Experimental Study On Detecting Diffuse Axonal Injury With FTIR Mapping

Background:In the fields of brain science,neurobiology,neuropathology,clinical medicine and forensic pathology,the diagnosis of diffuse axonal injury(DAI)is a research hotspot.For cases in which the survival time of DAI is short,it is necessary to find the cause early,objectively characterize the lesion,and accurately analyze the pathology.The aim of this study is to explore the specific spectral indices of DAI detected by Fourier transform infrared spectroscopy mapping(FTIR-mapping).It is also aimed at exploring the pathological changes of DAI from the perspective of the chemical bond infrared spectrum.This paper tries to establish a more accurate DAI forensic pathology detection method.In the current practice,especially in forensic identification,the DAI is mainly identified by gross specimens,tissue sections and damage lesions.As the pathological changes of DAI are often complicated in morphology,this raises high technical requirements for accurate judgment of lesions and injury.On the one hand,tissue section production and dyeing require high quality.It is necessary to observe the morphological changes of axons after DAI,or detect changes in the expression of some biomarkers caused by DAI,which mainly use detection method of immuneohistochemical staining,silver staining at the subcellular level.The high accuracy of these methods depends on scientific sampling of the samples,the standard production of the slices and the fine operation of the dyeing process,which requires an experienced technician to complete.On the other hand,it requires high level of image analysis under the microscope.It is a subjective histopathological examination to analyze the staining section and the positive staining intensity under the microscope.It is difficult to make an accurate identification in minor changes for an inexperienced researcher.In addition,there are shortcomings of DAI diagnosis in traditional methods.For example,after immunohistochemical,a variety of biomarkers staining may show axotomy,but it fails to indicate whether the system is primary or secondary,and if the axonal cord has not yet apparently changed,the marker cannot be positively expressed,so it doesn't has diagnostic value.Each immunohistochemical staining only analyzes a single biomarker,and fails to diagnosed simultaneously with a marker system.The axonal injury of acute DAI cannot be reflected in silver staining method.Therefore,this study plans to find a convenient,sensitive and repeatable DAI detection method at the molecular level used by animal experiments and infrared spectroscopy to break the limitations of DAIs that rely on axonal morphological diagnosis.Brain cells mainly include neurons and glial cells which are composed of cell membrane,cytoplasm,and cell nucleus.The main chemical components are proteins,lipids,glycogen,and nucleic acids.The content,configuration,and conformation of these chemical components behave differently in different tissue cells.In the course of the development of many diseases and injuries,such components will undergo different changes and may show differences in the infrared spectrum.Like nuclear magnetic resonance spectroscopy,mass spectrometry and ultraviolet spectroscopy,infrared spectroscopy,a kind of molecular spectroscopy,becomes a powerful tool for determining molecular composition and structure.Fourier transform infrared spectroscopy is the infrared spectrum formed by Fourier transform of interferograms,which can be used for qualitative analysis,quantitative analysis,and analysis of unknown substances.Based on the peak position,peak shape,and intensity of the absorption peak in the infrared spectrum of the unknown,it is possible to determine which groups are contained in the unknown molecule and to infer the chemical structure of the unknown.Infrared spectroscopy technology is non-destructive testing featured by simple sample preparation,convenient operation,high sensitivity,high luminous flux,low noise,fast measurement speed,high wave number accuracy,wide frequency measurement range,low operating costs,good repeatability,and it has wide range of applications.The use of infrared spectroscopy accessories such as infrared microscopes can analyze nano-,microgram-level,even nano-scale samples,It is widely applied in many fields such as chemistry,pharmaceuticals,jewelry appraisal,and forensic science.At present,there are few reports on the use of FTIR technology to detect DAI molecular changes,and the study of surface mapping techniques to determine the molecular characteristics of DAI has only been reported on the amide II band of proteins.In this study,the molecular characteristics of DAI are studied by using FTIR,infrared microscopy,and surface imaging technology,which expands the scope of research on the molecular level of brain tissue sample.It also reflects the distribution and changes of macromolecules such as proteins,lipids,and nucleic acids with chemical images.This study is an innovation of chemical technology,forensic pathology and neuroscience.Objective:To establish a rat DAI model to observe the post-injury behavioral responses in rats and to study the characteristics of ?-APP immunohistochemical staining in brain tissue of DAI rats.To find specific infrared spectra according to the chemical composition of white matter in DAI rats detected by FTIR microscope based on the study of changes of DAI lesions in rats after brain injury.To study the correspondence between chemical images and histopathology and to establish a foundation for the final application of FTIR to diagnose DAI at the molecular level based on the results of infrared spectroscopy.Method: Experimental materials and instrumentsSD rats;A weight-drop hitting device;Fourier transform infrared spectrometer(NicoletTM 7500-II);Infrared Microscope(Nicolet continuum microscope);Infrared Spectroscopy Workstation(OMNIC 6.0);Virtual Microscopic Imaging System(Dotslide 2.1);Dewatering Machine,Embedding machine,Slicer,Exhibition Machine,Roasting machine,et al;Hematoxylin,eosin,natural gum,xylene,ethanol,ammonia,et al.Research methods and experimental methodsThe first part of the experiment: to establish an axonal injury model.A total of 24 male and female SD rats were selected and randomly divided into 2 groups according to the random sampling principle,12 in each group.This aims to establish a Marmarou injury model.The rats were killed 24 hours after the injury and the brains were taken directly after the sham operation.The second part of the experiment: To detect brain tissue and analyze data by infrared spectroscopy,biomarker detection.(1)To scan samples of brain tissue by FTIR infrared spectrum and to draw the mapping image:(2)To select DAI-specific infrared spectrum parameters after staining brain slices with ?-APP,confirming the axonal injury area and extracting spectral information from the damaged area.Result:After FTIR-mapping,the amide A protein secondary amine NH stretching vibration and the collagen triple helix structure in the experimental group coincided with the diffuse axonal damage area verified by ?-APP.There was no significant difference between the absorbance of the symmetric stretching vibration of the phospholipid lipid PO2 and the validation group.The chemical images drawn by the FTIR-mapping surface scanning imaging technique can basically reflect the distribution characteristics of the DAI macromolecules,and are basically consistent with the diffuse axonal injury areas verified by ?-APP.Conclusion:Fourier Transform Infrared(FT-IR)microscopy is a powerful technique for obtaining chemical information from tissue sections.Infrared spectroscopy chemical images can visually demonstrate the corresponding relationship with histopathology.The application of FTIR-mapping technology can reveal the molecular characteristics of DAI,analyze the pathological changes of biomacromolecules,and interpret the damage and repair process of DAI from the perspective of the formation of molecular chemical bonds.From the morphological point of view,the location of DAI lesions was diagnosed,and the extent of DAI damage in brain tissue was judged according to the changes of infrared absorbance values.This technique is expected to be applied to forensic pathological diagnosis of other diseases and injuries.