Biomarkers And Mechanisms Of Early Traumatic Axonal Injury In The Brainstem Based On Proteomics And Untargeted Metabolomics

BACKGROUNDAs one of the primary injuries in traffic accidents traumatic axonal injury(TAI)can occur when the head is subjected to rapid hyperflexion/extension or rotational movements following an external force.When TAI occurs in the brainstem,it often affects the respiratory and/or cardiovascular centers and even causes irreversible respiratory and circulatory dysfunction,ultimately leading to rapid death.However,few or no pathological changes can be detected by gross observation and microscopy.Therefore,it is generally accepted that TAI is one of the main types of primary brainstem injury(PBSI)and that PBSI-related deaths are a major challenge for forensic identification.However,studies have not yet completely revealed the molecular mechanisms and biomarkers of TAI,and little attention has been paid to the important pathological alteration of brainstem TAI,or to the short survival time after injury,and there is a lack of in-depth studies of diagnostic biomarkers and early changes in the microenvironment.OBJECTIVEBy establishing a brainstem TAI model in rat,the biomarkers of early brainstem TAI and the mechanisms of injury were investigated at the proteomic and metabolomic levels,which will provide a theoretical basis for forensic identification.METHODS(1)A modified impact acceleration injury model was used to establish a brainstem TAI model in Sprague-Dawley rats,and the model was evaluated by vital sign measurements,HE staining,silver-plating staining,β-amyloid precursor protein immunohistochemical staining,and pathological features in the brainstem compared to PBSI death cases.(2)TMT combined with LC-MS/MS was used to analyse the differential expression proteins(DEPs)in brainstem tissues from 0.5 h after TAI.The biological functions of DEPs and potential molecular mechanisms in the hyperacute phase of TAI were analysed by bioinformatics techniques,and candidate biomarkers were validated using western blotting and immunohistochemistry on brainstem tissues from animal models and human.(3)A UPLC/TripleTOF-MS system was used to analyse metabolite changes in brainstem tissues from 0.5 h after TAI.,to screen for diffential metabolites,and to analyse metabolic pathways.RESULTS Chapter One:Construction and evaluation of a traumatic axonal injury model of the brainstem in rats(1)Immediately after the injury,the rats were in various degrees of unconscious with disturbances in vital signs.(2)Diffuse axonal injury to the brainstem was found in rats up to 7 days post-injury or at death.(3)Rats have chronological axonal pathological features after injury similar to those seen in cases of PBSI death.Chapter Two:Proteomics of early traumatic axonal injury in the brainstem(1)TMT-based proteomics identified 65 DEPs,and bioinformatics analysis indicated that the hyperacute phase of TAI involves multiple stages of biological processes including inflammation,oxidative stress,energy metabolism,neuronal excitotoxicity and apoptosis.(2)CBR1,EPHX2 and CYP2U1 were found to be significantly expressed in brainstem tissue 30 min-7 days after TAI in both animal models and humans.Chapter Three:Untargeted metabolomics of early traumatic axonal injury in the brainstem(1)In the hyperacute phase of TAI,significant changes in the levels of lauryl diethanolamide,monoethylhexyl phthalate,PE(20:5(5Z,8Z,11Z,14Z,17Z)/24:1(15Z)),PC(16:0/16:1(9Z)),(2E,6Z)-dodecadienoic acid methyl ester and altretamine.(2)In the hyperacute phase of TAI,the retrograde endocannabinoid signaling,autophagy-animal,arachidonic acid metabolism,glycosylphosphatidylinositol(GPI)-anchored biosynthesis,glycerophospholipid metabolism and glycerolipid metabolism pathways were activated.CONCLUSION(1)The successful establishment of brainstem TAI model in rat was confirmed by assessing both functional and structural aspects.(2)CBR1,EPHX2 and CYP2U1 can be used as biomarkers of early TAI in brainstem.(3)The molecular mechanisms underlying the hyperacute phase of brainstem TAI are complex and include the involvement of multiple pathways such as disturbances in lipid metabolism,arachidonic acid metabolism inflammation,oxidative stress,energy metabolism,neuronal excitotoxicity and apoptosis,etc.