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Altered astrocytic swelling in the cortex of ?-syntrophin-negative GFAP/EGFP mice.

ABSTRACT: Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K(+) and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of ?-syntrophin was shown to delay edema formation and K(+) clearance, we aimed to elucidate the impact of ?-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (?) in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking ?-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with ?-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that ?-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K(+). As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K(+), ?-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative ? changes showed a significantly smaller decrease in ?-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of ?-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K(+).

SUBMITTER: Anderova M

PROVIDER: S-EPMC4245134 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice.

Anderova Miroslava M Benesova Jana J Mikesova Michaela M Dzamba David D Honsa Pavel P Kriska Jan J Butenko Olena O Novosadova Vendula V Valihrach Lukas L Kubista Mikael M Dmytrenko Lesia L Cicanic Michal M Vargova Lydia L

PloS one 20141126 11

Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K(+) and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K(+) clearance, we aimed to elucidate the impact of α-syntrophin kn ...[more]

PMID: 25426721

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Project description:Aquaporin-4 (AQP4) is the primary cellular water channel in the brain and is abundantly expressed by astrocytes along the blood-brain barrier and brain-cerebrospinal fluid interfaces. Water transport via AQP4 contributes to the activity-dependent volume changes of the extracellular space (ECS), which affect extracellular solute concentrations and neuronal excitability. AQP4 is anchored by ?-syntrophin (?-syn), the deletion of which leads to reduced AQP4 levels in perivascular and subpial membranes. We used the real-time iontophoretic method and/or diffusion-weighted magnetic resonance imaging to clarify the impact of ?-syn deletion on astrocyte morphology and changes in extracellular diffusion associated with cell swelling in vitro and in vivo. In mice lacking ?-syn, we found higher resting values of the apparent diffusion coefficient of water (ADCW) and the extracellular volume fraction (?). No significant differences in tortuosity (?) or non-specific uptake (k'), were found between ?-syn-negative (?-syn -/-) and ?-syn-positive (?-syn +/+) mice. The deletion of ?-syn resulted in a significantly smaller relative decrease in ? observed during elevated K(+) (10 mM) and severe hypotonic stress (-100 mOsmol/l), but not during mild hypotonic stress (-50 mOsmol/l). After the induction of terminal ischemia/anoxia, the final values of ADCW as well as of the ECS volume fraction ? indicate milder cell swelling in ?-syn -/- in comparison with ?-syn +/+ mice. Shortly after terminal ischemia/anoxia induction, the onset of a steep rise in the extracellular potassium concentration and an increase in ? was faster in ?-syn -/- mice, but the final values did not differ between ?-syn -/- and ?-syn +/+ mice. This study reveals that water transport through AQP4 channels enhances and accelerates astrocyte swelling. The substantially altered ECS diffusion parameters will likely affect the movement of neuroactive substances and/or trophic factors, which in turn may modulate the extent of tissue damage and/or drug distribution.

Project description:Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green-fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K? and Cl? channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K(2P)) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K(2P) channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K? effluxes. Additionally, the inhibition of Na?-K?-Cl? co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K? channels (Kir4.1), K(2P) channels (TREK-1 and TWIK-1) and Cl? channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K(2P) channels.

Project description:BackgroundNeuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Gαs, termed XLαs, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated.ResultsRNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive α-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3(rd) ventricle, while β-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes.ConclusionGnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since α-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections.

Dataset Information

Altered astrocytic swelling in the cortex of ?-syntrophin-negative GFAP/EGFP mice.

Publications

Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice.

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