According to recent research, brain injury after premature birth often includes impaired growth of the cerebellum. However, causes of cerebellar injury in this population are poorly understood. In this study, we analyzed whether postnatal hyperoxia perturbs white matter development of the cerebellum, and whether cerebellar glial damage can be prevented by minocycline. We used a hyperoxia model in neonatal rats providing 24h exposure to 4-fold increased oxygen concentration (80% O2) from P6 to P7, followed by recovery in room air until P9, P11, P15, P30. Injections with minocycline were performed at the beginning and 12h into hyperoxia exposure. Hyperoxia induced oxidative stress in the cerebellum at P7 as evidenced by increased nitrotyrosine concentrations. Numbers of proliferating, NG2+Ki67+ oligodendroglial precursor cells were decreased at P7 after hyperoxia and at P11 following recovery in room air. Numbers of mature, CC1+ oligodendrocytes were diminished in recovering hyperoxia rats, and MBP expression was still decreased at P30. Electron microscopy analysis of myelinated fibers at P30 revealed thinner myelin sheath after hyperoxia. Long-term injury of the cerebellum by neonatal hyperoxia was confirmed by reduced volumes in MRI measurements at P30. In response to 80% O2, expression of PDGF-A was largely reduced in cerebellar tissue and also in cultured cerebellar astrocytes. Treatment with minocycline during hyperoxia prevented oxidative stress, attenuated oligodendroglial injury, and improved astroglial PDGF-A levels. In conclusion, early hyperoxia causes white matter damage in the cerebellum with astroglial dysfunction being involved, and both can be prevented by treatment with minocycline.

Impaired postnatal brain development of preterm infants often results in neurological deficits. Besides pathologies of the forebrain, maldeveolopment of the cerebellum is increasingly recognized to contribute to psychomotor impairments of many former preterm infants. However, causes are poorly defined. We used a hyperoxia model to define neonatal damage in cerebellar granule cell precursors (GCPs) and in Purkinje cells (PCs) known to be essential for interaction with GCPs during development. We exposed newborn rats to 24 h 80% O from age P6 to P7 to identify postnatal and long-term damage in cerebellar GCPs at age P7 after hyperoxia and also after recovery in room air thereafter until P11 and P30. We determined proliferation and apoptosis of GCPs and immature neurons by immunohistochemistry, quantified neuronal damage by qPCR and Western blots for neuronal markers, and measured dendrite outgrowth of PCs by CALB1 immunostainings and by Sholl analysis of Golgi stainings. After hyperoxia, proliferation of PAX6+ GCPs was decreased at P7, while DCX + CASP3+ cells were increased at P11. Neuronal markers Pax6, Tbr2, and Prox1 were downregulated at P11 and P30. Neuronal damage was confirmed by reduced NeuN protein expression at P30. Sonic hedgehog (SHH) was significantly decreased at P7 and P11 after hyperoxia and coincided with lower CyclinD2 and Hes1 expression at P7. The granule cell injury was accompanied by hampered PC maturation with delayed dendrite formation and impaired branching. Neonatal injury induced by hyperoxia inhibits PC functioning and impairs granule cell development. As a conclusion, maldevelopment of the cerebellar neurons found in preterm infants could be caused by postnatal oxygen toxicity.

Although brain injury of preterm infants has widely been ascribed to the cerebrum, recent studies demonstrate that injury of the cerebellum often occurs, too. However, mechanisms of the cerebellar injury are hardly understood. In general, hypoxia-ischemia, infection/inflammation, and hyperoxia are supposed to be toxic stimuli in the immature brain. We used a hyperoxia model established in rats by using 80% O2 exposure for 24 h from P6 to P7, and determined development and maturation of oligodendroglial cells in the cerebellum during recovery in room air until P30. Expression of platelet derived growth factor (PDGF)-A as a crucial factor for oligodendroglial development was determined in cerebellar tissue and in cultures of cerebellar astroglia. Volume of the cerebellum measured by MRI was significantly reduced in P30 rats after hyperoxia as compared to control litters (208 vs. 232 µL respectively, p < 0.05). Myelination measured by MBP in immunostainings and Western blots was significantly reduced during recovery from P9 to P30. Numbers of NG2+TUNEL+ oligodendroglial precursor cells were increased by hyperoxia, and oligodendroglial maturation towards CC1+ stages was significantly diminished. Expression of PDGF-A was reduced after hyperoxia by 46% at P7 and by 27% at P30 (n = 4; p < 0.01 each). In astrocyte cultures, exposure to 80% O2 also caused a significant downregulation of PDGF-A expression by 42% (n = 6; p < 0.01). Hyperoxia resembles critical features of the cerebellar pathology found in preterm infants. Astroglial changes caused by hyperoxia include reduced PDGF synthesis and are likely to contribute to oligodendroglial damage in the cerebellum.