Testosterone Have Neuronal Protection Role To Free Radical Damaged Primary Cultivated Hippocampal Neuron.

1.Rat primary hippocampal neuron's cultivation, identification and morphological investigationObjective: New born 1 d SD rat hippocampal neuron cultivation, master the method of rat primary hippocampal neuron cultivation in vitro. Immunocytochemistry to identify the neuron, Scanning electron microscopy, Hematoxylin and eosin, Nissl staining show the morphological characteristic of primary hippocampal neurons, detect the growth curve of hippocampal neuronsMethods: New born 1 d SD rat puppies, weight 7±1g, get the brain, put it into pre-colded D-hanks and on the ice plate, dissect hippocampus and divest the meninges and vessel under stereomicroscope, add 0.125% trypsin 37℃about 20 min, 10%FBS DMEM wash it 3 times. With pasture pipette resuspend for several times, 1-5×105 density cultivate in glass covers which were precovered with poly-l-lysine, put it in 37℃,5% CO2 incubator. 1 d exchange with Neurobasal(2% B27), 8-10 d use it in experiment. With 10 d neuron, 4% PFA fixed, 0.25% Triton 15 min, 3% H₂O₂ 15 min, goat serum block, add the mouse anti rat MAP-2 monoclone antibody, rabbit anti rat polyclone antibody 4℃overnight, biovidin labeled goat anti rabbit or goat anti mouse antibody 37℃1 h, with fresh HRP 20 min, DAB staining, ethanol dyehydration, xylene transparent, gummi mounting. Investigate MAP-2 and NSE expression with light microscopy, choose 10 visual field randomly and count the positive ratio and photography. Cell cultivate in 96 well plate, detect the absorbance every 2 d, total detect 7 times. 10 d cultivated neuron, fixed and dyed with hematoxylin 10 min, 1% hydrochloric acid ethanol separation, 1% dilute ammonia water, eosin staining, dyehydration and transparent, gummi mounting and observe. Cultivated 10 d hippocampal neuron, 1% glutara fixed, ethanol dyehydration, dryed with tert-butyl alcohol. Vacuum dyehydration and glid, Hitachi S3500 SEM observe and photography. 10 d cultivated hippocampal neuron, fixed, 1% toluidin blue staining, ethanol dyehydration, xylene transparent, gummi mounting and observe.Results: 1 Primary cultivated hippocampal neuron, 6-8 h start adhere, 1 d most of neuron adhere. 3 d cell aggregate group, prominence is elongate and enlarge, can form sparse connection, 5-7 d body showed chubbiness. With time extend, cell body enlarge furthermore, prominence enlarge and elongate, connection each other, cell show clearly. Cell body show multipolar neuron, it's pyramid, triangle, spindle, ellipse shape. After cultivated for 3 w, cell begin degenerate, there are granule vacuoles and prominence regression, nuclus pycnosis and disintergration. 2 Primary cultivated hippocampal neuron staining with MAP-2 and NSE antibody show positive, MAP-2 staining show cell body and prominence are brown color, while NSE show only cytoplasm is dyed with brown. Photography and count the positive cell ratio is about 90%. 3 Primary cultivated hippocampal neuron show it's growth curve is undergo a latency period(2-4 d), exponential growth phase(4-8 d), this period cell growth extremely fast, then growth plateau phase(8-14 d), cell in stasis. The primary cultivated neuron growth circle about 8-10 d. 4 Primary hippocampal neuron H-E staining show neuclear is deep blue dye, cytoplasm is pink. 5 Hippocampal neuron Nissl staining show Nissl body in cell body, in the prominence seldom find Nissl body. 6 Scanning electron microscopy show most of cell body are pyramid, others show triangle and spindle.Conclusions: 1 Primary cultivated SD puppy neuron, 10 d is mature, cell body show triangle, spindle and oval shape under phase inverted contrast microscope. 2 Primary cultivated hippocampal neuron immnunocytochemistry show MAP-2, NSE positive, purity about 90%. 3 Primary cultivated neuron under go a latency period, exponential growth phase, plateau phase, growth circle about 8-10 d. 4 Hippocampal neuron H-E staining is nuclear dark blue, cytoplasm is pink. 5 Nissl staining show Nissl body in cytoplasm, seldom find in prominence. 6 Primary cultivated neuron cell body show triangle, spindle, pyramid shape under SEM, prominence is abundant.2.The neuronal protection role of testosterone to primary cultivated neuron's after exposed to free radical.Objective: Primary cultivated hippocampal neuron, exposed to free rdical, observe the oxidative damage of hippocampal neuron, pretreated with testosterone and investigate if the testosterone have neuronal protection role of exposed free radical hippocampal neuron.Methods: Use primary cultivated hippocampal neuron, add different concentration of H₂O₂, cultivate for 1 d, observe the cell viability and MTT value, Trypan blue count the cell number, freedomize and count the positive cells in 10 visual fields, which are not dyeing, calculate the cell viability. Cell viability=(cell sum-blue dyeing cell(dead cell))/sum. Hippocampal neuron randomize grouped as control group, H₂O₂ group, administrate testosterone group(T group), pretreated with testosterone group and then exposed to H₂O₂ group(T pre and H₂O₂ group). Control group didn't add H₂O₂, add the original medium. H₂O₂ group add different concentration of H₂O₂, culture with neurobasal medium 1 d and detect the cell viability. Testosterone group add 10 nM testosterone 1 d before, and then detect the viability. T pre and H₂O₂ group add Testosterone 1 d ahead and then exposed to different concentration of H₂O₂, coculture and then detect. In order to obseve the time of testosterone to exert effect, as the previous H₂O₂ concentration, 10 d cells grouped in 4: 1'st group is before exposed to H₂O₂ 1 d add testosterone and last for 2 d, detect cell viability(T pre T post group); 2'nd group is before exposed to H₂O₂ 1 d administrate testosterone, add H₂O₂ and at the same time remove testosterone, detect the cell viability(T pre 0 post group); 3'rd group is add testosterone and H₂O₂ together, coculture for 1 d and detect(0 pre T post group). The last group is after add H₂O₂ 8 h then administrate testosterone, culture for 1 d and detect(0 pre T post 8 h), compare 4 groups cell viability. Detect the NOS, SOD and MDA in 4 groups as the manual illustration, still water reset, detect each tube absorbance.Results: 1 Add different concentration of H₂O₂, have different influence to hippocampal neuron, 10μM H₂O₂ have no obviously influence to hippocampal neuron's viability, while 20-200μM H₂O₂ have different degree damage on neuron(P<0.05), each group cell viability have the H₂O₂ concentration decrease trendency, H₂O₂ 100μM influence the cell viability and MTT value most. Expose to H₂O₂, cell body is shrink under phase contrast inverted microscope, process is attenuate, MTT is decrease, especially at 100μM H₂O₂ is the most, as the cell viability result. 2 Without testosterone add 100μM H₂O₂, show the number is less, process is reduce, attenuate and atrophy, there are more cell debris in medium, more vaculos in cells, before exposed to H₂O₂, treated with testosterone is better, though there are a few decrease in cell number, but compare with no testosterone group have statistically difference, testosterone have neuronal protection role to hippocampal neuron after exposed to H₂O₂, Testosterone can increase cell viability and MTT value of exposed H₂O₂ neuron. 3 Among 4 groups, different time administrate testosterone have vary effect. Cell viability are higher in both testosterone pre add and last after give H₂O₂ group (T pre T post group) and give testosterone at the same time with H₂O₂ group(0 pre T post group), compare with other groups have difference(P<0.05), while in other groups cell viability is lower. 4 Detect each group NOS, SOD and MDA content after give different concentration of H₂O₂, compare control group and only administrate testosterone group, difference is not obvious (P>0.05), without pre add testosterone, only exposed to H₂O₂ group, NOS, MDA content is increase, SOD is decrease versus control group, have statistically difference (P<0.05). Before exposed to H₂O₂, administrate with testosterone have significant difference compare to H₂O₂ group(P<0.05).Conclusions: 1 H₂O₂ have oxidative damage effect to primary cultivated hippocampal neuron, especially 100μM H₂O₂ have severely influence on cell viability. 2 Add 10 nM testosterone have neuronal protection role to exposed free radical damaged cell, compare with no testosterone group, difference is significant. 3 Testosterone's neuronal protection role is transient action. 4 Exposed to free radical, primary cultivated hippocampal neuron SOD contant is decrease, while NOS, MDA is increase, pre add testosterone can protected H₂O₂ neuronal damage effect of hippocampal neuron.3.The investigation of apoptosis in testosterone's protection role of free radical induce oxidative damage in primary cultivated neuronObjective: Testosterone have protection role in primary cultivated neuron, hippocampal neuron after exposed to free radical damage, with fluroesence dyeing and Western blot to observe if there are apoptosis mechanism involve in the neuron protection role of testosterone.Methods: Primary cultivated hippocampal neuron 10 d, as control group, H₂O₂ group and pre-add testosterone group, add H₂O₂ to each group beside control group, 4% stationary liquid fixed for 5 min, Hoechst 33258 dyeing for 10 min, filter paper absorb residum, mounting. Fluoresence microscope, wave length is 364-400 nm Ultraviolet to irritate, oberve and photography. Pirmary cultivated hippocampal neuron Bcl-2 and Bax expression with Western blot assay, primary cultivated cells, with different group add H₂O₂, collect the cell and add 200μL Lysate, add Aprotinin, LEUPTIN, PMSF, DTT cleavage for 60 min, ultrasonic cleavage, get the suspension. 4℃centrifuge get the suspension, subpackage and reserve the protein at -80℃, quantitate the protein with Coomassie blue. Make the separation gal and spacer gal, till the spacer gal is completely polymerize, take out the comb. Put the gal with correct direction into electrophoresis chamber, pour the electrophoresis buffer solution, make the liquid level is higher than sample application chamber. Trimming the sample application chamber, make it direct and wash it for several times, in order to remove remaining gal. Get 150μg protein and adequacy physiological saline, add sample buffer and mixed, use low molecular weight marker, centrifuge and boiled for 8 min, denaturate. Put the smaple and electrophoresis, till the sample arrive the boundary of separation gal and spacer gal, adjust the voltage is 120V. When indicator is about 1 cm to the bottom of gal, turn off the power. After rinse and put it in 3 pieces of filter paper, NC membrane, gal, 3 pieces of filter paper direction, transmembrane, with voltage is 100V, about 60 min stop. 5% defatted milk blocked for 1 h. Get the membrane, the primary antibody(1:80), control with PBS instand antibody, put it into sealed plastic bag, 4℃over night. TBS rinse, add HRP labeled 2'nd antibody, TBS rinsed, immunodeveloping, get X ray film and developing and fixing. With Hema analysis system to assay the strap, us AU( Darea·Ddensity) represent protein area multiple grew value. Detect GAPDH as reference, use target protein's AU and the GAPDH AU ratio to illustrate each protein's expression. All data use Mean±SEM, with SPSS14.0 software, as one way ANOVA, compare with control and experiment use Dunnett-t test.Results:1 Primary cultivated hippocampal neuron exposed to H₂O₂, with Hoechst 33258 dyeing show, cell dyeing blue, nuclear is obvious, H₂O₂ group can see the apoptosis cell, nuclear is deep dye demilune or debris, process is not clear. Pre add testosterone group exposed H₂O₂, hippocampal neuron dyeing with Hoechst 33258 show nuclear clearly, few debris is seen obviously. 2 Western blot show control group have a few Bcl-2 and Bax expression, add H₂O₂ group Bax is increase, but Bcl-2 decrease, compare with control group have significant difference(P<0.01). Pre add testosterone group, then exposed to 100μM H₂O₂, Bcl-2 expression increase, and Bax decrease, compare with H₂O₂ group, have statistically difference (P<0.05).Conclusion:1 Free radical damage to primary cultivated hippocampal neuron have cell apoptosis mechanism involved, pre add testosterone can protect against cell damage. 2 There are few Bcl-2 and Bax expression in hippocampal neurons, free radical damaged hippocampal neuron Bax expression increase while Bcl-2 decrease, pre administration testosterone increase Bcl-2 expression and decrease Bax expression.4.Intracellular calcium changes of free radical exposed hippocampal neurons after administrate testosterone.Objective: Primary cultivated hippocampal neurons expose to H₂O₂, observe if calcium changing involved in testosterone's neuronal protection role.Methods: Cultivated 10 d hippocampal neurons, randomize as control group, H₂O₂ group, pre-add testosterone group. Control group add original culture medium, H₂O₂ group add 10,20,40,60,80,100,200μM H₂O₂, detect the intracellular calcium before and after. Testosterone group add 10 nM testosterone before exposed to H₂O₂ 1 d, then expose to 100μM H₂O₂ and detect the cell calcium content. Add Fluo-3, AM solution to 10 d primary cultivated hippocampal neuron for 60 min, H₂O₂ group deal with the same method as in H₂O₂ group. HEPES wash and Laser confocal scanning microscope(LCSM) observe. Put the cell in plate and with different concentration of H₂O₂ and Neurobasal medium mixed together, radiated wave length is 488 nm, and emitted with 528 nm to detect the fluorescence value. Each group add H₂O₂ before and after 2 h to test intracellular value, 30 frames every min, constant scan 15 min to test inctracellular calcium change. Analysis with LCSM software and detect F0 and Fi, with Fi/F0 as the ratio to index intracellular calcium change.Results: 1. In control group, there is no calcium change. In H₂O₂ group, compare with control group, FI value have statistically different(P<0.05). 2. In H₂O₂ group, intracellular calcium change in certain level, and especially in 100μM change obviously, under the 60μM, there are no statistically differences. While up to 80μM compare with control group, there are statistically difference (P<0.05). 3. Pre added testosterone group, compare with control group, there are no differences. Add testosterone and then exposed to 100μM H₂O₂ group and compare without testosterone group, intracellular calcium content have decrease obviously, have statistically differences(P<0.01).Conclusions: 1. Primary cultivated hippocampal neuron, add different concentration of H₂O₂, intracellular calcium change obvious, and especially 100μM and 200μM is typical. 2. Testosterone can attenuate free radical caused intracellular calcium change in primary cultivated hippocampal neuron.