| With increase of Alzheimer's disease patients, many researchers begin to focus on changes of somatostatin (SS) in AD and its prevention and treatment, which is important substance of learning and memory as a kind of neurotransmitter in the central nervous system. Ginsenosides (GS) as the main active components of the traditional Chinese herb, although its effects on lengthening people' life have been generally accepted by researchers, its roles for SS neurons and contents in Alzheimer's disease (AD) model rats haven't been reported. We adopted immunohistochemistry ABC technique and in situ hybridization to detect the numbers of hippocampal SS neurons and treatment effectiveness of GS in AD rats induced by D-galactose and ibotenic acid (IBO). The study indicated GS might prevent and treat AD with morphological data.Materials and Methods 1. Animal groupsThirty-five male SD rats weighing 160~180g were divided into five groups randomly, including: AD model group, prevention group, GS treatment group, prevention control group (abbreviated control group) and normal control group (abbreviated normal group).2. Alzheimer's disease model construction and medicationAD model rats were induced by D-galactose subcutaneous injection for 6 weeks and lesion of Nucleus Basalis Magnocelluaris (NBM) with IBO. D-galactose and IBO for control rats and normal rats were replaced by isodose saline. The following parts were the medication of all animals: (1)Model group: isodose saline intraperitoneal injection (2.4%GS, 50 mg/Kg.d) from 8-11 weeks;(2)Prevention group: 2.4%GS (50 mg/Kg.d) intraperitoneal injection from 1-4 weeks; (3)Treatment group: 2.4%GS intraperitoneal injection( 50 mg/Kg.d)from 8-11 weeks; (4) Control group: 2.4 %GS intraperitoneal injection (50 mg/Kg.d) from 8-11 weeks; (5)Normal group: isodose saline intraperitoneal injection (2.4%GS, 50 mg/Kg.d) from 8-11 weeks.The following table was the program of medication:3. Detect SS and SSmRNA with ABC immunohistochemistry technique and in situ hybridication (1) Make materials and slicesRats' brains were perfused and taken out when they had been injected IBO after 5 weeks. All animals were anethitized with 2% sodium pentobarbital (35mg/Kg) by intraperitoneal injection. Opened rats' cranial cavity and poured 150ml sterile saline through ascending aorta, then poured 4% citromint buffeKpH 7.2)for 20-30min. Taking out of telencephalon (including hippocampus), brains were in 4% precool citromint buffer for 6-12h. After routine paraffin dressing, continuous cronal sections of hippocampus were made with Leitz 1512 microtome (thickness 8μm). Taking one among three sections carried out the experiment.(2) Experimental procedure(1) Detect SS neurons with ABC immunohistochemistry techniqueHippocampal slices were detected with ABC Kit from HuaMei biological Co. and anti-somatostatin antibody from the department of neurobiology of Second Military Medical University. The slices containing SS antibody had been incubated for Ih at room temperature, then transferred to 4癈 for 18-20h, whose dilution was 1: 1200. The following was performed with routine procedures. DAB Stainning, heamatoxylin restainning slices had dehydration and clearing routinely. At last, slices were sealed with Dimethylbenzene.(2) Detect SSmRNA with in situ hybridizationSlices with pretreatment were incubated at 43C for 20h containing DIG-Iabelling SScRNA probe hybridization buffer (0.5ug/ml) and at room temperature for 5h containing DIG-antibody Fab fragments marked with Alkaline phosphatase (1: 800 ) . SScRNA probe was supplied by the department of histology and embryology in Second Military Medical University; NBT and BCIP staining for 3-5h at room temperature; some were restained with nuclear fast red. After desiccation and clearing, sealed slices with Dimethylbenzene.The slices had positive contrast (distribution of SS in temporal cortex) and negative contrast (SS antibody and SScRNA probe being taken place with PBS buffer).(3) Image analysis and statisticsTo...
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