Potassium Buffering in the Neurovascular Unit: Models and Sensitivity Analysis

AbstractAstrocytes are critical regulators of neural and neurovascular network communication. Potassium transport is a central mechanism behind their many functions. Astrocytes encircle synapses with their distal processes, which express two potassium pumps (Na-K and NKCC) and an inward rectifying potassium channel (Kir), whereas the vessel-adjacent endfeet express Kir and BK potassium channels. We provide a detailed model of potassium flow throughout the neurovascular unit (synaptic region, astrocytes, and arteriole) for the cortex of the young brain. Our model reproduces several phenomena observed experimentally: functional hyperemia, in which neural activity triggers astrocytic potassium release at the perivascular endfoot, inducing arteriole dilation; K+ undershoot in the synaptic space after periods of neural activity; neurally induced astrocyte hyperpolarization during Kir blockade. Our results suggest that the dynamics of the vascular response during functional hyperemia are governed by astrocytic Kir for the fast onset and astrocytic BK for maintaining dilation. The model supports the hypothesis that K+ undershoot is caused by excessive astrocytic uptake through Na-K and NKCC pumps, whereas the effect is balanced by Kir. We address parametric uncertainty using high-dimensional stochastic sensitivity analysis and identify possible model limitations

Impact of Subarachnoid Hemorrhage on Astrocyte Calcium Signaling: Implications for Impaired Neurovascular Coupling

Deficits within the brain microcirculation contribute to poor patient outcome following aneurysmal subarachnoid hemorrhage (SAH). However, the underlying pathophysiology is not well understood. Intra-cerebral (parenchymal) arterioles are encased by specialized glial processes, called astrocyte endfeet. Ca2+ signals in the endfeet, driven by the ongoing pattern of neuronal activity, regulate parenchymal arteriolar diameter and thereby influence local cerebral blood flow. In the healthy brain, this phenomenon, called neurovascular coupling (NVC), matches focal increases in neuronal activity with local arteriolar dilation. This ensures adequate delivery of oxygen and other nutrients to areas of the brain with increased metabolic demand. Recently, we demonstrated inversion of NVC from vasodilation to vasoconstriction in brain slices obtained from SAH model animals. This pathological change, which would restrict blood flow to active brain regions, was accompanied by an increase in the amplitude of spontaneous Ca2+ events in astrocyte endfeet. It is possible that the emergence of higher amplitude endfoot Ca2+ events shifts the polarity of NVC after SAH by elevating levels of vasoactive agents (e.g. K+ ions) within the perivascular space. In the first aim of this dissertation we tested whether altered endfoot Ca2+ signaling underlies the inversion of NVC after SAH. Brain injury is often associated with increased levels of extracellular purine nucleotides (e.g. ATP). A recent study found that ATP levels in the cerebrospinal fluid of aneurysmal SAH patients were roughly 400-fold higher than that of non-SAH controls. Astrocytes express a variety of purinergic (P2) receptors that, when activated, could trigger a spike in intra-cellular Ca2+. It is possible that enhanced signaling via astrocyte P2 receptors underlies the change in endfoot Ca2+ signaling after SAH. In the second aim of this dissertation we determined the role of purinergic signaling in the generation of high-amplitude spontaneous endfoot Ca2+ events after SAH. Parenchymal arteriolar diameter and endfoot Ca2+ dynamics were recorded simultaneously in fluo-4-loaded rat brain slices using combined infrared-differential interference contrast and multi-photon fluorescence microscopy. We report that SAH led to a time-dependent emergence of spontaneous endfoot high-amplitude Ca2+ signals (eHACSs) that were only present in brain slices exhibiting inversion of NVC. Depletion of intracellular Ca2+ stores abolished spontaneous endfoot Ca2+ signals, including eHACSs, and restored arteriolar dilation in SAH brain slices to two downstream elements in the NVC signaling cascade, (1) increased endfoot Ca2+ and (2) elevated extracellular K+. We next tested the role of purinergic signaling in the generation of SAH-induced eHACSs by recording endfoot activity before and after treatment with the broad-spectrum purinergic receptor antagonist, suramin. Remarkably, suramin selectively abolished eHACSs and restored vasodilatory NVC in SAH brain slices. Desensitization of Ca2+-permeable ionotropic purinergic (P2X) receptors had no effect on eHACSs after SAH. However, eHACSs were selectively blocked using a cocktail of inhibitors targeting Gq-coupled purinergic (P2Y) receptors. Collectively, our results support a model in which SAH leads to an emergence of P2Y receptor-mediated eHACSs that cause inversion of NVC. Further, we identify the FDA-approved drug, suramin, as a potential therapy to be used in the treatment of aneurysmal SAH

Vasculo-neuronal coupling and neurovascular coupling at the neurovascular unit: impact of hypertension

Components of the neurovascular unit (NVU) establish dynamic crosstalk that regulates cerebral blood flow and maintain brain homeostasis. Here, we describe accumulating evidence for cellular elements of the NVU contributing to critical physiological processes such as cerebral autoregulation, neurovascular coupling, and vasculo-neuronal coupling. We discuss how alterations in the cellular mechanisms governing NVU homeostasis can lead to pathological changes in which vascular endothelial and smooth muscle cell, pericyte and astrocyte function may play a key role. Because hypertension is a modifiable risk factor for stroke and accelerated cognitive decline in aging, we focus on hypertension-associated changes on cerebral arteriole function and structure, and the molecular mechanisms through which these may contribute to cognitive decline. We gather recent emerging evidence concerning cognitive loss in hypertension and the link with vascular dementia and Alzheimer’s disease. Collectively, we summarize how vascular dysfunction, chronic hypoperfusion, oxidative stress, and inflammatory processes can uncouple communication at the NVU impairing cerebral perfusion and contributing to neurodegeneration.Fil: Presa, Jessica Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Augusta University Medical Center. Medical College of Georgia; Estados UnidosFil: Saravia, Flavia Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bagi, Zsolt. Augusta University Medical Center. Medical College of Georgia; Estados UnidosFil: Filosa, Jessica A.. Augusta University Medical Center. Medical College of Georgia; Estados Unido

Multiscale Model of Cerebral Blood Flow Control: Application to Small Vessel Disease and Cortical Spreading Depression

An in-time delivery of oxygen-rich blood into areas of high metabolic demand is pivotal in proper functioning of the brain and neuronal health. This highly precise communication between neuronal activity and cerebral blood flow (CBF) is termed as neurovascular coupling (NVC) or functional hyperemia. NVC is disrupted in major pathological conditions including Alzheimer’s disease, dementia, small vessel pathologies (SVD) and cortical spreading depression. Despite the utmost importance of NVC, its underlying mechanisms are not fully understood. This dissertation presents a multiscale mathematical modeling framework for studying unresolved mechanisms of NVC with major focus on K+ ions as a mediator of this process. To this end, models of single-cell electrophysiology are developed for endothelial (EC) and smooth muscle (SMC) cells of capillaries and parenchymal arterioles (PAs). Cells are electrically coupled, and large-scale geometrically-accurate models of microvascular networks are constructed. Model simulations predict an important role of capillary inward rectifying potassium channels (Kir) to sense neuronally-induced changes in extracellular potassium concentrations ([K+]o) and conduct hyperpolarizing signals over long distances to upstream PAs. Simulation results demonstrate a “tug-of-war” dynamic between Kir and voltage-gated potassium (Kv) channels in determining the Vm and myogenic tone of PA SMCs during NVC in SVD. Results also predict a key role of Kir channels in the experimentally observed multiphasic vascular response during high elevations of [K+]o in cortical spreading depression. The multiscale models presented in this study were able to accurately capture several experimentally observed responses during NVC and provided insights into their potential underlying mechanisms in health and disease. These models provide a theoretical platform where macroscale, tissue-level responses can be related to microscale, single-cell signaling pathways

Subarachnoid Hemorrhage, Spreading Depolarizations and Impaired Neurovascular Coupling

Aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences on brain function including profound effects on communication between neurons and the vasculature leading to cerebral ischemia. Physiologically, neurovascular coupling represents a focal increase in cerebral blood flow to meet increased metabolic demand of neurons within active regions of the brain. Neurovascular coupling is an ongoing process involving coordinated activity of the neurovascular unit—neurons, astrocytes, and parenchymal arterioles. Neuronal activity can also influence cerebral blood flow on a larger scale. Spreading depolarizations (SD) are self-propagating waves of neuronal depolarization and are observed during migraine, traumatic brain injury, and stroke. Typically, SD is associated with increased cerebral blood flow. Emerging evidence indicates that SAH causes inversion of neurovascular communication on both the local and global level. In contrast to other events causing SD, SAH-induced SD decreases rather than increases cerebral blood flow. Further, at the level of the neurovascular unit, SAH causes an inversion of neurovascular coupling from vasodilation to vasoconstriction. Global ischemia can also adversely affect the neurovascular response. Here, we summarize current knowledge regarding the impact of SAH and global ischemia on neurovascular communication. A mechanistic understanding of these events should provide novel strategies to treat these neurovascular disorders

Development of zebrafish and computational models of neurovascular coupling in health and disease

In this thesis, I have developed a novel zebrafish model of neurovascular coupling. Combining lightsheet imaging, compound transgenic zebrafish models and custom MATLAB based analysis pipelines, I characterised the neurovascular responses (neuronal calcium increases and change in red blood cell speed) in the optic tectum in response to visual stimulation. I determined the development stage at which neurovascular coupling in zebrafish larvae develops, followed by testing the requirement for nitric oxide or astrocyte cyclo-oxygenase in my model. I then used this model to investigate factors influencing neurovascular function. I first characterized the effect of glucose exposure and the role of nitric oxide in modulating neurovascular coupling. I then examined the effect of genetic mutation of Guanosine Triphosphate cyclohydrolase (an enzyme involved in nitric oxide and dopamine production in the brain) on neurovascular coupling. Finally, I have developed a minimal mathematical model of the neurovascular unit. To demonstrate the potential of this model I have simulated the effect of high blood glucose and low nitric oxide on neurovascular coupling and show this conforms with experimental data obtained in zebrafish

Úloha Aquaporin 4 kanálů a Transient Receptor Potential Vanilloid 4 kanálů při objemových změnách astrocytů

Astrocyty mají v mozku celou řadu funkcí. V ischemických podmínkách zvětšují svůj objem v důsledku zvýšeného příjmu osmolytů a jsou tedy z velké části zodpovědné za rozvoj cytotoxického edému mozku. Jsou ale také schopné regulovat svůj objem uvolňováním osmolytů společně s vodou. Tento proces je nazýván regulované snižování buněčného objemu (RVD). Za kanály, které se regulace objemu astrocytů významně účastní, jsou považovány Aquaporin 4 (AQP4) a Transient receptor potential vanilloid 4 (TRPV4). Cílem této diplomové práce bylo objasnit úlohu obou těchto kanálů při objemových změnách astrocytů in situ. Experimenty byly prováděny na populaci astrocytů, které byly značené zeleným flourescenčním proteinem a pocházely z AQP4-deficientních (AQP4-/- ), TRPV4-deficientních (TRPV4-/- ) a kontrolních (Ctrl) myší. Objemové změny byly vyvolány v mozkových řezech hypoosmotickým stresem a kyslíkovo-glukózovou deprivací (OGD). Data byla analyzována na základě změn intensity fluorescence v celých buňkách a v astrocytálních patkách. Při aplikaci hypoosmotického stresu jsme mezi našimi experimentálními skupinami nenašli žádné rozdíly ve zvětšování objemu buněk nebo následném snižování buněčného objemu. Naopak objemové změny vyvolané aplikací OGD se mezi jednotlivými skupinami významně lišily. Astrocyty z AQP4-/-...Astrocytes posses a wide range of functions within the brain. In response to ischemic conditions they swell due to increased uptake of osmolytes and they are mainly responsible for cytotoxic edema formation. However, they are also able to regulate their volume by releasing osmolytes together with water via the process of regulatory volume decrease (RVD). The Aquaporin 4 (AQP4) channel and Transient receptor potential vanilloid 4 (TRPV4) channel are suspected to be strongly involved in these processes of astrocytic volume regulation. The goal of the present diploma thesis was to clarify the role of both channels in astrocytic swelling in situ. For our experiments we used a subpopulation of green fluorescent protein-labelled astrocytes from AQP4-deficient (AQP4-/- ), TRPV4-deficient (TRPV4-/- ) and control (Ctrl) mice. Cell volume alterations were induced in acute brain slices by hypoosmotic stress or by oxygen-glucose deprivation (OGD). Data were quantified using fluorescence intensity-based approach in the whole cells and in astrocytic endfeet. Our results indicate, that there is no difference in astrocytic swelling or cell volume recovery between astrocytes from AQP4-/- , TRPV4-/- and control mice when exposed to hypoosmotic stress. On the contrary, volume changes induced by OGD varied...Katedra fyziologieDepartment of PhysiologyPřírodovědecká fakultaFaculty of Scienc

Computational model of an Astrocyte as spatial potassium buffer at the neurovascular unit

Gli astrociti sono cellule presenti nel cervello e nel midollo spinale con una forma caratteristica radiale, la quale richiama una stella. Queste cellule contribuiscono all'omeostasi e alla regolazione del sistema nervoso centrale, inoltre, dimostrano notevoli capacità di adattamento all'ambiente che le circonda. Quest'ultima caratteristica permette di attribuire alla loro presenza la capacità di mantenimento funzionale del sistema nervoso durante l'invecchiamento. Gli astrociti svolgono molte funzioni attive e di supporto, incluse il controllo biochimico delle cellule endoteliali, la fornitura di nutrienti al tessuto nervoso, il mantenimento dell'equilibrio ionico e la regolazione del flusso sanguigno cerebrale. Negli ultimi anni, si è assistito ad un crescente interesse per le comunicazioni neurone-glia, considerando la loro partecipazione alle funzioni cognitive e al loro coinvolgimento in molti disturbi cerebrali e malattie neurodegenerative. Gli astrociti agiscono sulla cosiddetta unità neurovascolare reagendo alla attività neurale locale, e mediando la concentrazione di potassio nello spazio perivascolare in prossimità delle cellule muscolari liscie. Alta attività neurale richiede un maggiore afflusso di nutrienti e ossigeno, il quale trova risposta in una propagazione di calcio attraverso gli astrociti vicini, che portano ad attivare scambi di potassio tra il cosiddetto endfoot e lo spazio perivascolare. Questo fenomeno si rappresenta in un controllo dell'attività di cellule muscolari liscie, le quali hanno il compito di regolare la dilatazione e costrizione dei vasi sanguigni vicini. \\ L'obiettivo è di sviluppare un modello computazione riproducibile di un astrocita in grado di cooperare con l'unità neurovasculare al livello della barriera emato-encefalica. Questo lavoro presenta un modello multi-compartimentale di stato in grado di descrivere l'astrocita come una cellula singola, in grado di interagire con ambienti perisinaptici e perivascolari. Il modello consiste in un insieme di equazioni differenziali, dipendenti tra loro, in grado di rappresentare la dinamica di ogni variabile di stato. Implementa svariati fenomeni biologici e integra caratteristiche di studi passati, concentrandosi sul fenomeno di attivazione di specifiche proteine di trasporto di potassio, una volta raggiunte da una propagazione di calcio. Tra le proteine studiate, un maggiore interesse è riposto sui canali $Kir_{4.1}$ e $BK$. In aggiunta, il modello permetterà di definire una organizzazione geometrica dell'astrocita, conferendo la possibilità di studiare pattern spaio temporali delle varie variabili di stato durante una stimolazione esterna di glutamato, la quale rappresenta un'alta attività nervosa locale. I risultati mostrano come, durante una stimulazione esterna di glutamato, sia presente un rilascio di potassio nell'ambiente perivascolare, controllato dalla dinamica del calcio proveniente da regioni distanti dell'astrocita. Tutte le analisi sono state implementate usando linguaggi di programmazione come \emph{Python} e \emph{MATLAB}.Astrocytes are sponge-like cells present in brains and spinal cord that provide homeostasis and regulation of the central nervous system. Astrocytes are highly heterogeneous in morphological appearance, demonstrating remarkable adaptive plasticity capabilities to their surroundings, the same ones that define the functional maintenance of the nervous system through aging. They perform many supporting and active functions, including biochemical control of endothelial cells, provision of nutrients to the nervous tissue, maintenance of ion balance and regulation of the cerebral blood flow. Recent years have witnessed an increasing interest in neuron–glia communication due to the realization of their participation in cognitive functions and information processing, as well as being involved in many brain disorders and neurodegenerative diseases. Astrocytes act in neurovascular coupling by reacting to neural activity and by mediating potassium concentration in the perivascular space around smooth muscle cells. High neural activity demands a larger supply of nutrients and oxygen, which is answered by a propagation of calcium through the astrocyte, that ultimately triggers potassium exchanges between endfeets and perivascular space. This results in a control of the local smooth muscle cell's activity, bringing to regulation of dilation and constriction of nearby blood vessels and therefore regulation of the cerebral blood flow. The aim of the thesis project is to develop a reproducible computational model of an astrocyte's endfoot cooperating with the neurovascular unit at the blood-brain barrier. This work presents a multi-compartmental state space model describing the astrocyte as a single cell that interacts with different domains, such as the perisynaptic and perivascular spaces. The model consists of a set of coupled ordinary differential equations that represent the dynamics of all states. It implements several biological phenomenon and merges together characteristics of past studies by focusing on how the calcium signaling would trigger the activation of specific potassium transporters, such as inward rectifying channel ($Kir_{4.1}$) and big conductance ($BK$) channels. Additionally, the model allows to define a geometrical organization of how many astrocyte's processes and how they are qualitatively distributed in space are present in a simulation. This, in order to investigate on spatial-temporal patterns during external glutamate stimulations, which simulate high neural activity. The results showed that there is actually, buffering of potassium during external glutamate stimuli, led by calcium dynamics that propagate the information from synaptic areas to the blood-brain barrier. All analysis are made implementing a computational model using \emph{Python} and \emph{MATLAB} as coding languages

Cell Volume Regulation Mechanisms in Differentiated Astrocytes

The ability of astrocytes to control extracellular volume homeostasis is critical for brain function and pathology. Uncovering the mechanisms of cell volume regulation by astrocytes will be important for identifying novel therapeutic targets for neurological conditions, such as those characterized by imbalances to hydro saline challenges (as in edema) or by altered cell volume regulation (as in glioma). One major challenge in studying the astroglial membrane channels involved in volume homeostasis in cell culture model systems is that the expression patterns of these membrane channels do not resemble those observed in vivo. In our previous study, we demonstrated that rat primary astrocytes grown on nanostructured interfaces based on hydrotalcite-like compounds (HTlc) in vitro are differentiated and display molecular and functional properties of in vivo astrocytes, such as the functional expression of inwardly rectifying K+ channel (Kir 4.1) and Aquaporin-4 (AQP4) at the astrocytic microdomain. Here, we take advantage of the properties of differentiated primary astrocytes in vitro to provide an insight into the mechanism underpinning astrocytic cell volume regulation and its correlation with the expression and function of AQP4, Transient Receptor Potential Vanilloid 4 (TRPV4), and Volume Regulated Anion Channel (VRAC)