Effects Of Androgen On Learning And Memory Ability And Hippocampus Neurons In SAMP8 Mouse

Objective: Observing the effect of castration and androgen replacement therapy on learning and memory ability and hippocampal neurons in senescence accelerated mouse-prone/8 (SAMP8). To explore the mechanism of androgen improving the learning and memory ability and to provide some experimental proof.Methods: Forty eight 7-month-old male SAMP8 were randomly divided into sham-operation control group (P8 group), castrated group, and androgen replacement therapy with castrated group(TU group). Chosen sixteen 7-month-old males SAMR1 as normal contrast of the consanguinity group. Castrated group and TU group were removed twin testis. TU group im 37.4mg?kg-1 TU once 15days after 1week. Other groups im equal asepsis sesame-seed oil, injecting 3 times, altogether 45days.1. Morris water maze(MWM) test. After injection, the SAM mice were raised 2days under the environment of labyrinth laboratory first. Training was progressed in every morning between 8:00~12:00. The fixed position location underway trials: The MWM was dividing equally for 4 quadrants; the plat was put among them in one quadrant. The mouse went into water from first quadrant midpoint towards the wall in pond, and the video frequency acquisition system tracken and recorded escape latency. After 5days removed the plat and undertaken exploring trial, and taken notes the times that the mouse acrossing span flat roof in 120s.2. Preparate tissue slices and observe. 10 mice of every group were taken out, broken right auricle and perfused quickly with normal saline and subsequently fixed with 4% parafom via left ventricle. The hippocampus was taken between superior colliculus and optic chiasma to make 4 sets of paraffin sections using for HE staining, Nissl staining, TUNEL and anti-Aβimmunohistochemical staining.3. Apoptotic cells were detected by flow cytometry (FCM). 10 mice of every group were taken out and killed by decapitation. The brains were removed. Then rapidly peeled off the hippocampus organization on the ices dish, fixed in 4℃, 70% alcohol. Adopting the net twisting to prepare unicellular suspension, dyed with PI and detected by FCM.4. The ultramicrostructure observation in CA1 region of hippocampal neurons. The hippocampus was dissociated, fixed in 4% precooling glutaraldehyde, and cutted into thin slice of 1mm, then into <1mm3 1h once more, 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, observed the ultrastructure change of neuron by using the transmission electron microscop.Results: 1. In the MWM test, the escape latency of castrated group was significantly longer than that in the other groups (P<0.05), and the times of span flat roof decreased (P<0.05). Androgen replacement therapy can improve the learning and memory ability. And there was no significant difference compared with sham-operation control group (P>0.05).2. With HE staining, neurons in hippocampal CA1 region of R1 group were well-arranged, with clear stratification. While neurons in P8 group were slightly deranged, with decreased cellular layers. And neurons in castrated group presented diffused vacuolar degeneration, swollen, sparsely and disorderedly arranged, with cell nucleuses karyochrome and pyknosis. Androgen replacement therapy can lessen the pathological lesion obviously.3. With Nissl staining, neurons in hippocampal CA1 region of R1 group were rich and dense, with statistical difference (P<0.01), while the neurons in the P8 group were fairly sparse. Neurons in hippocampal CA1 region of castrated group were much fewer and presented karyopyknosis, turbidness and anachromasis, with less and even desolved Nissl bodies. The neuron number increased with androgen replacement therapy, with significant statistical difference compared with castrated group (P<0.05) but no statistical difference compared with P8 group (P>0.05).4. With Tunel staining, small amount of loose Tunel-positve cells existed in the R1 group, with obviously increased Tunel-positve cells in P8 (P<0.05). In the castrated group, many different-sized Tunel-positive cells presented, with distinct nuclear boundary and perinuclear halo, more cells than in P8, with statistical difference (P<0.05). The Tunel-positive neurons expressed weakly compared with castrated group (P<0.05), while with no statistical difference compared with other groups (P>0.05).5. With anti-Aβimmunohistochemistry, the Aβimmune positive neurons in hippocampal CA1 region of castrated group were deeply dyed and the number and the optical density of were markedly higher than those in other groups(P<0.05). While the number and the optical density of Aβimmune positive neurons in Testosterone group were obviously lower than those in the castrated group (P<0.05).6. With FCM, flow cytometry revealed the hypodiploid apex and the apoptotic rate showed to be 32.66±4.60% in the castrated group, with statistics significance compared with P8 (21.19±3.96%) and TU group (20.78±3.08%). But it had no statistical significance between P8 group and TU group.7. Electronic microscope ultramicro examination: the nuclear membrane of neurocyte in R1 group was clear, smooth, with proportionate cytoplasm, low electron density and rich mitochondria. Double-deckered nuclear membrane was clear in P8 group, normal or slightly swollen mitochondria, a little lipofuscins deposited, and slightly higher electron density of mitochondria. Nuclear membrane disappeared in castrated group, with mitochondria swollen and degenerated, cristae broked, lipofuscin deposited, chromatin condensed and marginated, nucleus fragmented and apoptosis body formed. In the androgen group, ultramicrostructure was harmed less severely than that in the castrated group, with clear nuclear membrane, insignificant margination of the chromatin, no obvious change of the mitochondria shape and less lipofuscin.Conclusions:1. Androgen deficiency after castrating in SAMP8 results in lowered learning and memory ability. Androgen replacement can alleviate the damage induced by endogenous estrogen decreasing and improve the learning and memory ability.2. Androgen deficiency after castrating in SAMP8 results in hippocampal neurons sparse and disordered, swollen and degenerated, the ultramicrostructure damaged, increased number of withered neurons, and many lost neurons. Androgen replacement can improve the pathological lesion and lessen the apoptosis and loss of hippocampal neurons.3. Androgen deficiency after castrating in SAMP8 results in deposition increase of Aβimmune positive neurons in hippocampal CA1 region. Androgen replacement can inhibit the formation of Aβ.