The fasciola cinereum of the hippocampal tail as an interventional target in epilepsy.

Targeted tissue ablation involving the anterior hippocampus is the standard of care for patients with drug-resistant mesial temporal lobe epilepsy. However, a substantial proportion continues to suffer from seizures even after surgery. We identified the fasciola cinereum (FC) neurons of the posterior hippocampal tail as an important seizure node in both mice and humans with epilepsy. Genetically defined FC neurons were highly active during spontaneous seizures in epileptic mice, and closed-loop optogenetic inhibition of these neurons potently reduced seizure duration. Furthermore, we specifically targeted and found the prominent involvement of FC during seizures in a cohort of six patients with epilepsy. In particular, targeted lesioning of the FC in a patient reduced the seizure burden present after ablation of anterior mesial temporal structures. Thus, the FC may be a promising interventional target in epilepsy.

Seizure forecasting using machine learning models trained by seizure diaries.

Objectives.People with refractory epilepsy are overwhelmed by the uncertainty of their next seizures. Accurate prediction of future seizures could greatly improve the quality of life for these patients. New evidence suggests that seizure occurrences can have cyclical patterns for some patients. Even though these cyclicalities are not intuitive, they can be identified by machine learning (ML), to identify patients with predictable vs unpredictable seizure patterns.Approach.Self-reported seizure logs of 153 patients from the Human Epilepsy Project with more than three reported seizures (totaling 8337 seizures) were used to obtain inter-seizure interval time-series for training and evaluation of the forecasting models. Two classes of prediction methods were studied: (1) statistical approaches using Bayesian fusion of population-wise and individual-wise seizure patterns; and (2) ML-based algorithms including least squares, least absolute shrinkage and selection operator, support vector machine (SVM) regression, and long short-term memory regression. Leave-one-person-out cross-validation was used for training and evaluation, by training on seizure diaries of all except one subject and testing on the left-out subject.Main results.The leading forecasting models were the SVM regression and a statistical model that combined the median of population-wise seizure time-intervals with a test subject's prior seizure intervals. SVM was able to forecast 50%, 70%, 81%, 84%, and 87% of seizures of unseen subjects within 0, 1, 2, 3 to 4 d of mean absolute forecasting error, respectively. The subject-wise performances show that patients with more frequent seizures were generally better predicted.Significance.ML models can leverage non-random patterns within self-reported seizure diaries to forecast future seizures. While diary-based seizure forecasting alone is only one of many aspects of clinical care of patients with epilepsy, studying the level of predictability across seizures and patients paves the path towards a better understanding of predictable vs unpredictable seizures on individualized and population-wise bases.

3D optogenetic control of arteriole diameter in vivo.

Modulation of brain arteriole diameter is critical for maintaining cerebral blood pressure and controlling regional hyperemia during neural activity. However, studies of hemodynamic function in health and disease have lacked a method to control arteriole diameter independently with high spatiotemporal resolution. Here, we describe an all-optical approach to manipulate and monitor brain arteriole contractility in mice in three dimensions using combined in vivo two-photon optogenetics and imaging. The expression of the red-shifted excitatory opsin, ReaChR, in vascular smooth muscle cells enabled rapid and repeated vasoconstriction controlled by brief light pulses. Two-photon activation of ReaChR using a spatial light modulator produced highly localized constrictions when targeted to individual arterioles within the neocortex. We demonstrate the utility of this method for examining arteriole contractile dynamics and creating transient focal blood flow reductions. Additionally, we show that optogenetic constriction can be used to reshape vasodilatory responses to sensory stimulation, providing a valuable tool to dissociate blood flow changes from neural activity.

Pericyte Control of Blood Flow Across Microvascular Zones in the Central Nervous System.

The vast majority of the brain's vascular length is composed of capillaries, where our understanding of blood flow control remains incomplete. This review synthesizes current knowledge on the control of blood flow across microvascular zones by addressing issues with nomenclature and drawing on new developments from in vivo optical imaging and single-cell transcriptomics. Recent studies have highlighted important distinctions in mural cell morphology, gene expression, and contractile dynamics, which can explain observed differences in response to vasoactive mediators between arteriole, transitional, and capillary zones. Smooth muscle cells of arterioles and ensheathing pericytes of the arteriole-capillary transitional zone control large-scale, rapid changes in blood flow. In contrast, capillary pericytes downstream of the transitional zone act on slower and smaller scales and are involved in establishing resting capillary tone and flow heterogeneity. Many unresolved issues remain, including the vasoactive mediators that activate the different pericyte types in vivo, the role of pericyte-endothelial communication in conducting signals from capillaries to arterioles, and how neurological disease affects these mechanisms.

Clinical Problem Solving: An Older Woman With Weakness from Head to Toe.

A 67-year-old woman was admitted to our hospital for progressive weakness, dysphagia, muscle pain, and weight loss. Here we detail the clinical problem solving involved in diagnosing and treating her immune-mediated necrotizing myopathy caused by anti-HMGCoA reductase autoantibodies. Interestingly, this diagnosis coincided with discovery of a gastrointestinal stromal tumor (GIST) and positivity for anti-nuclear matrix protein (anti-NXP2), another myositis specific autoantibody.

VasoMetrics: unbiased spatiotemporal analysis of microvascular diameter in multi-photon imaging applications.

BACKGROUND: Multi-photon imaging of the cerebrovasculature provides rich data on the dynamics of cortical arterioles, capillaries, and venules. Vascular diameter is the major determinant of blood flow resistance, and is the most commonly quantified metric in studies of the cerebrovasculature. However, there is a lack of accessible and easy-to-use methods to quantify vascular diameter in imaging data. METHODS: We created VasoMetrics, a macro written in ImageJ/Fiji for spatiotemporal analysis of microvascular diameter. The key feature of VasoMetrics is rapid analysis of many evenly spaced cross-sectional lines along the vessel of interest, permitting the extraction of numerous diameter measurements from individual vessels. Here we demonstrated the utility of VasoMetrics by analyzing in vivo multi-photon imaging stacks and movies collected from lightly sedated mice, as well as data from optical coherence tomography angiography (OCTA) of human retina. RESULTS: Compared to the standard approach, which is to measure cross-sectional diameters at arbitrary points along a vessel, VasoMetrics accurately reported spatiotemporal features of vessel diameter, reduced measurement bias and time spent analyzing data, and improved the reproducibility of diameter measurements between users. VasoMetrics revealed the dynamics in pial arteriole diameters during vasomotion at rest, as well as changes in capillary diameter before and after pericyte ablation. Retinal arteriole diameter was quantified from a human retinal angiogram, providing proof-of-principle that VasoMetrics can be applied to contrast-enhanced clinical imaging of microvasculature. CONCLUSIONS: VasoMetrics is a robust macro for spatiotemporal analysis of microvascular diameter in imaging applications.

Brain capillary pericytes exert a substantial but slow influence on blood flow.

The majority of the brain's vasculature is composed of intricate capillary networks lined by capillary pericytes. However, it remains unclear whether capillary pericytes influence blood flow. Using two-photon microscopy to observe and manipulate brain capillary pericytes in vivo, we find that their optogenetic stimulation decreases lumen diameter and blood flow, but with slower kinetics than similar stimulation of mural cells on upstream pial and precapillary arterioles. This slow vasoconstriction was inhibited by the clinically used vasodilator fasudil, a Rho-kinase inhibitor that blocks contractile machinery. Capillary pericytes were also slower to constrict back to baseline following hypercapnia-induced dilation, and slower to dilate towards baseline following optogenetically induced vasoconstriction. Optical ablation of single capillary pericytes led to sustained local dilation and a doubling of blood cell flux selectively in capillaries lacking pericyte contact. These data indicate that capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain.

Mild pericyte deficiency is associated with aberrant brain microvascular flow in aged PDGFRß+/- mice.

The receptor tyrosine kinase PDGFRß is essential for pericyte migration to the endothelium. In mice lacking one allele of PDGFRß (PDGFRß+/-), previous reports have described an age-dependent loss of pericytes in the brain, leading to cerebrovascular dysfunction and subsequent neurodegeneration reminiscent of that seen in Alzheimer's disease and vascular dementia. We examined 12-20-month-old PDGFRß+/- mice to better understand how pericyte loss affects brain microvascular structure and perfusion in vivo. We observed a mild reduction of cortical pericyte number in PDGFRß+/- mice (27% fewer cell bodies) compared to controls, but no decrease in pericyte coverage of the endothelium. This mild degree of pericyte loss caused no discernable change in cortical microvascular density, length, basal diameter or reactivity to hypercapnia. Yet, it was associated with an increase in basal blood cell velocity, primarily in pre-capillary arterioles. Taken together, our results suggest that mild pericyte loss can lead to aberrant cerebral blood flow despite a lack of apparent effect on microvascular structure and reactivity.

Organizational hierarchy and structural diversity of microvascular pericytes in adult mouse cortex.

Smooth muscle cells and pericytes, together called mural cells, coordinate many distinct vascular functions. Canonically, smooth muscle cells are ring-shaped and cover arterioles with circumferential processes, whereas pericytes extend thin processes that run longitudinally along capillaries. In between these canonical mural cell types are cells with features of both smooth muscle cells and pericytes. Recent studies suggest that these transitional cells are critical for controlling blood flow to the capillary bed during health and disease, but there remains confusion on how to identify them and where they are located in the brain microvasculature. To address this issue, we measured the morphology, vascular territory, and α-smooth muscle actin content of structurally diverse mural cells in adult mouse cortex. We first imaged intact 3D vascular networks to establish the locations of major gradations in mural cell appearance as arterioles branched into capillaries. We then imaged individual mural cells occupying the regions within these gradations. This revealed two transitional cells that were often similar in appearance, but with sharply contrasting levels of α-smooth muscle actin. Our findings highlight the diversity of mural cell morphologies in brain microvasculature, and provide guidance for identification and categorization of mural cell types.

Pericyte Structural Remodeling in Cerebrovascular Health and Homeostasis.

The biology of brain microvascular pericytes is an active area of research and discovery, as their interaction with the endothelium is critical for multiple aspects of cerebrovascular function. There is growing evidence that pericyte loss or dysfunction is involved in the pathogenesis of Alzheimer's disease, vascular dementia, ischemic stroke and brain injury. However, strategies to mitigate or compensate for this loss remain limited. In this review, we highlight a novel finding that pericytes in the adult brain are structurally dynamic in vivo, and actively compensate for loss of endothelial coverage by extending their far-reaching processes to maintain contact with regions of exposed endothelium. Structural remodeling of pericytes may present an opportunity to foster pericyte-endothelial communication in the adult brain and should be explored as a potential means to counteract pericyte loss in dementia and cerebrovascular disease. We discuss the pathophysiological consequences of pericyte loss on capillary function, and the biochemical pathways that may control pericyte remodeling. We also offer guidance for observing pericytes in vivo, such that pericyte structural remodeling can be more broadly studied in mouse models of cerebrovascular disease.

Dynamic Remodeling of Pericytes In Vivo Maintains Capillary Coverage in the Adult Mouse Brain.

Direct contact and communication between pericytes and endothelial cells is critical for maintenance of cerebrovascular stability and blood-brain barrier function. Capillary pericytes have thin processes that reach hundreds of micrometers along the capillary bed. The processes of adjacent pericytes come in close proximity but do not overlap, yielding a cellular chain with discrete territories occupied by individual pericytes. Little is known about whether this pericyte chain is structurally dynamic in the adult brain. Using in vivo two-photon imaging in adult mouse cortex, we show that while pericyte somata were immobile, the tips of their processes underwent extensions and/or retractions over days. The selective ablation of single pericytes provoked exuberant extension of processes from neighboring pericytes to contact uncovered regions of the endothelium. Uncovered capillary regions had normal barrier function but were dilated until pericyte contact was regained. Pericyte structural plasticity may be critical for cerebrovascular health and warrants detailed investigation.

Does pathology of small venules contribute to cerebral microinfarcts and dementia?

Microinfarcts are small, but strikingly common, ischemic brain lesions in the aging human brain. There is mounting evidence that microinfarcts contribute to vascular cognitive impairment and dementia, but the origins of microinfarcts are unclear. Understanding the vascular pathologies that cause microinfarcts may yield strategies to prevent their occurrence and reduce their deleterious effects on brain function. Current thinking suggests that cortical microinfarcts arise from the occlusion of penetrating arterioles, which are responsible for delivering oxygenated blood to small volumes of tissue. Unexpectedly, pre-clinical studies have shown that the occlusion of penetrating venules, which drain deoxygenated blood from cortex, lead to microinfarcts that appear identical to those resulting from arteriole occlusion. Here we discuss the idea that cerebral venule pathology could be an overlooked source for brain microinfarcts in humans. This article is part of the Special Issue "Vascular Dementia". Cover Image for this Issue: doi: 10.1111/jnc.14167.

Functional deficits induced by cortical microinfarcts.

Clinical studies have revealed a strong link between increased burden of cerebral microinfarcts and risk for cognitive impairment. Since the sum of tissue damage incurred by microinfarcts is a miniscule percentage of total brain volume, we hypothesized that microinfarcts disrupt brain function beyond the injury site visible to histological or radiological examination. We tested this idea using a mouse model of microinfarcts, where single penetrating vessels that supply mouse cortex were occluded by targeted photothrombosis. We found that in vivo structural and diffusion MRI reliably reported the acute microinfarct core, based on spatial co-registrations with post-mortem stains of neuronal viability. Consistent with our hypothesis, c-Fos assays for neuronal activity and in vivo imaging of single vessel hemodynamics both reported functional deficits in viable peri-lesional tissues beyond the microinfarct core. We estimated that the volume of tissue with functional deficit in cortex was at least 12-fold greater than the volume of the microinfarct core. Impaired hemodynamic responses in peri-lesional tissues persisted at least 14 days, and were attributed to lasting deficits in neuronal circuitry or neurovascular coupling. These data show how individually miniscule microinfarcts could contribute to broader brain dysfunction during vascular cognitive impairment and dementia.

Pericytes as Inducers of Rapid, Matrix Metalloproteinase-9-Dependent Capillary Damage during Ischemia.

Blood-brain barrier disruption (BBB) and release of toxic blood molecules into the brain contributes to neuronal injury during stroke and other cerebrovascular diseases. While pericytes are builders and custodians of the BBB in the normal brain, their impact on BBB integrity during ischemia remains unclear. We imaged pericyte-labeled transgenic mice with in vivo two-photon microscopy to examine the relationship between pericytes and blood plasma leakage during photothrombotic occlusion of cortical capillaries. Upon cessation of capillary flow, we observed that plasma leakage occurred with three times greater frequency in regions where pericyte somata adjoined the endothelium. Pericyte somata covered only 7% of the total capillary length in cortex, indicating that a disproportionate amount of leakage occurred from a small fraction of the capillary bed. Plasma leakage was preceded by rapid activation of matrix metalloproteinase (MMP) at pericyte somata, which was visualized at high resolution in vivo using a fluorescent probe for matrix metalloproteinase-2/9 activity, fluorescein isothiocyanate (FITC)-gelatin. Coinjection of an MMP-9 inhibitor, but not an MMP-2 inhibitor, reduced pericyte-associated FITC-gelatin fluorescence and plasma leakage. These results suggest that pericytes contribute to rapid and localized proteolytic degradation of the BBB during cerebral ischemia. SIGNIFICANCE STATEMENT: Pericytes are a key component of the neurovascular unit and are essential for normal BBB function. However, during acute ischemia, we find that pericytes are involved in creating rapid and heterogeneous BBB disruption in the capillary bed. The mechanism by which pericytes contribute to BBB damage warrants further investigation, as it may yield new therapeutic targets for acute stroke injury and other neurological diseases involving capillary flow impairment.

Pericyte structure and distribution in the cerebral cortex revealed by high-resolution imaging of transgenic mice.

Pericytes are essential for normal brain function, but many aspects of their physiology remain enigmatic due to a lack of tools to genetically target this cell population. Here, we characterize brain pericytes using two existing Cre-recombinase driver mouse lines that can serve distinct purposes in cerebrovascular research. One line expresses an inducible version of Cre under the NG2 proteoglycan promoter, which provides the sparse labeling necessary to define the morphology of single cells. These mice reveal structural differences between pericytes adjacent to arterioles versus those broadly distributed in the capillary bed that may underlie differential roles in control of vessel caliber. A second line expresses Cre constitutively under the platelet-derived growth factor receptor ß promoter and provides continuous, highly specific and near-complete labeling of pericytes and myocytes along the entire cerebrovasculature. This line provides a three-dimensional view of pericyte distribution along the cortical angioarchitecture following optical clearing of brain tissue. In combination with recent reporter lines for expression of optogenetic actuators and activity-sensitive probes, these mice may be key tools for studying pericyte biology in the intact brain.

A murine toolbox for imaging the neurovascular unit.

The neurovascular unit (NVU) coordinates many essential functions in the brain including blood flow control, nutrient delivery, and maintenance of BBB integrity. These functions are the result of a cellular and molecular interplay that we are just beginning to understand. Cells of the NVU can now be investigated in the intact brain through the combined use of high-resolution in vivo imaging and non-invasive molecular tools to observe and manipulate cell function. Mouse lines that target transgene expression to cells of the NVU will be of great value in future work. However, a detailed evaluation of target cell specificity and expression pattern within the brain is required for many existing lines. The purpose of this review was to catalog mouse lines available to cerebrovascular biologists and to discuss their utility and limitations in future imaging studies.