Fredrik Blomstrand

Protective role of reactive astrocytes in brain ischemia

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP−/−Vim−/− mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP−/−Vim−/− than in wild-type (WT) mice; GFAP−/−, Vim−/− and WT mice had the same infarct volume. Endothelin B receptor (ETBR) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP−/−Vim−/− astrocytes. In WT astrocytes, ETBR colocalized extensively with bundles of IFs. GFAP−/−Vim−/− astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP−/−Vim−/− than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ETBR-mediated control of gap junctions, and PAI-1 expression.

Astrocytes and stroke: Networking for survival?

Astrocytes are now known to be involved in the most integrated functions of the central nervous system. These functions are not only necessary for the normally working brain but are also critically involved in many pathological conditions, including stroke. Astrocytes may contribute to damage by propagating spreading depression or by sending proapoptotic signals to otherwise healthy tissue via gap junction channels. Astrocytes may also inhibit regeneration by participating in formation of the glial scar. On the other hand, astrocytes are important in neuronal antioxidant defense and secrete growth factors, which probably provide neuroprotection in the acute phase, as well as promoting neurogenesis and regeneration in the chronic phase after injury. A detailed understanding of the astrocytic response, as well as the timing and location of the changes, is necessary to develop effective treatment strategies for stroke patients.

Astroglia and glutamate in physiology and pathology: aspects on glutamate transport, glutamate-induced cell swelling and gap-junction communication

Astroglia have the capacity to monitor extracellular glutamate (Glu) and maintain it at low levels, metabolize Glu, or release it back into the extracellular space. Glu can induce an increase in astroglial cell volume with a resulting decrease of the extracellular space, and thereby alter the concentration of extracellular substances. Many lines of evidence show that K(+) can be buffered within the astroglial gap-junction-coupled network, and recent results show that gap junctions are permeable for Glu. All these events occur dynamically: the astroglial network has the capacity to interfere actively with neurotransmission, thereby contributing to a high signal-to-noise ratio for the Glu transmission. High-quality neuronal messages during normal physiology can then be maintained. With the same mechanisms, astroglia might exert a neuroprotective function in situations of moderately increased extracellular Glu concentrations, i.e., corresponding to conditions of pathological hyper-excitability, or corresponding to early stages of an acute brain injury. If the astroglial functions are failing, neuronal dysfunction can be reinforced.

Extent of intercellular calcium wave propagation is related to gap junction permeability and level of connexin-43 expression in astrocytes in primary cultures from four brain regions

Astrocytes are coupled via gap junctions, predominantly formed by connexin-43 proteins, into cellular networks. This coupling is important for the propagation of intercellular calcium waves and for the spatial buffering of K+. Using the scrape-loading/dye transfer technique, we studied gap junction permeability in rat astrocytes cultured from four different brain regions. The cultures were shown to display regional heterogeneity with the following ranking of the gap junction coupling strengths: hippocampus = hypothalamus > cerebral cortex = brain stem. Similar relative patterns were found in connexin-43 messenger RNA and protein levels using solution hybridization/RNase protection assay and western blots, respectively. The percentages of the propagation area of mechanically induced intercellular calcium waves for cortical, brain stem and hypothalamic astrocytes compared with hippocampal astrocytes were approximately 77, 42, and 52, respectively. Thus, the extent of calcium wave propagation was due to more than just gap junctional permeability as highly coupled hypothalamic astrocytes displayed relatively small calcium wave propagation areas. Incubation with 5-hydroxytryptamine decreased and incubation with glutamate increased the calcium wave propagation area in hippocampal (67% and 170% of the control, respectively) and in cortical astrocytes (82% and 163% of the control, respectively). Contrary to hippocampal and cortical astrocytes, the calcium wave propagation in brain stem astrocytes was increased by 5-hydroxytryptamine incubation (158% of control), while in hypothalamic astrocytes, no significant effects were seen. Similar effects from 5-hydroxytryptamine or glutamate treatments were observed on dye transfer, indicating an effect on the junctional coupling strength. These results demonstrate a strong relationship between connexin-43 messenger RNA levels, protein expression, and gap junction permeability among astroglial cells. Furthermore, our results suggest heterogeneity among astroglial cells from different brain regions in intercellular calcium signaling and in its differential modulation by neurotransmitters, probably reflecting functional requirements in various brain regions.

Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca2+signaling

Astrocytes represent a major target for endothelins (ETs), a family of peptides that have potent and multiple effects on signal transduction pathways and can be released by several cell types in the brain. In the present study we have investigated the involvement of different ET receptor subtypes on intercellular dye diffusion, intracellular Ca2+homeostasis, and intercellular Ca2+ signaling in cultured rat astrocytes from hippocampus and striatum. Depending on the ET concentration and the receptor involved, ET-1- and ET-3-induced intracellular Ca2+ increases with different response patterns. Both ET-1 and ET-3 are powerful inhibitors of gap junctional permeability and intercellular Ca2+ signaling. The nonselective ET receptor agonist sarafotoxin S6b and the ETB receptor-selective agonist IRL 1620 mimicked these inhibitions. The ET-3 effects were only marginally affected by an ETA receptor antagonist but completely blocked by an ETB receptor antagonist. However, the ET-1-induced inhibition of gap junctional dye transfer and intercellular Ca2+ signaling was only marginally blocked by ETA or ETB receptor-selective antagonists but fully prevented when these antagonists were applied together. The ET-induced inhibition of gap junction permeability and intercellular Ca2+ signaling indicates that important changes in the function of astroglial communication might occur when the level of ETs in the brain is increased.

Astrocytes play a critical role in transient heterosynaptic depression in the rat hippocampal CA1 region

Active synapses can reduce the probability of transmitter release at neighbouring synapses. Depending on whether such heterosynaptic depression is mediated by intersynaptic diffusion of transmitter or by release of gliotransmitters, astrocytes should either hinder or promote the heterosynaptic depression. In the present study we have examined the developmental profile and astrocytic involvement in a transient heterosynaptic depression (tHeSD) in the CA1 region of the rat hippocampal slice preparation. A short stimulus burst (3 impulses at 50 Hz) to one group of synapses elicited a depression of the field EPSP evoked in another group of synapses that amounted to about 25% 0.5 s after the conditioning burst. This tHeSD was associated with an increase in the paired‐pulse ratio of about 30%. The tHeSD was not present in slices from rats younger than 10 postnatal days and developed towards the adult magnitude between postnatal days 10 and 20. The tHeSD was totally prevented by the glia‐specific toxin fluoroacetate (FAC), by carbenoxolone, a general blocker of connexin‐based channels, and by endothelin, an endogenous peptide that has been shown to block astrocytic connexin‐based channels. Antagonists to GABAB receptors and group II/III metabotropic glutamate receptors (mGluRs) abolished the tHeSD whereas antagonists to NMDA‐ and adenosine A1 receptors, and to group I mGluRs, did not affect the tHeSD. These results suggest that the tHeSD relies on GABAB receptors, group II/III mGluRs and on gliotransmitter release from functionally mature astrocytes.

5-Hydroxytryptamine and glutamate modulate velocity and extent of intercellular calcium signalling in hippocampal astroglial cells in primary cultures

The effects of 5-hydroxytryptamine or glutamate treatment on mechanically induced intercellular calcium waves were studied in gap junction-coupled astroglial cells using rat astroglial-neuronal primary cultures from hippocampus. Imaging software was developed to study amplitude, velocity and extent of wave propagation. Velocity software was designed to find the cell contours automatically and to calculate travelled distance and time-delay of the calcium wave as it propagates from the stimulated cell to all other cells. Propagation analyses were performed to calculate the area of wave propagation. Mechanical stimulation of a single astroglial cell induced an intercellular calcium wave spreading from cell to cell in the astroglial syncytium. When registering the appearances of calcium signals in individual cells along the wave path upon re-stimulation of the same cell, 44.7% of the cells responded with similar calcium signal appearances the second time as the first time. A second wave from the opposite direction resulted in similar calcium signal appearances in 27.3% of the studied cells. Both amplitude and velocity of the calcium signal decreased most prominently in the first part and showed a later flattening out. Treatment with 5-hydroxytryptamine or glutamate for 20-30 s before mechanical stimulation increased the velocity of the calcium waves. 5-Hydroxytryptamine treatment for varying times decreased the propagation area of the calcium waves. In contrast, glutamate treatment increased the propagation area.

Stimulated efflux of amino acids and glutathione from cultured hippocampal slices by omission of extracellular calcium: likely involvement of connexin hemichannels.

Omission of extracellular Ca2+ for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X7 receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-d -aspartate left the efflux by Ca2+ omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca2+ omission was combined with the glutamate uptake blocker l-trans-pyrrolidine-2,4-dicarboxylate and when both Ca2+ and Mg2+ were omitted from the medium. Omission of Ca2+ for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca2+ stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels. This type of efflux may have protective functions via glutathione efflux but can aggravate toxicity in situations when glutamate reuptake is impaired, such as following a stroke.

Gap junction blockage limits intercellular spreading of astrocytic apoptosis induced by metabolic depression

Astrocytes are highly coupled by gap junction channels, which allow transfer of intracellular signalling molecules and metabolites between connected cells. Astrocytic gap junctions remain open during ischemic conditions as previously demonstrated in vitro and in situ. In this study, we investigated the effect of gap junction blockage on iodoacetate‐induced ATP depression and cell death progression in astrocytes in primary rat hippocampal cultures. We demonstrated that blockage of gap junctions during iodoacetate‐induced inhibition of the glycolysis induced an earlier onset of the ATP depression. Moreover, initiation of apoptotic processes, demonstrated by binding of Annexin V, was critically dependent on the ATP levels. The apoptotic event was also shown to spread and involve neighbouring cells, a process that was inhibited by blockage of gap junction communication. Chelating intracellular calcium using BAPTA‐AM decelerated the iodoacetate‐induced ATP depression. The chelation also decelerated the spreading of apoptotic processes. Inhibition of caspases did not alter the expansion of cell groups being Annexin V positive. However, the proportion of Annexin V positive cells also being propidium iodide positive was increased after caspase inhibition. The results show that inhibition of gap junctions during cellular metabolic depression interferes with the metabolic status and cell death progression in astrocytes.

5-Hydroxytryptamine2B receptors stimulate Ca2+ increases in cultured astrocytes from three different brain regions.

The expression of 5-hydroxytryptamine-2B (5-HT2B) receptor mRNA has recently been shown in cultured astrocytes. Here the expression of functional 5-HT2B receptors has been studied in cultured astrocytes from rat cerebral cortex, hippocampus, and brain stem. Fluo-3- and fura-2-based microspectrofluorometry was used for measuring changes in intracellular free calcium concentrations ([Ca2+]i). The 5-HT2B agonist alpha-methyl 5-HT (40 nM) produced rapid transient increases in [Ca2+]i in astrocytes from all three brain regions studied, and these responses were blocked by the selective 5-HT2B antagonist rauwolscine (1 microM). The specificity of the responses to alpha-methyl 5-HT was further demonstrated by the failure of 4-(4-fluorobenzoyl)-1-(4-phenylbutyl)-piperidine oxalate (1 microM), a specific 5-HT2A/5-HT2C antagonist, to block these responses. The 5-HT2B-induced increases in [Ca2+]i persisted in Ca2+-free buffer, indicating that the increase in [Ca2+]i results from mobilization of intracellular Ca2+ stores. The expression of 5-HT2B receptors on astroglial cells was further verified immunohistochemically and by Western blot analysis. These results provide evidence of the existence of 5-HT2B receptors on astrocytes in primary culture.