The Rapid Role Of Dihydrostestosterone On Dendritic Spines Of Hippocampal Neurons In SAMP8

Objective: Apply the SAMP8mouse for study to observe the rapid roleof castration and androgen replacement therapy on hippocampal dendriticspines.Further investigate the effect non-genetic mechanism of androgen andto provide some experimental proof.Methods: Ninety7-month-old male SAMP8mice were selected.(1)Selected twenty SAMP8mice for serum androgen levels.They wererandomly divided into sham-operation group(Sham group) and castrated group.Castrated group were all removed twin testis. Then according to the differentcastration time,they were divided into castrated1d group(C-1d)、2d group（C-2d）and3d group（C-3d），and five mice in each group. Mice in eachgroup to take the blood of decapitated, after standing,3000RPM centrifugedfor20minutes at room temperature to get supernatant. Then they were savedin-20℃to the test. By radioimmunoassay for the determination of androgenlevels to determine the time of castration androgen depleted.(2) Seventy7-month-old male SAMP8mice were randomly divided intosham-operation group(Sham group), castrated group, and dihydrotestosteronereplacement therapy with castrated group(DHT group) Then according to thedifferent therapy time, they were divided into DHT-15min group、DHT-30mingroup、DHT-1h group、DHT-2h group and DHT-21d group, and10mice ineach group. Selected ten7-month-old male SAMR1mice as normal contrastof the consanguinity group(Control group). Castrated group and DHT groupwere removed twin testis. DHT group hypodermic injection1mg/(kg·day)DHT after3day. Sham groups were injected equal asepsismedical maize oil,1time/day.Tissue preparation and staining observation:5mice of each group were taken out, broken right auricle and perfused quickly with normal saline andsubsequently fixed with4%paraform via left ventricle. From superiorcolliculus to optic chiasma segment, the brain made the same two partsthrough middle sagittal viewing by razor blade. One was made use for Golgistaining, and count each group of mice hippocampal CA1region of2to3apical dendrites dendritic spine density; the other was made of paraffinsections using for anti-drebrin immunohistochemical staining, and analysis ofthe optical density of each group of mice hippocampal CA1region.Protein extraction and Western blot:5mice of each group were taken out,rapid decapitation after anesthesia with6%chloral hydrate aldehyde, removethe skull and stripped meninges to exposed the brain tissue, isolated thehippocampus. After PBS washing, quickly placed in lysis buffer, fullyhomogenized on ice at rest30min; the12000RPM centrifuge for20minutesat4°C and discard the pellet to save the supernatant. A small part of thesupernatants were used for determination of protein concentration (CoomassieBrilliant Bluemethod), the rest of the supernatant for the Western Blot at-80℃storage.Results:1. Radioimmunoassay results of serum androgen levels: serum androgenconcentrations in the sham-operation group was597.53±19.99ng/dl,C-1dgroup was97.14±9.88ng/dl, C-2d group was28.83±6.08ng/dl and C-3dgroup was0.03±0.01ng/dl.2. The results of Golgi Staining: the dendritic spines in hippocampal CA1region of Control group were well-arranged and intensive, the number of theapical dendritic thorns density was1.314±0.028/μm, with statistical differencecomparing with others group(P<0.05); The castrated group reducedsignificantly and the number of was0.887±0.022/μm. The number of dendriticspine of each DHT group increased, DHT-15min group was1.158±0.025/μm,DHT-30min group was1.149±0.016/μm, comparing with castrated group、DHT-1h group and DHT-2h group were increased(P<0.05). DHT-1h groupwas1.042±0.018/μm, DHT-2h group was1.015±0.021/μm, comparing with castrated group were higher, but less than the DHT-15min group andDHT-30min group (P<0.05). DHT-21d group(1.153±0.024) compared withSham group(1.167±0.024) was no statistically (P>0.05).3. The results of anti-drebrin immunohistochemistry: Control group inhippocampal CA1region stained deep, its optical density value was0.374±0.018, significantly higher than other groups (P<0.05); the Castratedgroup in hippocampal CA1region stained light, its optical density value was0.171±0.003, significantly lower than other group (P<0.05). DHT-15mingroup was0.256±0.011, DHT-30min group was0.249±0.008, comparing withcastrated group、DHT-1h group and DHT-2h group were increased(P<0.05).DHT-1h group was0.224±0.013, DHT-2h group was0.206±0.007, comparingwith castrated group were higher(P<0.05), but less than the DHT-15min groupand DHT-30min group (P<0.05). The optical density value of DHT-21d group(0.251±0.006) compared with Sham group (0.262±0.014) was no statistically(P>0.05).4. The results of Western blot: the drebrin of hippocampal dendritic spinesin control group expression rich, its IOD value was3.123±0.054,significantly higher than other groups (P<0.05); the drebrin of hippocampaldendritic spines in Castrated group expression less, its IOD value was1.279±0.069, significantly lower than other groups (P<0.05). DHT-15mingroup was2.744±0.043, DHT-30min group was2.729±0.036, comparing withcastrated group、DHT-1h group and DHT-2h group were increased(P<0.05).DHT-1h group was2.361±0.045, DHT-2h group was1.933±0.052, comparingwith castrated group were higher(P<0.05), but less than the DHT-15min groupand DHT-30min group(P<0.05). The IOD value of DHT-21d group(2.731±0.047) compared with Sham group(2.751±0.047) was no statistically(P>0.05).Conclusions:1. Androgen deficiency after castrating in SAMP8mice, the levels ofserum androgen were decreased with castration time. After castrated the thirdday, the levels of serum androgen levels were almost exhausted in SAMP8 mice. It was the best time to add androgen.2. Androgen deficiency after castrating in SAMP8mice, androgendeficiency lead to the reduced of the number of dendritic spines in thehippocampus, and the decreased expression of the drebrin in dendritic spines.After a rapid DHT replacement therapy, it increased the dendritic spine densityin hippocampal and the expression of drebrin in dendritic spines. Thisindicates that androgen affected the morphological plasticity of dendriticspines may related with its actin-binding protein-drebrin, and it may beachieved through the rapid non-genomic effects.