Distribution And Morphological Study Of Nestin, Sox2and BrdU Positive Cells Of The Median Eminence In The Rat

Objective: To observe and study distribution and morphology of Nestin,Sox2and BrdU positive cells of the median eminence in the rats.Methods:30healthy SD rats weight from180g to200g, were randomlydivided into two groups:①normal group (n=15);②BrdU model group(n=15): injected with BrdU.1Preparation of BrdU labeling experimental animal models: Modelanimals were injected intraperitoneally with BrdU dissolution in normal salineaccordance with50mg/kg per injection, at16:00and18:00per day,respectively, and continuous for three days.2Sampling and preparation:9normal animals were anesthetized andfixed by transcardial perfusion. After dissection, post-fixation, paraffinembedding, tissues contain the median eminence were sectioned into5μmslices. Toluidine blue staining and Nestin and Sox2immunohistochemistrywere performed respectively.9BrdU model animals were anesthetized andfixed by transcardial perfusion. After dissection, post-fixation, paraffinembedding, tissues contain the median eminence were sectioned into5μmslices and BrdU immunohistochemistry were performed. All sections wereimaged with the Olympus BX-UCB microscope system.3Nestin&Sox2double immunofluorescence histochemical staining:6normal animals were anesthetized and fixed by transcardial perfusion. Afterdissection, post-fixation, the part of the sections contain the median eminencewere soaked in sucrose, frozen sectioned into8μm slices. Nestin&Sox2double immunofluorescent staining, added50%glycerol and place coverslips.All sections were imaged with Olympus BX-UCB microscope system.4BrdU&Sox2double immunofluorescence histochemical staining:6BrdU model animals were anesthetized and fixed by transcardial perfusion. After dissection, post-fixation, the part of the sections contain the medianeminence were soaked in sucrose, frozen sectioned into8μm slices. BrdU&Sox2double immunofluorescent staining, added50%glycerol and placecoverslips. All sections were imaged with Olympus BX-UCB microscopesystem.6Statistical analysis: using specialized image analysis software Image-Pro Plus6.0counted positive cells.Results:1Toluidine blue staining: Coronary sections of normal animals show thatthe median eminence of the hypothalamus is located on the floor of the thirdventricle. After toluidine blue staining, nucleus are dyed as blue and Nisslbodies are dyed as bluish violet. The median eminence consists of five layers:ependymal, subependymal, fiber, reticular, and palisade layers. Ependymalcells in the median eminence and lateral wall of third ventricle are dividedfour types of morphology. The median eminence is covered by pars tuberalis.The median eminence has a large number of capillaries.2Immunohistochemistry staining2.1Nestin staining：In two third of the central ependymal layer andsubependymal layer of the median eminence, nucleu of Nestin positive cell islarge, cytoplasms is Nestin strong positive. Thick Nestin positive processscattered distribute in the fiber and reticular layers and nucleu of Nestinpositive cell is large. Occasionally can see Nestin positive cell in palisadelayer and its nucleus is large. Nestin-positive rate of unilateral wall of thirdventricle:6.01±5.30%. Nestin-positive rate of the median eminence:42.45±3.42%.2.2Sox2staining: Sox2immunoreactive cells are arranged closely inependymal and subendymal layers, and is sparse in fiber and reticular layers.Sox2-positive rate of unilateral wall of third ventricle:32.77±7.24%.Sox2-positive rate of the median eminence:48.30±5.66%.2.3BrdU staining: Distribution of BrdU-positive cells is sporadic in fiberand reticular layers and a little in the palisade layer and occasionally in subependymal layer. BrdU-positive rate of unilateral wall of third ventricle:0.12±0.08%. BrdU-positive rate of the median eminence:4.17±0.38%.3Immunofluorescence histochemistry staining3.1Nestin, Sox2double immunofluorescence histochemistry staining:Nestin-positive cytoplasm are dyed as green, Sox2-positive nucleus are dyedas red. In two third of the central ependymal layer and subependymal layer ofthe median eminence, distribution of Nestin and Sox2double-immunolabledcells is dense and in fiber and reticular layers is spare.3.2BrdU, Sox2double immunofluorescence histochemistry staining:BrdU-positive nucleus are dyed as green, Sox2-positive nucleus are dyed asred. BrdU and Sox2double-immunolabled cells that their nucleus are dyed asyellowgreen, is sporadic in fiber and reticular layers and a little in the palisadelayer and occasionally in subependymal layer.Conclusions:1Distribution of Nestin positive cells is dense in ependymal layer andsubependymal layer of the median eminence and is spare in fiber and reticularlayers.2Distribution of Sox2positive cells is dense in ependymal andsubependymal layers of the median eminence and is spare in fiber andreticular layers. Distribution of BrdU positive cells is sporadic in fiber andreticular layers.3Distribution of Nestin and Sox2double-immunolabled cells is dense inependymal and subependymal layers. Distribution of BrdU and Sox2double-immunolabled cells is sporadic in fiber and reticular layers.