Astrocytes remember inflammation.

Astrocytes respond to all forms of central nervous system maladies. In a recent issue of Nature, Lee et al. demonstrate that astrocytes encode inflammatory stimuli as epigenetic memory, which strengthens responses to subsequent stimuli and exacerbates pathology in disease models.

Histone serotonylation regulates ependymoma tumorigenesis.

Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment3. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.

Remote neuronal activity drives glioma progression through SEMA4F.

The tumour microenvironment plays an essential role in malignancy, and neurons have emerged as a key component of the tumour microenvironment that promotes tumourigenesis across a host of cancers1,2. Recent studies on glioblastoma (GBM) highlight bidirectional signalling between tumours and neurons that propagates a vicious cycle of proliferation, synaptic integration and brain hyperactivity3-8; however, the identity of neuronal subtypes and tumour subpopulations driving this phenomenon is incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumours promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity-dependent infiltrating population present at the leading edge of mouse and human tumours that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified SEMA4F as a key regulator of tumourigenesis and activity-dependent progression. Furthermore, SEMA4F promotes the activity-dependent infiltrating population and propagates bidirectional signalling with neurons by remodelling tumour-adjacent synapses towards brain network hyperactivity. Collectively our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, and also show new mechanisms of glioma progression that are regulated by neuronal activity.

Comparative Transcriptomic Analysis of Cerebellar Astrocytes across Developmental Stages and Brain Regions.

Astrocytes are the most abundant glial cell type in the central nervous system, and they play a crucial role in normal brain function. While gliogenesis and glial differentiation occur during perinatal cerebellar development, the processes that occur during early postnatal development remain obscure. In this study, we conducted transcriptomic profiling of postnatal cerebellar astrocytes at postnatal days 1, 7, 14, and 28 (P1, P7, P14, and P28), identifying temporal-specific gene signatures at each specific time point. Comparing these profiles with region-specific astrocyte differentially expressed genes (DEGs) published for the cortex, hippocampus, and olfactory bulb revealed cerebellar-specific gene signature across these developmental timepoints. Moreover, we conducted a comparative analysis of cerebellar astrocyte gene signatures with gene lists from pediatric brain tumors of cerebellar origin, including ependymoma and medulloblastoma. Notably, genes downregulated at P14, such as Kif11 and HMGB2, exhibited significant enrichment across all pediatric brain tumor groups, suggesting the importance of astrocytic gene repression during cerebellar development to these tumor subtypes. Collectively, our studies describe gene expression patterns during cerebellar astrocyte development, with potential implications for pediatric tumors originating in the cerebellum.

Segmentation-Less, Automated, Vascular Vectorization.

Recent advances in two-photon fluorescence microscopy (2PM) have allowed large scale imaging and analysis of blood vessel networks in living mice. However, extracting network graphs and vector representations for the dense capillary bed remains a bottleneck in many applications. Vascular vectorization is algorithmically difficult because blood vessels have many shapes and sizes, the samples are often unevenly illuminated, and large image volumes are required to achieve good statistical power. State-of-the-art, three-dimensional, vascular vectorization approaches often require a segmented (binary) image, relying on manual or supervised-machine annotation. Therefore, voxel-by-voxel image segmentation is biased by the human annotator or trainer. Furthermore, segmented images oftentimes require remedial morphological filtering before skeletonization or vectorization. To address these limitations, we present a vectorization method to extract vascular objects directly from unsegmented images without the need for machine learning or training. The Segmentation-Less, Automated, Vascular Vectorization (SLAVV) source code in MATLAB is openly available on GitHub. This novel method uses simple models of vascular anatomy, efficient linear filtering, and vector extraction algorithms to remove the image segmentation requirement, replacing it with manual or automated vector classification. Semi-automated SLAVV is demonstrated on three in vivo 2PM image volumes of microvascular networks (capillaries, arterioles and venules) in the mouse cortex. Vectorization performance is proven robust to the choice of plasma- or endothelial-labeled contrast, and processing costs are shown to scale with input image volume. Fully-automated SLAVV performance is evaluated on simulated 2PM images of varying quality all based on the large (1.4×0.9×0.6 mm3 and 1.6×108 voxel) input image. Vascular statistics of interest (e.g. volume fraction, surface area density) calculated from automatically vectorized images show greater robustness to image quality than those calculated from intensity-thresholded images.

A Window of Vascular Plasticity Coupled to Behavioral Recovery after Stroke.

Stroke causes remodeling of vasculature surrounding the infarct, but whether and how vascular remodeling contributes to recovery are unclear. We established an approach to monitor and compare changes in vascular structure and blood flow with high spatiotemporal precision after photothrombotic infarcts in motor cortex using longitudinal 2-photon and multiexposure speckle imaging in mice of both sexes. A spatially graded pattern of vascular structural remodeling in peri-infarct cortex unfolded over the first 2 weeks after stroke, characterized by vessel loss and formation, and selective stabilization of a subset of new vessels. This vascular structural plasticity was coincident with transient activation of transcriptional programs relevant for vascular remodeling, reestablishment of peri-infarct blood flow, and large improvements in motor performance. Local vascular plasticity was strongly predictive of restoration of blood flow, which was in turn predictive of behavioral recovery. These findings reveal the spatiotemporal evolution of vascular remodeling after stroke and demonstrate that a window of heightened vascular plasticity is coupled to the reestablishment of blood flow and behavioral recovery. Our findings support that neovascularization contributes to behavioral recovery after stroke by restoring blood flow to peri-infarct regions. These findings may inform strategies for enhancing recovery from stroke and other types of brain injury.SIGNIFICANCE STATEMENT An improved understanding of neural repair could inform strategies for enhancing recovery from stroke and other types of brain injury. Stroke causes remodeling of vasculature surrounding the lesion, but whether and how the process of vascular remodeling contributes to recovery of behavioral function have been unclear. Here we used longitudinal in vivo imaging to track vascular structure and blood flow in residual peri-infarct cortex after ischemic stroke in mice. We found that stroke created a restricted period of heightened vascular plasticity that was associated with restoration of blood flow, which was in turn predictive of recovery of motor function. Therefore, our findings support that vascular remodeling facilitates behavioral recovery after stroke by restoring blood flow to peri-infarct cortex.

Rehabilitation Augments Hematoma Clearance and Attenuates Oxidative Injury and Ion Dyshomeostasis After Brain Hemorrhage.

BACKGROUND AND PURPOSE: We assessed the elemental and biochemical effects of rehabilitation after intracerebral hemorrhage, with emphasis on iron-mediated oxidative stress, using a novel multimodal biospectroscopic imaging approach. METHODS: Collagenase-induced striatal hemorrhage was produced in rats that were randomized to enriched rehabilitation or control intervention starting on day 7. Animals were euthanized on day 14 or 21, a period of ongoing cell death. We used biospectroscopic imaging techniques to precisely determine elemental and molecular changes on day 14. Hemoglobin content was assessed with resonance Raman spectroscopy. X-ray fluorescence imaging mapped iron, chlorine, potassium, calcium, and zinc. Protein aggregation, a marker of oxidative stress, and the distribution of other macromolecules were assessed with Fourier transform infrared imaging. A second study estimated hematoma volume with a spectrophotometric assay at 21 days. RESULTS: In the first experiment, rehabilitation reduced hematoma hemoglobin content (P=0.004) and the amount of peri-hematoma iron (P<0.001). Oxidative damage was highly localized at the hematoma/peri-hematoma border and was decreased by rehabilitation (P=0.004). Lipid content in the peri-hematoma zone was increased by rehabilitation (P=0.016). Rehabilitation reduced the size of calcium deposits (P=0.040) and attenuated persistent dyshomeostasis of Cl- (P<0.001) but not K+ (P=0.060). The second study confirmed that rehabilitation decreased hematoma volume (P=0.024). CONCLUSIONS: Rehabilitation accelerated clearance of toxic blood components and decreased chronic oxidative stress. As well, rehabilitation attenuated persistent ion dyshomeostasis. These novel effects may underlie rehabilitation-induced neuroprotection and improved recovery of function. Pharmacotherapies targeting these mechanisms may further improve outcome.

Microvascular plasticity in mouse stroke model recovery: Anatomy statistics, dynamics measured by longitudinal in vivo two-photon angiography, network vectorization.

This manuscript quantitatively investigates remodeling dynamics of the cortical microvascular network (thousands of connected capillaries) following photothrombotic ischemia (cubic millimeter volume, imaged weekly) using a novel in vivo two-photon angiography and high throughput vascular vectorization method. The results suggest distinct temporal patterns of cerebrovascular plasticity, with acute remodeling peaking at one week post-stroke. The network architecture then gradually stabilizes, returning to a new steady state after four weeks. These findings align with previous literature on neuronal plasticity, highlighting the correlation between neuronal and neurovascular remodeling. Quantitative analysis of neurovascular networks using length- and strand-based statistical measures reveals intricate changes in network anatomy and topology. The distance and strand-length statistics show significant alterations, with a peak of plasticity observed at one week post-stroke, followed by a gradual return to baseline. The orientation statistic plasticity peaks at two weeks, gradually approaching the (conserved across subjects) stroke signature. The underlying mechanism of the vascular response (angiogenesis vs. tissue deformation), however, is yet unexplored. Overall, the combination of chronic two-photon angiography, vascular vectorization, reconstruction/visualization, and statistical analysis enables both qualitative and quantitative assessments of neurovascular remodeling dynamics, demonstrating a method for investigating cortical microvascular network disorders and the therapeutic modes of action thereof.

Subventricular zone cytogenesis provides trophic support for neural repair in a mouse model of stroke.

Stroke enhances proliferation of neural precursor cells within the subventricular zone (SVZ) and induces ectopic migration of newborn cells towards the site of injury. Here, we characterize the identity of cells arising from the SVZ after stroke and uncover a mechanism through which they facilitate neural repair and functional recovery. With genetic lineage tracing, we show that SVZ-derived cells that migrate towards cortical photothrombotic stroke in mice are predominantly undifferentiated precursors. We find that ablation of neural precursor cells or conditional knockout of VEGF impairs neuronal and vascular reparative responses and worsens recovery. Replacement of VEGF is sufficient to induce neural repair and recovery. We also provide evidence that CXCL12 from peri-infarct vasculature signals to CXCR4-expressing cells arising from the SVZ to direct their ectopic migration. These results support a model in which vasculature surrounding the site of injury attracts cells from the SVZ, and these cells subsequently provide trophic support that drives neural repair and recovery.

Diamond Raman laser and Yb fiber amplifier for in vivo multiphoton fluorescence microscopy.

Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for two-photon excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. With our system we demonstrate compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.

Reactive astrocytes facilitate vascular repair and remodeling after stroke.

Brain injury causes astrocytes to assume a reactive state that is essential for early tissue protection, but how reactive astrocytes affect later reparative processes is incompletely understood. In this study, we show that reactive astrocytes are crucial for vascular repair and remodeling after ischemic stroke in mice. Analysis of astrocytic gene expression data reveals substantial activation of transcriptional programs related to vascular remodeling after stroke. In vivo two-photon imaging provides evidence of astrocytes contacting newly formed vessels in cortex surrounding photothrombotic infarcts. Chemogenetic ablation of a subset of reactive astrocytes after stroke dramatically impairs vascular and extracellular matrix remodeling. This disruption of vascular repair is accompanied by prolonged blood flow deficits, exacerbated vascular permeability, ongoing cell death, and worsened motor recovery. In contrast, vascular structure in the non-ischemic brain is unaffected by focal astrocyte ablation. These findings position reactive astrocytes as critical cellular mediators of functionally important vascular remodeling during neural repair.

Multimodal mapping of neural activity and cerebral blood flow reveals long-lasting neurovascular dissociations after small-scale strokes.

Neurovascular coupling, the close spatial and temporal relationship between neural activity and hemodynamics, is disrupted in pathological brain states. To understand the altered neurovascular relationship in brain disorders, longitudinal, simultaneous mapping of neural activity and hemodynamics is critical yet challenging to achieve. Here, we use a multimodal neural platform in a mouse model of stroke and realize long-term, spatially resolved tracking of intracortical neural activity and cerebral blood flow in the same brain regions. We observe a pronounced neurovascular dissociation that occurs immediately after small-scale strokes, becomes the most severe a few days after, lasts into chronic periods, and varies with the level of ischemia. Neuronal deficits extend spatiotemporally, whereas restoration of cerebral blood flow occurs sooner and reaches a higher relative value. Our findings reveal the neurovascular impact of ministrokes and inform the limitation of neuroimaging techniques that infer neural activity from hemodynamic responses.

Acetazolamide Mitigates Intracranial Pressure Spikes Without Affecting Functional Outcome After Experimental Hemorrhagic Stroke.

Increased intracranial pressure (ICP) after stroke can lead to poor outcome and death. Novel treatments to combat ICP rises are needed. The carbonic anhydrase inhibitor acetazolamide diminishes cerebrospinal fluid (CSF) production, reduces ICP in healthy animals, and is beneficial for idiopathic intracranial hypertension patients. We tested whether acetazolamide mitigates ICP elevations by presumably decreasing CSF volume after collagenase-induced striatal hemorrhage in rats. We confirmed that acetazolamide did not adversely affect hematoma formation in this model or physiological variables, such as temperature. Then, we assessed the effects of acetazolamide on ICP. Lastly, we tested the effects of acetazolamide on behavioral and histological outcome. Acetazolamide reduced the magnitude and occurrence of short-timescale ICP spikes, assessed as disproportionate increases in ICP (sudden ICP increases > 10 mmHg), 1-min peak ICP, and the magnitude of spikes > 20 mmHg. However, mean ICP was unaffected. In addition, acetazolamide reduced ICP variability, reflecting improved intracranial compliance. Compliance measures were strongly correlated with high peak and mean ICP, whereas ipsilateral hemisphere water content was not correlated with ICP. Despite effects on ICP, acetazolamide did not improve behavioral function or affect lesion size. In summary, we show that intracerebral hemorrhage creates an impaired compliance state within the cranial space that can result in large, transient ICP spikes. Acetazolamide ameliorates intracranial compliance and mitigates ICP spikes, but does not improve functional outcome, at least for moderate-severity ICH in rats.

Functions of subventricular zone neural precursor cells in stroke recovery.

The proliferation and ectopic migration of neural precursor cells (NPCs) in response to ischemic brain injury was first reported two decades ago. Since then, studies of brain injury-induced subventricular zone cytogenesis, primarily in rodent models, have provided insight into the cellular and molecular determinants of this phenomenon and its modulation by various factors. However, despite considerable correlational evidence-and some direct evidence-to support contributions of NPCs to behavioral recovery after stroke, the causal mechanisms have not been identified. Here we discuss the subventricular zone cytogenic response and its possible roles in brain injury and disease, focusing on rodent models of stroke. Emerging evidence suggests that NPCs can modulate harmful responses and enhance reparative responses to neurologic diseases. We speculatively identify four broad functions of NPCs in the context of stroke: cell replacement, cytoprotection, remodeling of residual tissue, and immunomodulation. Thus, NPCs may have pleiotropic functions in supporting behavioral recovery after stroke.

Artery targeted photothrombosis widens the vascular penumbra, instigates peri-infarct neovascularization and models forelimb impairments.

The photothrombotic stroke model generates localized and reproducible ischemic infarcts that are useful for studying recovery mechanisms, but its failure to produce a substantial ischemic penumbra weakens its resemblance to human stroke. We examined whether a modification of this approach, confining photodamage to arteries on the cortical surface (artery-targeted photothrombosis), could better reproduce aspects of the penumbra. Following artery-targeted or traditional photothrombosis to the motor cortex of mice, post-ischemic cerebral blood flow was measured using multi-exposure speckle imaging at 6, 48, and 120 h post-occlusion. Artery-targeted photothrombosis produced a more graded penumbra at 48 and 120 h. The density of isolectin B4+ vessels in peri-infarct cortex was similarly increased after both types of infarcts compared to sham at 2 weeks. These results indicate that both models instigated post-ischemic vascular structural changes. Finally, we determined whether the strength of the traditional photothrombotic approach for modeling upper-extremity motor impairments extends to the artery-targeted approach. In adult mice that were proficient in a skilled reaching task, small motor-cortical infarcts impaired skilled-reaching performance for up to 10 days. These results support that artery-targeted photothrombosis widens the penumbra while maintaining the ability to create localized infarcts useful for modeling post-stroke impairments.

Measurement of Intracranial Pressure in Freely Moving Rats.

Brain injury, such as from stroke and trauma, can be complicated by elevated intracranial pressure (ICP). Although raised ICP can be a significant determinant of morbidity and mortality, clinical studies often report widely varying ICP measurements depending on location of measurement and technique used. For the same reasons, reported ICP measurements also vary widely in animal models. The need for anesthesia or tethered connections with some methods of ICP measurement in animals may introduce additional confounds. Moreover, these methods are not well suited for prolonged, continuous measurement. Here, we describe an approach to continually measure ICP in awake, freely moving rats for several days. This technique uses a commercially available, wireless pressure sensor mounted on the head to measure ICP from the epidural space via a fluid-filled catheter. We have demonstrated that this approach reliably detects elevations in ICP that last for several days after ischemic and hemorrhagic strokes in rat.

Evidence for Decreased Brain Parenchymal Volume After Large Intracerebral Hemorrhages: a Potential Mechanism Limiting Intracranial Pressure Rises.

Potentially fatal intracranial pressure (ICP) rises commonly occur after large intracerebral hemorrhages (ICH). We monitored ICP after infusing 100-160 µL of autologous blood (vs. 0 µL control) into the striatum of rats in order to test the validity of this common model with regard to ICP elevations. Other endpoints included body temperature, behavioral impairment, lesion volume, and edema. Also, we evaluated hippocampal CA1 sector and somatosensory cortical neuron morphology to assess whether global ischemic injury occurred. Despite massive blood infusions, ICP only modestly increased (160 µL 10.8 ± 2.1 mmHg for <36 h vs. control 3.4 ± 0.5 mmHg), with little peri-hematoma edema at 3 days. Body temperature was not affected. Behavioral deficits and tissue loss were infusion volume-dependent. There was no histological evidence of hippocampal or cortical injury, indicating that cell death was confined to the hematoma and closely surrounding tissue. Surprisingly, the most severe hemorrhages significantly increased cell density (~15-20%) and reduced cell body size (~30%) in regions outside the injury site. Additionally, decreased cell size and increased density were observed after collagenase-induced ICH. Parenchymal volume is seemingly reduced after large ICH. Thus, in addition to well-known compliance mechanisms (e.g., displacement of cerebrospinal fluid and cerebral blood), reduced brain parenchymal volume appears to limit ICP rises in rodents with very large mass lesions.

Localized hypothermia aggravates bleeding in the collagenase model of intracerebral hemorrhage.

Animal studies testing whether therapeutic hypothermia is neuroprotective after intracerebral hemorrhage (ICH) have been inconclusive. In rodents, ICH is often produced in the striatum by infusing collagenase, which causes prolonged hemorrhaging from multiple vessels. Our previous data shows that this bleeding (hematoma) is worsened by systemic hypothermia given soon after collagenase infusion. In this study we hypothesized that localized brain hypothermia would also aggravate bleeding in this model (0.2 U of collagenase in 1.2 µL of saline). We also evaluated cooling after intrastriatal thrombin infusion (1 U in 30 µL of saline)-a simplified model of ICH thought to cause bleeding. Focal hypothermia was achieved by flushing cold water through an implanted cooling device attached to the skull underneath the temporalis muscle of adult rats. Previous work and data at this time shows this method cools the striatum to ∼33°C, whereas the body remains normothermic. In comparison to normothermic groups, cooling significantly worsened bleeding when instituted at 6 hours (∼94 vs. 42 µL, p=0.018) and 12 hours (79 vs. 61 µL, p=0.042) post-ICH (24-hour survival), but not after a 24-hour delay (36-hour survival). Rats were cooled until euthanasia when hematoma size was determined by a hemoglobin-based spectrophotometry assay. Cooling did not influence cerebral blood volume after just saline or thrombin infusion. The latter is explained by the fact that thrombin did not cause bleeding beyond that caused by saline infusion. In summary, local hypothermia significantly aggravates bleeding many hours after collagenase infusion suggesting that bleeding may have confounded earlier studies with hypothermia. Furthermore, these findings serve as a cautionary note on using cooling even many hours after cerebral bleeding.

Use of an improvised pneumatic anti-shock garment and a non-pneumatic anti-shock garment to control pelvic blood flow.

BACKGROUND: Pelvic bleeding from trauma and postpartum hemorrhage is often difficult to treat successfully by emergency providers particularly in low resource environments, when hospital presentation is delayed or there is a lack of immediate surgical, anesthesia, and transfusion capabilities. Pneumatic anti-shock garments (PASG) decrease pelvic blood flow and hemorrhage. A tightly fitted neoprene non-pneumatic anti-shock garment (NASG) has been shown to decrease blood loss and improve survival rates from postpartum hemorrhage. AIMS: The objective of this study was to determine whether blood flow to the pelvis is decreased by use of the NASG or by an improvised PASG. METHODS: A PASG was made using three bicycle tubes, placing one tube on each leg and one on the lower abdomen/pelvis, wrapping firmly with sheets and inflating the tubes to approximately 3.5 bar (45 psi). A Doppler ultrasound was used to measure distal aortic blood flow in 12 healthy adults at baseline and in both devices. Data were analyzed with one sample and paired t tests. RESULTS: Mean flow was 1.99 l/min at baseline. Mean flow decrease was 1.11 [95% confidence interval (CI): 0.64-1.57, p = 0.0003 for the difference] for the PASG and 0.65 (95% CI: 0.03-1.26, p = 0.04) for the NASG. The PASG decreased blood flow more than the NASG (mean difference: 0.46, 95% CI: 0.02-0.90, p = 0.04). CONCLUSIONS: Both devices decreased distal aortic blood flow, but the improvised PASG device decreased it by a larger margin.