The Effects Of Estradiol On The Expression Of β-amyloid Precursor Protein And Amyloid Beta-ptotein Immunocreactivity In Rat Brain After Diffuse Axonal Injury

Diffuse axonal injury(DAI) is one of the serious type of primary and closing brain injury.It is most associated with high mortality and morbidity rates, and become the hot spot in the research of brain injury at present.The characteristic of DAI is the broken neuronal axon in pathology and consciousness obstacle in clinical manifestation.The dignosis and therapy of DAI is difficult, and the prognosis is poor.With the deepening of study on diffuse axonal injury, the recent research indicate that the generation and accumulation of β -amyloid precursor protein(APP) and amyloid beta-protein(A β) increase in the neuron axon after DAI, which were considered as the mainly mechanism and the characteristic pathological marker of DAI.Axonal injury can be evaluated and dignosised early by detection of APP and A β. In recent years, it verifies that estrogen has protective effects on the central nervous system that foundation, clinic and epidemic disease are studied.Its effects show as:1.Resist the function of apoptosis:through combine with its receptor thus transform and activate the receptor, and improve it with particular target ability that combine gene together, thus induce or inhibit the duplicating of peculiar gene of target cell, and promote the expression of bcl-2 and inhibit the apoptosis of cell.2.Impact on some brain blood flow of ischemic district:estrogen can promote damaged brain rCBF to recover and reduce the effect of area of no blood flow in brain.3.Estrogen can resist the excitability amino acid toxicity and stable the solubility of calcium ion in cells.4.Resist the function of oxidation.5.Inhibit the inflammation reacts after injury, expand the blood vessel and improve the glucosemetabolism, etc.Through studying in Alzheimer's disease(AD) patient, estrogen can regulate the metabolism of APP.Schonlnecht with his researcher carry on the relation of estrogen level and the density of A P in cerebrospinal fluid between the AD petient with non AD patient find that estrogen level and the density of A P is relevant to negative.Thus verified the impact on the metabolism of A 3 by estrogen.Recent discover that estrogen can inhibit the nerve toxicity of A 3 through regulating the transfer route of activate cell signal by kinase CBut the effect of estrogen on the traumatic brain injury , especially DAI, has not been totally expounded yet at presenkTherefore we should study further by defining its brain protects mechanism and instructing clinical pratice.This text regards Marmarou's animal diffuse axonal injury model as the target, through employ estrogen treat the rats after DAI, observe the effect of estrogen on the expression of APP and A 3 immunocreactivity in rat brain after DAI, study the interreaction among DAI, APP and A P, thus explore the possible mechanism of estrogen to treat DAI and offer a new route and theoretical foundation for clinical treatment for DAI.Experiment method1.Experimental animal and dividing into groupsxhoose 130 health grow upSprague-Dawley male rats, the weight is 250-300g, raise under the diet condition offreedom(offered by animal'scentre of academy of medical sciences of ZhejiangProvince).Divide animal at random into normal control group, trauma group,treatment group. The trauma group and treatment group are divided into 12 groupsaccording to different time:l hour, 6 hours, 12 hours.24 hours, 48 hours, 72 hours.Theanimal of every group is lO.There was no significant difference among the averagevalues of body weight in all groups by ANOVAtest(p>0.05).2.Reagent:a purified rabbit's polyclone antibody of APP, A 0 mo, which have highaffinity.The antibody and SABC-POD reagent box buy from Boster company ofWuhan.3.Experiment step:(DSetting-up of the animal model:make DAI model according to the method ofMarmorou's.(2)Experiment method: 1.Normal control group:fetch 10 male rats, abdominal cavity injects 1% barbital sodium(40mg/kg), open chest, aorta intubate, cut ofT right atrium, pour into physiological saline until the liquid flow out from the right atrium to be clear, pour into 4% formaldehyde solution to fix the brain, fetch brain rapidly and cut the belly side organize of the left brain stem:2 XIX 0.5cm then fixed.Carry on the sample fixed and dehydrating, wrapping up, burying, and dyeing according to the conventional method.Slice the wax lump into coronary piece of 5 u m thickness, carry on HE dye and immunohistochemical staining of APP and A p" (the operation of SABC, DAB dye according to the method explaining in the reagent box of Boster company of Wuhan).Take out the right side brain tissue and draw the superficial liquid with the filter paper, Weigh wet weight on the electronic scale, Put the brain tissue in the electric heat while toasting the case (temperature is 100 X2) for 24 hours subsequently , call dry weight again, the water content of the braincalculated by Elliott formula:The water content of the brain%= (wet weight—dry weight) -S-wet weight X 100%.2.Trauma group and treatment group:divide 120 rats into trauma group and treatment group at random.The two groups is made DAI model according to the method of Marmarou's.The trauma group put to death in batches in 1 hour, 6 hours, 12 hours.24 hours, 48 hours, 72 hours after attacking, cut and fetch the belly side of the left brain stem and then carry on HE dye and immunohistochemical staining of APP and A p" .Take out the right side brain tissue, then weigh and calculation the water content of brain.The treatment group is injected 17 0 estradiol(E2X0-8mg/kg) in femoral vein after attacking immediately, then put to death in batches just as the same of the trauma group.4.The result judges and picture analysis:the positive standard of immunohistochemical staining of APP and A 0 is the brown particle appeared in the neuron cytoplasm.Adopt HPIAS1000 pathologic picture analyticl system, put on the slice under the optical microscope, which is been connected with computer picture analyze process system, enlarge by 40 times, orientate as belly side vertebra body bunch of brain stem, choose 3 visual fields to observe at random, the picture spreads into the computer to calculate the area percentage of the positive signal after punished by digitization. 5.Statistics:All data were expressed as x + s and analyzed with "spssl3.0 forwindows" software.Data in the group are compared by analysis of one-way ANOVAtestBecause the data is not homogeneous by analysis of homogeneity of variance test,so we adopt the method of Dunnett T3 testThe relation with APP and A P wereanalyzed by multiple linear regression.Result1 .The change of pathologic histology in HENormal control group:The pyramidal cell is clearly dyed in the brain stem area, it isintensive and neat to arrange.Trauma group:The inflectional and spiral neurons could be observed after injury,whose cytoplasm is pink, the core is dyed densly, the body of cell creases and thecells are protruding after one hour.The neuron damages time to lengthen andaggravate, the survive neuron quantity obviously reduce at the same time, the necroticneuron obvivously increases and the cell structure disappears.Treatment group.The quantity of necrotic neuron in treatment group decreaseobviously, comparing with the trauma group.The swelling cell lighten and distributein the area.2.The change of immunohistochemical staining of APP and A P?The change of APPNormal control group:We can observe several neuron cytoplasm present weakpositive reaction in the brain stem.Trauma group:We can see the brown particle presented in the neuron cytoplasm in thebrain stem under the microscope.An hour after being wounded, the positive signalarea of APP begins to be crescent, increase obviously 12 hours later, reach the peak 24hours later, fall gradually hereafter, but is still higher than the normal group.Treatment group:The positive signal area of treatment group after 6hours, 12hours,24hours, 48hours, 72hours decreased obviously, comparing with the trauma group atthe same time, there are prominent differences(P<0.01).?The change of A ￡Normal control group.The neuron cytoplasm in the brain stem is expressed on a smallquantity.Trauma group:Positive signal area begins to appear an hour after being wounded,lengthen and present the parabola change with time when being wounded, reach thepeak in 24 hours, fall gradually hereafter. The increase that AP expresses and variation tendency of time course of increasing of APP expression are similar, which present positive correlation.Treatment group:The positive signal area of treated group after 12hours, 24hours, 48hours, 72hours decreased obsiviously, comparing with the trauma group at the sametime.(P<0.01). (3)The result of water content of brain: Normal control group: (76.58 + 1.29) %.Trauma group: It rises obviously 1 hour when being wounded compared with normal group (P<0.01), the water content increases continuously, reach the peak in 24 hours, drop gradually subsequently, basically drop to the normal level in 72 hours. Treatment group:The water content of brain of treated group decreased obsiviously after 6 hours but still is higher than the normal group. Conclusion1 .The expression of APP and A P increase early in the rats after DAI, and the trend present fluctuation, which can be regarded as the biological index of measuring early brain injury.2.The expression of APP and A P after DAI is raised as time lengthens and presents the parabola change.The increase that A P expresses and variation tendency of time course of increasing of APP expression are similar, which present positive correlation and reach the peak after 24 hours.The cooperative effect of the two aggravates brain injury.3.The expression of APP and A P decrease obviously with the estrogen treatment in the 12hours, 24hours, 48hours, 72hours groups, and the secondary brain edma also lighten, which indicate that estrogen play a protective role in the treatment of DAI by inhibiting the toxic function of APP and A P, then lightening the injury effect of neuron.