The Impact of Alpha-Syntrophin Deletion on the Changes in Tissue Structure and Extracellular Diffusion Associated with Cell Swelling under Physiological and Pathological Conditions

<div><p>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 α-syntrophin (α-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 α-syn deletion on astrocyte morphology and changes in extracellular diffusion associated with cell swelling <i>in vitro</i> and <i>in vivo</i>. In mice lacking α-syn, we found higher resting values of the apparent diffusion coefficient of water (ADC_W) and the extracellular volume fraction (α). No significant differences in tortuosity (λ) or non-specific uptake (<i>k′</i>), were found between α-syn-negative (α-syn −/−) and α-syn-positive (α-syn +/+) mice. The deletion of α-syn resulted in a significantly smaller relative decrease in α 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 ADC_W as well as of the ECS volume fraction α indicate milder cell swelling in α-syn −/− in comparison with α-syn +/+ mice. Shortly after terminal ischemia/anoxia induction, the onset of a steep rise in the extracellular potassium concentration and an increase in λ was faster in α-syn −/− mice, but the final values did not differ between α-syn −/− and α-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.</p></div

Changes in astrocyte morphology evoked by hypotonic stress and elevated extracellular K⁺.

<p>Changes in astrocyte morphology, based on changes in glial fibrillary acidic protein immunoreactivity, were determined in the cortex (layers III–IV) of α-syn +/+ and α-syn −/− mice: 1) prior to (control), 2) after a 30 minute application of hypotonic solutions (H-50 and H-100) or 10 mM K⁺ and 3) following a 60 minute washout. The bar graphs on the right side indicate cell volume changes expressed as changes in the area corresponding to GFAP immunoreactivity; n represents number of cells.</p

Representative TMA⁺-diffusion curves under resting conditions and during cell swelling.

<p>TMA⁺-diffusion curves with the corresponding values of the extracellular volume fraction (α), tortuosity (λ) and non-specific uptake (<i>k′</i>) obtained in the cortex of α-syn +/+ and α-syn −/− mice under resting conditions (<b>A, E</b>) as well as during acute cell swelling evoked by the application of hypotonic solutions H-50 (<b>B</b>), H-100 (<b>C</b>) or increased potassium (<b>D</b>) <i>in vitro</i> or by ischemia/anoxia <i>in vivo</i> (<b>F</b>). The values of the ECS diffusion parameters were determined by a non-linear curve fitting algorithm operating on the diffusion curve. The amplitude of the curves is inversely proportional to the ECS volume fraction while the shape of the curves reflects tortuosity.</p

Changes in the ECS diffusion parameters, ADC_W and [K⁺]_e evoked by terminal ischemia/anoxia <i>in vivo</i>.

<p>Each data point represents mean ± S.E.M. The control values as well as the final values of the ECS volume fraction α (<b>A</b>) and ADC_W (<b>C</b>) reached during terminal ischemia/anoxia evoked by cardiac arrest (CA) were significantly smaller in α-syn +/+ mice than in α-syn −/− animals and the time course of ADC_W changes was slower in the α-syn −/− mice. There was no significant difference in the control values of λ between α-syn +/+ and α-syn −/− mice (<b>B</b>). After the onset of terminal ischemia/anoxia, tortuosity (<b>B</b>) as well as [K⁺]_e (<b>D</b>) were significantly higher in α-syn −/− mice compared to α-syn +/+ animals, but the final values did not differ<b>.</b></p

Experimental set-up and representative TMA⁺-diffusion curves in agar gel and healthy cortex.

<p>To stabilize the intertip distance of the electrode array, an iontophoretic micropipette and TMA⁺-selective microelectrode were glued together with dental cement (left). In the brain, where diffusion is constrained by various barriers and restricted to the extracellular space, the amplitude of the diffusion curve is much higher and its shape differs from the diffusion curve measured in agar gel, where by definition α = λ = 1 and <i>k′</i> = 0 (right).</p

Effect of hypotonic stress and elevated K⁺ on the ECS diffusion parameters in α-syn +/+ and α-syn −/− mice <i>in vitro</i>.

<p>The values are presented as mean ± S.E.M. Asterisks (*-p<0.05; **-p<0.01;***-p<0.001) indicate significant differences between the values in α-syn +/+ and α-syn −/− animals; crosshatches (#-p<0.05; ##-p<0.01; ###-p<0.001) indicate significant differences between control values and those obtained under experimental conditions in the same group of animals. The control values of the ECS diffusion parameters from each individual experiment were calculated as the average values extracted from three diffusion curves before application. The mean control value presented in the table is the average of the control values from all in vitro experiments. The mean values of the maximum change during application correspond to the time-point of the maximum decrease of α (i.e., the 25^th min in H-50 and H-100 and the 30^th min in 10 mM K⁺); the values for washout correspond to the data point at the 90^th min. Abbreviations: extracellular space volume fraction (α), tortuosity (λ), non-specific uptake (k<i>′</i>), number of animals (N), number of slices (n), hypotonic solutions (H-50 and H-100).</p

Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice

<div><p>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 knockout on volume changes in individual astrocytes <i>in situ</i> evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) <i>in situ</i> and <i>in vivo</i> 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 α-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 α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-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⁺, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K⁺.</p></div

The effect of hypotonic stress or elevated K⁺ on the ECS volume <i>in situ</i>.

<p>The left side: The control values of all experiments were set to 100%, and the relative changes of the values of the extracellular volume fraction α were calculated at 5 min intervals during a 30 min application and a subsequent 60 min washout of mild (<b>A</b>) or severe (<b>B</b>) hypotonic stress or 10 mM K⁺ (<b>C</b>). Each data point represents mean ± S.E.M. The right side: volume regulation during washout at 20 min intervals is expressed as changes in the values reached in the 30^th minute of application, set as 0%. Asterisks indicate significant (*, p<0.05) and very significant (**, p<0.01) differences between GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice.</p

Astrocytic volume changes in the cortex of GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice during hypoosmotic stress.

<p>(<b>A, B</b>) Superimposed confocal images of EGFP-labeled cortical astrocytes from GFAP/EGFP/α-Syn^−/− mice obtained in aCSF (control), during a 30-minute exposure to hypoosmotic stress of 250 mOsm/kg (aCSF_H-50; <b>A</b>) or 205 mOsm/kg (aCSF_H-100; <b>B</b>) and during a 60-minute washout. The astrocytic volume was quantified every 10 minutes during exposure to hypoosmotic stress and every 20 minutes during washout. (<b>C, D</b>) Time-dependent changes in the total astrocytic volume in GFAP/EGFP (red) and GFAP/EGFP/α-Syn^−/− mice (green) during a 30-minute exposure to aCSF_H-50 (<b>C left</b>) or to aCSF_H-100 (<b>D left</b>) and during a 60-minute washout. The changes of astrocytic volume during washout were evaluated in each individual cell and expressed as an average percent of cell volume increase/decrease related to the maximal volume after 30 minutes of aCSF_H-50 (<b>C right</b>) or aCSF_H-100 (<b>D right</b>) exposure; these values were set as 0%. Note that α-syntrophin deletion results in smaller astrocyte swelling only during aCSF_H-100 application and in smaller volume changes following mild hypoosmotic stress. Asterisks indicate significant (*, p<0.05), very significant (**, p<0.01) and extremely significant (***, p<0.001) differences between GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice.</p

Volume changes in the astrocytic soma and processes during hypotonic stress, increased extracellular K⁺ concentration and OGD.

<p>(<b>A–C</b>) Time-dependent changes in the volume of the astrocytic soma (<b>top</b>) and processes (<b>bottom</b>) in GFAP/EGFP (red) and GFAP/EGFP/α-Syn^−/− mice (green) during a 30-minute application of aCSF_H-100 (<b>A</b>), a 20-minute application of aCSF_K+50 (<b>B</b>) or 20-minute OGD (<b>C</b>), followed by a 60- or 40-minute washout. (<b>D–F</b>) The contribution of the astrocytic soma and processes to the total astrocyte volume changes was expressed as a ratio of the volume changes of both compartments (V_processes/V_soma) after 30 minutes of hypotonic stress and a subsequent 60-minute washout (<b>D</b>), after a 20-minute exposure to aCSF_K+50 and a subsequent 40-minute washout (<b>E</b>), and after 20 minutes of OGD and a subsequent 40-minute washout (<b>F</b>). Note that in GFAP/EGFP mice the swelling of the astrocytic processes prevails (V_processes/V_soma = ∼1.2), while in GFAP/EGFP/α-Syn^−/− the ratio declines towards 1, indicating that the astrocytic processes swell less and the contribution of the cell soma to total astrocyte volume increases. The time-points at which the ratio was calculated are indicated by arrows in A–C. Asterisks indicate significant (*, p<0.05), very significant (**, p<0.01) and extremely significant (***, p<0.001) differences between GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice.</p