| Objective:Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the most common neonatal diseases, and is one of major reasons caused neonatal acute death and chronic nervous system damage, and there is no effective treatment for HIE at present. Currently, the treatment of enhancement of neural protection mechanisms and neural repair is gradually concerned by the medical profession, and that should be a new development trend. Our study established hypoxic-ischemic brain damage (HIBD) model and gave treatment with activin A (ACT A), to observe the pathological changes of brain tissue, and to determine the expression of nestin in brain tissue at different time points, thereby to reveal the possible mechanism of neural protection and neural repair of ACT A, and develop new method for the therapy of HIE.Methods:120 seven-day-old Wistar rats with the birth weight of 14±1 gram were divided into three groups randomly (n=40 in each group):sham-operated group, hypoxic-ischemic brain damage (HIBD) group and Activin A-treated group, each group were divided into five subgroups (n=8 in each subgroup) based on different time points after HIBD (6h,24h, 3d,7d,14d). The hypoxic-ischemic brain injury (HIBD) model was established by Rice's method:Rats in sham-operated group were given median neck incision and free left common carotid artery, without hypoxic-ischemic, rats in HIBD model group and Activin A-treated group were prepared by keeping rats into hypoxic environment of 8% O2 concentration and 92% nitrogen for 2 hours after freed and ligated left common carotid artery, and rats of Activin A-treated group were instantly given single intraperitoneal injection Activin A lml (0.005μg/ml) after hypoxic-ischemic brain damage. Rats in three groups were sacrificed at different time points and brains tissue were collected. The general morphologic changes of brain tissue were obtained through macroscopic observation, the pathological changes of left brain tissue were observed under light microscope, and expressions of nestin in left brain tissue were detected with immunohistochemistry.Results:(1) Neonatal rats had various abnormal behaviors after HIBD. (2) There were various general morphologic changes of brain tissue of rats in HIBD model group at different time points, but the general morphologic changes of brain tissue of rats in Activin A-treated group were reduced. (3) HE staining:Cellular layer of brain tissue in sham operation group was distinct and compact, cellular structure was normal and there were no obvious neuron loss. In HIBD model group, the brain cortical cells of left side were swelling in different degrees at 6h,24h after HIBD, neurons arrangement was disorder, cellular gap was increasing, neurons of hippocampus was sparse. At 3d after HIBD, nerve cells necrosis was found, glial cells and inflammatory cells increased significantly. At 7d after HIBD, neurons necrosis was found in cortex and hippocampus, and at 14d after HIBD, a large number of neurons disappeared in the cortex and hippocampus, glial scar formation was found. Compared with HIBD group, at each time point, the injury degree of brain tissues in ACT A treatment group was significantly reduced, the number of survival nerve cell was more, and cell arrangement still regular. (4) Immunohistochemical staining: Positive staining mainly located in the cytoplasm and processes, nestin-positive staining was brown, fine particle deposition. There were no obvious nestin positive cell in brain tissue in sham operation group at each time point, compared with sham operation group, nestin positive cells increased gradually in brain tissue in HIBD model group after HIBDand the particle color deeper than that of sham operation group, the peak time of positive expression was 7d after HIBD, afterward positive expressions decreased gradually, the average gray value of positive cells was lower compared with sham-operated, number of positive cells was more, the differences at 24h,3d,7d and 14d time point had statistical significance (P<0.01), ACT A-positive cells expression in the treatment group increased at different time points compared with HIBD group, the difference was statistically significant (P<0.01), and the expressions of nestin positive cells were increased significantly compared with HIBD model group and sham operation group respectively at 24h,3d,7d and 14d after HIBD (P<0.01).Conclusions:(1) Via observation of capacity of neonatal rats and pathological changes of brain tissues after hypoxic-ischemic, confirmed that the hypoxic-ischemic brain damage (HIBD) model was successfully established. ACT A was effective to reduce brain damage induced by hypoxic-ischemic. (2) ACT A may be can reduce the the pathological damage after HIBD via increasing the number of neural stem cells that marked nestin and maintaining its expression at a high level, simultaneously promoting nerve reparation and nerve regeneration. The experiment provided a new treatment idea for clinical treatment and prognosis improvement of neonatal hypoxic-ischemic encephalopathy.
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