Hypoxia-induced developmental delays of inhibitory interneurons are reversed by environmental enrichment in the postnatal mouse forebrain.

Infants born premature experience hypoxic episodes due to immaturity of their respiratory and central nervous systems. This profoundly affects brain development and results in cognitive impairments. We used a mouse model to examine the impact of hypoxic rearing (9.5-10.5% O2) from postnatal day 3 to 11 (P3-P11) on GABAergic interneurons and the potential for environmental enrichment to ameliorate these developmental abnormalities. At P15 the numbers of cortical interneurons expressing immunohistochemically detectable levels of parvalbumin (PV), somatostatin (SST), and vasoactive intestinal peptide were decreased in hypoxic-reared mice by 59%, 32%, and 38%, respectively, compared with normoxic controls. Hypoxia also decreased total GABA content in frontal neocortex by 31%. However, GAD67-EGFP knock-in mice reared under hypoxic conditions showed no changes in total number of GAD67-EGFP(+) cells and no evidence of increased interneuron death, suggesting that the total number of interneurons was not decreased, but rather, that hypoxic-rearing decreased interneuron marker expression in these cells. In adulthood, PV and SST expression levels were decreased in hypoxic-reared mice. In contrast, intensity of reelin (RLN) expression was significantly increased in adult hypoxic-reared mice compared with normoxic controls. Housing mice in an enriched environment from P21 until adulthood normalized phenotypic interneuron marker expression without affecting total interneuron numbers or leading to increased neurogenesis. Our data show that (1) hypoxia decreases PV and SST and increases RLN expression in cortical interneurons during postnatal cortical development and (2) enriched environment has the capacity to normalize the interneuron abnormalities in cortex.

Age-dependent fate and lineage restriction of single NG2 cells.

NG2-expressing glia (NG2 cells, polydendrocytes) appear in the embryonic brain, expand perinatally, and persist widely throughout the gray and white matter of the mature central nervous system. We have previously reported that NG2 cells generate oligodendrocytes in both gray and white matter and a subset of protoplasmic astrocytes in the gray matter of the ventral forebrain and spinal cord. To investigate the temporal changes in NG2 cell fate, we generated NG2creER™BAC transgenic mice, in which tamoxifen-inducible Cre is expressed in NG2 cells. Cre induction at embryonic day 16.5, postnatal day (P) 2, P30 and P60 in mice that were double transgenic for NG2creER™BAC and the Cre reporter revealed that NG2 cells in the postnatal brain generate only NG2 cells or oligodendrocytes, whereas NG2 cells in the embryonic brain generate protoplasmic astrocytes in the gray matter of the ventral forebrain in addition to oligodendrocytes and NG2 cells. Analysis of cell clusters from single NG2 cells revealed that more than 80% of the NG2 cells in the P2 brain give rise to clusters consisting exclusively of oligodendrocytes, whereas the majority of the NG2 cells in the P60 brain generate clusters that contain only NG2 cells or a mixture of oligodendrocytes and NG2 cells. Furthermore, live cell imaging of single NG2 cells from early postnatal brain slices revealed that NG2 cells initially divide symmetrically to produce two daughter NG2 cells and that differentiation into oligodendrocytes occurred after 2-3 days.

Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity.

NG2 cells (also known as polydendrocytes) are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes and microglia. They can be identified by the expression of the proteoglycan NG2, have a highly branched morphology and are distributed throughout the grey and white matter. They differentiate into oligodendrocytes in vitro and have often been equated with oligodendrocyte precursor cells. However, whether polydendrocytes are multipotential cells that can give rise to neurons and astrocytes as well as oligodendrocytes is now highly debated. Furthermore, electrophysiological studies indicate that polydendrocytes receive synaptic input from neurons, suggesting that they are integrated in the neural network. This Review highlights recent findings and unresolved questions related to the lineage and function of polydendrocytes in the CNS.

Environmental enrichment increases the GFAP+ stem cell pool and reverses hypoxia-induced cognitive deficits in juvenile mice.

Premature children born with very low birth weight (VLBW) can suffer chronic hypoxic injury as a consequence of abnormal lung development and cardiovascular abnormalities, often leading to grave neurological and behavioral consequences. Emerging evidence suggests that environmental enrichment improves outcome in animal models of adult brain injury and disease; however, little is known about the impact of environmental enrichment following developmental brain injury. Intriguingly, data on socio-demographic factors from longitudinal studies that examined a number of VLBW cohorts suggest that early environment has a substantial impact on neurological and behavioral outcomes. In the current study, we demonstrate that environmental enrichment significantly enhances behavioral and neurobiological recovery from perinatal hypoxic injury. Using a genetic fate-mapping model that allows us to trace the progeny of GFAP+ astroglial cells, we show that hypoxic injury increases the proportion of astroglial cells that attain a neuronal fate. In contrast, environmental enrichment increases the stem cell pool, both through increased stem cell proliferation and stem cell survival. In mice subjected to hypoxia and subsequent enrichment there is an additive effect of both conditions on hippocampal neurogenesis from astroglia, resulting in a robust increase in the number of neurons arising from GFAP+ cells by the time these mice reach full adulthood.

Cortical glial fibrillary acidic protein-positive cells generate neurons after perinatal hypoxic injury.

Glial fibrillary acidic protein-positive (GFAP(+)) cells give rise to new neurons in the neurogenic niches; whether they are able to generate neurons in the cortical parenchyma is not known. Here, we use genetic fate mapping to examine the progeny of GFAP(+) cells after postnatal hypoxia, a model for the brain injury observed in premature children. After hypoxia, immature cortical astroglia underwent a shift toward neuronal fate and generated cortical excitatory neurons that appeared synaptically integrated into the circuitry. Fate-mapped cortical GFAP(+) cells derived ex vivo from hypoxic, but not normoxic, mice were able to form pluripotent, long-term self-renewing neurospheres. Similarly, exposure to low oxygen conditions in vitro induced stem-cell-like potential in immature cortical GFAP(+) cells. Our data support the conclusion that hypoxia promotes pluripotency in GFAP(+) cells in the cortical parenchyma. Such plasticity possibly explains the cognitive recovery found in some preterm children.

NG2 cells are not a major source of reactive astrocytes after neocortical stab wound injury.

NG2 cells are an abundant glial cell type in the adult brain. They are distinct from astrocytes, mature oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes and a subpopulation of protoplasmic astrocytes in the ventral forebrain during development. To determine whether NG2 cells generate reactive astrocytes in the lesioned brain, stab wound injury was created in adult NG2creBAC:ZEG double transgenic mice, in which enhanced green fluorescent protein (EGFP) is expressed in NG2 cells and their progeny, and the phenotype of the EGFP(+) cells was analyzed at 10 and 30 days post lesion (dpl). The majority (>90%) of the reactive astrocytes surrounding the lesion that expressed glial fibrillary acidic protein (GFAP) lacked EGFP expression, and conversely the majority (>90%) of EGFP(+) cells were GFAP-negative. However, 8% of EGFP(+) cells co-expressed GFAP at 10 dpl. Most of these EGFP(+) GFAP(+) cells were morphologically distinct from hypertrophic reactive astrocytes and exhibited weak GFAP expression. NG2 was detected in a fraction of the EGFP(+) GFAP(+) cells found at 10 dpl. By 30 dpl the number of EGFP(+) GFAP(+) cells had decreased more than four-fold from 10 dpl. A similar transient appearance of EGFP(+) GFAP(+) cells with simple morphology was observed in NG2creER™:ZEG double transgenic mice in which EGFP expression had been induced in NG2 cells prior to injury. NG2 cell-specific deletion of the oligodendrocyte lineage transcription factor Olig2 using NG2creER™:Olig2(fl/fl) :ZEG triple transgenic mice did not increase the number of EGFP(+) reactive astrocytes. These findings suggest that NG2 cells are not a major source of reactive astrocytes in the neocortex.

Enriched environment increases neural stem/progenitor cell proliferation and neurogenesis in the subventricular zone of stroke-lesioned adult rats.

BACKGROUND AND PURPOSE: The subventricular zone in the adult brain is identified as an endogenous resource of neuronal precursors that can be recruited to adjacent lesioned areas. The hypothesis was tested that postischemic environmental enrichment might enhance subventricular zone cell genesis. METHODS: A cortical infarct was induced in adult spontaneously hypertensive rats by ligating the middle cerebral artery distal to the striatal branches, after which animals were housed in either standard or enriched environment and allowed to survive for 5 weeks. The thymidine analogue bromodeoxyuridine was administered during the first postischemic week. The generation of neural stem/progenitor cells and neuronal precursors in the subventricular zone were studied with cell specific markers such as Ki67 and phosphorylated histone H3 (cell proliferation), Sox-2 (neural stem/progenitor cells), bromodeoxyuridine (slowly cycling, nonmigratory putative neural stem cells), and doublecortin (newborn immature neurons). RESULTS: Proliferating cells in the subventricular zone were identified as chiefly neural progenitors but also putative neural stem cells and neuronal precursors. Five weeks after stroke, proliferation in the subventricular zone was lower in stroke-lesioned rats housed in standard environment compared with nonlesioned rats. Postischemic environmental enrichment normalized cell proliferation levels, increased the numbers of putative neural stem cells as assessed with bromodeoxyuridine, and increased doublecortin-positive neuroblasts, which extended in migratory chains toward the infarct. CONCLUSIONS: Enriched environment increased the neural stem/progenitor cell pool and neurogenesis in the adult subventricular zone 5 weeks after a cortical stroke. This might be of potential importance for tissue regeneration.

Contribution of maternal oxygenic state to the effects of chronic postnatal hypoxia on mouse body and brain development.

1-2% of live births are to very low birth weight, premature infants that often show a developmental trajectory plagued with neurological sequelae including ventriculomegaly and significant decreases in cortical volume. We are able to recapitulate these sequelae using a mouse model of hypoxia where early postnatal pups are exposed to chronic hypoxia for one week. However, because the timing of hypoxic exposure occurs so early in development, dams and pups are housed together in the hypoxic chamber, and therefore, dams are also subjected to the same hypoxic conditions as the pups. To understand the relative contribution of hypoxia directly on the pups as opposed to the indirect contribution mediated by the effects of hypoxia and potential alterations in the dam's care of the pups, we examined whether reducing the dams exposure to hypoxia may significantly increase pup outcomes on measures that we have found consistently changed immediately following chronic hypoxia exposure. To achieve this, we rotated dams between normoxic and hypoxic conditions, leaving the litters untouched in their respective conditions and compared gross anatomical measures of normoxic and hypoxic pups with non-rotating or rotating mothers. As we expected, hypoxic-rearing decreased pup body weight, brain weight and cortical volume. Reducing the dam's exposure to hypoxic conditions actually amplified the effects of hypoxia on body weight, such that hypoxic pups with rotating mothers showed significantly less growth. Interestingly, rotation of hypoxic mothers did not have the same deleterious effect on brain weight, suggesting the presence of compensatory mechanisms conserving brain weight and development even under extremely low body weight conditions. The factors that potentially contribute to these compensatory changes remain to be determined, however, nutrition, pup feeding/metabolism, or changes in maternal care are important candidates, acting either together or independently to change pup body and brain development.

Effects of cortical ischemia and postischemic environmental enrichment on hippocampal cell genesis and differentiation in the adult rat.

The study aimed to elucidate the effects of cortical ischemia and postischemic environmental enrichment on hippocampal cell genesis. A cortical infarct was induced by a permanent ligation of the middle cerebral artery distal to the striatal branches in 6-month-old spontaneously hypertensive rats. Bromodeoxyuridine (BrdU) was administered as 7 consecutive daily injections starting 24 hours after surgery and animals were housed in standard or enriched environment. Four weeks after completed BrdU administration, BrdU incorporation and its co-localization with the neuronal markers NeuN and calbindin D28k, and the astrocytic marker glial fibrillary acidic protein in the granular cell layer and subgranular zone of the hippocampal dentate gyrus were determined with immunohistochemistry and were quantified stereologically. Compared with sham-operated rats, rats with cortical infarcts had a five-to sixfold ipsilateral increase in BrdU-labeled cells. About 80% of the new cells were neurons. Differential postischemic housing did not influence significantly the total number of surviving BrdU-labeled cells or newborn neurons. However, postischemic environmental enrichment increased the ipsilateral generation of astrocytes normalizing the astrocyte-to-neuron ratio, which was significantly reduced in rats housed in standard environment postischemically.

Sox-2 is expressed by neural progenitors and astroglia in the adult rat brain.

Sox-2 is a transcription factor that is expressed by self-renewing and multipotent stem cells of the embryonic neuroepithelium. Very little is however known about Sox-2 expression in the adult brain and therefore we used immunohistochemistry to examine its distribution and co-localization with specific cell markers. We found that Sox-2 was expressed by actively dividing neural progenitor cells in the neurogenic regions in the adult rat brain, the subventricular zone of the forebrain and the subgranular zone of the dentate gyrus in the hippocampus. Cells expressing immature neuronal markers were essentially Sox-2 immunonegative. Sox-2 was also found to be expressed by glial fibrillary acidic protein immunopositive astroglia, widely distributed in the brain parenchyma. Given the fact that several studies have established the neurogenic capacity of a specialized type of astroglia in the adult brain, the findings of Sox-2 expression in parenchymal astroglia are of potential interest. We conclude that Sox-2 might, in combination with appropriate cell-specific markers, constitute a useful marker to study the in vivo dynamics of the neural progenitor cell compartment also in the adult brain.

NG2 cells are distinct from neurogenic cells in the postnatal mouse subventricular zone.

NG2 cells express the chondroitin sulfate proteoglycan NG2 and are a fourth type of glia distinct from astrocytes, oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes but have also been reported to represent neuronal progenitor cells in the postnatal mouse subventricular zone (SVZ). We performed a detailed immunohistochemical analysis of NG2 cells in the mouse SVZ, rostral migratory stream (RMS), and olfactory bulb granule cell layer (OB GCL), which constitute a neurogenic niche in the postnatal forebrain. NG2 cells in the SVZ and RMS expressed the oligodendrocyte precursor cell antigen platelet-derived growth factor receptor-alpha but did not express antigens known to be expressed by neuronogenic cells in the SVZ, such as doublecortin, PSA-NCAM, beta-tubulin, Dlx2, or GFAP. More than 99.5% of the proliferating cells in the SVZ were NG2 negative. In the olfactory bulb, NG2 cells were found to generate primarily oligodendrocytes and a small number of astrocytes but not neurons. In the SVZ and RMS, NG2 cells were sparse and made up a much smaller fraction of the cells compared with the surrounding nonneurogenic parenchyma. Parenchymal NG2 cells were often located along the border of the SVZ and RMS. The abundance of NG2 cells increased in the distal parts of the RMS and especially in the OB GCL, where NG2 cell processes were seen in close proximity to many maturing interneurons. Our findings indicate that NG2 cells do not represent neuronal progenitor cells in the postnatal SVZ but are likely to be oligodendrocyte precursor cells.

On neural plasticity, new neurons and the postischemic milieu: an integrated view on experimental rehabilitation.

This review discusses actual and potential contributors to functional improvement after stroke injuries. Topics that will be covered are neuronal re-organization and sprouting, neural stem/progenitor cell activation and neuronal replacement, as well as the neuronal milieu defined by glia, inflammatory cells and blood vessel supply. It is well established that different types of neuronal plasticity ultimately lead to post-stroke recovery. However, an untapped potential which only recently has started to be extensively explored is neuronal replacement through endogenous or exogenous resources. Major experimental efforts are needed to achieve progress in this burgeoning area. The review stresses the importance of applying neurodevelopmental principles as well as performing a characterization of the role of the postischemic milieu when studying adult brain neural stem/progenitor cells. Integrated and multifaceted experimentation, incorporating actual and possible poststroke function modulators, will be necessary in order to determine future strategies that will ultimately enable considerable progress in the field of neurorehabilitation.

Enriched environment after focal cortical ischemia enhances the generation of astroglia and NG2 positive polydendrocytes in adult rat neocortex.

Environmental enrichment (EE) alleviates sensorimotor deficits after brain infarcts but the cellular correlates are not well-known. This study aimed to test the effects of postischemic EE on neocortical cell genesis. A neocortical infarct was caused by distal ligation of the middle cerebral artery in adult spontaneously hypertensive rats, subsequently housed in standard environment or EE. Bromodeoxyuridine (BrdU) was administered during the first postischemic week to label proliferating cells and BrdU incorporation was quantified 4 weeks later in the periinfarct, ipsilateral medial and contralateral cortex. Immunohistochemistry and confocal microscopy were used to analyze co-localization of BrdU with neuronal (calbindin D28k, calretinin, parvalbumin, glutamic acid decarboxylase, tyrosine hydroxylase), astrocytic (glial fibrillary acidic protein, glutamine synthetase, vimentin, nestin), microglia/macrophage (CD11b/Ox-42, CD68/ED-1), oligodendrocyte progenitor/polydendrocyte (NG2, platelet-derived growth factor alpha receptor) or mature oligodendrocyte (myelin basic protein) markers. BrdU positive cells were increased in all analyzed cortical regions in stroke EE rats compared with stroke standard environment rats. Newly born cells in the periinfarct cortex were mostly reactive astroglia. Occasionally, BrdU positive cells in the periinfarct cortex that were negative for glial or microglia/macrophage markers co-expressed markers typical for interneurons but did not express appropriate functional markers. The majority of BrdU positive cells in intact cortical regions, ipsi- and contralaterally, were identified as NG2 positive polydendrocytes. Perineuronally situated newly born cells and polydendrocytes were found to be brain-derived neurotrophic factor immunoreactive. In conclusion, EE enhanced newborn glial scar astroglia and NG2+ polydendrocytes in the postischemic neocortex which might be beneficial for brain repair and poststroke plasticity.

Postischemic exercise attenuates whereas enriched environment has certain enhancing effects on lesion-induced subventricular zone activation in the adult rat.

Experimental stroke increases cell proliferation and neurogenesis in the subventricular zone (SVZ) and in the dentate gyrus subgranular zone (SGZ) in the adult mammalian brain. This study examined the effects of postischemic voluntary exercise (running wheel) and environmental enrichment on the SVZ and SGZ 1 week after focal cortical ischemia in adult spontaneously hypertensive rats. Immunohistochemical labeling was performed for incorporation of specific cell markers such as Ki67 and 5-bromodeoxyuridine (proliferating and newborn cells), terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling (apoptotic cells), Sox-2 and glial fibrillary acidic protein (neural stem and progenitor cells), polysialylated neural cell adhesion molecule and doublecortin (neuroblasts). Postischemic exercise and environmental enrichment differentially modulated SVZ cell genesis but lacked effects on the SGZ. Lesion-induced proliferation of neural stem/progenitor cells and neuronal precursors was attenuated in stroke runners without any effects on apoptosis or neuronal migration in the forebrain. Running activity did not affect the SVZ in intact rats. In contrast to postischemic wheel running, postischemic environmental enrichment did not have attenuating effects on the ipsilateral SVZ and increased proliferating putative neural stem cells and neuronal precursors contralaterally. A significant functional improvement, assessed using a rotating pole, was observed only in the postischemically enriched group and was likely due to other types of plasticity than neuronal replacement at this early time point. It may be concluded that in contrast to enriched environment, exercise during the first postischemic week might be detrimental for regenerative processes initiated in the SVZ after stroke.