Exploring the dynamics of adult Axin2 cell lineage integration into dentate gyrus granule neurons.

The Wnt pathway plays critical roles in neurogenesis. The expression of Axin2 is induced by Wnt/ß-catenin signaling, making this gene a reliable indicator of canonical Wnt activity. We employed pulse-chase genetic lineage tracing with the Axin2-CreERT2 allele to follow the fate of Axin2+ lineage in the adult hippocampal formation. We found Axin2 expressed in astrocytes, neurons and endothelial cells, as well as in the choroid plexus epithelia. Simultaneously with the induction of Axin2 fate mapping by tamoxifen, we marked the dividing cells with 5-ethynyl-2'-deoxyuridine (EdU). Tamoxifen induction led to a significant increase in labeled dentate gyrus granule cells three months later. However, none of these neurons showed any EdU signal. Conversely, six months after the pulse-chase labeling with tamoxifen/EdU, we identified granule neurons that were positive for both EdU and tdTomato lineage tracer in each animal. Our data indicates that Axin2 is expressed at multiple stages of adult granule neuron differentiation. Furthermore, these findings suggest that the integration process of adult-born neurons from specific cell lineages may require more time than previously thought.

Focused Ultrasound Blood-Brain Barrier Opening Arrests the Growth and Formation of Cerebral Cavernous Malformations.

BACKGROUND: Cerebral cavernous malformations (CCM) are vascular lesions within the central nervous system, consisting of dilated and hemorrhage-prone capillaries. CCMs can cause debilitating neurological symptoms, and surgical excision or stereotactic radiosurgery are the only current treatment options. Meanwhile, transient blood-brain barrier opening (BBBO) with focused ultrasound (FUS) and microbubbles is now understood to exert potentially beneficial bioeffects, such as stimulation of neurogenesis and clearance of amyloid-ß. Here, we tested whether FUS BBBO could be deployed therapeutically to control CCM formation and progression in a clinically-representative murine model. METHODS: CCMs were induced in mice by postnatal, endothelial-specific Krit1 ablation. FUS was applied for BBBO with fixed peak-negative pressures (PNPs; 0.2-0.6 MPa) or passive cavitation detection-modulated PNPs. Magnetic resonance imaging (MRI) was used to target FUS treatments, evaluate safety, and measure longitudinal changes in CCM growth after BBBO. RESULTS: FUS BBBO elicited gadolinium accumulation primarily at the perilesional boundaries of CCMs, rather than lesion cores. Passive cavitation detection and gadolinium contrast enhancement were comparable in CCM and wild-type mice, indicating that Krit1 ablation does not confer differential sensitivity to FUS BBBO. Acutely, CCMs exposed to FUS BBBO remained structurally stable, with no signs of hemorrhage. Longitudinal MRI revealed that FUS BBBO halted the growth of 94% of CCMs treated in the study. At 1 month, FUS BBBO-treated lesions lost, on average, 9% of their pre-sonication volume. In contrast, non-sonicated control lesions grew to 670% of their initial volume. Lesion control with FUS BBBO was accompanied by a marked reduction in the area and mesenchymal appearance of Krit mutant endothelium. Strikingly, in mice receiving multiple BBBO treatments with fixed PNPs, de novo CCM formation was significantly reduced by 81%. Mock treatment plans on MRIs of patients with surgically inaccessible lesions revealed their lesions are amenable to FUS BBBO with current clinical technology. CONCLUSIONS: Our results establish FUS BBBO as a novel, non-invasive modality that can safely arrest murine CCM growth and prevent their de novo formation. As an incisionless, MR image-guided therapy with the ability to target eloquent brain locations, FUS BBBO offers an unparalleled potential to revolutionize the therapeutic experience and enhance the accessibility of treatments for CCM patients.

Dynamics of Adult Axin2 Cell Lineage Integration in Granule Neurons of the Dentate Gyrus.

The Wnt pathway plays critical roles in neurogenesis. The expression of Axin2 is induced by Wnt/ß-catenin signaling, making this gene a sensitive indicator of canonical Wnt activity. We employed pulse-chase genetic lineage tracing with the Axin2-CreERT2 allele to follow the fate of Axin2 -positive cells in the adult hippocampal formation. We found Axin2 expressed in astrocytes, neurons and endothelial cells, as well as in the choroid plexus epithelia. Simultaneously with tamoxifen induction of Axin2 fate mapping, the dividing cells were marked with 5-ethynyl-2'-deoxyuridine (EdU). Tamoxifen induction resulted in significant increase of dentate gyrus granule cells three months later; however, none of these neurons contained EdU signal. Conversely, six months after the tamoxifen/EdU pulse-chase labeling, EdU-positive granule neurons were identified in each animal. Our data imply that Axin2 is expressed at several different stages of adult granule neuron differentiation and suggest that the process of integration of the adult-born neurons from certain cell lineages may take longer than previously thought.

Intraarterial Transplantation of Mitochondria After Ischemic Stroke Reduces Cerebral Infarction.

Background-: Transplantation of autologous mitochondria into ischemic tissue may mitigate injury caused by ischemia and reperfusion. Methods-: Using murine stroke models of middle cerebral artery occlusion, we sought to evaluate feasibility of delivery of viable mitochondria to ischemic brain parenchyma. We evaluated the effects of concurrent focused ultrasound activation of microbubbles, which serves to open the blood-brain barrier, on efficacy of delivery of mitochondria. Results-: Following intra-arterial delivery, mitochondria distribute through the stroked hemisphere and integrate into neural and glial cells in the brain parenchyma. Consistent with functional integration in the ischemic tissue, the transplanted mitochondria elevate concentration of adenosine triphosphate in the stroked hemisphere, reduce infarct volume and increase cell viability. Additional of focused ultrasound leads to improved blood brain barrier opening without hemorrhagic complications. Conclusions-: Our results have implications for the development of interventional strategies after ischemic stroke and suggest a novel potential modality of therapy after mechanical thrombectomy.

Preclinical models of middle cerebral artery occlusion: new imaging approaches to a classic technique.

Stroke remains a major burden on patients, families, and healthcare professionals, despite major advances in prevention, acute treatment, and rehabilitation. Preclinical basic research can help to better define mechanisms contributing to stroke pathology, and identify therapeutic interventions that can decrease ischemic injury and improve outcomes. Animal models play an essential role in this process, and mouse models are particularly well-suited due to their genetic accessibility and relatively low cost. Here, we review the focal cerebral ischemia models with an emphasis on the middle cerebral artery occlusion technique, a "gold standard" in surgical ischemic stroke models. Also, we highlight several histologic, genetic, and in vivo imaging approaches, including mouse stroke MRI techniques, that have the potential to enhance the rigor of preclinical stroke evaluation. Together, these efforts will pave the way for clinical interventions that can mitigate the negative impact of this devastating disease.

Magnetic Resonance Imaging of Mouse Cerebral Cavernomas Reveal Differential Lesion Progression and Variable Permeability to Gadolinium.

BACKGROUND: Cerebral cavernous malformations, also known as cavernous angiomas, are blood vessel abnormalities comprised of clusters of grossly enlarged and hemorrhage-prone capillaries. The prevalence in the general population, including asymptomatic cases, is estimated to be 0.5%. Some patients develop severe symptoms, including seizures and focal neurological deficits, whereas others remain asymptomatic. The causes of this remarkable presentation heterogeneity within a primarily monogenic disease remain poorly understood. METHODS: We established a chronic mouse model of cerebral cavernous malformations, induced by postnatal ablation of Krit1 with Pdgfb-CreERT2, and examined lesion progression in these mice with T2-weighted 7T magnetic resonance imaging (MRI). We also established a modified protocol for dynamic contrast-enhanced MRI and produced quantitative maps of gadolinium tracer gadobenate dimeglumine. After terminal imaging, brain slices were stained with antibodies against microglia, astrocytes, and endothelial cells. RESULTS: These mice develop cerebral cavernous malformations lesions gradually over 4 to 5 months of age throughout the brain. Precise volumetric analysis of individual lesions revealed nonmonotonous behavior, with some lesions temporarily growing smaller. However, the cumulative lesional volume invariably increased over time and after about 2 months followed a power trend. Using dynamic contrast-enhanced MRI, we produced quantitative maps of gadolinium in the lesions, indicating a high degree of heterogeneity in lesional permeability. MRI properties of the lesions were correlated with cellular markers for endothelial cells, astrocytes, and microglia. Multivariate comparisons of MRI properties of the lesions with cellular markers for endothelial and glial cells revealed that increased cell density surrounding lesions correlates with stability, whereas denser vasculature within and surrounding the lesions may correlate with high permeability. CONCLUSIONS: Our results lay a foundation for better understanding individual lesion properties and provide a comprehensive preclinical platform for testing new drug and gene therapies for controlling cerebral cavernous malformations.

Microglia Modulate Cortical Spreading Depolarizations After Ischemic Stroke: A Narrative Review.

Cortical spreading depolarizations (CSDs) are characterized by waves of diminished electroencephalography activity that propagate across the cortex with subsequent loss of ionic homeostasis. CSDs have been found in many pathological conditions, including migraine, traumatic brain injury, and ischemic stroke. Because of CSD-associated ionic and metabolic disturbances at the peri-infarct area after ischemic stroke, it is thought that CSDs exacerbate tissue infarction and worsen clinical outcomes. Microglia, the main innate immune cells in the brain, are among the first responders to brain tissue damage. Recent studies demonstrated that microglia play a critical role in CSD initiation and propagation. In this article, we discuss the significance of CSD in the setting of ischemic stroke and how microglia may modulate peri-infarct CSDs, also known as iso-electric depolarizations. Finally, we discuss the significance of microglial Ca2+ and how it might be used as a potential therapeutic target for patients with ischemic stroke.

Biomarkers Predictive of Long-Term Outcome After Ischemic Stroke: A Meta-Analysis.

BACKGROUND AND OBJECTIVE: The goal of this study was to systematically review the usefulness of serum biomarkers in the setting of ischemic stroke (IS) to predict long-term outcome. METHODS: A systematic literature review was performed using the PubMed and MEDLINE databases for studies published between 1986 and 2018. All studies assessing long-term functional outcome (defined as ≥30 days) after IS with respect to serum biomarkers were included. Data were extracted and pooled using a meta-analysis of odds ratios. RESULTS: Of the 2928 articles in the original literature search, 183 studies were selected. A total of 127 serum biomarkers were included. Biomarkers were grouped into several categories: inflammatory (n = 32), peptide/enzymatic (n = 30), oxidative/metabolic (n = 28), hormone/steroid based (n = 23), and hematologic/vascular (n = 14). The most commonly studied biomarkers in each category were found to be CRP, S100ß, albumin, copeptin, and D-dimer. With the exception of S100ß, all were found to be statistically associated with >30-day outcome after ischemic stroke. CONCLUSIONS: Serum-based biomarkers have the potential to predict functional outcome in patients with IS. This meta-analysis has identified C-reactive protein, albumin, copeptin, and D-dimer to be significantly associated with long-term outcome after IS. These biomarkers have the potential to serve as a platform for prognosticating stroke outcomes after 30 days. These serum biomarkers, some of which are routinely ordered, can be combined with imaging biomarkers and used in artificial intelligence algorithms to provide refined predictive outcomes after injury. These tools will assist physicians in providing guidance to families regarding long-term independence of patients.

Microglial Calcium Waves During the Hyperacute Phase of Ischemic Stroke.

BACKGROUND AND PURPOSE: Ischemic injury triggers multiple pathological responses in the brain tissue, including spreading depolarizations across the cerebral cortex (cortical spreading depolarizations [CSD]). Microglia have been recently shown to play a significant role in the propagation of CSD. However, the intracellular responses of myeloid cells during ischemic stroke have not been investigated. METHODS: We have studied intracellular calcium activity in cortical microglia in the stroke model of the middle cerebral artery occlusion, using the murine Polr2a-based and Cre-dependent GCaMP5 and tdTomato reporter (PC::G5-tdT). High-speed 2-photon microscopy through cranial windows was employed to record signals from genetically encoded indicators of calcium. Inflammatory stimuli and pharmacological inhibition were used to modulate microglial calcium responses in the somatosensory cortex. RESULTS: In vivo imaging revealed periodical calcium activity in microglia during the hyperacute phase of ischemic stroke. This activity was more frequent during the first 6 hours after occlusion, but the amplitudes of calcium transients became larger at later time points. Consistent with CSD nature of these events, we reproducibly triggered comparable calcium transients with microinjections of potassium chloride (KCl) into adjacent cortical areas. Furthermore, lipopolysaccharide-induced peripheral inflammation, mimicking sterile inflammation during ischemic stroke, produced significantly greater microglial calcium transients during CSD. Finally, in vivo pharmacological analysis with CRAC (calcium release-activated channel) inhibitor CM-EX-137 demonstrated that CSD-associated microglial calcium transients after KCl microinjections are mediated at least in part by the CRAC mechanism. CONCLUSIONS: Our findings demonstrate that microglia participate in ischemic brain injury via previously undetected mechanisms, which may provide new avenues for therapeutic interventions.

Systematic Review of Functional Mapping and Cortical Reorganization in the Setting of Arteriovenous Malformations, Redefining Anatomical Eloquence.

Objective: The goal of this study was to systematically review functional mapping and reorganization that takes place in the setting of arteriovenous malformations (AVMs) and its potential impact on grading and surgical decision making. Methods: A systematic literature review was performed using the PubMed database for studies published between 1986 and 2019. Studies assessing brain mapping and functional reorganization in AVMs were included. Results: Of the total 84 articles identified in the original literature search, 12 studies were ultimately selected. This includes studies evaluating the impact of cortical reorganization on patient outcomes and factors impacting and triggering cortical reorganization in AVM. Conclusion: These studies demonstrate the utility of preoperative brain mapping and acknowledgment of functional reorganization in the setting of AVMs. While these findings led to alterations in Spetzler-Martin grading and subsequent surgical decision making, it remains unclear the clinical utility of this information when assessing patient outcomes. While promising, more research is required before recommendations can be made regarding functional brain mapping and cortical reorganization with respect to AVM surgery involving eloquent brain tissue.

From Bench to Bedside, the Current State of Oncolytic Virotherapy in Pediatric Glioma.

Glioma continues to be a challenging disease process, making up the most common tumor type within the pediatric population. While low-grade gliomas are typically amenable to surgical resection, higher grade gliomas often require additional radiotherapy in conjunction with adjuvant chemotherapy. Molecular profiling of these lesions has led to the development of various pharmacologic and immunologic agents, although these modalities are not without great systemic toxicity. In addition, the molecular biology of adult glioma and pediatric glioma has been shown to differ substantially, making the application of current chemotherapies dubious in children and adolescents. For this reason, therapies with high tumor specificity based on pediatric tumor cell biology that spare healthy tissue are needed. Oncolytic virotherapy serves to fill this niche, as evidenced by renewed interest in this domain of cancer therapy. Initially discovered by chance in the early 20th century, virotherapy has emerged as a viable treatment option. With promising results based on preclinical studies, the authors review several oncolytic viruses, with a focus on molecular mechanism and efficacy of these viruses in tumor cell lines and murine models. In addition, current phase I clinical trials evaluating oncolytic virotherapy in the treatment of pediatric glioma are summarized.

Arteriovenous malformation presenting with epilepsy: a multimodal approach to diagnosis and treatment.

Arteriovenous malformation (AVM) presenting with epilepsy significantly impacts patient quality of life, and it should be considered very much a seizure disorder. Although hemorrhage prevention is the primary treatment aim of AVM surgery, seizure control should also be at the forefront of therapeutic management. Several hemodynamic and morphological characteristics of AVM have been identified to be associated with seizure presentation. This includes increased AVM flow, presence of long pial draining vein, venous outflow obstruction, and frontotemporal location, among other aspects. With the advent of high-throughput image processing and quantification methods, new radiographic attributes of AVM-related epilepsy have been identified. With respect to therapy, several treatment approaches are available, including conservative management or interventional modalities; this includes microsurgery, radiosurgery, and embolization or a combination thereof. Many studies, especially in the domain of microsurgery and radiosurgery, evaluate both techniques with respect to seizure outcomes. The advantage of microsurgery lies in superior AVM obliteration rates and swift seizure response. In addition, by incorporating electrophysiological monitoring during AVM resection, adjacent or even remote epileptogenic foci can be identified, leading to extended lesionectomy and improved seizure control. Radiosurgery, despite resulting in reduced AVM obliteration and prolonged time to seizure freedom, avoids the risks of surgery altogether and may provide seizure control through various antiepileptic mechanisms. Embolization continues to be used as an adjuvant for both microsurgery and radiosurgery. In this study, the authors review the latest imaging techniques in characterizing AVM-related epilepsy, in addition to reviewing each treatment modality.

Cortical Spreading Depression in the Setting of Traumatic Brain Injury.

Cortical spreading depression (CSD) is a pathophysiologic phenomenon that describes an expanding wave of depolarization within the cortical gray matter. Originally described over 70 years ago, this spreading depression disrupts neuronal and glial ionic equilibrium, leading to increased energy demands that can cause a metabolic crisis. This results in secondary insult, further perpetuating brain injury and neuronal death. Initially not thought to be of clinical significance, the view of CSD was modified with the advent of intracranial electroencephalography, or electrocorticography. With these improved monitoring techniques, CSD has been identified as a major mechanism by which traumatic brain injury (TBI) imparts its negative sequalae. TBI is a heterogenous disease process that runs the gamut of clinical presentations. This includes concussion, epidural and subdural hematoma, diffuse axonal injury, and subarachnoid hemorrhage. Nonetheless, CSD appears to be frequently occurring among the various types of TBI, thus allowing for the potential development of targeted therapies in an otherwise ill-fated patient cohort. Although a complete understanding of the interplay between CSD and TBI has not yet been achieved, the authors recount the efforts that have been employed over the last several decades in an effort to bridge this gap. In addition, our current understanding of the role neuroimmune cells play in CSD is discussed in the context of TBI. Finally, current therapeutic strategies using CSD as a pharmacologic target are explored with respect to their clinical use in patients with TBI.

Calcium Imaging of Microglial Network Activity in Stroke.

Calcium signaling plays a significant role in microglial activation. Genetically encoded calcium indicators (GECI) have been widely used for calcium imaging studies in many brain cell types, including neurons, astrocytes, and oligodendrocytes. However, microglial calcium imaging approaches have been hampered by idiosyncrasies of their gene expression and malleable cell properties. The generation of PC::G5-tdT, a Polr2a locus-based conditional mouse reporter of calcium, facilitated the deployment of GECI in microglia. When crossed with the Iba1(Aif1)-IRES-Cre line, all brain microglia of the progeny are labeled with the calcium indicator variant GCaMP5G and the red fluorescent protein tdTomato. This reporter system has enabled in vivo studies of intracellular calcium in large microglial cell populations in cerebral pathologies such as ischemic stroke. In this chapter, we outline specific guidelines for genetic, surgical, imaging, and data analysis aspects of microglial calcium monitoring of the ischemic cortex following middle cerebral artery occlusion.