| Objective:White matter damage, as the predominant form of cerebral injury in premature infants, is the leading cause of neurodevelopmental impairment. It considerably contributes to neonatal mortality and long-term neurological deficits. The upstream pathogenesis of white matter damage is mainly related to hypoxia-ischemia (HI) and infection/inflammation in sick premature infants. Premyelinating oligodendrocyte has been confirmed to be the main target cell of white matter injury. Unfortunately, there is currently no promising intervention for white matter injury in preterm neonates. Ethyl pyruvate (EP), a stable derivative of pyruvate, has been shown to be therapeutically effective in a series of experimental models. Its neuroprotective effects have also been investigated. Nonetheless, the influence of EP on oligodendrocytes in neonatal white matte damage after hypoxia-ischemia is still unknown. This study was designed to assess whether EP administration could protect the neonatal rat brain from white matter damage, using a postnatal day3rat model of preterm hypoxia-ischemia. And the potential mechanisms underlying the effects of EP were also evaluated.Methods:The study was conducted on postnatal day3(P3) premature rat pups. Rats were allocated randomly into three groups:control group; HI group (right common carotid artery ligation and6%O2for60min); and HI+EP group (50mg/kg, intraperitoneally, injected10min,1h and24h after HI insult). Examination of body weight was started from postnatal day3and was carried out until postnatal day17. HE staining was used to observe the pathological changes of white matter after HI insult. Immunohistochemistry and double-labeling immunofluorescence were employed to assess the injury of oligodendrocytes in different stages and the activation of inflammatory glial cells. In addition, the concentrations of tumor necrosis factor (TNF-a) and interleukin (IL-1β) at the time interval between6h and6d after HI insult were assessed by enzyme-linked immunosorbent assay (Elisa). Meanwhile, the expression of cleaved caspase-3, Bax and Bcl-2protein was analyzed by Western blot2d following HI.Results:1.EP significantly alleviated hypoxia-ischemia-induced white matte injury in premature rat brain.1) Ethyl pyruvate improved the survival rate of neonatal rats after hypoxia-ischemia, and effectively reversed the retardation of somatic growth (p<0.01).2) Pathological changes of white matter under light microscope showed rarefaction, liquefaction, activation of glial cells, and obvious lateral ventricle dilation. Ethyl pyruvate showed effective protection against these pathological changes.3) Oligodendrocytes in different stages showed obvious damage after hypoxia-ischemia, leading to the number of oligodendrocytes markedly lower than the control group (p<0.01). The expression of myelin-related protein also had marked reduction which meant hypomyelination. EP treatment significantly decreased the loss of oligodendrocytes and hypomyelination following hypoxia-ischemia insult (p<0.01).2. Ethyl pyruvate effectively inhibited the inflammatory response after hypoxia-ischemia. Compared with the control group, hypoxia-ischemia induced obvious activation of microglia and astrocytes in white matter (p<0.01), while administration of ethyl pyruvate greatly blocked such activation of glial cells (p<0.01). Meantime, ethyl pyruvate significantly reduced hypoxia-ischemia-stimulated elevations in TNF-α and IL-1β in different time points (p<0.01). 3. Ethyl pyruvate protected oligodendrocytes from apoptosis after hypoxia-ischemia. Ethyl pyruvate effectively alleviated the number of apoptotic late oligodendrocyte precursor in white matter. Results of Western blot showed that ethyl pyruvate down-regulated the expression of cleaved caspase-3and Bax (p<0.01), while up-regulated the anti-apoptotic protein’level of Bcl-2after hypoxia-ischemia exposure (p<0.01).Conclusions:1. A3-day-old premature rat model for white matter damage that was similar to the pathological changes in human preterm infants had been successfully established. Oligodendrocytes were significantly damaged and lost in this model.2. EP conferred potent protection against hypoxia-ischemia-induced white matte injury in neonatal rat brain, as well as improved the somatic growth retardation.3. The protective effect of EP was associated with its suppression of cerebral inflammatory responses, indicated by the inhibition of glial cells and the expression of inflammatory cytokines.4. The protective effect of EP could be partly ascribed to its anti-apoptotic properties induced by modulating the apoptotic death program of oligodendrocytes and apoptosis-related proteins.5. The current study helped us better understand the pathologic changes of white matter damage in premature infants and provided a potential neuroprotective agent in neonatal brain injury. |
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