Dynamic Volume Changes in Astrocytes Are an Intrinsic Phenomenon Mediated by Bicarbonate Ion Flux

Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in extracellular potassium ([K⁺]_o) and pH homeostasis. Pathological brain states that result in [K⁺]_o and pH dysregulation have been shown to cause astrocyte swelling. However, whether astrocyte volume changes occur under physiological conditions is not known. In this study we used two-photon imaging to visualize real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were observed to swell by 19.0±0.9% in response to a small physiological increase in the concentration of [K⁺]_o (3 mM). Astrocyte swelling was mediated by the influx of bicarbonate (HCO₃₋) ions as swelling was significantly decreased when the influx of HCO₃₋ was reduced. We found: 1) in HCO₃₋ free extracellular solution astrocytes swelled by 5.4±0.7%, 2) when the activity of the sodium-bicarbonate cotransporter (NBC) was blocked the astrocytes swelled by 8.3±0.7%, and 3) in the presence of an extracellular carbonic anhydrase (CA) inhibitor astrocytes swelled by 11.4±0.6%. Because a significant HCO₃₋ efflux is known to occur through the γ-amino-butyric acid (GABA) channel, we performed a series of experiments to determine if astrocytes were capable of HCO₃₋ mediated volume shrinkage with GABA channel activation. Astrocytes were found to shrink −7.7±0.5% of control in response to the GABA_A channel agonist muscimol. Astrocyte shrinkage from GABA_A channel activation was significantly decreased to −5.0±0.6% of control in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ). These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.     

Amyloid Beta1-40-Induced Astrogliosis and the Effect of Genistein Treatment in Rat: A Three-Dimensional Confocal Morphometric and Proteomic Study

Astrocytes are highly involved in regulation and homeostasis of the extracellular environment in the healthy brain. In pathological conditions, these cells play a major role in the inflammatory response seen in CNS tissues, which is called reactive astrogliosis and includes hypertrophy and proliferation of astrocytes. Here, we performed 3D confocal microscopy to evaluate the morphological response of reactive astrocytes positive for glial fibrillary acidic protein (GFAP) in rats, to the presence of Aβ_1–40 in the rat brain before and after treatment with genistein. In 50 astrocytes per animal, we measured the volume and surface area for the nucleus, cell body, the entire cell, the tissue covered by single astrocytes and quantified the number and length of branches, the density of the astrocytes and the intensity of GFAP immunoreactivity. Injecting Aβ_1–40 into the brain of rats caused astrogliosis indicated by increased values for all measured parameters. Mass spectrometric analysis of hippocampal tissue in Aβ_1–40-injected brain showed decreased amounts of tubulins, enolases and myelin basic protein, and increased amounts of dihydropyrimidinase-related protein 2. In Aβ_1–40-injected rats pretreated with genistein, GFAP intensity was decreased to the sham-operated group level, and Aβ_1–40-induced astrogliosis was significantly ameliorated.     

HCO3?-independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase

The mechanisms of HCO₃⁻-independent intracellular pH (pH_i) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pH_i in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H⁺ pump by Cl⁻ removal or via the selective inhibitor bafilomycin had only a small effect upon resting pH_i and recovery following an acid load. In contrast, resting pH_i in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pH_i recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO₃⁻-independent pH_i regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.     

A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B³C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B³C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B³C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B³C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B³C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10⁻⁶ cm/s to 2.9±1.0 x 10⁻⁶ cm/s or 1.1±0.4 x 10⁻⁶ cm/s, respectively). Measurement of electrical resistance in B³C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B³C exhibits significantly improved barrier characteristics compared to the transwell model and B³C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B³C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.     

Astrocytic gap junctional communication is reduced in amyloid-β-treated cultured astrocytes,but not in Alzheimer's disease transgenic mice

Alzheimer''s disease is characterized by accumulation of amyloid deposits in brain, progressive cognitive deficits and reduced glucose utilization. Many consequences of the disease are attributed to neuronal dysfunction, but roles of astrocytes in its pathogenesis are not well understood. Astrocytes are extensively coupled via gap junctions, and abnormal trafficking of metabolites and signalling molecules within astrocytic syncytia could alter functional interactions among cells comprising the neurovascular unit. To evaluate the influence of amyloid-β on astrocyte gap junctional communication, cultured astrocytes were treated with monomerized amyloid-β_1–40 (1 μmol/l) for intervals ranging from 2 h to 5 days, and the areas labelled by test compounds were determined by impaling a single astrocyte with a micropipette and diffusion of material into coupled cells. Amyloid-β-treated astrocytes had rapid, sustained 50–70% reductions in the area labelled by Lucifer Yellow, anionic Alexa Fluor® dyes and energy-related compounds, 6-NBDG (a fluorescent glucose analogue), NADH and NADPH. Amyloid-β treatment also caused a transient increase in oxidative stress. In striking contrast with these results, spreading of Lucifer Yellow within astrocytic networks in brain slices from three regions of 8.5–14-month-old control and transgenic Alzheimer''s model mice was variable, labelling 10–2000 cells; there were no statistically significant differences in the number of dye-labelled cells among the groups or with age. Thus amyloid-induced dysfunction of gap junctional communication in cultured astrocytes does not reflect the maintenance of dye transfer through astrocytic syncytial networks in transgenic mice; the pathophysiology of Alzheimer''s disease is not appropriately represented by the cell culture system.     

miR-21a-5p Promotes Inflammation following Traumatic Spinal Cord Injury through Upregulation of Neurotoxic Reactive Astrocyte (A1) Polarization by Inhibiting the CNTF/STAT3/Nkrf Pathway

Reactive astrocytes are implicated in traumatic spinal cord injury (TSCI). Interestingly, naïve astrocytes can easily transform into neurotoxic reactive astrocytes (A1s) with inflammatory stimulation. Previous studies demonstrated that microRNA(miR)-21a-5p was up-regulated in spinal cord tissue after TSCI; however, it is not clear whether this affected reactive astrocyte polarization. Here, we aim to detect the effects of miR-21a-5p on the induction of A1 formation and the underlying mechanisms. Our study found that the expression of miR-21a-5p was significantly increased while that of Cntfr α was decreased, since naïve astrocytes transformed into A1s 3 days post-TSCI; the binding site between miR-21a-5p and Cntfr α was further confirmed in astrocytes. After treatment with CNTF, the levels of A1 markers decreased while that of A2 increased. The expression of A1 markers significantly decreased with the downregulation of miR-21a-5p, while Cntfr α siRNA treatment caused the opposite both in vitro and in vivo. To summarize, miR-21a-5p/Cntfr α promotes A1 induction and might enhance the inflammatory process of TSCI; furthermore, we identified, for the first time, the effect and potential mechanism by which CNTF inhibits naïve astrocytes transformation into A1s. Collectively, our findings demonstrate that targeting miR-21a-5p represents a prospective therapy for promoting the recovery of TSCI.     

Drimanes from Drimys brasiliensis with leishmanicidal and antimalarial activity

This paper evaluates CHCl₃ and CH₃OH extracts of the stem bark, branches and leaves of Drimys brasiliensis and drimane sesquiterpenes isolated from the stem bark against strains of Leishmania amazonensis and Leishmania braziliensis promastigotes and Plasmodium falciparum trophozoites. All of the extracts and compounds were tested in cell lines in comparison with reference standards and cell viability was determined by the XTT method. The CHCl₃ and CH₃OH extracts from the stem bark and branches yielded promising results against two strains of Leishmania, with 50% inhibitory concentrations (IC₅₀ ) values ranging from 39-100 µg/mL. The CHCl₃ extract of the stem bark returned IC₅₀ values of 39 and 40.6 µg/mL for L. amazonensis and L. braziliensis, respectively. The drimanes were relatively effective: 1-β-(p-coumaroyloxy)-polygodial produced IC₅₀ values of 5.55 and 2.52 µM for L. amazonensis and L. braziliensis, respectively, compared with 1-β-(p-methoxycinnamoyl)-polygodial, which produced respective IC₅₀ values of 15.85 and 17.80 µM. The CHCl₃ extract demonstrated activity (IC₅₀ of 3.0 µg/mL) against P. falciparum. The IC₅₀ values of 1-β-(p-cumaroyloxyl)-polygodial and 1-β-(p-methoxycinnamoyl)-polygodial were 1.01 and 4.87 µM, respectively, for the trophozoite strain. Therefore, the results suggest that D. brasiliensis is a promising plant from which to obtain new and effective antiparasitic agents.     

Hypoxia Inducible Factor-2α Regulates the Development of Retinal Astrocytic Network by Maintaining Adequate Supply of Astrocyte Progenitors

Here we investigate the role of hypoxia inducible factor (HIF)-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26^CreERT2, Tie2^Cre, and GFAP^Cre. Global Hif-2α disruption by Rosa26^CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2^Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAP^Cre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP⁺ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2⁺ and PDGFRα⁺ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other''s development, mediated at least in part by HIF-2α.     

CAG*CTG repeat instability in cultured human astrocytes

Cells of the central nervous system (CNS) are prone to the devastating consequences of trinucleotide repeat (TNR) expansion. Some CNS cells, including astrocytes, show substantial TNR instability in affected individuals. Since astrocyte enrichment occurs in brain regions sensitive to neurodegeneration and somatic TNR instability, immortalized SVG-A astrocytes were used as an ex vivo model to mimic TNR mutagenesis. Cultured astrocytes produced frequent (up to 2%) CAG·CTG contractions in a sequence-specific fashion, and an apparent threshold for instability was observed between 25 and 33 repeats. These results suggest that cultured astrocytes recapitulate key features of TNR mutagenesis. Furthermore, contractions were influenced by DNA replication through the repeat, suggesting that instability can arise by replication-based mechanisms in these cells. This is a crucial mechanistic point, since astrocytes in the CNS retain proliferative capacity throughout life and could be vulnerable to replication-mediated TNR instability. The presence of interruptions led to smaller but more frequent contractions, compared to a pure repeat, and the interruptions were sometimes deleted to form a perfect tract. In summary, we suggest that CAG·CTG repeat instability in cultured astrocytes is dynamic and replication-driven, suggesting that TNR mutagenesis may be influenced by the proliferative capacity of key CNS cells.     

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.     

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

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⁺.     

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm² on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10^-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.     

All astrocytes are not created equal—the role of astroglia in brain injury

In two recent papers published in Nature Neuroscience and Cell Stem Cells, Magdalena Götz and colleagues shed new light on the in vivo response of glial cells to brain injury and characterize a highly heterogeneous behavior of astrocytes to chronic and acute brain injury.Astrocytes have important roles in the brain, for example by regulating neurotransmitter clearance, controlling the formation and maintenance of synapses, and by contributing to the blood–brain barrier (BBB; for a review see [1]). In addition, astrocytes respond to acute and chronic injury by hypertrophy and induced proliferation. Notably, astrocytes in the mammalian brain represent a highly heterogeneous population and the exact cellular identity of the astrocytic response in the damaged brain remains largely unknown (for a review see [2]). Thus, live-imaging and single-cell studies are required to unravel the complexity of astrocyte behaviour and distinguish between the good and the bad effects of astrocytic activation on brain function and tissue homeostasis in response to acute and chronic injury.It is thought that astrocytes respond to injury through hypertrophy of cell bodies and processes, upregulation of the intermediate filaments GFAP and vimentin, extension of processes, proliferation and gradual overlapping of astrocytic domains (for a review see [3]). Interestingly, it is known that although some aspects of the astrocyte response to injury can be detrimental—such as the formation of a glial scar—it can also be beneficial by limiting the invasion of immune cells into the brain parenchyma [4,5,6]. However, our understanding of the response of astrocytes to injury assumes a global homogeneous response, and an unawareness of the more complex and diverse in vivo situation. Two papers from the group of Magdalena Götz, published in Nature Neuroscience and Cell Stem Cell, begin to unmask the heterogeneity of the astrocyte response to injury through in vivo live imaging after brain injury and by using multiple lesion models and comparing their effects on astroglial behaviour and properties within the injured brain.In the first study, Bardehle et al used in vivo two-photon laser-scanning microscopy to monitor individual astrocytes for up to 28 days after a stab wound to the somatosensory cortex [7]. To visualize single cells, astrocytes were labelled using different lines: GLAST^CreERT2/eGFP or Confetti reporter, labelling 60–80% of all astrocytes; Aldh1l1-eGFP mice, labelling all astrocytes; and hGFAP-eGFP mice, labelling only those astrocytes with the highest GFAP expression. The authors found that most GFP⁺ astrocytes maintained their morphology after injury and that only subsets showed signs of hypertrophy and polarization towards the injury site. Interestingly, only a small population of astrocytes divided, all of which had their somata apposed to blood vessels (juxtavascular) and depended on proper functioning of the small RhoGTPase Cdc42 for their proliferative response. Strikingly, none of the labelled astrocytes migrated towards the lesion site, suggesting that the increase in GFAP reactivity often seen at the site of injury is not due to astrocyte migration, but rather is due to increased GFAP expression through hypertrophy, an increased number of proliferative cells and the upregulation of GFAP in cells that might not express detectable levels of GFAP before injury. Notably, migration of other glial cells (microglia and NG2⁺ glia) to the injury site was observed, suggesting that the migratory properties in response to injury in the brain might not be general to all glia. Thus, the contribution of activated astrocytes to the formation of a glial scar in the brain following injury might be limited and need to be reconsidered. In addition, the location of proliferating astroglial cells at juxtavascular positions, and their limited movement, suggest that these proliferating astrocytes might be a subset that is responsible for the ‘beneficial'' astrocytic response to injury by tightening the BBB, preventing the invasion of cells into the lesioned brain parenchyma. Thus, observing the glial response after brain injury in real time within their in vivo environment identified a highly selective and cell-specific astrocyte response, challenging previously held concepts of astroglial migration and massive astrocyte proliferation after injury.In the next study, Sirko et al analysed how the astroglial response varies between different types of acute or more chronic brain injury [8]. To this end the authors used four different models of injury: MCAo lesion (invasive), stab wound (invasive), APPPS1 mutation (non-invasive) and ectopic p25 activation in neurons (non-invasive). They analysed comparative data for reactive gliosis and induction of stem cell properties in activated astroglia found after brain injury (Figure 1). Interestingly, the two non-invasive, chronic lesion models induced the least response from astrocytes, with astrocytes undergoing hypertrophy but having low levels of proliferation and virtually no neurosphere-forming capacity, indicating that chronic injury in these models does not enhance astrocyte proliferation or acquisition of stem cell properties. In contrast, a much larger astrocytic response occurred in the invasive models, in which astrocytes not only underwent hypertrophy but also had a relatively high proliferative rate and formed multipotent and self-renewing neurospheres in vitro. The authors then showed that Sonic hedgehog (SHH) levels increased dramatically, but only in invasive models, and that SHH levels correlated with in vivo astrocyte proliferation rates and in vitro stem cell potential between injury conditions. By using pharmacological and genetic gain- and loss-of-function strategies, SHH signalling could indeed be identified as a crucial mediator of injury-induced acquisition of stem cell properties in astrocytes. Thus, Sirko et al identified substantial differences with respect to glial response between chronic and acute injury models and identified a molecular pathway (SHH) that at least partly accounts for enhanced astroglial response in invasive injury models.Open in a separate windowFigure 1Glial cell response, stem cell potential and extracellular Sonic hedgehog (SHH) levels vary depending on the type of brain injury. Astrocytes (yellow), NG2⁺ glial cells (blue) and microglia (red) reside in the uninjured intact brain, in which only NG2⁺ cells usually proliferate. When this tissue is studied in vitro to measure its stem cell potential, virtually no neurospheres are formed. After different types of injury, however, morphological and proliferative changes occur to all cells and their in vitro stem cell potential can be reactivated. In six-month-old APPPS1 mice, all glial cells change their morphology, with astrocytic and NG2⁺ hypertrophy of cell body and processes, and hypertrophy and reduction of processes in microglia. While few astrocytes proliferate, large amounts of proliferation ocurrs in both NG2⁺ glia and microglia. This tissue in vitro can form a few spheres that are self-renewing and multipotent, generating astrocytes, neurons and oligodendrocytes. In a model of neuronal death (CK/p25; overexpressing p25 in the postnatal forebrain), astrocytes and microglia change their morphology as described above. Astrocytes and NG2⁺ glia do not have any increase in proliferation rates, whereas microglia proliferate greatly. This tissue has little stem cell potential and makes only a few primary multipotent spheres. Finally, in the more invasive stab wound injury to the cortex, all glial cells become morphologically reactive, and astrocytes, NG2⁺ glia and microglia all proliferate in response. This tissue has the largest stem cell potential, capable of making both primary and secondary spheres with multipotent progeny. In each situation, the levels of SHH (green) can be correlated with the proliferation rates of astrocytes and in vitro stem cell potential, such that only in stab wound injury are SHH levels significantly upregulated. APPPS1, co-expresses mutated amyloid precursor protein 1 and mutated presenilin 1; NG2⁺, neuron-glial antigen 2.The two papers by the Götz group shed new light on the in vivo response of glial cells to brain injury and characterize a highly heterogeneous behaviour of astrocytes to chronic and acute brain injury. Surprisingly, only subsets of astrocytes proliferate or polarize, and none of them migrate towards the lesion. The juxtavascular position of proliferating astrocytes suggests that these cells might have access to the increase in SHH after invasive injury, which can regulate their division. However, it is not clear whether this proliferation is through their de-differentiation and acquisition of neural stem cell potential, or whether it is a result of a mature astrocyte division. That the astrocyte progeny remains with the original cell at the juxtavascular location suggests that they might be acting in a positive way to limit the migration of invading immune cells into the brain. Further studies on whether the increase in juxtavascular, astroglial proliferation affects the BBB permeability or decreases the number of invading cells will be important to understand this effect. If it turns out that enhanced astroglial proliferation might be generally beneficial for the injured brain, it is also tempting to speculate that for other brain injuries where the proliferation rates and SHH levels are reduced, enhanced glial proliferation in close proximity to blood vessels might help to reduce tissue damage and to improve regeneration and repair. Thus, SHH could represent a future therapeutic target to activate glial proliferation in the context of non-invasive, chronic brain injury. In any case, the acquisition of stem cell properties allowing astrocytes to form neurospheres in vitro is not directly tied to the in vivo use of these stem cell properties (for a review, see [9]). Whether the de-differentiation of astrocytes and proliferation of stem cells in vivo is beneficial or detrimental remains unclear. However, the new data have set the cellular framework for future studies to understand injury-induced astroglial stem cell characteristics in vivo and whether this in vitro potential might be unleashed for regenerative strategies in vivo.     

Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease.     

Radiobiological comparison of two radiotherapy treatment techniques for high-risk prostate cancer

Background

To make a radiobiological comparison, for high risk prostate cancer (T3a, PSA > 20 ng/ml or Gleason > 7) of two radiotherapy treatment techniques. One technique consists of a treatment in three phases of the pelvic nodes, vesicles and prostate using a conventional fractionation scheme of 2 Gy/fraction (SIMRT). The other technique consists of a treatment in two phases that gives simultaneously different dose levels in each phase, 2 Gy/fraction, 2.25 Gy/fraction and 2.5 Gy/fraction to the pelvic nodes, vesicles and prostate, respectively (SIBIMRT).

Materials and methods

The equivalent dose at fractionation of 2 Gy (EQD₂), calculated using the linear quadratic model with α/β_prostate = 1.5 Gy, was the same for both treatment strategies. For comparison the parameters employed were D95, mean dose and Tumour Control Probabilities for prostate PTV and D15, D25, D35, D50, mean dose and Normal Tissue Complication Probabilities for the rectum and bladder, with physical doses converted to EQD₂. Parameters were obtained for α/β_prostate = 1.5, 3 and 10 Gy and for α/β_oar = 1, 2, 3, 4, 6 and 8.

Results

For prostate PTV, both treatment strategies are equivalent for α/β_prostate = 1.5 Gy but for higher α/β_prostate, EQD₂ and TCP, decrease for the SIBIMRT technique. For the rectum and bladder when α/β_oar ≤ 2 Gy, EQD₂ and NTCP are lower for the SIMRT technique or equal in both techniques. For α/β_oar ≥ 2–3 Gy, EQD₂ and NTCP increase for the SIMRT treatment.

Conclusions

A comparison between two radiotherapy techniques is presented. The SIBIMRT technique reduces EQD₂ and NTCP for α/β_oar from 2 to 8 Gy.     

BMP Signaling in Astrocytes Downregulates EGFR to Modulate Survival and Maturation

Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.     

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca²⁺ concentration fluctuations ([Ca²⁺]_i) and in several settings respond to neuronal excitation with enhanced [Ca²⁺]_i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca²⁺]_i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca²⁺]_i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca²⁺]_i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca²⁺]_i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca²⁺]_i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca²⁺]_i signals in the striatal microcircuitry.     

Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ_1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ_1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ_1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.