| [Background]Neuonatal hypoxic-ischimic brain damage(HIBD) is fetus and newborn infant's brain damage dease by absence of oxgen and blood,it is an intercurrent disease with asphyxiation during perinatal period,it is a main reason resulting in neural sequela and resulting in newborn death,cerebral paralysis, epilepsy , mental retardation and so on.It is one of the main causes of invalidity in children and often bring a heavy burden to society and family. But its pathogenesis is not so obvious, and its clinic effect is not satisfying.So, replicating a animals' model of human HIBD,it is important to research its pathology,physiology, pathogenesis and therapy. From the studies, we can make out that the one of the reasons, to which NSCs is the most sensitive, and brain hypoxia and ischemia results in the necrosis and apoptosis of cells.We use the traditional Rice method, ligate the bilateral uterus arteries and isolate the uterus to stop blood in order to make HIBD models of the newborn rat. We make comparisons among these models by testing the survival rate, rate of the weight put on, the change of the behavior capacity, the index of pathological variation and so on to lay a foundation for the studies of the neonatal HIBD. Based on thenewborn HIBD models, we explain after the HBID, by immunohistochemistry, the culture of cells and the technology of light microscope, to provide theoretical and experimental foundation for the clinic therapy of HIBD.Hyperbaric oxygenation (HBO) is the breathing of pure oxygen in a high pressure environment. Many scholjars think HBO can raise the oxygen cotent and pressure of blood,the oxygen capacity of dispersion, improve the supply of oxygen,metabolism and microcirculation of brain, shrink cerebral blood vessel and reduce speed of blood stream, reduce the brain edema and pressure.So,HBO has significant effect on some diseases such as CO poisoning, decompression sickness, some infections caused by anaerobes, craniocerebral injury, spinal cord injury, and tissues damage by hypoxic ischemia. However, scholars have two different kinds of opinions, some of them think HBO is effective to such kind of diseases, through such mechanism; others believe that HBO can increase the production of oxygen-derived free radical, have poison effect for nerve cells, accelerate the necrosis and apoptosis of nerve cells and make the HIBD become severe. So this essay provides some theoretical bases for the clinic treatment of HIBD using HBO, by discussion of the rules of the change of the nerve stem cells. [Objectives]1. To investigate the method to make newborn rat models for HIBD by comparison of different methods,and find a simple , convenient, cheap, practical and credible method for HIBD.2. To observe the regulation of the HBO treatment for the HIBD rat model nerve cells, illustrate the mechanism of HBO treatment for the HIBD, so as to induce the multiplication and differentiation of neural stem cells(NSCs) ,to provide experimental and theoretic evidences for treating HIBD validly and applying NSCs treat HIBD at the right moment.[Methods]1. Experimental object: healthy 7-days-old Sprague-Dawley (SD) pregnant and newborn rat coincidence with the national standard of grade 2 animal provided by experimental animal center of the First Military Medical College.2. Groups: SD pregnant rats were 32 and male rats were 8,they were feeded in cage according to the proportion of 4:1 .All animals were divided into four groups in random: uterus isolated (A), uterus blood vessel ligated (B), ligating the left common carotid artery(C)and normal parturition(D). According to the difference of time-point of blood stop,A group was divided into three team:A5,A10,A15. B group was divided into three team: B5, BIO, B15. The newborn rats of C group were ligated the left common carotid artery at the 7th day and then put into in the mixed gases of N2 and O2 into hypoxic cabin during hypoxia for 2 hours, rats of every group were divided randomly into 6 groups,each including 1st, 3rd, 5th, 7th, 14th and 21st time point, according to the time to put to death.The rats at A and B groups were put to death at 1st, 3rd, 5th, 7th, 14th and 21st day and take out their brains. The rats at D group were put to death at 1st, 3rd, 5th, 7th, 14th and 21st day and 8st, 10rd, 12th, 14th, 21th and 28st days respectively,in order to match with the rats of A,B and C groups.The rats at the group of uterus blood vessel ligated 10-minutes and the rats were born successfully will accept the treatment of 2.0 ATA HBO for an hour per time per day for 7 days,and put them to death and put out their brain atlst, 3rd, 5th, 7th, 14th and 21st days after finishing the treatmentTake out 2 rats randomly,inject Brdu into abdominal cavity before putting them to death in the light of 200 mg per kilogramme.The neonatal rats were divided into 4 groups randomly, HIBD+HBO group,HIBD group, HBO group and Normal group.A group were divided into 6,48,96h group according to the temporal differnce of HBO,named them Aa, Ab,Ac. B group rats accept HBO treatment at 6-hour-old. One rat of each team were takenout for cell culture. HBO is the interventional measure, NSCs from brain tissues of rats in four groups were determined with HE staining and immunohistochemistry method under light microscope.NSCs from brain tissues of rats in four groups were isolated, cultured,passed and differentiated respectively,then the cells including cultured NSCs and differentiated cells were identified with immunocytochemistry method. [Items of observation]1. After the model-making:1.1 The numbers of the alive & dead and the mortality1.2 Behavior observation: at the 1st, 3rd, 5th, 7th, 14th and 21st day time point after the model, to observe the capacity of behavior of every rat, including: the ability to turn over, to keep balance, to crawl, and the tension of the muscle.1.3 The measurement of the body, after the model making, weigh every rat at 8 to 10 o'clock every morning and to weigh the wet net by the electronic scale.2 . After the HBO treatment:2.1 HE dying2.2 SABC immunohistochemistry method to detect the positive Nestin NSCs of SD rats' brain organism and take count of them.2.3 SABC immunohistochemistry method to detect the positive Brdu celles of SD rats' brain organism and take count of them.2.4 SABC immunohistochemistry method to detect the positive celles of Brdu and Nestin NSCS in SD rats' brain organism and take count of them.2.5 The culture of the NSCS of SD rats in vitro and identified them. [Results]1. The mortality of the newborn rats of the group A and B during the perinatal period was higher than group D. In addition, the longer the times were isolated, thehigher the mortality. The mortality of the rats group A was much higher than the other three groups at the same time point. Most of them died in 24 hours after birth, while the mortalities of the 5-minute of group B and the rats of group D were not so distinct at the certain time (p>0.05).2. For the rats of group B, the movement of the fetus increased after one minute the isolation of the blood, five minutes later the movement decreased, and at the 8th minute, there were stasis of blood on the placenta. Ten minutes later (on average 12 minutes), the placenta bled, even was separated from the uterus and resulted in the death of the pregrant rat because of missing a lot of blood.3. They display different degree anoxia symptom when they enter airtight hypoxic cabin: they appear dysphoria and rolling about after 3~5 minutes, 10 minutes later they display cyanopathy of the whole body skin, the breath become deeper and faster, they stand unsteady after 20-30 minutes, the right hind legs drag along and gradual emergence the whole body muscle vibrate, can't turn over the body, go round and round spontaneously to left or have muscular spasms, even collapsed and fainted etc. Their activity decrease obviously after 35-60 minutes,60 minutes latter or more, a few of them die. A lot of them still display circumgyratetion to the left spontaneously.4. The rats of uterus isolated group(A) and uterus blood vessel ligated (B)10, 15 minutes group,their body weight increase slowly,even their body weight don't increase.However, their weight have no difference at correspond time when B5 group compare with Dgroup (P>0.05).5. Behavioral development of A,B and C group get behind obviously D group. Their reaction is bad, irascible, the eye pupil contract or the dissymmetry, the tension of the muscle is increased or decreased,even eclampsia and death,the symptoms are more obvious in A group. However, they have no difference at correspond timewhen B5 group compare with D group(P>0.05).6. The structures of nerve cells on normal group under microscope were and clear.We found that the brain ditch deepen at 7th in A and B10,B15 group by naked eyes.HE staining we can see a large range of nerve cells were degenerated, putrescence and broken,the cell's body swell, cytoclasis and disappearance after 72 hours.The cell body contract, karyon condense to dye deeply, the structure disappear.The neural-gum-cell surroundings dropsy, cell interspace aggrandizement, accomply with putrescence and blood. The neural-gum-cells are hyperplasia and cicatrization with the disappearance of nerve cell.But the degree in D group is light.7. Tth NSCs of Nestin positive cells display brown-yellow colour , that included large or small round and elliptic, and multiangle cells were existed in brain tissues .The cells amount at 1th and 3th are more than others time point, the amount is least at 21th.The amounts of rat which accept HBO are more higher than others. The amounts of rat which accept HBO and HIBD are more less than others D group,and obviously less than HBO group,but higher than HIBD group. The quantities of cell which accept HBO at 6-hour-old are more than those at 48-hour-old and 96-hour-old.8. Tth NSCs of Brdu positive cells display brown-yellow colour ,the cell body is more bigger than other cells,cell nucleus were stained into brown-yellow.The cells amount at 1th , 3th and 5th are more than others time point, the amount is least at 21th.9.The NSCs from brain tissues can success fully isolated and cultured with serum midium,they formed typical neuraospheres in suspension.These NSCs could be cloned and passed continuously,expressing nestin antigen.But the clonal neuraospheres are decreasing gradually with the progress of time. [Conclusions]Uterus isolated, uterus blood vessel ligated, ligating the left common carotid arteryand normal parturition can made animal model successfully of HIBD.But the group which uterus blood vessel ligated lasting for 10 minutes can copy thehypoxic-ischemia process of embryo, is a suitable method which copy HIBD model of newborn animalWe can culture nerve stem cells from corresponding period brains, but the NSCs decrease gradualy with the progress of time. HBO therapy on HIBD model can decrease the incidence rates and the degree of lesion of brain , relieve the injury of pathology ,stimulate the increment of the NSCs, and promote the abruption of the nerve cell, propagate. There will be a better therapy effect after hypoxic-ischemia 48 hours.
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