Isolation and characterization of microvessels from normal brain and brain tumors

Silbergeld, Daniel L.; Ali‐Osman, Francis

doi:10.1007/bf00166997

Cited by 23 publications

(15 citation statements)

References 19 publications

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“…To understand the state of tight junction proteins in our model of CNS vascular permeability we utilized a well established method of rodent brain microvessel isolation [31]–[33]. Microvessel isolation from brain tissue enabled the analysis of BBB tight junction protein levels in the CNS.…”

Section: Discussionmentioning

confidence: 99%

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

et al. 2008

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Disruption of the blood brain barrier (BBB) is a hallmark feature of immune-mediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis. Although current models hypothesize that immune cells promote vascular permeability in human disease, the role CD8 T cells play in BBB breakdown remains poorly defined. Our laboratory has developed a novel murine model of CD8 T cell mediated central nervous system (CNS) vascular permeability using a variation of the Theiler's virus model of multiple sclerosis. In previous studies, we observed that MHC class II−/− (CD4 T cell deficient), IFN-γR−/−, TNF-α−/−, TNFR1−/−, TNFR2−/−, and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTβ activity were susceptible to CNS vascular permeability. Therefore, the objective of this study was to determine the extent immune effector proteins utilized by CD8 T cells, perforin and FasL, contributed to CNS vascular permeability. Using techniques such as fluorescent activated cell sorting (FACS), T1 gadolinium-enhanced magnetic resonance imaging (MRI), FITC-albumin leakage assays, microvessel isolation, western blotting and immunofluorescent microscopy, we show that in vivo stimulation of CNS infiltrating antigen-specific CD8 T cells initiates astrocyte activation, alteration of BBB tight junction proteins and increased CNS vascular permeability in a non-apoptotic manner. Using the aforementioned techniques, we found that despite having similar expansion of CD8 T cells in the brain as wildtype and Fas Ligand deficient animals, perforin deficient mice were resistant to tight junction alterations and CNS vascular permeability. To our knowledge, this study is the first to demonstrate that CNS infiltrating antigen-specific CD8 T cells have the capacity to initiate BBB tight junction disruption through a non-apoptotic perforin dependent mechanism and our model is one of few that are useful for studies in this field. These novel findings are highly relevant to the development of therapies designed to control immune mediated CNS vascular permeability.

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Section: Discussionmentioning

confidence: 99%

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

et al. 2008

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“…Anterior, middle cerebral, and basilar arteries were isolated, cleaned, and used for mitochondrial respiration measurements and for Western blotting. Brain microvessel isolation was then performed as previously described (42,43,49) with minor modifications. Cerebellum, brain stem, choroid plexus, and large superficial arteries and meninges were all removed.…”

Section: Methodsmentioning

confidence: 99%

The mitochondrial function of the cerebral vasculature in insulin-resistant Zucker obese rats

Merdžo

Rutkai

Tokes

et al. 2016

American Journal of Physiology-Heart and Circulatory Physiology

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Little is known about mitochondrial functioning in the cerebral vasculature during insulin resistance (IR). We examined mitochondrial respiration in isolated cerebral arteries of male Zucker obese (ZO) rats and phenotypically normal Zucker lean (ZL) rats using the Seahorse XFe24 analyzer. We investigated mitochondrial morphology in cerebral blood vessels as well as mitochondrial and nonmitochondrial protein expression levels in cerebral arteries and microvessels. We also measured reactive oxygen species (ROS) levels in cerebral microvessels. Under basal conditions, the mitochondrial respiration components (nonmitochondrial respiration, basal respiration, ATP production, proton leak, and spare respiratory capacity) showed similar levels among the ZL and ZO groups with the exception of maximal respiration, which was higher in the ZO group. We examined the role of nitric oxide by measuring mitochondrial respiration following inhibition of nitric oxide synthase with Nω-nitro-l-arginine methyl ester (l-NAME) and mitochondrial activation after administration of diazoxide (DZ). Both ZL and ZO groups showed similar responses to these stimuli with minor variations. l-NAME significantly increased the proton leak, and DZ decreased nonmitochondrial respiration in the ZL group. Other components were not affected. Mitochondrial morphology and distribution within vascular smooth muscle and endothelium as well as mitochondrial protein levels were similar in the arteries and microvessels of both groups. Endothelial nitric oxide synthase (eNOS) and ROS levels were increased in cerebral microvessels of the ZO. Our study suggests that mitochondrial function is not significantly altered in the cerebral vasculature of young ZO rats, but increased ROS production might be due to increased eNOS in the cerebral microcirculation during IR.

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“…These purified brain microvessels consist of vascular endothelium ensheathed by a basement membrane containing pericytes and offcuts of astrocytic endfeet. The purification procedure consists of a combination of mechanical homogenization, enzymatic dissociation, filtration, and density gradient centrifugation followed by column filtration (85). Visual inspection by light/fluorescence microscopy as well as scanning electron microscopy and transmission electron microscopy (85) for positive expression of typical BBB biomarkers (such us glucose transporter-1 (glut-1); transferrin receptor (OX-26); ZO-1, claudin-5 and 12, (86)) are concomitantly used to assess the purity of the preparation.…”

Section: Isolated Brain Microvesselsmentioning

confidence: 99%

“…The purification procedure consists of a combination of mechanical homogenization, enzymatic dissociation, filtration, and density gradient centrifugation followed by column filtration (85). Visual inspection by light/fluorescence microscopy as well as scanning electron microscopy and transmission electron microscopy (85) for positive expression of typical BBB biomarkers (such us glucose transporter-1 (glut-1); transferrin receptor (OX-26); ZO-1, claudin-5 and 12, (86)) are concomitantly used to assess the purity of the preparation. Lack of typical smooth muscle cell biomarkers such as desmin and calponin (87, 88) in isolates is generally indicative of a preparation free of post-capillary fragment contaminants.…”

Section: Isolated Brain Microvesselsmentioning

confidence: 99%

In Vitro Cerebrovascular Modeling in the 21st Century: Current and Prospective Technologies

et al. 2014

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The blood-brain barrier (BBB) maintains the brain homeostasis and dynamically responds to events associated with systemic and/or rheological impairments (e.g., inflammation, ischemia) including the exposure to harmful xenobiotics. Thus, understanding the BBB physiology is crucial for the resolution of major central nervous system CNS) disorders challenging both health care providers and the pharmaceutical industry. These challenges include drug delivery to the brain, neurological disorders, toxicological studies, and biodefense. Studies aimed at advancing our understanding of CNS diseases and promoting the development of more effective therapeutics are primarily performed in laboratory animals. However, there are major hindering factors inherent to in vivo studies such as cost, limited throughput and translational significance to humans. These factors promoted the development of alternative in vitro strategies for studying the physiology and pathophysiology of the BBB in relation to brain disorders as well as screening tools to aid in the development of novel CNS drugs. Herein, we provide a detailed review including pros and cons of current and prospective technologies for modelling the BBB in vitro including ex situ, cell based and computational (in silico) models. A special section is dedicated to microfluidic systems including micro-BBB, BBB-on-a-chip, Neurovascular Unit-on-a-Chip and Synthetic Microvasculature Blood-Brain Barrier.

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Isolation and characterization of microvessels from normal brain and brain tumors

Cited by 23 publications

References 19 publications

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

The mitochondrial function of the cerebral vasculature in insulin-resistant Zucker obese rats

In Vitro Cerebrovascular Modeling in the 21st Century: Current and Prospective Technologies

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