Intercellular Signaling Pathways as Therapeutic Targets for Vascular Dementia Repair.

Vascular dementia (VaD) is a white matter ischemic disease and the second-leading cause of dementia, with no direct therapy. Within the lesion site, cell-cell interactions dictate the trajectory towards disease progression or repair. To elucidate the underlying intercellular signaling pathways, a VaD mouse model was developed for transcriptomic and functional studies. The mouse VaD transcriptome was integrated with a human VaD snRNA-Seq dataset. A custom-made database encompassing 4053 human and 2032 mouse ligand-receptor (L-R) interactions identified significantly altered pathways shared between human and mouse VaD. Two intercellular L-R systems, Serpine2-Lrp1 and CD39-A3AR, were selected for mechanistic study as both the ligand and receptor were dysregulated in VaD. Decreased Seprine2 expression enhances OPC differentiation in VaD repair. A clinically relevant drug that reverses the loss of CD39-A3AR function promotes tissue and behavioral recovery in the VaD model. This study presents novel intercellular signaling targets and may open new avenues for VaD therapies.

The emergence of multiscale connectomics-based approaches in stroke recovery.

Stroke is a leading cause of adult disability. Understanding stroke damage and recovery requires deciphering changes in complex brain networks across different spatiotemporal scales. While recent developments in brain readout technologies and progress in complex network modeling have revolutionized current understanding of the effects of stroke on brain networks at a macroscale, reorganization of smaller scale brain networks remains incompletely understood. In this review, we use a conceptual framework of graph theory to define brain networks from nano- to macroscales. Highlighting stroke-related brain connectivity studies at multiple scales, we argue that multiscale connectomics-based approaches may provide new routes to better evaluate brain structural and functional remapping after stroke and during recovery.

Theta Frequency Electromagnetic Stimulation Enhances Functional Recovery After Stroke.

Extremely low-frequency, low-intensity electromagnetic field (ELF-EMF) therapy is a non-invasive brain stimulation method that can modulate neuroprotection and neuroplasticity. ELF-EMF was recently shown to enhance recovery in human stroke in a small pilot clinical trial (NCT04039178). ELF-EMFs encompass a wide range of frequencies, typically ranging from 1 to 100 Hz, and their effects can vary depending on the specific frequency employed. However, whether and to what extent the effectiveness of ELF-EMFs depends on the frequency remains unclear. In the present study, we aimed to assess the efficacy of different frequency-intensity protocols of ELF-EMF in promoting functional recovery in a mouse cortical stroke model with treatment initiated 4 days after the stroke, employing a series of motor behavior tests. Our findings demonstrate that a theta-frequency ELF-EMF (5 Hz) effectively enhances functional recovery in a reach-to-grasp task, whereas neither gamma-frequency (40 Hz) nor combination frequency (5-16-40 Hz) ELF-EMFs induce a significant effect. Importantly, our histological analysis reveals that none of the ELF-EMF protocols employed in our study affect infarct volume, inflammatory, or glial activation, suggesting that the observed beneficial effects may be mediated through non-neuroprotective mechanisms. Our data indicate that ELF-EMFs have an influence on functional recovery after stroke, and this effect is contingent upon the specific frequency used. These findings underscore the critical importance of optimizing the protocol parameters to maximize the beneficial effects of ELF-EMF. Further research is warranted to elucidate the underlying mechanisms and refine the protocol parameters for optimal therapeutic outcomes in stroke rehabilitation.

A toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences.

Astrocytes, one of the most prevalent cell types in the central nervous system (CNS), are critically involved in neural function. Genetically manipulating astrocytes is an essential tool in understanding and affecting their roles. Adeno-associated viruses (AAVs) enable rapid genetic manipulation; however, astrocyte specificity of AAVs can be limited, with high off-target expression in neurons and sparsely in endothelial cells. Here, we report the development of a cassette of four copies of six miRNA targeting sequences (4x6T) which triggers transgene degradation specifically in neurons and endothelial cells. In combination with the GfaABC1D promoter, 4x6T increases astrocytic specificity of Cre with a viral reporter from <50% to >99% in multiple serotypes in mice, and confers astrocyte specificity in multiple recombinases and reporters. We also present empty vectors to add 4x6T to other cargo, independently and in Cre/Dre-dependent forms. This toolbox of AAVs allows rapid manipulation of astrocytes throughout the CNS, is compatible with different AAV serotypes, and demonstrates the efficacy of using multiplexed miRNA targeting sequences to decrease expression in multiple off-target cell populations simultaneously.

Motor Activity-Induced White Matter Repair in White Matter Stroke.

Subcortical white matter stroke (WMS) is a progressive disorder which is demarcated by the formation of small ischemic lesions along white matter tracts in the CNS. As lesions accumulate, patients begin to experience severe motor and cognitive decline. Despite its high rate of incidence in the human population, our understanding of the cause and outcome of WMS is extremely limited. As such, viable therapies for WMS remain to be seen. This study characterizes myelin recovery following stroke and motor learning-based rehabilitation in a mouse model of subcortical WMS. Following WMS, a transient increase in differentiating oligodendrocytes occurs within the peri-infarct in young male adult mice, which is completely abolished in male aged mice. Compound action potential recording demonstrates a decrease in conduction velocity of myelinated axons at the peri-infarct. Animals were then tested on one of three distinct motor learning-based rehabilitation strategies (skilled reach, restricted access to a complex running wheel, and unrestricted access to a complex running wheel) for their capacity to induce repair. These studies determined that unrestricted access to a complex running wheel alone increases the density of differentiating oligodendrocytes in infarcted white matter in young adult male mice, which is abolished in aged male mice. Unrestricted access to a complex running wheel was also able to enhance conduction velocity of myelinated axons at the peri-infarct to a speed comparable to naive controls suggesting functional recovery. However, there was no evidence of motor rehabilitation-induced remyelination or myelin protection.SIGNIFICANCE STATEMENT White matter stroke is a common disease with no medical therapy. A form of motor rehabilitation improves some aspects of white matter repair and recovery.

Recognizing and Responding to the Needs of Future Child and Adult Neurology Care Through the Evolution of Residency Training.

Recent insights into the frequency of occurrence and the genetic and mechanistic basis of nervous system disease have demonstrated that neurologic disorders occur as a spectrum across all ages. To meet future needs of patients with neurologic disease of all ages and prepare for increasing implementaton of precision therapies, greater integration of child and adult neurology residency training is needed. ANN NEUROL 2023;94:1005-1007.

Cardiac pericytes mediate the remodeling response to myocardial infarction.

Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-ß signaling. Deleting TGF-ß receptor 1 in chondroitin sulfate proteoglycan 4-expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.

Subcortical White Matter Stroke in the Mouse: Inducing Injury and Tracking Cellular Proliferation.

Here, we describe a method for inducing subcortical white matter stroke in mice, as well as tracking cellular proliferation through drinking water administration of EdU and ex vivo labeling.

Quantitative Spatial Mapping of Axons Across Cortical Regions to Assess Axonal Sprouting After Stroke.

Neurological disease such as a stroke causes death of brain tissue and loss of connectivity. Paradoxically, the stroke itself induces growth of new axonal collaterals, a phenomenon that is restrained in the normal adult brain. Enhancements in sprouting of axons have been linked with enhancements in motor function. Here, we describe a method developed in-house using standard reagents to map and quantitatively assess differential sprouting responses in stroke and following treatment with candidate molecular or pharmacological targets. This method allows for measurements of axonal growth responses that act as structural correlates for neural repair processes in the brain that aid in stroke recovery.

A Funeral for a Neurologist.

This essay describes the author's experience at a funeral for a neurologist.

The Ties That Bind: Glial Transplantation in White Matter Ischemia and Vascular Dementia.

White matter injury is a progressive vascular disease that leads to neurological deficits and vascular dementia. It comprises up to 30% of all diagnosed strokes, though up to ten times as many events go undiagnosed in early stages. There are several pathologies that can lead to white matter injury. While some studies suggest that white matter injury starts as small infarcts in deep penetrating blood vessels in the brain, others point to the breakdown of endothelial function or the blood-brain barrier as the primary cause of the disease. Whether due to local endothelial or BBB dysfunction, or to local small infarcts (or a combination), white matter injury progresses, accumulates, and expands from preexisting lesions into adjacent white matter to produce motor and cognitive deficits that present as vascular dementia in the elderly. Vascular dementia is the second leading cause of dementia, and white matter injury-attributed vascular dementia represents 40% of all diagnosed dementias and aggravates Alzheimer's pathology. Despite the advances in the last 15 years, there are few animal models of progressive subcortical white matter injury or vascular dementia. This review will discuss recent progress in animal modeling of white matter injury and the emerging principles to enhance glial function as a means of promoting repair and recovery.

IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury.

Cerebral small vessel disease and brain white matter injury are worsened by cardiovascular risk factors including obesity. Molecular pathways in cerebral endothelial cells activated by chronic cerebrovascular risk factors alter cell-cell signaling, blocking endogenous and post-ischemic white matter repair. Using cell-specific translating ribosome affinity purification (RiboTag) in white matter endothelia and oligodendrocyte progenitor cells (OPCs), we identify a coordinated interleukin-chemokine signaling cascade within the oligovascular niche of subcortical white matter that is triggered by diet-induced obesity (DIO). DIO induces interleukin-17B (IL-17B) signaling that acts on the cerebral endothelia through IL-17Rb to increase both circulating and local endothelial expression of CXCL5. In white matter endothelia, CXCL5 promotes the association of OPCs with the vasculature and triggers OPC gene expression programs regulating cell migration through chemokine signaling. Targeted blockade of IL-17B reduced vessel-associated OPCs by reducing endothelial CXCL5 expression. In multiple human cohorts, blood levels of CXCL5 function as a diagnostic and prognostic biomarker of vascular cognitive impairment.

A molecular interactome of the glioblastoma perivascular niche reveals integrin binding sialoprotein as a mediator of tumor cell migration.

Glioblastoma (GBM) is characterized by extensive microvascular hyperproliferation. In addition to supplying blood to the tumor, GBM vessels also provide trophic support to glioma cells and serve as conduits for migration into the surrounding brain, promoting recurrence. Here, we enrich CD31-expressing glioma vascular cells (GVCs) and A2B5-expressing glioma tumor cells (GTCs) from primary GBM and use RNA sequencing to create a comprehensive molecular interaction map of the secreted and extracellular factors elaborated by GVCs that can interact with receptors and membrane molecules on GTCs. To validate our findings, we utilize functional assays, including a hydrogel-based migration assay and in vivo mouse models to demonstrate that one identified factor, the little-studied integrin binding sialoprotein (IBSP), enhances tumor growth and promotes the migration of GTCs along the vasculature. This perivascular niche interactome will serve as a resource to the research community in defining the potential functions of the GBM vasculature.

PDE2A Inhibition Enhances Axonal Sprouting, Functional Connectivity, and Recovery after Stroke.

Phosphodiesterase (PDE) inhibitors have been safely and effectively used in the clinic and increase the concentration of intracellular cyclic nucleotides (cAMP/cGMP). These molecules activate downstream mediators, including the cAMP response element-binding protein (CREB), which controls neuronal excitability and growth responses. CREB gain of function enhances learning and allocates neurons into memory engrams. CREB also controls recovery after stroke. PDE inhibitors are linked to recovery from neural damage and to stroke recovery in specific sites within the brain. PDE2A is enriched in cortex. In the present study, we use a mouse cortical stroke model in young adult and aged male mice to test the effect of PDE2A inhibition on functional recovery, and on downstream mechanisms of axonal sprouting, tissue repair, and the functional connectivity of neurons in recovering cortex. Stroke causes deficits in use of the contralateral forelimb, loss of axonal projections in cortex adjacent to the infarct, and functional disconnection of neuronal networks. PDE2A inhibition enhances functional recovery, increases axonal projections in peri-infarct cortex, and, through two-photon in vivo imaging, enhances the functional connectivity of motor system excitatory neurons. PDE2A inhibition after stroke does not have an effect on other aspects of tissue repair, such as angiogenesis, gliogenesis, neurogenesis, and inflammatory responses. These data suggest that PDE2A inhibition is an effective therapeutic approach for stroke recovery in the rodent and that it simultaneously enhances connectivity in peri-infarct neuronal populations.SIGNIFICANCE STATEMENT Inhibition of PDE2A enhances motor recovery, axonal projections, and functional connectivity of neurons in peri-infarct tissue. This represents an avenue for a pharmacological therapy for stroke recovery.

Connecting the lines after a stroke.

In mice, stimulating cortical areas in the undamaged hemisphere of a brain affected by stroke impairs recovery.

Single-nucleus transcriptome analysis reveals disease- and regeneration-associated endothelial cells in white matter vascular dementia.

BACKGROUND: Vascular dementia (VaD) is the accumulation of vascular lesions in the subcortical white matter of the brain. These lesions progress and there is no direct medical therapy. AIMS: To determine the specific cellular responses in VaD so as to provide molecular targets for therapeutic development. MATERIALS AND METHODS: Single-nucleus transcriptome analysis was performed in human periventricular white matter (PVWM) samples of VaD and normal control (NC) subjects. RESULTS: Differential analysis shows that cell type-specific transcriptomic changes in VaD are associated with the disruption of specific biological processes, including angiogenesis, immune activation, axonal injury and myelination. Each cell type in the neurovascular unit within white matter has a specific alteration in gene expression in VaD. In a central cell type for this disease, subcluster analysis of endothelial cells (EC) indicates that VaD contains a disease-associated EC subcluster that expresses genes associated with programmed cell death and a response to protein folding. Two other subpopulations of EC in VaD express molecular systems associated with regenerative processes in angiogenesis, and in axonal sprouting and oligodendrocyte progenitor cell maturation. CONCLUSION: This comprehensive molecular profiling of brain samples from patients with VaD reveals previously unknown molecular changes in cells of the neurovascular niche, and an attempt at regeneration in injured white matter.

Retrospective comparison of outcomes following tibial plateau levelling osteotomy and lateral fabello-tibial suture stabilisation of cranial cruciate ligament disease in small dogs with high tibial plateau angles.

AIMS: To compare short and long-term outcomes after tibial plateau levelling osteotomy (TPLO) and lateral fabello-tibial suture (LFTS) techniques for the management of cranial cruciate ligament disease in small dogs with high tibial plateau angles (TPA). METHODS: In this retrospective study, the medical records of two veterinary specialist practices in the United Kingdom were searched for dogs (<20 kg) that underwent TPLO or LFTS between 2000 and 2010, and had a preoperative radiographic TPA >30° with either short-term (6 weeks) and/or long-term (>3 months) follow-up data. Data collected at a 6-week post-surgical re-examination was derived from orthopaedic examination and radiographic assessment and included the incidence of major and minor complications and scoring of the short-term outcome. Long-term outcome was scored based on results of a subjective owner questionnaire and veterinary orthopaedic examination. RESULTS: A total of 61 (84 stifles) dogs were included in the study: 24 (30 stilfes) and 37 (54 stifles) dogs underwent LFTS and TPLO, respectively. Long-term clinical outcome was different (p = 0.017) between groups; 15/15 stifles in the TPLO group had a good or excellent long-term clinical outcome, compared to 4/8 (50%) in the LFTS group. There was no evidence of a difference in short-term post-operative outcome or owner subjective long-term outcome between treatment groups.Stifles in the LFTS group were more likely (p = 0.027) to have palpable stifle pain at long-term follow-up. Owners reported that 5/16 (31.3%) dogs in the LFTS group required oral non-steroidal anti-inflammatory drug (NSAID) treatment at least monthly (4/5 required daily treatment), whereas no dogs in the TPLO group required treatment with NSAID more frequently than three times per year (p = 0.011).No correlation was found between short-term outcome and owner subjective long-term outcome but there was a positive correlation between short-term outcome and long-term clinical outcome.There was no evidence of a difference in overall major complication rates between treatment groups. The occurrence of complications was associated with heavier body weight at the time of surgery. No other variables were shown to be risk factors for complications. CONCLUSION AND CLINICAL RELEVANCE: Small breed dogs with high TPA that underwent TPLO had better long-term clinical outcomes and were less likely to require NSAID administration than those that underwent LFTS. The risk of complication increased with the weight of the dog at surgery. There was a positive correlation between short-term outcome and long-term clinical outcome.

Particle hydrogels decrease cerebral atrophy and attenuate astrocyte and microglia/macrophage reactivity after stroke.

Increasing numbers of individuals live with stroke related disabilities. Following stroke, highly reactive astrocytes and pro-inflammatory microglia can release cytokines and lead to a cytotoxic environment that causes further brain damage and prevents endogenous repair. Paradoxically, these same cells also activate pro-repair mechanisms that contribute to endogenous repair and brain plasticity. Here, we show that the direct injection of a hyaluronic acid based microporous annealed particle (MAP) hydrogel into the stroke core in mice reduces the percent of highly reactive astrocytes, increases the percent of alternatively activated microglia, decreases cerebral atrophy and preserves NF200 axonal bundles. Further, we show that MAP hydrogel promotes reparative astrocyte infiltration into the lesion, which directly coincides with axonal penetration into the lesion. This work shows that the injection of a porous scaffold into the stroke core can lead to clinically relevant decrease in cerebral atrophy and modulates astrocytes and microglia towards a pro-repair phenotype.