Neonatal hyperoxia in mice triggers long-term cognitive deficits via impairments in cerebrovascular function and neurogenesis.

Preterm birth is the leading cause of death in children under 5 years of age. Premature infants who receive life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD), a chronic lung disease. Infants with BPD are at a high risk of abnormal neurodevelopment, including motor and cognitive difficulties. While neural progenitor cells (NPCs) are crucial for proper brain development, it is unclear whether they play a role in BPD-associated neurodevelopmental deficits. Here, we show that hyperoxia-induced experimental BPD in newborn mice led to lifelong impairments in cerebrovascular structure and function as well as impairments in NPC self-renewal and neurogenesis. A neurosphere assay utilizing nonhuman primate preterm baboon NPCs confirmed impairment in NPC function. Moreover, gene expression profiling revealed that genes involved in cell proliferation, angiogenesis, vascular autoregulation, neuronal formation, and neurotransmission were dysregulated following neonatal hyperoxia. These impairments were associated with motor and cognitive decline in aging hyperoxia-exposed mice, reminiscent of deficits observed in patients with BPD. Together, our findings establish a relationship between BPD and abnormal neurodevelopmental outcomes and identify molecular and cellular players of neonatal brain injury that persist throughout adulthood that may be targeted for early intervention to aid this vulnerable patient population.

Podoplanin: a marker for reactive gliosis in gliomas and brain injury.

Reactive astrogliosis is associated with many pathologic processes in the central nervous system, including gliomas. The glycoprotein podoplanin (PDPN) is upregulated in malignant gliomas. Using a syngeneic intracranial glioma mouse model, we show that PDPN is highly expressed in a subset of glial fibrillary acidic protein-positive astrocytes within and adjacent to gliomas. The expression of PDPN in tumor-associated reactive astrocytes was confirmed by its colocalization with the astrocytic marker S100ß and with connexin43, a major astrocytic gap junction protein. To determine whether the increase in PDPN is a general feature of gliosis, we used 2 mouse models in which astrogliosis was induced either by a needle injury or ischemia and observed similar upregulation of PDPN in reactive astrocytes in both models. Astrocytic PDPN was also found to be coexpressed with nestin, an intermediate filament marker for neural stem/progenitor cells. Our findings confirm that expression of PDPN is part of the normal host response to brain injury and gliomas, and suggest that it may be a novel cell surface marker for a specific population of reactive astrocytes in the vicinity of gliomas and nonneoplastic brain lesions. The findings also highlight the heterogeneity of glial fibrillary acidic protein-positive astrocytes in reactive gliosis.

Histological assessment of angiogenesis in the hypoxic central nervous system.

Angiogenesis, the sprouting of new capillaries from preexisting vessels, is an integral part of both normal development and numerous pathological conditions such as tumor growth, inflammation, and stroke. The development of angiogenesis assays has been critical in understanding this process in both the context of disease and normal physiology. With the growing availability of antibodies against angiogenic markers as well as advances in microscopy and imaging analysis software, a more comprehensive assessment of the angiogenesis process is beginning to take form (Milner et al., Stroke 39:191-197, 2008; Freitas-Andrade et al., J Cereb Blood Flow Metab 32:663-675, 2012; Li et al., Glia 58:1157-1167, 2010; Dore-Duffy and LaManna, Antioxid Redox Signal 9:1363-1371, 2007). This chapter describes an in vivo method of inducing brain angiogenesis in mice by chronic exposure to mild hypoxia. In addition, a detailed procedure of quantifying angiogenesis using multiple immunofluorescent labeling of mouse brain tissue sections is also presented.

PlGF knockout delays brain vessel growth and maturation upon systemic hypoxic challenge.

In this study, we have investigated the potential role of placental growth factor (PlGF) in hypoxia-induced brain angiogenesis. To this end, PlGF wild-type (PlGF(+/+)) and PlGF knockout (PlGF(-/-)) mice were exposed to whole body hypoxia (10% oxygen) for 7, 14, and 21 days. PlGF(+/+) animals exhibited a significant ~40% increase in angiogenesis after 7 days of hypoxia compared with controls, while in PlGF(-/-) this effect only occurred after 14 days of hypoxia. No differences in pericyte/smooth muscle cell (SMC) coverage between the two genotypes were observed. After 14 days of hypoxia, PlGF(-/-) microvessels had a significant increase in fibrinogen accumulation and extravasation compared with those of PlGF(+/+), which correlated with endothelial cell disruption of the tight junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked both pericyte/SMC coverage and endothelial vascular endothelial growth factor expression, and regressed after 21 days of hypoxia. Vascular endothelial growth factor expression levels were found to be significantly lower in the frontal cortex of PlGF(-/-) compared with those in PlGF(+/+) animals during the first 5 days of hypoxia, which in combination with the lack of PlGF may have contributed to the delayed angiogenic response and the prothrombotic phenotype observed in the PlGF(-/-)animals.

VEGFR-2-mediated increased proliferation and survival in response to oxygen and glucose deprivation in PlGF knockout astrocytes.

In hypoxic/ischemic conditions, astrocytes are involved in neuroprotection and angiogenesis. Vascular endothelial growth factor (VEGF) induces angiogenesis and exhibits neuroprotective and neurotrophic properties. However, the role of placental growth factor (PlGF), a VEGF homolog, in these processes is unclear. Therefore, proliferation and survival studies were performed on PlGF knockout (PlGF-/-) and wild-type (PlGF+/+) mouse astrocytes. A significant increase in cell proliferation and survival to oxygen and glucose deprivation (OGD) was observed in PlGF-/- compared to PlGF+/+ astrocytes. Interestingly, no PlGF protein expression was detected in PlGF+/+ astrocytes and no changes in VEGF protein levels were observed between the two genotypes. Real-time PCR and immunocytochemistry showed over-expression of VEGF receptor-2 (VEGFR-2) in PlGF-/- compared with PlGF+/+ astrocytes. Confocal microscopy revealed nuclear, membrane, and cytoplasmic localization of VEGFR-2. In vivo over-expression of VEGFR-2 mRNA was also detected in PlGF-/- compared with PlGF+/+ astrocytes. Stimulation with VEGF165 resulted in increased proliferation in PlGF-/- compared with PlGF+/+ astrocytes. This effect was blocked by the VEGFR-2 antagonist, VEGF165b. The enhanced proliferation of PlGF-/- astrocytes correlated with increased phospho-extracellular-signal-regulated kinase-1/2 levels, while the resistance to OGD was independent of the phosphatidylinositol 3'-kinase/Akt pathway. These results suggest that VEGFR-2 mediates the enhanced proliferative/OGD resistant phenotype observed in PlGF-/- astrocytes.