| Hypoxic-ischemic encepholopathy(HIE), due to asphyxia before confinement, is one of the biggest and most familiar diseases during the neonatal period, mortality and the rate of being disabled are high. The latest research shows that the activin (ACT) has the nerve protection action while leaving the circumstance of ketone body, and it is also beneficial to the repair of interstitial cell. The method of this research adopts animal experiment, it studies the protective effects and mechanisms of activin (ACT) in neonatal rats with hypoxic-ischemic brain damage (HIBD), it aims to provide experimental gist to the valid prevention and cure measure for further study of HIE.1 Object and method1. 1 ObjectNeonatal 60 seven-day-old Wistar rats, with average weight of (15.6 ± 3.2) g, regardless of female or male, being offered by University of Shandong Experimental Animal Center.1. 2 HIBD model preparation and grouping60 seven-day-old Wistar rats are divided into 6 groups randomly, 10 for each. Thegroups are normal control group, HIBD model group, fake surgery group and treatment group-I, treatment group-II, treatment group-III ( respectively inject into the belly cavity with high, inside, low amount of ACT)HIBD model preparation: After inhalating aether to be anesthesia ,the control group and treatment groups of HIBD rats are asepticly ligated in left side common carotidarteroy. 1 hour later put into the 3000 ml oxygenous nitrogen in 8% odour depository to anoxia for 2 hours, monitoring oxygen concentration is(8 ± 0.5)%, and control depository temperature (36 ± 1) °C . For false surgical group, after the left side neck main artery is separated, sew up the skin instantly in order not to cause the hypoxic-ischemic ( HI).1.3 CureActivin A( ACT A) is offered by the United States R& D System Company . After HI Treatment groups( I, II, III) are given lml belly cavity injection of ACT A(the thickness is respectively 0.025 % 0.005% 0.001 u g/ml ), which is 5ml/kg(the thickness is respectively 0. 25% 0.05% 0.01 u g/ml); other groups are given lml belly cavity injection of lml physiological saline, which is 5ml/kg.1.4 Specimen collectionafter HI, each one is broken necks to death( 10 for each time ) at different time(0 hs,2 hs,6 hs,24 hs,48 hs ). Then measure their left and right brain ,take left side brain tissue to do pathology check, ultrastructure observation and cell apoptosis check; before their death take their heart blood 2 mls,0.5 ml, separate the serum, conservant at -20 °C in order to check the density of FGF and TNF- a .1.5 Tissue section and pathology observationThe brain tissue is fixed with 10% formalin aqua and embed with paraffin, cut into coronal section(4 fimof thickness) continually, dyed with normal HE and observed tissue pathology change with light microscope.1.6 Ultrastructure observation with electron microscopeFrom the similar part with electron microscope, Take cortex, hippocampi round Tissue(lmm3), place them into 2.5% Glutaral aqua(0~4°C) to fix for several days, them normally fixed, embedded and dyed, observant ultrastructure with electron microscope.1.7 Serum TNF-a , FGF level examinationUsing a solid phase sandwich Enzyme Linked-Sorbent Assay (ELISA) to detect the concentration of TNF-a and FGF in serum. The TNF-a and FGF ELISA Kit are bought from French Diaclone Research; the operation procedure abided by the manual.1.8 Apoptosis examinationTaking fresh brain tissue, applying the flow cytometry, using FCM based on the DNA deal analysis, to do the apoptosis analysis, regardingappearing of the second Gl peak(apoptosis peak) as feature. The apoptosis examination elixir boxes are provided by the United States BD Biosciences Pharmingen, following the step of the manual.1.9 Effect analysis and statistics processData are expressed as mean ± standard variance ( x ± s).Use Kruskai-Wallis rank sum test and proceed with the software named as Statistica5.0. P<0.05 means that the analysis has statistics meaning.2 Effect2.1 External appearance of brain tissue , brain weight and the different weight comparison between left and right brain2.1.1 External appearance of brain tissueAfter building the mold(Oh), all the neonatal rats' brain tissue in every groups shows white and moistening, the shape and texture is normal. There is no blood, lump or other abnormality. Each time of hereafter orders, different amount of brain tissue extravasated blood from the neonatal rats of the model group and treatment groups, as can be seen without the aid of instruments. Among them, the model group is obvious and aggravate gradually; Each treatment group is weaker than model in extravasated blood, and turn for the better gradually in each time after building mold for 6hours.Among them, the inside dosage treatment group is obviously higher than the low dosage and high dosage treatment group.2.1.2 Brain weight comparisonAfter building mold(Oh), all the neonatal rats' brain tissue in every groups show not much difference in weight. Each time of hereafter orders, the normal control group always has no obvious variety with the false surgical group. While the treatment groups and model group brain weight increase gradually, among them the model group is of the most obvious; The increment of brain weight in each treatment group shows obvious difference comparing with the model group( P<0.05); Among the treatment groups,the middle dosage group shows some degree obviosity than the lower dosage and higher dosage treatment group,but not much .(P>0.05)( see table 1)2.1.3 Different weight comparison between left and right brainAfter building mold(Oh), all the neonatal rats' left and right brain tissue in every groups show not much difference in weight(P>0.05). Each time of hereafter orders, the normal control group always has no obvious variety with the false surgical group. While the treatment groups and model group difference between left and right brain weight increase gradually, among them the model group is the most obvious;The difference between left and right brain weight in each treatment group shows obviousdifference comparing with the model group( PO.01); The difference between left and right brain weight in each treatment group is not much obvious by examination. ( P>0.05)( see table 1)table 1 .- After building mold Each time of hereafter orders neonatal rats' brain weight and the different weight comparison between left and right brain (x±s)groupnumberbrain weight(g)Pidif.wt of l&r brain(g)P2normal control101.16 + 0.05*0.021 ±0.012*fake surgery101.17 + 0.08? *0.027±0.015**model101.29 + 0.11*?*0.092 ±0.063?**treatment I101.21 ±0.09****0.062 + 0.037****treatment II101.20+0.060.051 ±0.031treatment III101.22 + 0.070.063 ±0.042* normal control vs fake surgery P/>0.05, P2 >0.05** fake surgery vs model and each treatment Pi <0.05, P2 <0.01 *** model vs each treatment P,< 0.05, P2 <0.01* * * * treatment vs treatment P, >0.05, P2 >0.052.2 Pathology changesThe normal control group, false surgical group rat's cerebral cortex cells are arranged orderly, the construction of the nerve cells are complete, the nerve host has no edema, and the colloidal cells are not immerged. The fundus node neuron's struction is normal and the nerve fiber tract has not swelledoIn model groups the cerebral cortex inner part vessel has extended, nerve cells have obviously decreased, the normal alignment construction has disappeared, nerve host has obvious edema and becomes bubble. The neuron is solved to death (appears red and purple), astrocyte have swelled, parts of focal foam cells have appeared andparts of colloidal cells have hyperplasis. Under the nearly arachnoid low cavity, large quantities of inflamed cells are infiltrated like sleeve. Inside the tapetum parenchyma are many coagulative necrosis and ghost cells. Colloidal node has formed and diffuse glial cell proliferated. The nerve fiber tract has swelled and resolved?The cerebral piamater, cerebrum, cerebellum, hippocampi area, choroids plexus are normal. Under the light microscope can see the volume of neuron has contracted and the cells have wrinkled, but the quantity has not reduced obviously, the construction of the nerve cells are complete ? the nerve host has no edema, and the colloidal cells have not obvious hyperplasis. Focal foam cells are very few. Only a small amount of neuron have wrinkled and there are few ghost cells, fundus node fiber tract has not swelled2.3 Ultrastructure changesNormal control group: The nucleus of neutron are obvious, the cell machine is plenty. Rough endoplasmic reticulum(RER) and dissociated nucleosidase are scattered inside the cytoplasm, Golgi body, mitochondria, lysosome, glycogen granule etc are obvious. The colloidal cells look like eggs, the electronics density is bigger than the nucleus of neutron, the chromatin has distributed evenly, colloidal fiber is inside the cytoplasm. Observed from the dendrite and the axon, much star polygon colloidal cells adhered to the blood capillary. The axon has aligned and encapsulated by sheath; the synapse has bubble and mitochondria; the dendrite diameter is longer and contains tidy polyribosome, the end is swelled like a ball and connected the synapses, there is mitochondria int the cytoplasm. Inside the blood capillary cells the endothelial cells are connected tightly, the membrane of the endothelial cells are dissolved to each other and become natural cover construction; outside the endothelial cells there is a piece of fundus membrane, much of which is wrapped up with star polygon colloidal dendrite, there are many RBC inside the antrum.Model group: The neutron is pale, the cytoplasm is transparent; the nucleus is contracted, dissolved, the membrane is broken and the structure is disappeared; smallbubbles are formed, the mitochondria has swelled, the cell machine is sliced evenly, the membrane of the colloidal cells are dissolved and destructed., the euchromatin has overflowed like bubble and the heterochromatin has clustered ;Endoplasmic reticulum has swelled and the mitochondria is misty, the dendrite and the axon are normal. Inside the blood capillary cells the endothelial cells have swelled and the membrane has cutting vestige.High dosage treatment group: The neutron is oval, the boundary is misty, the euchromatin has reduced and endoplasm net has swelled , the mitochondria has swelled obviously. The glial cell and vessel are normal.The middle dosage treatment group: The nucleus of neutron is obvious, the cell machine is plenty. Rough endoplasmic reticulum(RER) and dissociated nucleosidase protein are scattered inside the cytoplasm, Golgi body, mitochondria, lysosome, glycogen granule etc are obvious. The colloidal cells are normal. The nucleus of them is round. The chromatin has distributed evenly. The dendrite and the axon are normal, inside the blood capillary cells the endothelial cells are normal and connected tightly.The low dosage treatment group: The neutron is pale, the cytoplasm is transparent; the nucleus is irregular, the cell machine is reduced and the mitochondria has swelled. The glial cells and vessel are normal, but not so regular as the middle dosage treatment group.The false surgery group: The neutron contain more mitochondria, endoplasmic reticulum, and glycogen granule, The nucleus of neutron is obvious, endoplasmic reticulum has not obviously swelled. The glial cells are normal. The nucleus look round, the electronics density is bigger than the nucleus of neutron, the chromatin has distributed evenly, The dendrite, axon and vessel cells are normal.2 .4 Apoptosis analysisAfter building the mold, compare each time the apoptosis mean of the rat's tissue cells, we found the mean of the false surgery group is obviously lower than themodel group and treatment groups(PO.Ol); the mean of the model group is obviously higher than the treatment groups(P<0.05); the mean of the middle treatment groups is obviously lower than the high and low treatment groups(P<0.05); the mean of the last two groups has no obvious difference.table 2 : After building mold Each time of hereafter orders neonatal rats' cells'apoptosis mean compare (x ± s)group number cells' apoptosis mean (%) Pnormal control100fake surgery100.76 ±0.09model107.46 + 0.12treatmentl105.91+0.15treatmentll103.47±0.08treatment!!!105.23 ±0.17***** fake surgery vs normal control P<0.01*** model vs each treatment P< 0.05**** treatment II vs treatment I,III P< 0.05 I vs III P >0.052.5 serum TNF-a , FGF level compareThe serum TNF-a, FGF level compare can be seen from table 3. The result shows that, the serum TNF-a level the model group is obviously higher than the normal control group and false surgery group; after given ACT, the serum TNF-a level reduced ,particularly in the treatment group II. The serum FGF level the model group is obviously lower than the normal control group and false surgery group; after given ACT, the serum FGF level increased particularly in the treatment group II. Through the examination in the treatment group II , the serum TNF-a level is obviously lower than the model group, and the FGF level higher. The two have no obvious differencewith the false surgery group and the normal control group.3 DiscussionHypoxic-ischemic encephalopathy (HIE), due to asphyxia before confinement, is one of the biggest and most familiar diseases during the neonatal period, its pathogenesis is complicated and it often cause the neonatal children's death or disorder of nervous system. So far, the treatment of neonatal HIE is still one of the problem of the confused clinical pediatrician. Although there are many methods, the effect are quarrelous and lack of objective experimental basis. After HIBD there are two parallel mechanism—cell death or survive, the two mechanism is a path to treat HIE. With the pathology development of ischemic nerve cells disorder, the method of exploring the mechanism or prevention of HIE from the molecule level and treating it with increasing the nerve protective mechanism has been widely concerned and started very well.Activin (ACT) is a sort of sugar proteinic hormone, belonging to the transforming growth factor (TGF) 3 super kindred. The research shows, ACT can participate to inflammation injury repairing respondence, a certain degree of ACT express is helpful to the proceeding of the inflammation injury repairing respondance. The out body experiment shows, ACT has nerve protection and anti- inflammation effect when leaving body, it is helpful to the survive of the neutron, to the protection of cells from nerve drug and to the repair of interstitial cell. The animal experiment shows, the tapetum injured part's ACT express increase obviously after 6 hours, this respondence is similar to growth factors. The abroad research shows, ACT can also participate the growth of neutron; the exogenous ACT can prevent the chorea in Huntingdon striatum neutron from transformation. Clarification of ACT of its action in these procession can not only deepen the comprehension of the neutron repairing mechanism, but also develop a new way to treat it.The research studies the protective effects and mechanisms of ACT to HIBDthrough animal experiment. The result shows, ACT can obviously decrease the neonatal rats HIBD brain tissue extravasated blood or lump; under the light microscope, the treatment groups show ideal reparative appearance, comparing to the model group's pathology change. The cerebral piamater, cerebrum, cerebellum , the hippocampi area, choroids plexus are normal. The quantity has not reduced obviously, the construction of the nerve cells are complete, the nerve host has no edema, and the colloidal cells have not obvious hyperplasia. Focal foam cells are very few, fundus node fiber tract has not swelled. Under the electron microscope, the treatment groups' ultrastructure breakage decreases obviously than the model groups' breakage, the dendrite, axon and colloidal cells are normal. The nucleus of neutron is obvious, The chromatin has distributed evenly, the cell machine is plenty. The check with flow cytometry shows, the mean of treatment groups is obviously lower than the model group (P<0.05); the mean of the middle dosage group is obviously lower than the higher and lower dosage treatment groups(P<0.05); the serum TNF-a level of the middle dosage group is obviously lower than the model group, and the FGF level higher. The two have no obvious difference with the normal control group and the false surgery group.The above result shows, ACT is helpful to the repair of neonatal rats' brain tissue after HIBD, the possible mechanism maybe: ? ACT can prevent the secret of acute period respond proteinic, is helpful to keep body from resulting in the damage because of responding excessively; (2) ACT has the anti-inflammation enzyme activity, can effectively restrain ischemic again the partial excessive inflammation responds in brain tissue change after ischemic ; (3) primar HIBD is cytotoxicity tapetum edema, because energy balance is exhausted, natrium, potassium- ATP enzyme has disturbance in transport the ion of natrium ,result to the formation of cell edema; ACT can alleviate the cytotoxicity tapetum edema, without aggravating the vessel source tapetum edema;? ACT has the anti-oxygenation effect, can repress the neutron and free radical aggregation inside the blood capillary after HIBD;(5)After...
|
PDF Full Text Request