Study Of Protection On Multiple Course Of Hyperbaric Oxygen Treatment In Hypoxic-ischemic Brain Damage Neonatal Rat

Hypoxic-ischemic encephalopathy (HIE) is a common but severe complication of asphyxia that occurs in the perinatal period with a series of abnormal manifestations of central nervus system. HIE is also the main reason of severe nervous sequela in children. At present, there are no effective means of repairing the hypoxic ischemic brain damage (HIBD), so it is necessary to develop a suitable therapy for HIE.The promising therapeutic methods for HIE include: (1) selective brain hypothermia, (2) hyperbaric oxygen (HBO) , and (3) stem cell transplantation.Renewal of neural stem cells (NSCs) and hyperbaric oxygen therapy (HBO) are known as more effective methods of HIE. Ever since a long time ago, people considered that the central lesion could not be recovered. But now we have found that there are many endogenous neural stem cells at subventricular zone (SVZ) and hippoacmpal dentate gyrus (DG) lifetimes. These cells can proliferate, differentiate by stimulation of much pathologic impairment [Maslov,2004] . It is found that NSCs were capable of being activated to proliferation and differentiation [Hayashi,2005]. However, the capacity for recruiting endogenous NSCs is limited. Thus, other strategies used to modify endogenous neurogensis after ischemic brain damage have been described.HBO treatment is one potential therapy that has been widely used for the treatment of neonatal diseases especially HIE for many years. A lot of literate reports confirmed the therapeutic effect of HBO in HIE [Hu, 2004; Gai, 2005] . Animal experiment also showed that HBO not only can promote the NSCs proliferate [Yu,2006], but also can promote the endogenous NSCs migrate to the lesion cerebrum and differentiate to full-grown neurons[Wang, 2008].The therapeutic window plays an important role in HBO treatment. Animal study has showed that HBO should be administered as early as possible. Our previous studies demonstrated that the protective effect of HBO was significant within 6-12 hours after HIBD [Wang , 2008]. But in clinical, many babies could not received HBO treatment so early because of instable conditions which may bring poor prognosis in HIE babies. Thus whether increasing the course of HBO treatment can lighten the brain damage of HIE under the condition of delayed therapeutic window? Our investigation was divided into the following three parts. Part I Effect of multiple course of HBO treatment on the long-lasting behavioral alterations and pathological changesin HIBD neonatal rat.Objective: To exam whether the multiple course of HBO can improve the long-lasting behavioral alterations of HIBD neonatal rat.Methods: Seven-day-old Sprague-Dawley (SD) rat pups were subjected to unilateral carotid ligation followed by 2 hours of hypoxia (8%O2). Multiple course of HBO was administered after 2 hours later (2 ATA, once daily for 7 days a course ). At 35 days, T-maze forced alternation, the radial arm maze and foot-fault tests were tested. Histological analysis such as TUNEL and Nissl were used to evaluated the neuron density of the brain tissue.Results: (1) There was great recovery of behavioral tests in the HBO group rats than the untreated HIBD group; (2) In the T-maze test, along with the increase of test days, the rate of correction in group of CON and HBO increased gradually; and in HBO groups, the rate of correction was increased along with the course of HBO; (3) In the radial arm maze test, the time of water-hunting , the average frequency of fault and repeat are all decreased gradually along with the course of HBO; (4) TUNEL staining showed that HBO groups had few TUNEL positive cells with no difference between different courses of HBO; (5) There was a significant conservation of CA1 neuron in HBO groups compared with that of HIBD group. But the neuron density among different HBO courses was indiscriminately.Conclusions: (1) Multiple course of HBO within short time window (2h) can increase the protection of long-lasting behavioral of rat along with the courses. (2) One course HBO can inhibit the apoptosis of neuron completely. Increasing the course of HBO can not enhance this protection.Part II Effect of multiple course of hyperbaric oxygen treatment onproliferation and differentiation of neural stem cells inhypoxic-ischemic brain damage neonatal ratObjective: To exam whether multiple course of HBO promotes proliferation and differentiation of neural stem cells (NSCs) in HIBD neonatal rat.Methods: Seven-day-old SD rat pups were subjected to unilateral carotid ligation followed by 2 hours of hypoxia (8%O2). Multiple course of HBO was administered after 2 hours later (2 ATA, once daily for 7 days a course). At 35 days, BrdU/nestin immunofluorescence in the injury subventricular zone (SVZ) and dentate gyrus (DG), BrdU/NSE, BrdU/GFAP immunofluorescence in the injury brain cortex and Nestin protein was detected by Western blot analysis. Results: (1) The number of BrdU⁺/nestin⁺ cells in SVZ of the HBO treatment groups increased along with the course of HBO, specially in group HBO-3; (2) There was no obvious increase of BrdU⁺/nestin⁺ cells in DG of HBO treatment groups; (3) The number of BrdU⁺/NSE⁺ cells in cortex increased along with the courses of HBO , (4) The number of GFAP⁺ cells in HIBD was higher than other groups, there are few BrdU⁺/GFAP⁺ cells in all groups; (5) Westen blot showed that the expression of nestin protein in group HBO-3 was higher than other groups.Conclusions: Multiple course of HBO treatment may promote neurogenesis of the endogenous NSCs in SVZ and neurons in cortex of HIBD neonatal rats.Part III Protective effect of multiple course of HBO treatment at different therapeutic window on hypoxic-ischemic braindamage in neonatal ratObjective: To investigate the protective effect of multiple course of HBO treatment at different therapeutic window on HIBD neonatal rats.Methods: Seven-day-old SD rat pups were divided into CON, HIBD and HBO groups. HIBD and HBO groups were subjected to unilateral carotid ligation followed by 2 hours of hypoxia (8% O₂). One to three courses of HBO was administered each after 2, 48 and 96 hours later ( 2 ATA, once daily for 7 days a course) in HBO group. TUNEL stain was administered to test the apoptosis in cortex and hippocampus, and NSE stain for the neuron number of cortex.Results: (1) The TUNEL positive cells in HIBD was higher sharply than CON group. And NSE positive cells was lower significantly than CON group. (2) Along with the prolonged therapeutic window, the inhibitory action of HBO became weaken, especially in 96H. At the same time, the number of NSE positive cells decreased along with the prolonged therapeutic window. But in 2H group, one course HBO can protect the neuron completely.Conclusions: (1) One course HBO within 2 hours after HIBD can protect the neuron completely. (2) The injury brain can be protected through increasing the courses of HBO even though delaying the therapeutic window.The main contents and conclusions of the research are summarized as following:1. HBO treatment within 2h after HIBD can attenuate brain damage. And the behavioral tests of HIBD neonatal rats can be improved gradually by the increasing of courses of HBO.2. Multiple course of HBO treatment within 2h after HIBD may promote neurogenesis of the endogenous NSCs in SVZ and neurons in cortex of HIBD neonatal rats.3. One course HBO within 2 hours after HIBD can protect the neuron completely. The injury brain can be protected through increasing the courses of HBO even though delaying the therapeutic window.