Browsing by Author "Mentor, Shireen"

Now showing 1 - 8 of 8
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    Are claudin-5 tight-junction proteins in the blood-brain barrier porous?
    (Wolters Kluwer Health, 2020) Fisher, David W.; Mentor, Shireen
    The capillaries of the brain are particularly special, as they are not simply conduits for blood, but are primarily responsible to ensure that the neurons function in a strictly regulated homeostatic interstitium. Brain endothelial cells (BECs) express a plethora of ion channels on its luminal and abluminal surfaces, namely: potassium (K+ ) channels (i.e., Kir2 and Kv1), chloride (Cl–)/bicarbonate (HCO3–) channels, as well as a number of ion-solute exchangers (Redzic et al., 2011). These channels essentially prioritize vectorial transendothelial transport, especially for the regulation of K+ flux across the blood-brain barrier (BBB) (Redzic et al., 2011). The differences between the K+ concentration of the brain interstitium and plasma is only 2 mM to 4 mM, but the maintenance of this ionic concentration difference provides a constancy for the neuronal resting membrane potential, their associated firing thresholds and the preservation of a constant level of neuronal excitability.
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    Exosomes form tunneling nanotubes (TUNTs) in the blood-brain barrier: A nano-anatomical perspective of barrier genesis
    (Frontiers Media, 2022) Mentor, Shireen; Fisher, David
    The blood-brain barrier (BBB) is a robust interface between the blood and the central nervous system. Barrier type endothelium is able to limit paracellular (PC) movement, relegating molecular flux to the transendothelial pathways of brain endothelial cells (BECs). It is, therefore, apparent that any leakage via the PC shunts would effectively nullify the regulation of molecular flux across the transcellular pathways. The application of higher-resolution scanning electron microscopy (HR-SEM) illuminates the heterogenous, morphological profile that exists on the surface of BEC membranes and the relationship between these ultrastructures during the molecular construction of the PC space between adjacent BECs. In this study developing BEC monolayers were grown on mixed, cellulose esters insert membranes in a bicameral system.
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    High-resolution insights into the in vitro developing blood-brain barrier: Novel morphological features of endothelial nanotube function
    (Frontiers Media, 2021) Mentor, Shireen; Fisher, David
    High-resolution electron microscopy (HREM) imaging of the in vitro blood-brain barrier (BBB), is a promising modality for investigating the dynamic morphological interplay underpinning BBB development. The successful establishment of BBB integrity is grounded in the brain endothelial cells (BEC’s) ability to occlude its paracellular spaces of brain capillaries through the expression of the intercellular tight junction (TJ) proteins. The impermeability of these paracellular spaces are crucial in the regulation of transcellular transport systems to achieve homeostasis of the central nervous system. To-date research describing morphologically, the dynamics by which TJ interaction is orchestrated to successfully construct a specialized barrier remains undescribed. In this study, the application of HREM illuminates the novel, dynamic and highly restrictive BEC paracellular pathway which is founded based on lateral membrane alignment which is the functional imperative for the mechanical juxtapositioning of TJ zones that underpin molecular bonding and sealing of the paracellular space.
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    In vitro modulatory effects of fermented rooibos extract (Aspalathus linearis) against ethanol-induced effects on the mouse blood-brain barrier
    (University of the Western Cape, 2014) Mentor, Shireen; Fisher, D; Cummings, F.; Gamieldien, K.
    Alcohol abuse is a growing crisis within South Africa, with severe health and socio-economic implications. Alcohol compromises the function of the blood-brain barrier (BBB), and thus its ability to regulate the homeostatic environment of the CNS is interrupted. In this study, an in vitro model of the BBB was utilized to study the effects of selected concentrations of alcohol (25mM-200mM) and the ameliorating effects of fermented rooibos (Aspalathus linearis) (0.003125%-1%), in an attempt to reverse the harmful oxidative effects of alcohol. The literature clearly states that alcohol (ethanol) compromises the BBB by reactive oxygen species (ROS) production and, therefore, rooibos, a shrub high in antioxidants and widely utilized nationally, was added to alcohol-exposed mouse brain endothelial (bEnd5) cells with the view to reverse the alcohol-induced effects on the BBB model. Alcohol-treated (25mM-400mM) bEnd5 monolayers expressed no toxicity, however, cell numbers were significantly suppressed (P<0.0274). To validate this finding, the activity of the mitochondria was investigated in order to understand if the cell’s metabolism was related to the decrease in cell division. Results showed that for both acute and chronic exposure there was a decrease in mitochondrial activity (MA) for a period of 24-48 hours, thereafter, the MA of the bEnd5 cells returned to normality. However, in experiments which chronically (600mM and 800mM) exposed cells to alcohol over a period of 96 hours, MA was suppressed and did not return to normal. Fermented rooibos caused a biphasic response to cellular proliferation at 24-72 hours, where the lower concentrations (0.0625-0.125 %) caused an increase in cellular proliferation and the higher concentrations (0.5-1%) resulted in a relative decrease in cellular proliferation. The long-term effect, after acute exposure, however, resulted in cell suppression at 96 hours (P<0.0073). With respect to the MA, bEnd5 cells exposed to fermented rooibos showed that lower concentrations (0.003125-0.0125%) were suppressed at 24 hours and was elevated at 48 hours and96 hours for all concentrations. The exception being the highest concentration (0.1%), which showed a depression in MA (P<0.05). Treating cells with both alcohol and rooibos, resulted in exacerbated suppressing of the MA. The physiological function of the BBB model was investigated by monitoring the permeability using transendothelial electrical resistance (TEER) studies and the in vitro model used in this study was endorsed for the first time using high resolution scanning electron microscopy. TEER indicated incidental changes in the permeability, only at 24 hours, for both acute and chronic exposure to alcohol and rooibos. A novel finding, within this study, was the increase in electrical resistance across the formation of the cell monolayer, after treatment with alcohol. The data lead to the hypothesis for the effect of ROS on resistivity and provides a rationale to explain the effects of combinatory treatments that were expected to ameliorate the negative effect of alcohol, however, this study showed synergistically negative effects on the bEnd5 cells. In summary the main findings in this study were: (a) alcohol was not toxic on bEnd5cells, (b) alcohol increased the permeability across monolayers of bEnd5 cells and(c) rooibos did not significantly reverse the ROS-induced effects of alcohol, but exacerbated the effects. Rooibos treatment caused the following: (i) biphasic effect on cellular proliferation, (ii) an increase in MA, and (iii) a cyclic effect in TEER studies.
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    Investigating novel aspects of the blood-brain barrier using high resolution electron microscopy
    (University of the Western Cape, 2022) Mentor, Shireen; Fisher, David
    The blood-brain barrier (BBB) is a restrictive interface located between the blood circulation and the central nervous system (CNS), regulating the homeostatic environment of the neuronal milieu, by controlling the permeability of the cerebrovasculature. Currently, we cannot fully comprehend the regulatory features and the complexity of BBB morphology to allow for intervention clinically. The thesis consists of four publications. The methodology paper proposes a novel experimental design to visualize the morphological architecture of immortalized mouse brain endothelial cell lines (bEnd3/bEnd5). The brain endothelial cells (BECs) were grown on cellulose matrices and fixed in 2.5 % glutaraldehyde in preparation for visualization of the paracellular (PC) spaces between adjacent BECs, employing high-resolution electron microscopy (HREM), with vested interest in the morphological profile of the developing BEC.
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    The Ism between endothelial cilia and endothelial nanotubules is an evolving concept in the genesis of the BBB
    (MDPI, 2022) Mentor, Shireen; Fisher, David
    The blood–brain barrier (BBB) is fundamental in maintaining central nervous system (CNS) homeostasis by regulating the chemical environment of the underlying brain parenchyma. Brain endothelial cells (BECs) constitute the anatomical and functional basis of the BBB. Communication between adjacent BECs is critical for establishing BBB integrity, and knowledge of its nanoscopic landscape will contribute to our understanding of how juxtaposed zones of tight-junction protein interactions between BECs are aligned. The review discusses and critiques types of nanostructures contributing to the process of BBB genesis. We further critically evaluate earlier findings in light of novel high-resolution electron microscopy descriptions of nanoscopic tubules.
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    Next generation precision medicine: Crispr-mediated genome editing for the treatment of neurodegenerative disorders
    (Springerlink, 2019) Mentor, Shireen
    Despite significant advancements in the field of molecular neurobiology especially neuroinflammation and neurodegeneration, the highly complex molecular mechanisms underlying neurodegenerative diseases remain elusive. As a result, the development of the next generation neurotherapeutics has experienced a considerable lag phase. Recent advancements in the field of genome editing offer a new template for dissecting the precise molecular pathways underlying the complex neurodegenerative disorders. We believe that the innovative genome and transcriptome editing strategies offer an excellent opportunity to decipher novel therapeutic targets, develop novel neurodegenerative disease models, develop neuroimaging modalities, develop next-generation diagnostics as well as develop patient-specific precision-targeted personalized therapies to effectively treat neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis, Frontotemporal dementia etc
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    The Role of Cytoskeletal Proteins in the Formation of a Functional In Vitro Blood-Brain Barrier Model
    (MDPI, 2022) Mentor, Shireen; Makhathini, Khayelihle Brian; David, Fischer
    The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain’s microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.