| [Background and Purpose]Neonatal hypoxic-ischemic brain damage(HIBD) is a perinatal disease as a result of hypoxia/ischemia of any cause and reduction or discontinuation in cerebral blood flow in neonates or fetus.The neonatal HIBD may cause metal impairment, seizures, motor deficits, cognize and learning disability. Neonatal HIBD is always associated with high rate of morbidity and mortality, and it is a heath concern highlighted all over the world.Previous reviews described that central nervous system(CNS) could not be restored once it was damaged. But in recent years, the study on neural stem cells(NSCs) have found that endogenous NSCs reside in two main locations throughout life in the brain:the subventricular zone (SVZ) lining the lateral ventricles, and the hippocampal dentate gyrus (DG), and the two areas are called nerve regeneration zone. And the germinal zones of NSCs proliferation, differentiation and self-repair occur after brain injury. However, the potential for self-repair of the brain is limited. If we can find some methods to promote the proliferation of NSCs will bring hope in the treatment of neonatal HIBD.Currently, the treatment on neonatal HIBD is still based on "three maintain" and "three symptomatic".Acute period symptom may get some relief by the basic care, but neurological sequelae can not be avoided.It is a common concern that we should find more effective treatment. In recent years, along with the deepening study of the pathogenesis of neonatal HIBD, people have found that the pathogenesis of neonatal HIBD is related to cytotoxicity of excitatory amino acids, oxidative stress and free radical damage,calcium overload and apoptosis, and some therapeutic measures appear accordingly. At present, there are several ways of approaching the problem such as Mild hypothermia, HBO therapy, Neural stem cell transplantation and Neuroprotective drugs. At present, mild hypothermia is considered to be the most effective method in the treatment of neonatal HIBD, and it has now been standard of care for treatment of HIBD in neonates. Efficacy of other treatments are required to be further extensive evaluated.Hyperbaric oxygenation (HBO) therapy is the medical use of 100% oxygen at a level higher than atmospheric pressure.It has significant therapeutic effect in adult animals and adult brain injury models. But due to the great difference in neonates and adults physiologically, the HBO therapy in neonates still exists controversy at home and abroad. Although many studies have proved that HBO exerts neuroprotective effects through a variety of mechanisms, and some researchers reported the HBO is an effective therapy for neonatal brain damage. However, due to inconsistent research methods, there is still lack of sufficient evidence.Nerve growth factor (NGF), the first type of typical neurotrophic factor to be described and studied, has been found to exert anti-oxidative effect, anti-apoptotic effect,inhibit the cytotoxicity of excitatory amino acids, and stabilize the intracellular calcium concentration.It can protect neurons and prompt the damaged nerve regeneration. It has been proved NGF can pass through the blood-brain barrier, and provides the possibility of exogenous NGF in the treatment of neonatal HIBD.However, its exact curative effect need to be further study.Nestin is an intermediate filament protein which is known as the marker of embryonic NSCs. Its expression decreased and disappeared soon after birt,It is only expressed in a few parts of keeping neurogenesis function. Nestin can be re-expression after brain injury, accompanied by infiltration of inflammatory cells, edema and tissue necrosis. It is a sensitive marker of reflection damaged central nervous system,and it may reflect the activation of endogenous NSCs.5-bromodeoxyuridine (BrdU) is a thymidine analogue. Served as a synthetic nucleoside, it can be incorporated into the newly synthesized DNA during the S phase of cell cycle, and can be kept for long periods in nucleus DNA. BrdU antibody does not cross react with thymine. We can observe that BrdU incorporation in the case of intracellular after immunohistochemical staining. Less than 1% of synthesis of DNA cells can also be detected. BrdU is also the ideal indicator that reflect regeneration of the nerve cell proliferation.The Morris water maze (MWM) experiment is a standard test of spatial learning and reference memory. We can judge experimental animal memory function by the analysis of the search time and the route using the platform in the water. Foot-fault test and limb-placing test are commonly used in sensorimotor function test. Morris water maze test and sensorimotor function test are used as the index of neurobehavioral evaluation in rats.The HIBD model was successfully constructed with 7-day-postnatal rate and widely used to investigate the mechanism underlying HIBD development in neonates. In this study, we constructed the HIBD model with Sprague-Dawley rats. Combined with BrdU (5-Bromo-2’-deoxyuridine) labeling technology, we assessed the Nestin and BrdU positive cells to trace the proliferation of NSCs. The learning and memory disability of HBO and NGF treatment rats were evaluated by MWM test, and sensorimotor function were measured by foot-fault test and limb placing test.The purpose of this work was to explore the therapeutic effect of HBO and NGF on HIBD neonates.[Methods]The HIBD model was made in one hundred and sixty seven-day-old Sprague-Dawley rats by ligation of right common carotid artery followed by 8%02+92%N2 for two hours. And then they were randomly divided into four groups equally:Control group of HIBD, HBO treated group, NGF treated group and NGF +HBO treated group. The other fourty rats, whose right common carotid artery were isolated but not ligated and they were no exposure to hypoxia, were taken into the sham operation group. Before every rat was sacrificed,5-bromodeoxyuridine (BrdU) was injected intraperitonally three times in order to label newly generated neurocytes. Expressions of Nestin, BrdU were examined with immunohistochemical staining and image quantitative analysis at 4,7,14,21 days after the operation. Pathological change of brain was investigated with microscope. Eight rats of each group’s were evaluated spatial cognitive capability by using the MWM at the age of 30 days after birth, and this experiment last six days. And sensorimotor function were measured by foot-fault test and limb-placing test at the age of 42 days after birth.[Results]1. Behavior of experimental animalsThe behavior of all neonatal rats was normal before operation.Sham operation group demonstrated no abnormal behavior,but after experiencing serious hypoxia,the rats of HIBD groups showed obvious dystrophy.2. Pathological change of brainThere were no changes in the sham operation group. A clear hierarchy of brain tissue, hippocampal pyramidal cells were multilayered distribution, arranged in neat rows, normal cell outline, nuclear center, nuclear stained bluish purple, clear nucleolus, cytoplasm pink. In the control group of HIBD, rats lesions mainly in the cortex and hippocampus CA1 and CA3 regions, and the hippocampus pyramidal cell layers were decreased and the cells arranged in disorder.The gliacyte proliferation at day 4 post hypoxia-ischemia(HI),inflammatory cell infiltration, neuronal degeneration, nuclear condensation was strongly basophilic, narrow part of the nerve cell body, blurred or disappeared structure was shaped voids or cavities to form, in addition visible swelling of endothelial cells, perivascular space to expand, and parts of neurocytes were karyopycnosis and nuclear fragmentation. After 7 days post HI the gliacyte proliferation obvious than before, a large number of neurons in the brain tissue necrosis, disintegration, mesh and cable strip scar formation.The glial scars were formed and large quantities of neurons were lost at 14-21 days post HI. These damages in HBO treated group, NGF treated group and HBO+NGF treated group were milder than those in control group of HIBD. The pyramidal cells arranged loosely and reduced unconspicuously, the phenomena of karyopyknosis and fragmentation were relatively less. Compared with the HBO treated group and NGF treated group,the lesion was alleviate after NGF+HBO combination therapy.3. Expression of NestinThe expression of Nestin in hippocampal dentate gyrus (DG) region of rats reached peak at day 4,and gradually decreased at day 7 to day 14, decreased significantly at day 21 after the operation in five groups, the difference was significant (P<0.01).The number of Nestin-positive cells in hippocampal DG region of control group of HIBD was increased obviously compared with the sham operation group at all time points (P<0.05). The Nestin-positive cells in hippocampal DG region of three treated group were more than those of the sham operation group and the control group of HIBD, there was a significant difference at all points (P<0.01). The Nestin-positive cells in hippocampal DG region of the NGF treated group were increased than those of the HBO treated group slightly at all time points, but there was significant difference between two groups only at day 21 (P<0.05). The Nestin-positive cells in hippocampal DG region of the NGF+HBO treated group were increased than those of the HBO treated group slightly at all time points, but there was a significant difference between two groups only at day 4 (P<0.01) and day 21 (P<0.05).The Nestin-positive cells in hippocampal DG region of the NGF+HBO treated group were increased than those of the NGF treated group slightly at all time points, but there was a significant difference between two groups only at day 4 (P<0.05)4. Expression of BrdUThe expression of BrdU in hippocampal dentate gyrus (DG) region of rats reached peak at day 4, and gradually decreased at day 7 to day 14, decreased significantly at day 21 after the operation in five groups. There was significant difference in the number of BrdU positive cells between different time points (P<0.01). The number of BrdU-positive cells in hippocampal DG region of control group of HIBD was increased compared with the sham operation group at all time points, and the BrdU-positive cells in hippocampal DG region of three treated group were more than those of the sham operation group and the control group of HIBD, there was a significant difference in each group at all points (P<0.01). The BrdU-positive cells in hippocampal DG region of the NGF treated group and NGF+HBO treated group were increased significantly than those of the HBO treated group at all time points (P<0.01). The BrdU-positive cells in hippocampal DG region of the NGF+HBO treated group were increased than those of the NGF treated group slightly at all time points, but there was no significant difference between two groups (P>0.05).5. Water maze test5.1 Place navigation testThe latency to reach the escape platform was significantly longer in HIBD control group than in other four groups (P<0.01).The latency to reach the escape platform was significantly longer in HBO treated group than in sham operation group, but there was significant difference between two groups only at day 1 and day 5 (P<0.05).The latency to reach the escape platform was significantly longer in NGF treated group and NGF+HBO treated group than in sham operation group,there was a significant difference at different time points except at day 2 (P<0.05 or P <0.01).The latency to reach the escape platform was no obviously change among the three threated groups (P>0.05).There was a gradual downward trend of escape latency of rats in each group with age and frequency of swimming increased (P<0.01).5.2 Spatial probe testThe frequency of passing the platform of control group was less than that of other four groups (P<0.01).The frequency of passing the platform was not significantly change among the three threated groups (JP>0.05).The frequency of passing the platform of sham operation group was higher than that of three treated groups, but there was no significant difference in each group (P>0.05).6. Sensorimotor function test6.1 Foot-fault testThe difference of left-right side in the foot-fault test of HIBD control group was larger than that of other four groups (P<0.01).The difference of left-right side in the foot-fault test was not significantly change among the three threated groups (P >0.05).The difference of left-right side in the foot-fault test of sham operation group was less than that of three treated groups, but there was no significant difference in each group (P> 0.05).6.2 Limb-placing testThe difference of left-right side in the limb-placing test of HIBD control group was larger than that of other four groups (P<0.01).The difference of left-right side in the limb-placing test was not significantly change among the three threated groups (P>0.05).The difference of left-right side in the limb-placing test of sham operation group was less than that of HBO treated groups(P<0.01). The difference of left-right side in the limb-placing test of sham operation group was less than that of NGF treated group and NGF+HBO treated group (P<0.05).[Conclusion]1. HBO treatment, NGF treatment and NGF+HBO treatment could mitigate the degeneration and necrosis of the neurocytes in hippocampal region of neonatal rats with HIBD.2. The expression of Nestin, BrdU increased in the brain of neonatal rats after HIBD, it may be concerned with the regeneration of neurocytes and activation of neural stem cells after HIBD.3. HBO treatment, NGF treatment and NGF+HBO treatment could enhance the expression of Nestin, BrdU in the brain of neonatal rats after HIBD, and could promote the nerve regeneration after hypoxic ischemic brain injury.4. HBO treatment, NGF treatment and NGF+HBO treatment could improve the ability of learning and memory and sensorimotor function in neonatal rats after HIBD. |
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