scholarly journals An Improved Method for Physical Separation of Cerebral Vasculature and Parenchyma Enables Detection of Blood-Brain-Barrier Dysfunction

NeuroSci ◽  
2021 ◽  
Vol 2 (1) ◽  
pp. 59-74
Author(s):  
Frank Matthes ◽  
Hana Matuskova ◽  
Kajsa Arkelius ◽  
Saema Ansar ◽  
Iben Lundgaard ◽  
...  
Keyword(s):  
Protein Expression ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Barrier Dysfunction ◽  
Brain Vasculature ◽  
Blood Brain ◽  
Junction Protein ◽  
Tight Association ◽  
Neurovascular Niche ◽  
Astrocytic Endfeet

The neurovascular niche is crucial for constant blood supply and blood-brain barrier (BBB) function and is altered in a number of different neurological conditions, making this an intensely active field of research. Brain vasculature is unique for its tight association of endothelial cells with astrocytic endfeet processes. Separation of the vascular compartment by centrifugation-based methods confirmed enrichment of astrocytic endfeet processes, making it possible to study the entire vascular niche with such methods. Several centrifugation-based separation protocols are found in the literature; however, with some constraints which limit their applicability and the scope of the studies. Here, we describe and validate a protocol for physically separating the neurovascular niche from the parenchyma, which is optimized for smaller tissue quantities. Using endothelial, neuronal, and astrocyte markers, we show that quantitative Western blot-based target detection can be performed of both the vessel-enriched and parenchymal fractions using as little as a single mouse brain hemisphere. Validation of our protocol in rodent stroke models by detecting changes in tight junction protein expression, serum albumin signals and astrocyte activation, i.e., increased glial fibrillary acidic protein expression, between the ipsilateral and the lesion-free contralateral hemisphere demonstrates this protocol as a new way of detecting BBB breakdown and astrogliosis, respectively.

scholarly journals An Improved Method for Physical Separation of Cerebral Vasculature and Parenchyma Enables Detection of Blood-Brain-Barrier Dysfunction

2020 ◽  
Author(s):  
Frank Matthes ◽  
Hana Matuskova ◽  
Kajsa Arkelius ◽  
Saema Ansar ◽  
Iben Lundgaard ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Barrier Dysfunction ◽  
Physical Separation ◽  
Brain Vasculature ◽  
Blood Brain ◽  
Stroke Models ◽  
Tight Association ◽  
Neurovascular Niche ◽  
Astrocytic Endfeet

The neurovascular niche is crucial for constant blood supply and blood-brain barrier (BBB) function and is altered in a number of different neurological conditions, making this an intensely active field of research. Brain vasculature is unique for its tight association of endothelial cells with astrocytic endfeet processes. Separation of the vascular compartment by centrifugation-based methods confirmed enrichment of astrocytic endfeet processes, making it possible to study the entire vascular niche with such methods. Several centrifugation-based separation protocols are found in the literature; however, with some constraints which limit their applicability and the scope of the studies. Here, we describe and validate a protocol for physically separating the neurovascular niche from the parenchyma, which is optimized for smaller tissue quantities. Using endothelial, neuronal and astrocyte markers, we show that quantitative Western blot-based target detection can be performed of both the vascular and parenchymal fractions using as little as a single mouse brain hemisphere. Validation of our protocol in rodent stroke models by detecting changes in serum albumin signals and astrocyte activation, i.e. increased glial fibrillary acidic protein expression, between the ipsilateral and the lesion-free contralateral hemisphere demonstrates this protocol as a new way of detecting BBB breakdown and astrogliosis, respectively.

scholarly journals Protein Kinase C Activation Modulates Reversible Increase in Cortical Blood–Brain Barrier Permeability and Tight Junction Protein Expression during Hypoxia and Posthypoxic Reoxygenation

2010 ◽  
Vol 30 (11) ◽  
pp. 1847-1859 ◽  
Author(s):  
Colin L Willis ◽  
Diana S Meske ◽  
Thomas P Davis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Expression ◽  
Tight Junction ◽  
Vascular Permeability ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Kinase C ◽  
Blood Brain ◽  
Junction Protein

Hypoxia (Hx) is a component of many disease states including stroke. Ischemic stroke occurs when there is a restriction of cerebral blood flow and oxygen to part of the brain. During the ischemic, and subsequent reperfusion phase of stroke, blood–brain barrier (BBB) integrity is lost with tight junction (TJ) protein disruption. However, the mechanisms of Hx and reoxygenation (HR)-induced loss of BBB integrity are not fully understood. We examined the role of protein kinase C (PKC) isozymes in modifying TJ protein expression in a rat model of global Hx. The Hx (6% O2) induced increased hippocampal and cortical vascular permeability to 4 and 10 kDa dextran fluorescein isothiocyanate (FITC) and endogenous rat-IgG. Cortical microvessels revealed morphologic changes in nPKC-θ distribution, increased nPKC-θ and aPKC-ζ protein expression, and activation by phosphorylation of nPKC-θ (Thr538) and aPKC-ζ (Thr410) residues after Hx treatment. Claudin-5, occludin, and ZO-1 showed disrupted organization at endothelial cell margins, whereas Western blot analysis showed increased TJ protein expression after Hx. The PKC inhibition with chelerythrine chloride (5 mg/kg intraperitoneally) attenuated Hx-induced hippocampal vascular permeability and claudin-5, PKC (θ and ζ) expression, and phosphorylation. This study supports the hypothesis that nPKC-θ and aPKC-ζ signaling mediates TJ protein disruption resulting in increased BBB permeability.

scholarly journals Interleukin-1β-induced barrier dysfunction is signaled through PKC-θ in human brain microvascular endothelium

2012 ◽  
Vol 302 (10) ◽  
pp. C1513-C1522 ◽  
Author(s):  
Robert R. Rigor ◽  
Richard S. Beard ◽  
Olesya P. Litovka ◽  
Sarah Y. Yuan
Keyword(s):  
Human Brain ◽  
Blood Brain Barrier ◽  
Molecular Mechanisms ◽  
Brain Barrier ◽  
Brain Diseases ◽  
Barrier Dysfunction ◽  
Microvascular Endothelium ◽  
Blood Brain ◽  
Pkc Isoforms ◽  
Junction Protein

Blood-brain barrier dysfunction is a serious consequence of inflammatory brain diseases, cerebral infections, and trauma. The proinflammatory cytokine interleukin (IL)-1β is central to neuroinflammation and contributes to brain microvascular leakage and edema formation. Although it is well known that IL-1β exposure directly induces hyperpermeability in brain microvascular endothelium, the molecular mechanisms mediating this response are not completely understood. In the present study, we found that exposure of the human brain microvascular endothelium to IL-1β triggered activation of novel PKC isoforms δ, μ, and θ, followed by decreased transendothelial electrical resistance (TER). The IL-1β-induced decrease in TER was prevented by small hairpin RNA silencing of PKC-θ or by treatment with the isoform-selective PKC inhibitor Gö6976 but not by PKC inhibitors that are selective for all PKC isoforms other than PKC-θ. Decreased TER coincided with increased phosphorylation of regulatory myosin light chain and with increased proapoptotic signaling indicated by decreased uptake of mitotracker red in response to IL-1β treatment. However, neither of these observed effects were prevented by Gö6976 treatment, indicating lack of causality with respect to decreased TER. Instead, our data indicated that the mechanism of decreased TER involves PKC-θ-dependent phosphorylation of the tight junction protein zona occludens (ZO)-1. Because IL-1β is a central inflammatory mediator, our interpretation is that inhibition of PKC-θ or inhibition of ZO-1 phosphorylation could be viable strategies for preventing blood-brain barrier dysfunction under a variety of neuroinflammatory conditions.

scholarly journals Alterations in blood-brain barrier ICAM-1 expression and brain microglial activation after λ-carrageenan-induced inflammatory pain

2006 ◽  
Vol 290 (2) ◽  
pp. H732-H740 ◽  
Author(s):  
J. D. Huber ◽  
C. R. Campos ◽  
K. S. Mark ◽  
T. P. Davis
Keyword(s):  
Protein Expression ◽  
Blood Brain Barrier ◽  
Proinflammatory Cytokines ◽  
Inflammatory Pain ◽  
Microglial Activation ◽  
Brain Barrier ◽  
Cell Counts ◽  
Blood Brain ◽  
Junction Protein ◽  
The Brain

Previous studies showed that peripheral inflammatory pain increased blood-brain barrier (BBB) permeability and altered tight junction protein expression and the delivery of opioid analgesics to the brain. What remains unknown is which pathways and mediators during peripheral inflammation affect BBB function and structure. The current study investigated effects of λ-carrageenan-induced inflammatory pain (CIP) on BBB expression of ICAM-1. We also examined the systemic contribution of a number of proinflammatory cytokines and microglial activation in the brain to elucidate pathways involved in BBB disruption during CIP. We investigated ICAM-1 RNA and protein expression levels in isolated rat brain microvessels after CIP using RT-PCR and Western blot analyses, screened inflammatory cytokines during the time course of inflammation, assessed white blood cell counts, and probed for BBB and central nervous system stimulation and leukocyte transmigration using immunohistochemistry and flow cytometry. Results showed an early increase in ICAM-1 RNA and protein expression after CIP with no change in circulating levels of several proinflammatory cytokines. Changes in ICAM-1 protein expression were noted at 48 h. Immunohistochemistry showed that the induction of ICAM-1 was region specific with increased expression noted in the thalamus and frontal and parietal cortices, which directly correlated with increased expression of activated microglia. The findings of the present study were that CIP induces increased ICAM-1 mRNA and protein expression at the BBB and that systemic proinflammatory mediators play no apparent role in the early response (1–6 h); however, brain region-specific increases in microglial activation suggest a potential for a central-mediated response.

Transcytosis within PVN capillaries - a mechanism determining both hypertension-induced blood-brain barrier dysfunction and exercise-induced correction

2021 ◽  
Author(s):  
Matheus Garcia Fragas ◽  
Vanessa Brito Candido ◽  
Gustavo Gastão Davanzo ◽  
Carla Rocha-Santos ◽  
Alexandre Ceroni ◽  
...  
Keyword(s):  
Protein Expression ◽  
Blood Brain Barrier ◽  
Brain Parenchyma ◽  
Autonomic Control ◽  
Brain Barrier ◽  
Barrier Dysfunction ◽  
Caveolin 1 ◽  
Blood Brain ◽  
Gene And Protein Expression ◽  
Exercise Induced

While hypertension disrupts the blood-brain barrier (BBB) integrity within the paraventricular nucleus of hypothalamus (PVN) and increases the leakage into the brain parenchyma, exercise training (T) was shown to correct it. Since there is scarce and contradictory information on the mechanism(s) determining hypertension-induced BBB deficit and nothing is known about T-induced improvement, we sought to evaluate the paracellular and transcellular transport across the BBB within the PVN in both conditions. SHR and WKY submitted to 4-weeks aerobic T or sedentary (S) protocol were chronically catheterized for hemodynamic recordings at rest and intra-arterial administration of dyes (Rhodamine-dextran 70kDa + FITC-dextran 10kDa). Brains were harvesting for FITC leakage examination, qPCR evaluation of different BBB constituents and protein expression of Caveolin-1 and Claudin-5, the main markers of transcytosis and paracellular transport, respectively. Hypertension was characterized by increased arterial pressure and heart rate, augmented sympathetic modulation of heart and vessels, and reduced cardiac parasympathetic control, marked FITC extravasation into the PVN which was accompanied by increased caveolin-1 gene and protein expression, without changes in claudin-5 and others tight junctions' components. SHR-T vs. SHR-S showed a partial pressure reduction, resting bradycardia, improvement of autonomic control of the circulation simultaneously with correction of both FITC leakage and caveolin-1 expression; there was a significant increase in claudin-5 expression. Caveolin-1 content was strongly correlated with improved autonomic control after exercise. Data indicated that within the PVN the transcytosis is the main mechanism governing both hypertension-induced BBB leakage as well as the exercise-induced correction.

EMP-induced alterations of tight junction protein expression and disruption of the blood–brain barrier

2010 ◽  
Vol 196 (3) ◽  
pp. 154-160 ◽  
Author(s):  
Gui-Rong Ding ◽  
Lian-Bo Qiu ◽  
Xiao-Wu Wang ◽  
Kang-Chu Li ◽  
Yong-Chun Zhou ◽  
...  
Keyword(s):  
Protein Expression ◽  
Tight Junction ◽  
Blood Brain Barrier ◽  
Tight Junction Protein ◽  
Brain Barrier ◽  
Tight Junction Protein Expression ◽  
Blood Brain ◽  
Junction Protein
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