A Case Study Of Tissue Clearing Technique Combined With 3D Imaging In Brain Injury Diseases

Tissue clearing technology is a method developed in recent years to remove lipids,pigments and other substances that affect imaging light path in biological tissue samples by using physical and chemical means,so that tissues become transparent to the naked eye.It is mainly used in combination with 3D structure imaging of biological tissue,which provides great convenience for the study of neural structure.However,there are many Tissue clearing technologys in the field,and their color removal principles are not the same,leading to their application in different scenarios in the experiment.Moreover,the compatibility of Tissue clearing technologys and the current histological labeling methods has not been confirmed.This paper aims to match several representative Tissue clearing technologys with immunofluorescence staining and endogenous fluorescence labeling methods,conduct specific three-dimensional data analysis on different types of imaging results,form a set of optimal tissue transparency combined with fluorescence labeling scheme for different research objects,and establish three-dimensional structure imaging analysis strategies.Three Tissue clearing technologys based on different principles,PACT(passive CLATRITY technique),MACS(MXDA-based Aqueous Clearing System)and commercial Visikol-Histol,were selected.The imaging effect of tissue hyalinization staining with different antigen types was compared,and the choice between specific target protein labeling methods and different tissue hyalinization schemes was explored.In this study,we used transgenic mice with endogenous fluorescence,small molecular fluorescent markers to label specific proteins,and immunofluorescent staining to label specific antigens to achieve tissue labeling of brain slices of mice.Tissue transparency treatment was performed in the thick brain sections of mice using PACT method based on hydrogel embedding,MACS method based on m-diphenylenediamine(MXDA),and Visikiol-Histol commercial kit based on solvent transparent technology.After the transparency of the samples was completed,the samples obtained by the above three transparency methods were imaged using confocal laser scanning microscope under the objective lens of appropriate refractive index.It has been observed that taking 150 μm thick slices of mouse brain as an example,three transparent methods make the tissue transparent the longest time PACT,Visiko-Histol followed,MACS the shortest time.After experiments with different endogenous fluorescence samples,it was found that PACT and MACS could complete tissue transparency while preserving endogenous fluorescence,while Visikol-Histol kit had serious fluorescence quenching phenomenon after tissue transparency.In the experiment of labeling vascular endothelial cells with fluorescein histolectins,the three methods all showed good effect,but the Viskol-Histol effect was the best.In the experiment of immunofluorescence staining on karyotype antigen and cytoplasmic antigen,the three methods also showed good labeling effect.PACT and MACS methods can be used in immunofluorescence staining of cell membrane antigens.By comparison,we determined the optimal transparency scheme for different antigen types and labeling methods with a certain thickness of mouse brain slices as research objects.On this basis,taking chronic hypoxia model of newborn mice as an example,three-dimensional structural imaging was conducted on the neuromorphological characteristics of mice after hypoxia by using tissue transparency technology,combined with appropriate target protein labeling methods,and threedimensional structural data analysis software Imaris.Microglia were labeled with Iba1 immunofluorescence staining and combined with Imaris surface tool and Filament tool to reconstruct the 3D structure of microglia and filament tool to map its protrusion morphology.The results of protrusion branching were obtained by sholl analysis.Oligodendrocytes were labeled by OLIG2 immunofluorescence staining,and mature oligodendrocytes were labeled by CC1.The spatial distribution and density of oligodendrocytes were analyzed by Imaris surface tool and Spot tool.PV neurons were labeled by AAV neurotropic virus and excitatory postsynaptic protein was labeled by PSD95,and myelin structure was labeled by MBP immunofluorescence staining.The synaptic afference of PV neurons was reconstructed by Spot tool,and the 3D myelin morphology of PV neuron axons was reconstructed by Imaris Surface tool.The synaptic distribution and number of PV neuron cell body and axon,as well as the volume of axon myelination were analyzed and studied by Matlab software.The results showed that the hypoxia model of neonatal mice caused morphological changes such as increased branching of microglia cells in early development,decreased number of mature oligodendrocytes in late development,decreased synaptic influx of PV neurons,and decreased myelination level through chronic hypoxia injury.To sum up,due to different principles,the three transparency methods show different effects on different types of target protein labeling methods and antigen categories,so optimizing the combination of antigen labeling scheme and Tissue clearing technology is crucial for the successful realization of three-dimensional imaging of brain tissue cell morphology within a certain thickness.In addition,through the imaging of the three-dimensional structure of the brain in pathological conditions,the quantitative analysis of the neuromorphologic structural changes caused by specific pathological injuries is realized,which provides important research ideas and methods for researchers in related fields.