| 1. background,significance and problem to be solved.The etiology and pathogenesy of hypertensive disorder complicating pregnancy are hot topic inland and oversea.Especialy the pathogenesy and treatment of preeclampsia and eclampsia were the difficulty in obstetrics field. Our study set up animal models of pathogenesy of hypertensive disorder complicating pregnancy encephalopathy through exert homocysteine and glutamate to pregnancy rats. This model not only analogue hypertensive disorder complicating pregnancy's symptom and sign from the blood pressure , urine protein and damage of important organs ,but also detect the change of nerve system structure ,function and pathophisiology throuth serum immunohistochemistry immunoblotting and hybrid in situ. The study indicate exert homocysteine and glutamate to pregnancy rats can induce hypertensive disorder complicating pregnancy, especially preeclampsia and eclampsia. And the base of the disease was the change of nerve system structure and function, was induced by up-regulation exciting neurotransmitter and down-regulation inhibiting neurotransmitter. Our study investigate pathology base of preeclampsia and eclampsia,and provide new ways to genisis and development of encephalopathy. At the same time we observed the treating effect of folic acid and MK801 to hypertensive disorder complicating pregnancy encephalopathy. To provide experimental base to treat preeclampsia and eclampsia by folic acid and MK801.2. Material and method :our study divide into two parts.(1) the study about mechanism of hypertensive disorder complicating pregnancy induced by homocysteine and glutamate.No .1: Establishment of hypertensive disorder complicating pregnancy animal model and study of its mechanism. a. Groups and factorsSelectingⅡgrade Wister adult sexual matural healty male and female rats, and randomly divide into 4 groups ,each group have 10. From tenth day ,pregnant control group(PN) , pregnant homocysteine group(PH) were injected into abdomen by saline and homocysteine separately, and injected saline subcutaneuly every two days. Pregnant homocysteine plus glutamate group (PHG) and pregnant glutamate group(PG) were injected into abdomen by saline and homocysteine separately, and injected sodium glutamate subcutaneuly every two day. The dose of homosteine was 200mg/kg/d, glutamate was 1g/kg/48h, till to pregnant 20days.b. Method and index2.1 Blood pressure: detect BP from 8th pregnant day, once a day ,till delivery. 2.2 Urine protein: At 8th, 15th, 19th day, put the pregnant rats into metablic cages, and collect 24 hours urine ,at the same time detect protein quantitation .2.3 Pregnant rats and baby rats : at 20th day , anaesthesia by ether, get the fetus out by U-D, write down the body weight ,length, number of fetus ,weight of placenta, death number and fatility of futus2.4 Serum level of ALT,AST,BUN and Cr: at 20th day, detect serum level of ALT,AST,BUN and Cr by AU-10000lympus. 2.5 PLT ,NO,NOS activity in plasma: at 20th day detect these enzyme activity.2.6 Example and pathological test: microstructure of kidney and endothelial cells.c. result3.1 contrasting of systolic blood pressure PH group and PHG group were all significantly higher than PN group. PH group and PHG group were no significantly difference.3.2 contrasting of urine proteine each group urine protein of PH group and PHG group were higher than PN group, PH with PHG,PG with PN were no significantly difference.3.3 contrasting of ALT, AST, BUN, Cr: BUN,Cr of PH group and PHG group were significantly higher than PN group and PG group , AST in PHG group was significantly different with PN and PG group.3.4 contrasting of PLT,NO,NOS: PLT in PH and PHG group was significantly higher than PN and PG group , but NO and NOS were lower than PN and PG group.3.5 contrasting of fetal baby rats and placenta Body weight of fetal rats, weight of placenta and length of fetal rats were significantly lower than PN group, and death fetus was increased , no significant abnormity by naked eye.3.6 pathohistological and electron microscope observation3.6.1 Microstructure change of kidney tissue: renal tubule cell was damaged in PHG group, nucleus apomorphosis, proximal convoluted tubule microvili destroyed, chondrosome engorged, rough endocytoplasmic reticulum dilated. Renal tubule in PH group was damaged acutely. No significantly different with PHG group. 3.6.2 Microstructure change of endothelial cells: endothelial cells in PHG group irregular, which were short of organ in cytoplasm and abundant of collagen fibers. endothelial cells in PHG group irregular too, also short of organ in cytoplasm,rich of euchromatin and short of heterochromatin.No.2 : study about mechanism of hypertensive disorder complicating pregnancy enthaphalopathy1. Goups and factors are the same with No.1.2. Detecting ways2.1 Ethology observation2.2 Pathology and electron microscope observation2.3 Immuno- histochemical staining and semiqutity assay : NMDAR-1,NF-κBp65 and nervous fibroin in brain cortex and hippocampus CA1 of pregnancy rats.2.4 Detect the expression of NMDAR-1,NF-κBp65 by western-blot.2.5 Detect the mRNA expression of GABA-A and NMDAR-1 .3. Result3.1 Ethology observation The rats in PHG group were paroxysmal gazing, nutating and rythmicitic chewing accompany with prosopotic and trembling by one side limb. 3.2 Pathohistology and electron microscope observation 3.2.1 light microscope: cortex nervous cells in frontal lobe appeared retrogression, atrophy of nervous cell in PHG group. 3.2.2 Electron microscope :colloid cell of cortex were round, organs in cytoplasm were disappear, mesenchyma was edema. 3.2.3 Immuno-histochenical staining and semiqutity assay of NF200,NMDAR-1 and NF-κBp65.Immuno-histochemical staining of NF-200 display that in PHG groups marked fibers and nervous cells in frontal cortex, molecular layer and outer granular cell layer were significantly decreased.NF(+) cells in inner granular cell layer,segment cell layer and fusiform cell layer were significantly decreased.Immuno-histochemical staining of NMDAR-1 display that in PHG group expression of NMDAR-1 was increasing in cortex frontal cell.Immuno-histochemical staining of NF-κBp65 display that in PHG group a large amount of NF-κBp65 move into cellular nucleus in frontal cortex and hippocampus cells, and had significantly different with PN group.3.4 Assay of western-blot :in PHG group ,NMDAR-1 and NF-κBp65 expression were enhancing in frontal cortex and hippocampus CA1 region , which have significantly different with PN group.3.5 hybride in situ and semiquantity assay Under light microscope , expression of NMDARmRNA in PHG group were significantly enhance than PN, PG and PH group. mRNA expression of GABA-A receptor in PHG group were significantly lower than that in PN, PG and PH group.(2) The study of treatment and mechanism by folic acid and MK801 in hypertensive disorder complicating pregnancy encephathology. . No.1 The study of treatment and mechanism by folic acid and MK801 in hypertensive disorder complicating pregnancy1.Animals and groups divide animals randomly into 5 groups, A group(PN), B group (PHG), C group(PHG+F),D group (PHG+D),E group(PHG+D+F), 10 rats per group. At 10th pregnant day, the way was the same as experiment one.2.Detect method and index were the same as the experiment one.3.Result:(1)Contrasting of systolic blood pressure in tail artery. B group and D group were all increased at 12th day, which were significantly difference with A group. D group was lower than B group ,but no significantly difference.And C group and E group were all significantly lower than B group.(2)Contrasting of urine proteine each group 24 hours urine protein of B and D group were significantly increased at 15th and 19th and higer than A group.D group were lower than B group, and C,E group were significantly lower than B group.(3)Contrasting of serum ALT, AST, BUN, Cr each group Serum ALT, AST, BUN, Cr of B and D group were increased at 20th day,which were significantly higher than A group. Serum ALT, AST, BUN, Cr of D group were lower than B group,but no significantly difference ,and C and E group were significantly lower than B group.(4)Contrasting of serum PLT,NO,NOS in each group Serum PLT,NO,NOS of B and D group were increased at 20th day,which were significantly higher than A group. Serum ALT, AST, BUN, Cr of D group were lower than B group,but no significantly difference ,and C and E group were significantly lower than B group.(5)Contrasting of fetal rats and placenta There are no significantly difference in number of fetal rats, body weight of fetal rats ,weight of placenta and lenth of fetal rats in B and D group were decresed,and have significantly difference with A group. Those in D group were higher than B group,but no significantly difference ,and C and E group were significantly lower than B group.(6)Pathology and electron microscopeThe change of renal microstructure: renal tubule epithelial cells and cellular organ in E and C group were normal. Neuclus in renal tubule were completed, mitochondria id exist.renal tubule in B and D group were damaged. The change of thorax artery endothelial cells: Euchromatin and heterochromatin distribute uniformity in E and C group. There are chondrosome RER and pinocytisis. There were short of cellular organ. Euchromatin were more and heterochromatin were little. No.2 The study of treatment and mechanism by folic acid and MK801 in hypertensive disorder complicating pregnancy encephathology.1..Animals ,group and modle:the same as experiment 2 , NO 1.2.Detect index: Ethology observation, immuno-histochemical, immuno blot and hybrid in situ staining were the same as experiment 2 , NO 1.3. result:3.1 Ethology observation The rats in B group were paroxysmal gazing, nutating and rythmicitic chewing accompany with prosopotic and trembling by one side limb.And E group were normal.3.2 Pathohistology and electron microscope observation3.2.1 light microscope: cortex nervous cells in frontal lobe appeared retrogression, atrophy of nervous cell in B group.3.2.2 Electron microscope :colloid cell of cortex were round, organs in cytoplasm were disappear, mesenchyma was edema in B group, colloid cell of cortex were normal round, a was edema in B group.Euchromatin and heterochromatin distribute uniformity, chondrosome and RER in cytoplasm.3.3 Immuno-histochenical staining and semiqutity assay of NF200,NMDAR-1 and NF-κBp65.Immuno-histochemical staining of NF-200 display that in B groups marked fibers and nervous cells in frontal cortex, molecular layer and outer granular cell layer were significantly decreased. A large mount of random nerve fibers in frontal cortex E group. marked nerve fiber in cortex fontal lobe appear trabe tapes and verge to the ecphyma near the cell, all these cells arranged in a crisscross pattern, distribute uniformity, texture clear.Immuno-histochemical staining of NMDAR-1 display that in B group expression of NMDAR-1 was increasing in cortex frontal cell,staining deeper ,number of cells increased, and member was deeper than plasma. Cortex fontal lobe nerve cells in E group , NMDAR-1 immunoreactor distribute uniformity, line around the cells.Immuno-histochemical staining of NF-κBp65 display that NF-Κb: cortex frontal lobe and nerve cells in hippocampus in B group can see NF-Κb move into nucleus,and highly expressing. But nearly expressing in cytoplasm in E group.3.4 Assay of western-blot :in B group ,NMDAR-1 and NF-κBp65 expression were enhancing in frontal cortex and hippocampus CA1 region , which have significantly different with A group.3.5 hybride in situ and semiquantity assay expression of NMDARmRNA in B group were significantly enhance ,There are significance between A and B, E and B group.mRNA expression of GABA-A receptor in B group were significantly down-regulating than that in A group. There are significance between E and B. 4. Conclution:(1) Homocysteine could make NO and NOS decreased , contract the blood vessel, BP increased, urine protein increased, critical organ damaged by destroying endothelium, and the symptom were the same as hypertensive disorder complicating pregnancy in human .(2) Glutamate can not induce the change ofhypertensive disorder complicating pregnancy and nervous system damaged.(3) Cooperation effect of Homocysteine and Glutamate can induce the change of hypertensive disorder complicating pregnancy and nervous system damaged , which can analysis the model of hypertensive disorder complicating pregnancy.(4) Homocysteine can induce the damagement of liver and kidney, especially the kidney ,which was the characteristic change of hypertensive disorder complicating pregnancy.(5) Cooperation of homocysteine and glutamate can induce the fetal rats growth disorder,especially IUGR.(6) Cooperation of homocysteine and glutamate can induce high expression of exciting nerve transmitter and lower expression of inhibiting nerve transmitter. in order to induce epilapsia seizure(7) Cooperation of homocysteine and glutamate can induce NF-κB increased through destroying NF-200, priming transcription system, up-regulating NMDAR-1 in order to induce epilapsia seizure.(8).folic acid can treat hypertensive disorder complicating pregnancy .through decrease homocysteine in serum.(9).NK801 act as glutame antagonism incompetition can effectively protect brain tissue through inhibiting calium overload to treat hypertensive disorder complicating pregnancy encephalopathy.
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