| Objective: The main aim of this study is to investigate the changes of cholesterol homeostasis and white matter development after neonatal hypoxic-ischemic brain injury, and to explore the potential mechanisms of these alterations.Hypoxic-ischemic encephalopathy (HIE) that occurs during the perinatal period is one of the most commonly recognized causes of severe, long-term neurological deficits in children, which leads to neonatal mortality and sequelae such as cerebral palsy, mental retardation, learning disability, and epilepsy. Although there is an increased understanding of its mechanisms, there is currently no clinically effective treatment for this common disorder. Therefore, further investigations to the pathogenesis of HIE are critical to developing preventive therapies.Cholesterol, a multifaceted molecule which serves as an essential component of mammalian plasma membranes, is highly enriched in the brain. Cholesterol has been implicated in several cellular functions in the developing brain. It is essential for patterning and development of the central nervous system (CNS) through the activation and propagation of hedgehog signaling, for dendrite outgrowth and axonal branching, for neurons to keep their viability, for astrocyte proliferation and for synaptogenesis. Furthermore, cholesterol is an indispensable component of myelin membranes and recently it has been suggested that cholesterol availability in oligodendrocytes is a rate-limiting factor for white matter maturation. Accordingly, it is extremely critical for the developing brains to keep their cholesterol at a homeostatic state.The first 3 weeks after birth is the time when CNS cholesterol increases most rapidly in rats, coincidentally, it is the peak time of CNS myelination, and neonatal hypoxia-ischemia (HI) also happens in this period. Since cholesterol plays an important role during myelination, the unilateral carotid ligation/hypoxia neonatal rat model was used to observe the changes of cholesterol homeostasis and white matter development after HI. As hypocholesterolemia had been correlated with high concentrations of cytokines in the early phases of critical illness, brain TNF-αand IL-6 concentrations were also detected in the present study for further investigations.Methods: Postnatal day 7 (P7) Sprague-Dawley rats were divided into three groups randomly, i.e. the naive group, the SH group and the HI injury group. Rats in the HI injury group were subjected to left common carotid artery occlusion followed by exposure to 8% oxygen for 2 hours. After body weights and brain weights were measured, brain cholesterol concentrations were acquired on P8, P9, P10, P14 and P21 using reverse-phase high performance liquid chromatography (HPLC). White matter integrity was evaluated by densitometric analysis of myelin basic protein (MBP) immunostaining on P10 and P14 and electron microscopy viewing on P21. At 12 hours post-HI, expression of TNF-αand IL-6 was also detected by enzyme-linked immunosorbent assay (ELISA).Results: Significant decreases were found in body weights on P21 (P<0.001) and in brain weights on P14 and P21 (P<0.001) of HI animals. Brain cholesterol levels decreased significantly over the first three days after HI (P<0.05). HI also elicited marked reductions in MBP immunostaining on both P10 and P14 (P<0.01), especially in the caudate putamen, external capsule, internal capsule and thalamus. Myelinated nerve fibers in the HI group animals exhibited a less number and a thinner thickness, and aberrant oligodendrocyte membrane compaction was also observed. In addition, there was a higher expression of TNF-αand IL-6 12h after the operation (P<0.05).Conclusions: 1. Neonatal rats suffered from low growth rates of body weight and brain weight after HI. 2. Cholesterol homeostasis was disputed by HI, early after the injury, brain cholesterol concentrations were significantly decreased. 3. Myelination times varied in different brain areas. The intensity and the extent of MBP positive staining reduced significantly after HI, especially in caudate putamen, external capsule, internal capsule and thalamus. 4. Selective vulnerability of white matter to HI may, at least in part, be caused by disturbed cholesterol homeostasis. 5. The unbalanced cholesterol levels may be mediated by the over-expressed proinflammatory cytokines produced in activated microglial cells.
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