The Protective Effects Of NRGβ1 On The Hypoxic Injured O2A Progenitors And Hypoxic-ischemic White Matter Damage Of Neonatal Rat

Hypoxic-ischemic brain damage is one of the main factors that might cuase children neurological injuries. It was approved that the oligodendrocyte damages and the diffuses myelination disturbances in perivericular white matter were the main pathologic changes. Neuregulin-1(NRG1) is a signaling protein among cells produced by glial cells and neurons. It plays a critical roles in the development, survival, migration and differentation of oligodendrocyte, and regulates the myelin sheath thickness. However, questions remain unknown: could NRG1 protect O2A progenitors after ischemic injury? And could NRG1 improve the hypoxic-ischemic white matter damages? In order to answer these questions, we have done as follows:Paper1: The purified cultivation and oxygen deprivated cultivation of oligodendrocyte/type-2 astrocyte progenitors from neonatal rat cerebrumObjective To obtain pure and numerous oligodendrocyte/type-2 astrocyte (O2A) progenitors from neonatal rat cerebrum. And to study the influence of oxygen and glucose deprivation on O2A progenitors.Methods O2A progenitors were isolated by twice orbital shaking and identified by immunofluorescence technique. O2A progenitors were cultured in oxygen deprivation combining with low glucose medium to mimic hypoxic injury. Electron microscope was used to study the ultrastructural changes of O2A progenitors. Trypan blue staining was used to measure O2A progenitors viability. Flow cytometry was used to measure apoptotic cells. Results O2A progenitors were small and have one or two fine processes, >90% of the total cells were A2B5-positive O2A progenitors. O2A progenitor cells had bipotential differentiation features, they could differentiate into oligodendrocytes or type-2-astrocytes according to the ingredient of the culture medium. Electron microscope showed a bundle of filament in the cytoplasma of O2A progenitor. The number of apoptotic O2A cells increased after oxygen deprivation combined with low glucose for 6 hours, and the apoptotic cells increased with time depenting (P<0.01). Most cells undergo necrosis after culturing in oxygen deprivation combined with low glucose medium for 12 hours.Conclusions 1. Proper culture medium was very import for purified cultivation of O2A progenitors. 2. Oxygen deprivation combined with low glucose on O2A cultivation can mimic the ischemic-hypoxic changes on O2A progenitor in vivo.Paper2: The effect of NRGβ1 and the role of PI3K-Akt signal on the cultured O2A progenitors after oxygen deprivationObjective To investigate the protective effect and the signal pathway of NRGβ1 in O2A progenitors after oxygen deprivation.Methods O2A progenitors were divided into 2 group, including oxygen deprivation combined with low glucose group and NRGβ1 treatment group. In NRGβ1 treatment group , NRGβ1 with 2 concentration was added at the beginning of oxygen deprivation. Trypan blue staining was used to measure cell viability. Hoechest 33258 labelling and flow cytometry was used to measure apoptotic cells. Western blotting was used to detect the expression of both Akt protein and p-Ak protein.Results Compared with the oxygen deprivated group , the survival percentage of O2A progenitors decreased significantly (P<0.05 or P<0.01), and the apoptotic cells decreased significantly (P<0.05 or P<0.01) in the NRGβ1 group. The expression of p-Akt protein was down regulated after hypoxic injury (P<0.01). After treating with NRGβ1 100ng/ml, the reduction of p-Akt was reversed (P<0.01), but this effect could be abrogated by Ly294002 (P<0.01).Conclusions NRGβ1 could protect O2A progenitors against hypoxic injury, and the mechanism may be associated with PI3K-Akt signal pathway.Paper3: The studies on model establishment and ultrapathologic changes of hypotic-ischemic white matter damage in 3-day-old neonatal ratsObjective To establish an experimental model of hypoxic-ischemic white matter damage in 3-day-old (P3) neonate rats. And to study the ultrastructure changes of oligodendrocyte cells and myelin sheaths.Methods P3 Sprague-Dawley (SD) neonatal rats were randomized into the sham operation group (n=40) and the hypoxic-ischemic (HI) group (n=40). The right common carotid artery of each pup was permanently ligated in HI group, and the rat pups were placed in airtight jars and exposed to a continuous flow of oxygen-nitrogen mixture in which the oxgen concentration was 6% for 3 hours. The physical development and neural behavior were evaluated(n=20, each). The brain samples of P14 (n=10) and P21 (n=10) rat pups were studied with HE staining and immunohistochemistry staining, ultrastructure changes of glia cells and myelin sheaths were studied by electron microscopy.Results HI insult affected physic development (including the body weight gain and eye opening) and neurobehavioral performance (cliff avoidance and anterior limb suspension), the difference were statistically significant (P<0.05 or P<0.01). It is found that 60%(6/10) of P14 and 50% (5/10) of P21 rats'right brains showed white matter rare faction at the corpus callosum and periventricular areas in HI group, immunohistochemistry staining showed MBP expression decreased in right side of rat's brains in HI group. In electron microscopy, both oncotic and apoptotic oligodendrocytes could be found in preiventricular zones. Myelin sheaths in HI injured corpus callorum of P21 rats presented looseing and focal electron-lucent of the lamellae, and their thickness was thinner than sham group (p<0.05).Conclusions The model of periventricular white matter damage (PWMD) could be established in P3 rats. Both apoptosis and oncosis of oligodendrocytes as well as both reducing synthesis and abnomal synthesis of myelin protein may contribute to HI induced PWMD.Paper4: The protective effects of NRGβ1 on white matter damage in 3-year-old neonatal ratsObjective To investigate the protective effects of NRGβ1 on periventricular white matter damage (PWMD) in P3 neonatal rats.Methods P3 Sprague-Dawley (SD) neonatal rats were randomized into the sham operation group (n=40) , the hypoxic-ischemic (HI) group (n=40), and NRGβ1 group (n=40). A previously developed neonatal rat model of white matter damage was used in this study. 2ul NRGβ1( NRGβ1 group) or 2ul NS (HI group) was injected in to the the right ventricle after artery ligation and before the hypoxic exposure. The physical development and neural behavior were evaluated (n=20). The brain samples of P21 rat pups (n=5) were studied with electron microscopy. The MBP expression in white matter of P7, P14 and P21 rat pups (n=5,each) in 3 groups were studied with western blot method.Results In electron microscopy, the damages of oligodendrocyte were alleviatived,both necrotic and apoptotic oligodendrocyte decreased in preiventricular white matter zones in NRGβ1 group. The thickness of myelin sheath of NRGβ1 group increased comparing with that of HI group. MBP expression increased along with the development of the rat pups. MBP expression in P7, P14 and P21 rat pups of HI group was lower respectively than that of sham group (P<0.01). NRGβ1 improved MBP expression in P7, P14 and P21 rat pups of HI injured group respectively, the difference was statistically significant (P<0.05). NRGβ1 improved physic development (body weight gain and eye opening) and neurobehavioral performance (cliff avoidance ) of HI injuried rat pups, the difference was statistically significant (P<0.05 or P<0.01).Conclusions NRGβ1 provided potent neuroprotection to oligodendrocyte , increased the synthesis of MBP and improved neurological functions following cerebral hypoxia-ischemia in P3 neonatal rat.