Efficient isolation of brain capillary from a single frozen mouse brain for protein expression analysis

Ogata, Seiryo; Ito, Shingo; Masuda, Takeshi; Ohtsuki, Sumio

doi:10.1177/0271678x20941449

Cited by 19 publications

(16 citation statements)

References 43 publications

(44 reference statements)

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“…We performed transcardial perfusion with ice-cold DEPC-treated water under deep anesthesia with the mixture of Zoletil (30 mg/kg) and xylazine (10 mg/kg). Capillaries were isolated from the extracted brain, as previously described ( Ogata et al, 2020 ). Total RNA was isolated using easy-BLUE reagent (iNtRON Biotechnology, 17061).…”

Section: Methodsmentioning

confidence: 99%

Pericyte Loss Leads to Capillary Stalling Through Increased Leukocyte-Endothelial Cell Interaction in the Brain

Choe

Yoon

Joo

et al. 2022

Front. Cell. Neurosci.

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The neurovascular unit is a functional unit composed of neurons, glial cells, pericytes, and endothelial cells which sustain brain activity. While pericyte is a key component of the neurovascular unit, its role in cerebral blood flow regulation remains elusive. Recently, capillary stalling, which means the transient interruption of microcirculation in capillaries, has been shown to have an outsized impact on microcirculatory changes in several neurological diseases. In this study, we investigated capillary stalling and its possible causes, such as the cerebral endothelial glycocalyx and leukocyte adhesion molecules after depleting pericytes postnatally in mice. Moreover, we investigated hypoxia and gliosis as consequences of capillary stalling. Although there were no differences in the capillary structure and RBC flow, longitudinal optical coherence tomography angiography showed an increased number of stalled segments in capillaries after pericyte loss. Furthermore, the extent of the cerebral endothelial glycocalyx was decreased with increased expression of leukocyte adhesion molecules, suggesting enhanced interaction between leukocytes and endothelial cells. Finally, pericyte loss induced cerebral hypoxia and gliosis. Cumulatively, the results suggest that pericyte loss induces capillary stalling through increased interaction between leukocytes and endothelial cells in the brain.

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Section: Methodsmentioning

confidence: 99%

Pericyte Loss Leads to Capillary Stalling Through Increased Leukocyte-Endothelial Cell Interaction in the Brain

Choe

Yoon

Joo

et al. 2022

Front. Cell. Neurosci.

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“…Brain microvessels were isolated from individual mouse brains as described in a recent report with modifications [ 37 ]. After removal of the brain stem and cerebellum, brain hemispheres were transferred to a 2 mL reinforced polypropylene tube (Cat.…”

Section: Methodsmentioning

confidence: 99%

Neurovascular protection by adropin in experimental ischemic stroke through an endothelial nitric oxide synthase-dependent mechanism

et al. 2021

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Adropin is a highly-conserved peptide that has been shown to preserve endothelial barrier function. Blood-brain barrier (BBB) disruption is a key pathological event in cerebral ischemia. However, the effects of adropin on ischemic stroke outcomes remain unexplored. Hypothesizing that adropin exerts neuroprotective effects by maintaining BBB integrity, we investigated the role of adropin in stroke pathology utilizing loss- and gain-of-function genetic approaches combined with pharmacological treatment with synthetic adropin peptide. Long-term anatomical and functional outcomes were evaluated using histology, MRI, and a battery of sensorimotor and cognitive tests in mice subjected to ischemic stroke. Brain ischemia decreased endogenous adropin levels in the brain and plasma. Adropin treatment or transgenic adropin overexpression robustly reduced brain injury and improved long-term sensorimotor and cognitive function in young and aged mice subjected to ischemic stroke. In contrast, genetic deletion of adropin exacerbated ischemic brain injury, irrespective of sex. Mechanistically, adropin treatment reduced BBB damage, degradation of tight junction proteins, matrix metalloproteinase-9 activity, oxidative stress, and infiltration of neutrophils into the ischemic brain. Adropin significantly increased phosphorylation of endothelial nitric oxide synthase (eNOS), Akt, and ERK1/2. While adropin therapy was remarkably protective in wild-type mice, it failed to reduce brain injury in eNOS-deficient animals, suggesting that eNOS is required for the protective effects of adropin in stroke. These data provide the first causal evidence that adropin exerts neurovascular protection in stroke through an eNOS-dependent mechanism. We identify adropin as a novel neuroprotective peptide with the potential to improve stroke outcomes.

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“…The highly pure brain capillaries were isolated as described in a previous report, with minor modifications. 37 The cortex (neocortex; gray matter) was collected with a razor less than 3 mm from the surface of the cerebrum, and the white matter was collected from the radial crown just under the cortex used. The weights of collected tissues are listed in The samples were immediately centrifuged (5800 g, 10 min, 4 C) and the supernatant was collected in a new 2 mL tube, while the pellets were stored on ice.…”

Section: Preparation Of Highly Pure Capillaries and Whole-tissue Lysa...mentioning

confidence: 99%

Upregulation of ribosome complexes at the blood-brain barrier in Alzheimer's disease patients

Suzuki

Tezuka

Handa

et al. 2022

J Cereb Blood Flow Metab

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The cerebrovascular-specific molecular mechanism in Alzheimer’s disease (AD) was investigated by employing comprehensive and accurate quantitative proteomics. Highly purified brain capillaries were isolated from cerebral gray and white matter of four AD and three control donors, and examined by SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. Of the 29 ribosomal proteins that were quantified, 28 (RPLP0, RPL4, RPL6, RPL7A, RPL8, RPL10A, RPL11, RPL12, RPL14, RPL15, RPL18, RPL23, RPL27, RPL27A, RPL31, RPL35A, RPS2, RPS3, RPS3A, RPS4X, RPS7, RPS8, RPS14, RPS16, RPS20, RPS24, RPS25, and RPSA) were significantly upregulated in AD patients. This upregulation of ribosomal protein expression occurred only in brain capillaries and not in brain parenchyma. The protein expression of protein processing and N-glycosylation-related proteins in the endoplasmic reticulum (DDOST, STT3A, MOGS, GANAB, RPN1, RPN2, SEC61B, UGGT1, LMAN2, and SSR4) were also upregulated in AD brain capillaries and was correlated with the expression of ribosomal proteins. The findings reported herein indicate that the ribosome complex, the subsequent protein processing and N-glycosylation-related processes are significantly and specifically upregulated in the brain capillaries of AD patients.

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Efficient isolation of brain capillary from a single frozen mouse brain for protein expression analysis

Cited by 19 publications

References 43 publications

Pericyte Loss Leads to Capillary Stalling Through Increased Leukocyte-Endothelial Cell Interaction in the Brain

Pericyte Loss Leads to Capillary Stalling Through Increased Leukocyte-Endothelial Cell Interaction in the Brain

Neurovascular protection by adropin in experimental ischemic stroke through an endothelial nitric oxide synthase-dependent mechanism

Upregulation of ribosome complexes at the blood-brain barrier in Alzheimer's disease patients

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