| Objective: Observing the effect of Ovariectomized and estrogen replacement therapy on learning and memory ability and hippocampal neurons and the nNOS positive neurons in senescence accelerated mouse-prone/8 (SAMP8). Further investigate the effects of estrogen to improve mechanism of the learning and memory ability and to provide some experimental proof.Methods: Forty five 6-month-old female SAMP8 were randomly divided into sham-operation group (SHAM group), Ovariectomized group(OVX group), and estrogen replacement therapy with Ovariectomized group(OVX-E group).Chosen fifteen 6-month-old females SAMR1 as normal contrast of the consanguinity group(R1 group). OVX group and OVX-E group were removed the two ovarians, OVX-E group im estradiol benzoate 5μg once every three days and other groups im equal asepsis sesame-seed oil, altogether three months.1. Morris water maze test: After injection, the SAM mice accommodated 1day under the environment of Morris water maze. Testing was progressed in every morning. The fixed position location underway trials: The Morris water maze was dividing equally for 4 quadrants, the plat was put among them in one quadrant. The mouse went into water from the 4 quadrants midpoint towards the wall in pond, and the video frequency acquisition system tracken and recorded escape latency. The plat was removed and undertaken space exploring trial in the 6days, and taken notes the times that the mouse acrossing span flat roof in 120s.2. The preparation and observation of example of transmission electron microscope: The mouse was anesthed using 6%chloral hydrate. And the mouse was broken right auricle and perfused quickly with normal saline and subsequently fixed with 2%parafom-2%glutaraldehyde precooling via left ventricle and lasting 1h. And to take the CA1 of hippocampus and fixed by osmium acid, dewated, embed and sectioned at a thickness of 50nm. Cut it using the LKB-5 ultramicrotome, thickness 50nm. Plumbum-uranium staining, and observed the ultrastructure change of neuron by using the transmission electron microscop.3. The observation of HE stain and Immunohistochemistry technique: The mouse was anesthed using 6%chloral hydrate. And the mouse was broken right auricle and perfused quickly with normal saline and subsequently fixed with 4% paraform via left ventricle and lasting 1h. The hippocampus was taken between superior colliculus and optic chiasma to make 2 sets of paraffin sections using for HE staining and anti-nNOS immunohistochemistry staining.4. The detection of flow cytometry: The mouse was taken out and killed by decapitation. The brains were removed and then rapidly peeled off the hippocampus organization on the ices dish, fixed in 4℃, 70% alcohol. Adopting the net twisting to prepare unicellular suspension, and using anti-nNOS incubation and detected by flow cytometry.Results:1. In the Morris water maze test: The result of the escape latency of the OVX group was longer than the SHAM group and OVX+E group(P<0.05). The result of the total number of crossing the platform of the OVX group was fewer than the SHAM group and OVX+E group(P<0.05). And there was no significant difference compared with SHAM group and OVX+E group (P>0.05).2. Electronic microscope ultramicro examination: In the OVX group, there were bulk ambiguous nuclear membrane structure, chromatin margination, large number of lipofuscin particle deposition and marked swelling of mitochondria of pyramidal cells in the hippocampal CA1. In the OVX+E group, the majority of pyramidal cells in the hippocampal CA1 have complete cells structure, Obviously nucleolus, chromatin distribution, normal mitochondrial size and morphology. Few cells can be seen fuzziness double-layer structure of nuclear membrane and chromatin unclear marginal phenomenon, as well as a small amount of lipofuscin particle deposition. SHAM group and OVX+E group are basically the same situation. In the R1 group, the Pyramidal cells in the CA1 have complete cells structure, obviously nucleolus, chromatin distribution, normal mitochondrial size, morphology, cytoplasmic rough endoplasmic reticulum, golgi apparatus and ribosome structure.3. With HE staining: In the OVX group, the cells hierarchy were floc chaos and cells gap increases and the number of cells were decreased significantly and a large number of pyramidal cells concentrated dye and vacuolation in the CA1.In the OVX+E group and SHAM group, the Cells hierarchy were regularity, cells gap increase were not obviously, and a small number of pyramidal cells concentrated dye in the CA1. SHAM group and OVX+E group are basically the same situation. In the R1 group, the Pyramidal cells hierarchy were clearly and arranged uniform and cells structural integrity and the nucleolus clearly visibled in the CA1.4. The observation immunohistochemistry test: The number of positive neurons and the optical density of the OVX group was significantly lower than SHAM group and the OVX+E group (P<0.05). And there was no significant difference compared with SHAM group and OVX+E group (P>0.05).5. The Flow cytometry :the FI number of the OVX group was significantly lower than SHAM group and the OVX+E group (P<0.05).The SHAM group and OVX+E group have no significant difference.Conclusions:1. Estrogen deficiency after ovariectomized in SAMP8 results in lowere learning and memory ability. Estrogen replacement can alleviate the damage induced by endogenous estrogen decreasing and improve the learning and memory ability.2. Estrogen deficiency after ovariectomized in SAMP8 results in hippocampal neurons sparse and disordered, swollen and degenerated, the ultramicrostructure damaged, reduce the number of neurons, and many neurons losted. Estrogen replacement can improve the pathological lesion and lessen the apoptosis and loss of hippocampal neurons.3. Estrogen deficiency after ovariectomized in SAMP8 results in decrease the expression of nNOS positive neurons in hippocampal CA1. Estrogen replacement can increase the expression of nNOS positive neurons. |
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