| Objective: To measure the character of castration and testosterone or dihydrotestosterone replacement treatment on NMDA receptor(NR1,NR2 A,NR2B)through the selection of SAMP8 mice for observation,and to explore the mechanism so as to provide some useful proof for neurodegenerative diseases associated with androgen.Methods:Forty six-month-old SAMP8(male)mice were randomly allocated into four groups.Each group has ten mice,including the control group(P8 group),castrated group(Cast group),testosterone replacement therapy for castrated mice(T group)and dihydrotestosterone replacement therapy for castrated mice(DHT group).10 six-month-old SAMR1(male)mice were treated as normal contrast(R1group).A total of 30 mice from Cast group,T group and DHT group were operated with the removal of testis(namely castration).Three days after castration,abdominal injection of physiological doses of testosterone and dihydrotestosterone were respectively opereated in the mice of T group and DHT group.While the same amount of corn oil solution were infected in mice of each test group.The infection was operated once each day,sequentially for 21 days.Immunohistochemical staining of hippocampal tissue: 5 mice were randomly taken out from each group,which were cut open right auricle and imported quickly with physiological saline and afterwards imported 4% paraform via left ventricle.Segment of brain tissue was taken out from mice to make paraffin section,for the anti-NR1,anti-NR2 A,anti-NR2 B immunohistochemical staining.Then we measured the average opitical density in hippocampal CA1 of mice.Western blotting of hippocampal tissue: 5 mice were randomly selected from each group,brains of which were removed after anesthesia to extract hippocampal tissue protein for the protein concentration measurement and tissue protein denaturation..We used SDS-poly acrylamide to analysis electrophoresis and immune response after trarsmembrane.Thus the relative levels of expression of protein with ?-actin as an internal control were tested.Results: 1.Results of immunohistochemical staining Results with anti-NR1 immunohistochemistry: the average optical density values of the Cast group(0.053±0.012)was significantly lower than R1 group(0.272±0.015),T group(0.193±0.009),DHT group(0.197± 0.011)(P<0.05).P8 group(0.186±0.013),T group and DHT group have no significant difference(P>0.05).Results with anti-NR2 A immunohistochemistry: the average optical densityvalues of the Cast group(0.014±0.002)was significantly lower than the R1 group(0.054±0.009),T group(0.046±0.007),the DHT group(0.034±0.011)(P<0.05).The P8 group(0.044±0.007),T group and DHT group have no significant difference(P>0.05).Results with anti-NR2 B immunohistochemistry: the average optical densityvalues of the Cast group(0.058±0.006)was significantly lower than R1 group(0.104±0.008),T group(0.077±0.004)and the DHT group(0.084±0.007)(P<0.05).The P8 group(0.087±0.007),T group and DHT group have no significant difference(P>0.05).2.Results of western blotting Results of NR1 Western blotting: its OD value in R1 group was 0.88±0.05,significantly higher than other groups(P<0.05);its OD value in Cast group was 0.28±0.05,significantly lower(P<0.05).OD value of P8 group was 0.71±0.06,and that of T group and DHT group was 0.63±0.05 and 0.66±0.04 respectively,significantly higher than Cast group(P<0.05).The results of NR2 A Western blotting: its OD value in R1 group was 0.76±0.02,significantly higher(P<0.05);its OD value in Cast group was 0.23±0.02,significantly lower(P<0.05).OD value of P8 group was 0.63±0.05,and that of T group and DHT group were 0.60±0.06 and 0.59±0.07 respectively,significantly higher than Cast group(P<0.05).The results of NR2 B Western blotting: its OD value in R1 group was 0.66±0.03,significantly higher(P<0.05);its OD value in Cast group was 0.21±0.02,significantly lower(P<0.05).OD value of P8 group was 0.52±0.01 and that of T group and DHT group were 0.53±0.03 and 0.52±0.04 respectively,significantly higher than Cast group(P<0.05).Conclusions:1 After castration,the NMDA receptor subunit NR1,NR2 A and NR2 B protein levels were significantly decreased in the hippocampal of SAMP8 mouse.2 Androgen replacement therapy can raise the protein level. |
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