44 research outputs found
The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia.
No full textThe polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult
Impact of global cerebral ischemia on K+ channel expression and membrane properties of glial cells in the rat hippocampus
No full textQuantification of astrocyte volume changes during ischemia in situ reveals two populations of astrocytes in the cortex of GFAP/EGFP mice
No full textCell Death/Proliferation and Alterations in Glial Morphology Contribute to Changes in Diffusivity in the Rat Hippocampus after Hypoxia—Ischemia
No full text To understand the structural alterations that underlie early and late changes in hippocampal diffusivity after hypoxia/ischemia (H/I), the changes in apparent diffusion coefficient of water (ADCW) were studied in 8-week-old rats after H/I using diffusion-weighted magnetic resonance imaging (DW-MRI). In the hippocampal CA1 region, ADCW analyses were performed during 6 months of reperfusion and compared with alterations in cell number/cell-type composition, glial morphology, and extracellular space (ECS) diffusion parameters obtained by the real-time iontophoretic method. In the early phases of reperfusion (1 to 3 days) neuronal cell death, glial proliferation, and developing gliosis were accompanied by an ADCW decrease and tortuosity increase. Interestingly, ECS volume fraction was decreased only first day after H/I. In the late phases of reperfusion (starting 1 month after H/I), when the CA1 region consisted mainly of microglia, astrocytes, and NG2-glia with markedly altered morphology, ADCW, ECS volume fraction and tortuosity were increased. Three-dimensional confocal morphometry revealed enlarged astrocytes and shrunken NG2-glia, and in both the contribution of cell soma/processes to total cell volume was markedly increased/decreased. In summary, the ADCW increase in the CA1 region underlain by altered cellular composition and glial morphology suggests that considerable changes in extracellular signal transmission might occur in the late phases of reperfusion after H/I. </jats:p
Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice.
No full textRecently, 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
Persistent reduction of mitral regurgitation by implantation of a transannular mitral bridge: Durability and effectiveness of the repair at 2 years - Results of a prospective trial
No full text© 2018 The Author(s). Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved. Objectives: Ring annuloplasty reduces the septal-lateral diameter (SLD) indirectly by circumferential annular cinching and frequently results in the recurrence of mitral regurgitation (MR) in patients with functional MR (FMR). Our goal was to report the results from the trial and the 2-year post-trial surveillance data. We evaluated whether direct reduction of the SLD with a transannular mitral bridge could achieve significant and durable MR reduction in patients with FMR. Methods: In a prospective trial, 34 consecutive patients with FMR had a mitral bridge implanted surgically. Primary end points were MR ≤1+ at 1, 3 and 6 months postimplant and freedom from subsequent surgical mitral valve repair or replacement. Results: Thirty-two of 34 (94.1%) patients met the primary end points with MR ≤1+ at 6 months. At 2 years, there were no strokes or device-related adverse events. At 2 years, MR was reduced from 3.32 ± 0.47 to 0.50 ± 0.83 (P ≤ 0.001) with ≤1+ MR in 33/34 patients, including 4 reinterventions for periprosthetic recurrent MR ≥3 without mitral bridge explants or conventional mitral repair or replacement. At 2 years, the mean mitral gradient was 2.15 ± 0.82 mmHg; the mitral annular SLD decreased from 40.4 ± 2.91 mm to 28.9 ± 1.55 mm (P ≤ 0.001). The left ventricular ejection fraction increased (57.9 ± 10.4-62.4 ± 9.7%; P ≤ 0.001). The New York Heart Association functional class improved (2.19 ± 0.76-1.41 ± 0.61; P ≤ 0.001). Conclusions: The single-centre trial data indicate that direct reduction in the SLD with a mitral bridge is feasible, safe and efficacious in patients with FMR. Validation in a larger population of patients and comparison to conventional annuloplasty ring are necessary
The TRPV4 antagonist RN1734 decreases 4αPDD-induced and spontaneous Ca<sup>2+</sup> oscillations in astrocytes of the hippocampal CA1 region 7 days after ischemia.
No full text<p>(<b>A</b>) Representative fluorescence traces of hippocampal astrocytes in slices prepared from sham-operated rats (CTRL) and rats 7 days after hypoxia/ischemia (7D H/I) before 4αPDD application (aCSF), during 5 µM 4αPDD application, during the application of 5 µM 4αPDD with 10 µM RN1734 and following washout (aCSF). (<b>B</b>) Histogram of the mean intracellular calcium transients per hour before 4αPDD application (aCSF), during the application of 5 µM 4αPDD, during the application of 5 µM 4αPDD +10 µM RN1734 and following washout in aCSF, in astrocytes in hippocampal slices prepared from the brains of sham-operated rats (CRTL, n = 43) and those 7 days after hypoxia/ischemia (7D H/I, n = 40). (<b>C</b>) Histogram of the mean spontaneous intracellular calcium transients per hour before RN1734 application (aCSF) and during the application of 10 µM RN1734 (RN1734), measured in astrocytes from acute hippocampal slices 7 days after hypoxia/ischemia (7D H/I, n = 10). The values are presented as mean ± S.E.M. Statistical significance was calculated using one-way ANOVA in (B) and a paired t-test in (C); ***p<0.001 extremely significant, **p<0.01 very significant.</p
Experiment II: gene expression profiling of distinct astrocytic subpopulations.
No full text<p><b>A:</b> Bar plot with SEM for all the expressed genes; significant differences are indicated with asterisks (<i>p</i><0.05 (*), <i>p</i><0.01 (**), <i>p</i><0.001 (***). <b>B:</b> Principal component analysis. The identification of 2 astrocytic subpopulations is along the first principal component, which accounts for most of the variation in the measured data. <b>C:</b> Clustering of astrocytes using Kohonen SOMs. The expression levels of all genes were mean-centered. Each dot represents one cell. <b>D:</b> Dendrogram based on all astrocytic genes. The y-axis shows the distance between groups.</p
4αPDD-induced currents in cultured astrocytes dissociated from the hippocampal CA1 region.
No full text<p>(<b>A</b>) An image of a Lucifer Yellow (LY)-loaded astrocyte taken by a digital camera immediately after patch-clamp recording and (<b>B</b>) the same astrocyte identified by immunostaining with glial fibrillary acidic protein (GFAP). The overlay image shows the co-localization of GFAP with LY. (<b>C</b>) “Complex” and “passive” current patterns in astrocytes <i>in vitro</i> evoked by membrane depolarization and hyperpolarization from the holding potential of −70 mV. The currents were recorded using K<sup>+</sup>- and Na<sup>+</sup>-containing intra- and extracellular solutions (Int1 and Ext1). The voltage step protocol is shown in the inset. (<b>D</b>) Current pattern evoked with a voltage step protocol (see the inset) in cultured astrocytes recorded in intra- and extracellular solutions in which K<sup>+</sup> and Na<sup>+</sup> were replaced by Cs<sup>+</sup> (Int2 and Ext2). Note the marked reduction in membrane conductance. (<b>E</b>) Time course of 4αPDD-evoked currents measured from the ramp protocol in astrocytes of controls (blue squares) and astrocytes 1H (green triangles) and 7D after H/I (red circles). Currents were measured at −100 mV (white arrowhead) and +100 mV (black arrowhead) in response to a voltage ramp stimulation protocol (see the inset). (<b>F</b>) Representative traces of steady state currents (same cells as in E) recorded prior to (in Ext2 solution, dashed line) and during 4αPDD application (full line) in cultured astrocytes of controls (left) and those 1H (middle) and 7D after H/I (right). Representative traces of steady state currents were obtained at the times indicated by the filled blue squares, green triangles and red circles in E. White and black arrowheads indicate the applied voltage ramp and the corresponding current traces (see the inset in E).</p
Composition of the extracellular and intracellular solutions for Ca<sup>2+</sup> imaging and patch-clamp measurements.
No full text<p>Abbreviations: extracellular solution (Ext); intracellular solution (Int). All concentrations are in mM. The pH of the extracellular solutions was adjusted to 7.4 with NaOH in Ext1 or with CsOH in Ext2 and Ext2<sub>ØCa</sub>, while the pH of the intracellular solutions was adjusted to 7.2 with KOH in Int1 or CsOH in Int2<sub>.</sub> Osmolality was adjusted with mannitol in Ext1, Ext2, Ext2<sub>ØCa</sub> and Int1 solutions.</p
