High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

In a series of previous studies, we demonstrated that the photodynamic therapy (PDT), as a widely used tool for treatment of glioblastoma multiforme (GBM), also site-specifically opens the blood–brain barrier (BBB) in PDT-dose and age-related manner via reversible disorganization of the tight junction machinery. To develop the effective protocol of PDT-opening of the BBB, here we answer the question of what kind of photosensitizer (PS) is the most effective for the BBB opening. We studied the PDT-opening of the BBB in healthy mice using commercial photosensitizers (PSs) such as 5-aminolevulenic acid (5-ALA), aluminum phthalocyanine disulfonate (AlPcS), zinc phthalocyanine (ZnPc) and new synthetized PSs such as galactose functionalized ZnPc (GalZnPc). The spectrofluorimetric assay of Evans Blue albumin complex (EBAC) leakage and 3-D confocal imaging of FITC-dextran 70 kDa (FITCD) extravasation clearly shows a revisable and dose depended PDT-opening of the BBB to EBAC and FITCD associated with a decrease in presence of tight junction (TJ) in the vascular endothelium. The PDT effects on the BBB permeability, TJ expression and the fluorescent signal from the brain tissues are more pronounced in PDT-GalZnPc vs. PDT-5-ALA/AlPcS/ZnPc. These pre-clinical data are the first important informative platform for an optimization of the PDT protocol in the light of new knowledge about PDT-opening of the BBB for drug brain delivery and for the therapy of brain diseases.

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High-Resolution Insights Into the in vitro Developing Blood-Brain Barrier: Novel Morphological Features of Endothelial Nanotube Function

Frontiers in Neuroanatomy ◽

10.3389/fnana.2021.661065 ◽

2021 ◽

Vol 15 ◽

Author(s):

Shireen Mentor ◽

David Fisher

Keyword(s):

High Resolution ◽

Blood Brain Barrier ◽

Cell Communication ◽

High Resolution Electron Microscopy ◽

Brain Barrier ◽

Transport Systems ◽

Functional Roles ◽

Blood Brain ◽

First Time

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. For the first time, we report on the secretion of a basement membrane in vitro, which allow BECs to orientate themselves into distinct basolateral and apicolateral domains and establish a 3-dimensional BEC construct. We report for the first time, on the expression of nanovesicles bound to the plasma membrane surfaces of the BECs. These membrane-bound vesicles are reported to possess an array of DNA/RNA constituents and chemotaxic properties affecting the formation of nanotubes that span the paracellular space between BECs, facilitating BBB construction, alluding to a functional role in mediating cell-to-cell communication. This study suggests that novel, ultrathin nanotubular (NT) structures are involved in functional roles in bringing into alignment the paracellular space of BECs. Immortalized mouse BECs (b.End3, b.End5) and primary rat cardiac microvascular ECs were used to further validate the in vitro BBB model by profiling variances in peripheral EC monolayer development. These cardiac capillary ECs presented with an opposite topographical profile: large fenestra and intercellular spaces, devoid of morphological ultrastructures. This comparative study alludes to the role of NT facilitation in TJ-induced hemifusion of apicolateral BEC membranes, as a structural event forming the basis for establishing a polarized BBB.

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Confocal imaging of xenobiotic transport across the blood-brain barrier

Journal of Experimental Zoology ◽

10.1002/jez.a.10313 ◽

2003 ◽

Vol 300A (1) ◽

pp. 84-90 ◽

Cited By ~ 8

Author(s):

David S. Miller

Keyword(s):

Blood Brain Barrier ◽

Confocal Imaging ◽

Brain Barrier ◽

Blood Brain

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A Novel Method to Stimulate Lymphatic Clearance of Beta-Amyloid from Mouse Brain Using Non-invasive Music-Induced Opening of the Blood-Brain Barrier with EEG Markers

10.20944/preprints202109.0320.v1 ◽

2021 ◽

Author(s):

Oxana Semyachkina-Glushkovskaya ◽

Alexander Khorovodov ◽

Ivan Fedosov ◽

Alexey Pavlov ◽

Alexander Shirokov ◽

...

Keyword(s):

Blood Brain Barrier ◽

Mouse Brain ◽

Protein A ◽

Magnetic Resonance Tomography ◽

Confocal Imaging ◽

Brain Barrier ◽

Fluctuation Analysis ◽

Non Invasive ◽

Blood Brain ◽

The Brain

The lymphatic system of the brain meninges and head plays a crucial role in the clearance of amyloid-β protein (Aβ), a peptide thought to be pathogenic in Alzheimer’s disease (AD), from the brain. The development of methods to modulate lymphatic clearance of Aβ from the brain coild be a revolutionary step in the therapy of AD. The opening of the blood-brain barrier (OBBB) by focused ultrasound is considered as a possible tool for stimulation of clearance of Aβ from the brain of humans and animals. Here, we propose an alternative method of non-invasive music-induced OBBB that is accompanied by the activation of clearance of fluorescent Aβ (Fαβ) from the mouse brain. Using confocal imaging, fluorescence microscopy and magnetic resonance tomography, we clearly demonstrate that OBBB by music stimulates the movement of Fαβ and Omniscan in the cerebrospinal fluid and lymphatic clearance of Fαβ from the brain. We propose the extended detrended fluctuation analysis (EDFA) as a promising method for the identification of OBBB markers in the electroencephalographic (EEG) patterns. These pilot results suggest that music-induced OBBB and the EDFA analysis of EEG can be a non-invasive, low cost, labelling free, clinical perspective and completely new approach for the treatment and monitoring of AD.

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The Proteasome-Ubiquitin System Is Required for Efficient Killing of Intracellular Streptococcus pneumoniae by Brain Endothelial Cells

mBio ◽

10.1128/mbio.00984-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 7

Author(s):

Federico Iovino ◽

Henrik Gradstedt ◽

Jetta J. Bijlsma

Keyword(s):

Endothelial Cells ◽

Streptococcus Pneumoniae ◽

Blood Brain Barrier ◽

Confocal Imaging ◽

Brain Barrier ◽

High Morbidity ◽

Content Type ◽

Blood Brain ◽

Ubiquitin System

ABSTRACTStreptococcus pneumoniae(pneumococcus) is a Gram-positive bacterium that causes serious invasive diseases, such as pneumonia, bacteremia, and meningitis, with high morbidity and mortality throughout the world. Before causing invasive disease,S. pneumoniaeencounters cellular barriers, which are often composed of endothelial cells, like the alveolar-capillary barrier and the blood-brain barrier.S. pneumoniaeadheres to endothelial cells and may invade them, which requires an efficient host response to the intracellular bacteria. The precise intracellular fate ofS. pneumoniaeduring infection still remains a subject of debate. The proteasome-ubiquitin system is largely responsible for the degradation of misfolded, damaged, or no-longer-useful proteins. Recently, the role of the proteasome-ubiquitin system in the clearing of invading bacteria and viruses has been more closely studied. In this study, we show that inhibition of the proteasome-ubiquitin system leads to a marked increase inS. pneumoniaesurvival inside host cells. Immunofluorescence analysis showed that intracellular pneumococci colocalized with proteasome and ubiquitin in human endothelial cellsin vitro. Confocal imaging analysis demonstrated that in the brains of mice intravenously infected withS. pneumoniae, the bacteria were inside endothelial cells, where they colocalized with proteasome and ubiquitin signals. In conclusion, our data indicate that a fully functional proteasome-ubiquitin system in endothelial cells is crucial for efficient killing of intracellularS. pneumoniae.IMPORTANCEBacterial meningitis is a serious invasive disease with high morbidity and mortality. How bacteria traverse the blood-brain barrierin vivoand what mechanisms are employed by the host to prevent invasion are still unclear. Our data show that inhibition of the proteasome-ubiquitin systemin vitroleads to a significant increase inS. pneumoniaesurvival inside brain endothelial cells. Confocal imaging analysis of brain tissue from mice intravenously infected with pneumococci demonstrated that the bacteria are inside brain microvascular endothelial cells, where they associate with the proteasome and ubiquitin. This is, as far as we know, the first report that demonstrates thatStreptococcus pneumoniaeinvades endothelial cells of the blood-brain barrierin vivo. The host requires the proteasome-ubiquitin system for an efficient decimation of intracellularS. pneumoniae.

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