Modulating endogenous regenerative processes may represent a suitable treatment for central nervous system (CNS) injuries, such as stroke or trauma. Neural stem/progenitor cells (NS/PCs), which naturally reside in the subventricular zone (SVZ) of the adult brain, proliferate and differentiate to other cell types, and therefore may compensate the negative consequences of ischemic injury. The fate of NS/PCs in the developing brain is largely influenced by Wingless/Integrated (Wnt) signaling; however, its role in the differentiation of adult NS/PCs under ischemic conditions is still enigmatic. In our previous study, we identified the Wnt/β-catenin signaling pathway as a factor promoting neurogenesis at the expense of gliogenesis in neonatal mice. In this study, we used adult transgenic mice in order to assess the impact of the canonical Wnt pathway modulation (inhibition or hyper-activation) on NS/PCs derived from the SVZ, and combined it with the middle cerebral artery occlusion (MCAO) to disclose the effect of focal cerebral ischemia (FCI). Based on the electrophysiological properties of cultured cells, we first identified three cell types that represented in vitro differentiated NS/PCs - astrocytes, neuron-like cells, and precursor cells. Following FCI, we detected fewer neuron-like cells after Wnt signaling inhibition. Furthermore, the immunohistochemical analysis revealed an overall higher expression of cell-type-specific proteins after FCI, indicating increased proliferation and differentiation rates of NS/PCs in the SVZ. Remarkably, Wnt signaling hyper-activation increased the abundance of proliferating and neuron-like cells, while Wnt pathway inhibition had the opposite effect. Finally, the expression profiling at the single cell level revealed an increased proportion of neural stem cells and neuroblasts after FCI. These observations indicate that Wnt signaling enhances NS/PCs-based regeneration in the adult mouse brain following FCI, and supports neuronal differentiation in the SVZ.

• Publikační typ
• časopisecké články MeSH

The canonical Wnt signaling pathway plays an important role in embryogenesis, and the establishment of neurogenic niches. It is involved in proliferation and differentiation of neural progenitors, since elevated Wnt/β-catenin signaling promotes differentiation of neural stem/progenitor cells (NS/PCs(1)) towards neuroblasts. Nevertheless, it remains elusive how the differentiation program of neural progenitors is influenced by the Wnt signaling output. Using transgenic mouse models, we found that in vitro activation of Wnt signaling resulted in higher expression of β-catenin protein and Wnt/β-catenin target genes, while Wnt signaling inhibition resulted in the reverse effect. Within differentiated cells, we identified three electrophysiologically and immunocytochemically distinct cell types, whose incidence was markedly affected by the Wnt signaling output. Activation of the pathway suppressed gliogenesis, and promoted differentiation of NS/PCs towards a neuronal phenotype, while its inhibition led to suppressed neurogenesis and increased counts of cells of glial phenotype. Moreover, Wnt signaling hyperactivation resulted in an increased incidence of cells expressing outwardly rectifying K(+) currents, together with inwardly rectifying Na(+) currents, a typical current pattern of immature neurons, while blocking the pathway led to the opposite effect. Taken together, our data indicate that the Wnt signaling pathway orchestrates neonatal NS/PCs differentiation towards cells with neuronal characteristics, which might be important for nervous tissue regeneration during central nervous system disorders. Furthermore, the transgenic mouse strains used in this study may serve as a convenient tool to manipulate β-catenin-dependent signaling in neural progenitors in the neonatal brain.

• MeSH
• beta-katenin genetika   metabolismus   MeSH
• imunohistochemie MeSH
• kultivované buňky MeSH
• membránové potenciály fyziologie   MeSH
• metoda terčíkového zámku MeSH
• mezibuněčné signální peptidy a proteiny genetika   metabolismus   MeSH
• mozek cytologie   metabolismus   MeSH
• myši transgenní MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neurogeneze fyziologie   MeSH
• neuroglie cytologie   metabolismus   MeSH
• neurony cytologie   metabolismus   MeSH
• signální dráha Wnt fyziologie   MeSH
• transkripční faktory BHLH-Zip genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Alzheimer's disease (AD) is a complex neurodegenerative disorder with major clinical hallmarks of memory loss, dementia, and cognitive impairment. Besides the extensive neuron-oriented research, an increasing body of evidence suggests that glial cells, namely astrocytes, microglia, NG2 glia and oligodendrocytes, may play an important role in the pathogenesis of this disease. In the first part of this review, AD pathophysiology in humans is briefly described and compared with disease progression in routinely used animal models. The relevance of findings obtained in animal models of AD is also discussed with respect to AD pathology in humans. Further, this review summarizes recent findings regarding the role/participation of glial cells in pathogenesis of AD, focusing on changes in their morphology, functions, proteins and gene expression profiles. As for astrocytes and microglia, they are fundamental for the progression and outcome of AD either because they function as effector cells releasing cytokines that play a role in neuroprotection, or because they fail to fulfill their homeostatic functions, ultimately leaving neurons to face excitotoxicity and oxidative stress. Next, we turn our attention towards NG2 glia, a novel and distinct class of glial cells in the central nervous system (CNS), whose role in a variety of human CNS diseases has begun to emerge, and we also consider the participation of oligodendrocytes in the pathogenesis and progression of AD. Since AD is currently an incurable disease, in the last part of our review we hypothesize about possible glia-oriented treatments and provide a perspective of possible future advancements in this field.

• MeSH
• Alzheimerova nemoc farmakoterapie   patologie   patofyziologie   MeSH
• lidé MeSH
• neuroglie účinky léků   patologie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Adherent, fibroblastic cells from different tissues are thought to contain subsets of tissue-specific stem/progenitor cells (often called mesenchymal stem cells). These cells display similar cell surface characteristics based on their fibroblastic nature, but also exhibit differences in molecular phenotype, growth rate, and their ability to differentiate into various cell phenotypes. The mechanisms underlying these differences remain poorly understood. We analyzed Ca(2+) signals and membrane properties in rat adipose-derived stromal cells (ADSCs) and bone marrow stromal cells (BMSCs) in basal conditions, and then following a switch into medium that contains factors known to modify their character. Modified ADSCs (mADSCs) expressed L-type Ca(2+) channels whereas both L- and P/Q- channels were operational in mBMSCs. Both mADSCs and mBMSCs possessed functional endoplasmic reticulum Ca(2+) stores, expressed ryanodine receptor-1 and -3, and exhibited spontaneous [Ca(2+)]i oscillations. The mBMSCs expressed P2X7 purinoceptors; the mADSCs expressed both P2X (but not P2X7) and P2Y (but not P2Y1) receptors. Both types of stromal cells exhibited [Ca(2+)]i responses to vasopressin (AVP) and expressed V1 type receptors. Functional oxytocin (OT) receptors were, in contrast, expressed only in modified ADSCs and BMSCs. AVP and OT-induced [Ca(2+)]i responses were dose-dependent and were blocked by their respective specific receptor antagonists. Electrophysiological data revealed that passive ion currents dominated the membrane conductance in ADSCs and BMSCs. Medium modification led to a significant shift in the reversal potential of passive currents from -40 to -50mV in cells in basal to -80mV in modified cells. Hence membrane conductance was mediated by non-selective channels in cells in basal conditions, whereas in modified medium conditions, it was associated with K(+)-selective channels. Our results indicate that modification of ADSCs and BMSCs by alteration in medium formulation is associated with significant changes in their Ca(2+) signaling and membrane properties.

• MeSH
• buňky kostní dřeně cytologie   MeSH
• buňky stromatu cytologie   účinky léků   metabolismus   MeSH
• evokované potenciály účinky léků   MeSH
• glutamátové receptory metabolismus   MeSH
• iontové kanály metabolismus   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• metoda terčíkového zámku MeSH
• oxytocin farmakologie   MeSH
• potkani Sprague-Dawley MeSH
• purinergní receptory metabolismus   MeSH
• tuková tkáň cytologie   MeSH
• vápník metabolismus   MeSH
• vápníkové kanály metabolismus   MeSH
• vasopresiny farmakologie   MeSH
• videomikroskopie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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 in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) 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 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(+).

• MeSH
• akvaporin 4 genetika   metabolismus   MeSH
• astrocyty metabolismus   patologie   MeSH
• biologický transport MeSH
• draslík metabolismus   MeSH
• draslíkové kanály genetika   metabolismus   MeSH
• edém mozku genetika   metabolismus   patologie   MeSH
• glukosa nedostatek   MeSH
• konfokální mikroskopie MeSH
• membránové proteiny nedostatek   genetika   MeSH
• mikrotomie MeSH
• mozková kůra metabolismus   patologie   MeSH
• myši transgenní MeSH
• myši MeSH
• osmolární koncentrace MeSH
• osmotický tlak MeSH
• promotorové oblasti (genetika) MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• proteiny vázající vápník nedostatek   genetika   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• stereotaktické techniky MeSH
• svalové proteiny nedostatek   genetika   MeSH
• techniky tkáňových kultur MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Astrocytes respond to ischemic brain injury by proliferation, the increased expression of intermediate filaments and hypertrophy, which results in glial scar formation. In addition, they alter the expression of ion channels, receptors and transporters that maintain ionic/neurotransmitter homeostasis. Here, we aimed to demonstrate the expression of Hcn1-4 genes encoding hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following focal cerebral ischemia (FCI) or global cerebral ischemia (GCI) and to characterize their functional properties. A permanent occlusion of the middle cerebral artery (MCAo) was employed to induce FCI in adult GFAP/EGFP mice, while GCI was induced by transient bilateral common carotid artery occlusion combined with hypoxia in adult rats. Using FACS, we isolated astrocytes from non-injured or ischemic brains and performed gene expression profiling using single-cell RT-qPCR. We showed that 2 weeks after ischemia reactive astrocytes express high levels of Hcn1-4 transcripts, while immunohistochemical analyses confirmed the presence of HCN1-3 channels in reactive astrocytes 5 weeks after ischemia. Electrophysiological recordings revealed that post-ischemic astrocytes are significantly depolarized, and compared with astrocytes from non-injured brains, they display large hyperpolarization-activated inward currents, the density of which increased 2-3-fold in response to ischemia. Their activation was facilitated by cAMP and their amplitudes were decreased by ZD7288 or low extracellular Na(+) concentration, suggesting that they may belong to the family of HCN channels. Collectively, our results demonstrate that regardless of the type of ischemic injury, reactive astrocytes express HCN channels, which could therefore be an important therapeutic target in poststroke therapy.

• MeSH
• AMP cyklický farmakologie   MeSH
• astrocyty účinky léků   metabolismus   MeSH
• gliový fibrilární kyselý protein genetika   metabolismus   MeSH
• ischemie patologie   MeSH
• kationtové kanály řízené cyklickými nukleotidy genetika   metabolismus   MeSH
• krysa rodu rattus MeSH
• membránové potenciály účinky léků   fyziologie   MeSH
• modely nemocí na zvířatech MeSH
• mozek cytologie   MeSH
• myši transgenní MeSH
• myši MeSH
• neurony účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• pyrimidiny farmakologie   MeSH
• regulace genové exprese účinky léků   fyziologie   MeSH
• sodík metabolismus   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The 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.

• MeSH
• astrocyty fyziologie   MeSH
• DNA primery MeSH
• hipokampus patologie   patofyziologie   MeSH
• imunohistochemie MeSH
• kationtové kanály TRPV fyziologie   MeSH
• krysa rodu rattus MeSH
• metoda terčíkového zámku MeSH
• mozková hypoxie a ischemie patologie   patofyziologie   MeSH
• polymerázová řetězová reakce MeSH
• potkani Wistar MeSH
• sekvence nukleotidů MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Astrocytes and NG2 glia respond to CNS injury by the formation of a glial scar. Since the changes in K(+) currents in astrocytes and NG2 glia that accompany glial scar formation might influence tissue outcome by altering K(+) ion homeostasis, we aimed to characterize the changes in K(+) currents in hippocampal astrocytes and NG2 glia during an extended time window of reperfusion after ischemic injury. Global cerebral ischemia was induced in adult rats by bilateral, 15-min common carotid artery occlusion combined with low-pressure oxygen ventilation. Using the patch-clamp technique, we investigated the membrane properties of hippocampal astrocytes and NG2 glia in situ 2 hours, 6 hours, 1 day, 3 days, 7 days or 5 weeks after ischemia. Astrocytes in the CA1 region of the hippocampus progressively depolarized starting 3 days after ischemia, which coincided with decreased Kir4.1 protein expression in the gliotic tissue. Other K(+) channels described previously in astrocytes, such as Kir2.1, Kir5.1 and TREK1, did not show any changes in their protein content in the hippocampus after ischemia; however, their expression switched from neurons to reactive astrocytes, as visualized by immunohistochemistry. NG2 glia displayed increased input resistance, decreased membrane capacitance, increased delayed outwardly rectifying and A-type K(+) currents and decreased inward K(+) currents 3 days after ischemia, accompanied by their proliferation. Our results show that the membrane properties of astrocytes after ischemia undergo complex alterations, which might profoundly influence the maintenance of K(+) homeostasis in the damaged tissue, while NG2 glia display membrane currents typical of proliferating cells.

• MeSH
• buněčná membrána metabolismus   patologie   MeSH
• down regulace genetika   fyziologie   MeSH
• draslíkové kanály dovnitř usměrňující antagonisté a inhibitory   biosyntéza   genetika   MeSH
• glióza genetika   metabolismus   patologie   MeSH
• hipokampální oblast CA1 metabolismus   patologie   patofyziologie   MeSH
• ischemie mozku metabolismus   patologie   patofyziologie   MeSH
• krysa rodu rattus MeSH
• membránové potenciály fyziologie   MeSH
• neuroglie metabolismus   patologie   MeSH
• polarita buněk fyziologie   MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

During the last decade, much progress has been made in developing protocols for the differentiation of human embryonic stem cells (hESCs) into a neural phenotype. The appropriate agent for cell therapy is neural precursors (NPs). Here, we demonstrate the derivation of highly enriched and expandable populations of proliferating NPs from the CCTL14 line of hESCs. These NPs could differentiate in vitro into functionally active neurons, as confirmed by immunohistochemical staining and electrophysiological analysis. Neural cells differentiated in vitro from hESCs exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. To analyze the population of the derived NPs, we used fluorescence-activated cell sorting (FACS) and characterized the expression of several pluripotent and neural markers, such as Nanog, SSEA-4, SSEA-1, TRA-1-60, CD24, CD133, CD56 (NCAM), beta-III-tubulin, NF70, nestin, CD271 (NGFR), CD29, CD73, and CD105 during long-term propagation. The analyzed cells were used for transplantation into the injured rodent brain; the tumorigenicity of the transplanted cells was apparently eliminated following long-term culture. These results complete the characterization of the CCTL14 line of hESCs and provide a framework for developing cell selection strategies for neural cell-based therapies.

• MeSH
• biologické markery metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• buněčný rodokmen MeSH
• embryonální kmenové buňky cytologie   MeSH
• fenotyp MeSH
• imunohistochemie MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mozek metabolismus   patologie   MeSH
• neurony cytologie   metabolismus   transplantace   MeSH
• průtoková cytometrie MeSH
• tkáňová terapie - dějiny MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Energy depletion during ischemia leads to disturbed ionic homeostasis and accumulation of neuroactive substances in the extracellular space, subsequently leading to volume changes in astrocytes. Confocal microscopy combined with 3D reconstruction was used to quantify ischemia-induced astrocyte volume changes in cortical slices of GFAP/EGFP transgenic mice. Twenty-minutes of oxygen-glucose deprivation (OGD) or oxygen-glucose deprivation combined with acidification (OGD(pH 6.8)) revealed the presence of two distinct astrocytic populations, the first showing a large volume increase (HR astrocytes) and the second displaying a small volume increase (LR astrocytes). In addition, changes in resting membrane potential (V(m)), measured by the patch-clamp technique, supported the existence of two astrocytic populations responding differently to ischemia. Although one group markedly depolarized during OGD or OGD(pH 6.8), only small changes in V(m) toward more negative values were observed in the second group. Conversely, acidification (ACF(pH 6.8)) led to a uniform volume decrease in all astrocytes, accompanied by only a small depolarization. Interestingly, two differently responding populations were not detected during acidification. Differences in the expression of inwardly rectifying potassium channels (Kir4.1), glial fibrillary acidic protein (GFAP), and taurine levels in cortical astrocytes were detected using immunohistochemical methods. We conclude that two distinct populations of astrocytes are present in the cortex of GFAP/EGFP mice, based on volume and V(m) changes during exposure to OGD or OGD(pH 6.8). Immunohistochemical analysis suggests that the diverse expression of Kir4.1 channels and GFAP as well as differences in the accumulation of taurine might contribute to the distinct ability of astrocytes to regulate their volume. 2008 Wiley-Liss, Inc.

• MeSH
• astrocyty fyziologie   klasifikace   patologie   MeSH
• draslíkové kanály dovnitř usměrňující metabolismus   MeSH
• elektrická stimulace MeSH
• financování organizované MeSH
• gliový fibrilární kyselý protein genetika   MeSH
• glukosa nedostatek   MeSH
• hypoxie MeSH
• ischemie patologie   MeSH
• koncentrace vodíkových iontů MeSH
• konfokální mikroskopie MeSH
• membránové potenciály fyziologie   MeSH
• metoda terčíkového zámku MeSH
• modely nemocí na zvířatech MeSH
• mozková kůra patologie   MeSH
• myši transgenní MeSH
• myši MeSH
• proteiny nervové tkáně metabolismus   MeSH
• taurin metabolismus   MeSH
• velikost buňky MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH