Three-Dimensional Reconstruction Of Cholinergic Neural System In Transgenic Mice With Alzheimer's Disease

Background: Cholinergic neural system is fundamental for many advanced brain functions such as learning and memory.Cholinergic neurons,especially in the basal forebrain,are more vulnerable to damage in some neurodegenerative diseases,such as Alzheimer's disease.In this study,five familial AD mutations(5×FAD)were used to establish Alzheimer's disease model mice to explore the effect of Alzheimer's disease on the injury of cholinergic neural system.Although cholinergic system has been widely studied in mice,the defects of cholinergic system in AD mice model and their changes in the occurrence and development of neurological diseases have not been systematically demonstrated in the whole brain.The relationship between the distribution and projection of cholinergic neural system in the specific lesion location remain unclear.Therefore,we used three-dimensional reconstruction technique combined with immunohistochemical method to provide a detailed overview of cholinergic neurons in the brain of AD mice,which will be helpful for understanding the pathological changes in the AD brain.Objective: To observe the difference of cholinergic neuron deletion and nerve fiber injury between 5 × FAD mice and control C57BL/6 mice with the same age at different brain regions and different nuclei by three-dimensional brain reconstruction technique.To explore the detailed situation of cholinergic neurons in the mice brain under the pathological condition of Alzheimer's disease.This may help us directly understand the area and severity of the lesion in the AD brain.Methods: After perfusion,the whole brain tissues of AD and control mice were taken and the sequential coronal sections were collected by frozen section technique.The whole mice brain imaging was obtained by confocal scanning microscope.High definition(high-definition,HD)three-dimensional reconstruction of the whole mouse brain was carried out by using three-dimensional reconstruction technology combined with immunohistochemical technique(3D reconstruction combined with immunohistochemistry,3D-IHC).The injury of cholinergic neurons and nerve fibers in AD mice and healthy control mice were counted and analyzed by Stereo Investigator 8.0 software.Results: 1.In aged 5 × FAD mice,cholinergic nerve fibers were seriously damaged in the middle of prefrontal lobe,lateral side of entorhinal cortex,dorsal hippocampus and amygdala,especially in amygdala area,cholinergic nerve fibers were significantly decreased(t test,P < 0.001).2.The number of cholinergic neurons in the basal forebrain of 5 × FAD mice were significantly lower than that of C57/BL6 mice at 9 months of age(t test,P < 0.05).3.The caudate-putamen region of aging C57/BL6 mice(20 months old)was higher than that of adult mice(3 months old).The number of multipolar cholinergic neurons decreased by about 30%(t test,P < 0.001),and the number of cholinergic neurons in Ch3 region(horizontal limb nucleus of the diagonal band of Broca,HDB,Ch3)decreased by 32%(t test,P < 0.001).Conclusion: 1.It was found that the injury of cholinergic nerve fibers occurred earlier than the deletion of cholinergic neurons in 5 × FAD mice.And the injury of cholinergic nerve fibers occurred in amygdala,cortex,hippocampus and basal forebrain.2.Basal forebrain was the first area where cholinergic neuron body injury was observed at9 months of age in 5 × FAD mice,while this phenomenon was first observed in basal forebrain of C57BL/6 control mice at 15 months of age.3.Multipolar cholinergic neurons are more likely to be damaged in aged mice.