| Mesencephalic Astrocyte Derived Neurotrophic Factor（MANF）,a kind of endoplasmic reticulum stress protein,is also a new kind of conservative Neurotrophic Factor,and belongs to the neurotrophlic factors（NTFS）family.MANF not only has neuroprotective effects on neurons and non-nerve cells but also retains functions such as promoting cell survival,participating in inflammation,immune regulation,and inhibiting cell proliferation.MANF was widely expressed in the nervous system such as the hypothalamus,cerebral cortex,hippocampus,cerebellum,caudate nucleus,and non-neuronal tissues such as liver,heart,eyes,blood,saliva,bones,cartilage,testis,spleen and pancreas.In recent years,important progress has been made in the study of MANF in brain-related diseases.So far,the distribution of MANF in the brain has not been systematically studied.In this study,we aimed at observing the distribution of MANF within the brain as a whole and comparing it across the species in human,mouse and rat.The immunohistochemistry technique was applied to investigate the expression of MANF-immunoreactivity（ir）in human,mouse,and rat brain.Objective:The purpose of this study was to observe the distribution of MANF in human,mouse and rat brain and compare it across the species with immunohistochemistry technique.Methods:Human,mouse and rat brain samples were obtained.Human brian samples without brain diseases were applied from Human Brain Tissue Resource Center,ranging in age from 39 to 82 years old.Dissection was performed within 6 hours after death,the skull was opened,the whole brain was removed,weighed,and the brain was evaluated and observed whether there are cerebrovascular abnormalities,brain atrophy,substantia nigra lesions and other disorders.Then fixed with formaldehyde solution.After that,the tissue was dehydrated,paraffin embedded and sectioned;After abdominal anesthesia,SD rats and C57 mice were subjected to cardiac perfusion,and the brains were removed for dehydration,paraffin embedding and section.The expression and distribution of MANF were studied by immunohistochemistry technique.Results:1.The expression of MANF protein in human brainThe expression of MANF protein was found in all human brain regions we concerned in this study.However,the staining of MANF varied in different brain regions.There was no significant difference in the expression level of MANF in different cerebral cortex regions and subcortical region of human brain.In the brainstem and cerebellum,such as red nucleus,the medulla oblongata,cerebellar cortex,dentate nucleus showed positive staining.Some nerve fiber staining and cell staining were shown in the red nucleus.However,most nerve fiber staining and very few cell staining were observed in the medulla oblongata.Strong positive staining was shown in the substantia nigra.The staining intensity was significantly higher in the substantia nigra than that of red nucleus,medulla oblongata and cerebellum regions.Overall,the expression level of MANF in the cortex was significantly higher than that in the subcortical regions,brainstem and cerebellum.2.The expression of MANF protein in mouse brainThe expression level of MANF protein was significantly higher than that of other brain regions except the hypothalamus in mouse brain.In subcortical areas of striatum,the staining was mostly distributed in nerve fibers,but rarely in cells,and its expression level was significantly lower than that of other subcortical areas.The expression level of MANF in the hypothalamus was significantly higher than that in the hippocampus;the thalamus and the striatum;Weakly positive staining of MANF was found in the cerebellar cortex,and its expression level was significantly lower than that in the cortex,hippocampus and hypothalamus.3.The expression of MANF protein in rat brainThe expression of MANF protein in SD rat brain was investigated.The results were similar to those reported.The expression level was significantly higher than that of other brain regions.In subcortical region,such as the striatum,similar to mice,MANF staining was mostly distributed in nerve fibers,but rarely in cells,and its expression level was significantly lower than that of other groups.The expression level of MANF protein in cerebellum was significantly lower than that in the cortex,hippocampus and hypothalamus.Conclusion:In this study,the expression and distribution of MANF in human,mouse and rat brain were studied by using immunohistochemistry technique.We found the distribution pattern of MANF was similar across the species,however,within a species,MANF expression levels varied in different brain regions in human,mouse and rat.Tissue Clearing technique refers to the application of hydrosoluble organic solvent or hydrophilic reagent to transparent treatment of fixed tissues by immersion,electrophoresis or perfusion and then the high refractive index medium was used to match the refractive index of the tissue to reduce light scattering and make the tissue optically transparent,thus increasing the imaging depth and image contrast.The principle was that biological tissue has strong light scattering and refraction ability due to the existence of lipid substances located in the membrane structure,resulting in light can not penetrate the tissue,presenting an opaque state.By removing the lipid structure or changing the refractive index of the tissue,light scattering is reduced,thus making the tissue transparent.It can observe the internal microstructure without destroying the integrity of the organ,avoid the loss of information between sections,and truly realize the three-dimensional imaging of cells and other structures.Hydrosoluble organic solvent or hydrophilic reagent was applied,the fixed organization through soaking,transparent,in the form of electrophoresis or perfusion and then apply high refractive index matching of medium organization in order to reduce the light scattering,make the organization achieve optical transparent and,in turn,increase the image contrast and imaging depth,the current organization transparent technology and methods are widely used in the study of vertebrates.In this study,the brain of human and mouse with the thickness of 100μm was transparent by using TMPDE（Trimethylolpropane triglycidyl Ether）technique.The expression and distribution of MANF was studied by using mmunofluorescent staining.Microscopic neuroanatomic networks of MANF in human and mice brain were presented.Objective: In this study,TMPDE technique was used to accomplish the three-dimensional microscopic anatomical structure of MANF protein and observe the distribution of MANF in human,mouse and rat brain and compare it across the species by using immunohistochemistry technique.Methods: Human and mice brain samples were obtained.Dissection was performed within 6 hours after death,the skull was opened,the whole brain was removed,weighed,and the brain was evaluated and observed whether there are cerebrovascular abnormalities,brain atrophy,substantia nigra lesions and other disorders.and then the prefrontal cortex were removed.For C57 mice,cardiac perfusion were performed after abdominal anesthesia.Then the mice brains were removed.Subsequently,accomplish the three-dimensional microscopic anatomical structure of MANF and observe the distribution of MANF in human,mouse and rat brain and compare it across the species by using immunohistochemistry technique.Results: 1.The TMPDE technique has proper compatibility with different antibodies.2.The brain of human and mouse with the thickness of 100μm were transparent by using TMPDE technique.3.The results showed that MANF positive cells were labeled by using immunofluorescence staining and the three-dimensional imaging thickness of MANF was increased compared with that of the uncleared tissue.But the specific fluorescence signal was weaker.Conclusion: The thickness of 100μm in human and mouse brain were cleared by using TMPDE technique.The positive rate of MANF labeling was higher of the cleared cortex in human and mouse brain than uncleared. |
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