methionine metabolism were significantly changed in ischemic core,which are related to the mechanisms of energy metabolism,oxidative stress,sodium and potassium balance and inflammatory response after ischemia.Metabolic enzyme activity was reconstructed with reactive pair of intensity ratio by substrate and product ions.ACO2 and LTA4 hydrolase were found upregulated in ischemic core,which was further verified by IHC.Besides,we found phosphatidylinositol signaling system,pentose phosphate pathway,glycolysis,inositol phosphate metabolism,starch and sucrose metabolism and fructose and mannose metabolism were significantly changed in penumbra.We also annotated dynamic changes of different injury regions by specific biomarker location and intensity.This study,for the first time,reveals metabolomic disorders of penumbra with normal tissue and ischemic core from both metabolite and enzyme level,providing new clues for the study of ischemic stroke.3.In vivo pharmaceutical analysis and metabolic localization and annotation of anti-stroke candidate CZ-7Liquid chromatography-mass spectrometry(LC-MS)and AFADESI-MSI technologies were used for in vivo CZ-7 analysis.In addition,the proposed localization and annotation of metabolome method is further developed for drug effect evaluation.In vivo metabolites of CZ-7 were analyzed and identified by LC-MS-IDA-MS/MS combined with quality deficit filtration method.A total of 17 metabolic forms of CZ-7 were found in vivo.After identification of fragmentation in MS/MS spectrum,3 metabolites of CZ-7 structure were identified.AFADESI-MSI analysis was performed on MCAO-CZ-7 treated rat whole body and brain slides to annotate CZ-7 distribution.The result shows CZ-7 was detected in most organs,among which kidney,liver and lung are major metabolized and excreted organs.In brain,CZ-7 is widespread and accumulated in cortex,ventricle and hypothalamus.In situ pharmacokinetic analysis of the ischemic core,penumbra and healthy regions shows the content of CZ-7 remains constant from 30 min to 6 h after treatment,and lasts longer in ischemic regions.With CZ-7 treatment,relative area of ischemic core is significantly decreased while the area of penumbra is significantly increased through Wilcoxon’s sign rank pairwise comparison test.In addition,energy-related metabolites were recovered,which illustrates therapeutic effect of CZ-7.In situ pharmacometabolomics provides clues for CZ-7 working as an effective antioxidant by reducing ROS production,activating endogenous anti-oxidative stress system and antagonizing oxidative stress damage to protect neurons. |
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