Universal method for the isolation of microvessels from frozen brain tissue: A proof-of-concept multiomic investigation of the neurovasculature

The neurovascular unit, comprised of vascular cell types that collectively regulate cerebral blood flow to meet the needs of coupled neurons, is paramount for the proper function of the central nervous system. The neurovascular unit gatekeeps blood-brain barrier properties, which experiences impairm...

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Published inBrain, behavior, & immunity. Health Vol. 34; p. 100684
Main Authors Wakid, Marina, Almeida, Daniel, Aouabed, Zahia, Rahimian, Reza, Davoli, Maria Antonietta, Yerko, Volodymyr, Leonova-Erko, Elena, Richard, Vincent, Zahedi, René, Borchers, Christoph, Turecki, Gustavo, Mechawar, Naguib
Format Journal Article
LanguageEnglish
Published Elsevier Inc 01.12.2023
Elsevier
Subjects
Blood-Brain Barrier
LC-MS/MS
Microvessels
Neurovascular unit
Postmortem
RNA sequencing
Study Protocol
RNA sequencing
Microvessels
LC-MS/MS
Neurovascular unit
Postmortem
Blood-Brain Barrier
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Abstract The neurovascular unit, comprised of vascular cell types that collectively regulate cerebral blood flow to meet the needs of coupled neurons, is paramount for the proper function of the central nervous system. The neurovascular unit gatekeeps blood-brain barrier properties, which experiences impairment in several central nervous system diseases associated with neuroinflammation and contributes to pathogenesis. To better understand function and dysfunction at the neurovascular unit and how it may confer inflammatory processes within the brain, isolation and characterization of the neurovascular unit is needed. Here, we describe a singular, standardized protocol to enrich and isolate microvessels from archived snap-frozen human and frozen mouse cerebral cortex using mechanical homogenization and centrifugation-separation that preserves the structural integrity and multicellular composition of microvessel fragments. For the first time, microvessels are isolated from postmortem ventromedial prefrontal cortex tissue and are comprehensively investigated as a structural unit using both RNA sequencing and Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Both the transcriptome and proteome are obtained and compared, demonstrating that the isolated brain microvessel is a robust model for the NVU and can be used to generate highly informative datasets in both physiological and disease contexts. •Method presents singular protocol to isolate microvessels from postmortem and frozen mouse cortex.•High yield of microvessels with preserved integrity were isolated, and compatible with high-throughput techniques.•The transcriptome and proteome of isolated microvessels from human vmPFC tissue were characterized.•Limitations of past isolation methods are overcome, and standardized cross-species comparisons are made possible.
AbstractList The neurovascular unit, comprised of vascular cell types that collectively regulate cerebral blood flow to meet the needs of coupled neurons, is paramount for the proper function of the central nervous system. The neurovascular unit gatekeeps blood-brain barrier properties, which experiences impairment in several central nervous system diseases associated with neuroinflammation and contributes to pathogenesis. To better understand function and dysfunction at the neurovascular unit and how it may confer inflammatory processes within the brain, isolation and characterization of the neurovascular unit is needed. Here, we describe a singular, standardized protocol to enrich and isolate microvessels from archived snap-frozen human and frozen mouse cerebral cortex using mechanical homogenization and centrifugation-separation that preserves the structural integrity and multicellular composition of microvessel fragments. For the first time, microvessels are isolated from postmortem ventromedial prefrontal cortex tissue and are comprehensively investigated as a structural unit using both RNA sequencing and Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Both the transcriptome and proteome are obtained and compared, demonstrating that the isolated brain microvessel is a robust model for the NVU and can be used to generate highly informative datasets in both physiological and disease contexts. • Method presents singular protocol to isolate microvessels from postmortem and frozen mouse cortex. • High yield of microvessels with preserved integrity were isolated, and compatible with high-throughput techniques. • The transcriptome and proteome of isolated microvessels from human vmPFC tissue were characterized. • Limitations of past isolation methods are overcome, and standardized cross-species comparisons are made possible.
The neurovascular unit, comprised of vascular cell types that collectively regulate cerebral blood flow to meet the needs of coupled neurons, is paramount for the proper function of the central nervous system. The neurovascular unit gatekeeps blood-brain barrier properties, which experiences impairment in several central nervous system diseases associated with neuroinflammation and contributes to pathogenesis. To better understand function and dysfunction at the neurovascular unit and how it may confer inflammatory processes within the brain, isolation and characterization of the neurovascular unit is needed. Here, we describe a singular, standardized protocol to enrich and isolate microvessels from archived snap-frozen human and frozen mouse cerebral cortex using mechanical homogenization and centrifugation-separation that preserves the structural integrity and multicellular composition of microvessel fragments. For the first time, microvessels are isolated from postmortem ventromedial prefrontal cortex tissue and are comprehensively investigated as a structural unit using both RNA sequencing and Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Both the transcriptome and proteome are obtained and compared, demonstrating that the isolated brain microvessel is a robust model for the NVU and can be used to generate highly informative datasets in both physiological and disease contexts. •Method presents singular protocol to isolate microvessels from postmortem and frozen mouse cortex.•High yield of microvessels with preserved integrity were isolated, and compatible with high-throughput techniques.•The transcriptome and proteome of isolated microvessels from human vmPFC tissue were characterized.•Limitations of past isolation methods are overcome, and standardized cross-species comparisons are made possible.
The neurovascular unit, comprised of vascular cell types that collectively regulate cerebral blood flow to meet the needs of coupled neurons, is paramount for the proper function of the central nervous system. The neurovascular unit gatekeeps blood-brain barrier properties, which experiences impairment in several central nervous system diseases associated with neuroinflammation and contributes to pathogenesis. To better understand function and dysfunction at the neurovascular unit and how it may confer inflammatory processes within the brain, isolation and characterization of the neurovascular unit is needed. Here, we describe a singular, standardized protocol to enrich and isolate microvessels from archived snap-frozen human and frozen mouse cerebral cortex using mechanical homogenization and centrifugation-separation that preserves the structural integrity and multicellular composition of microvessel fragments. For the first time, microvessels are isolated from postmortem ventromedial prefrontal cortex tissue and are comprehensively investigated as a structural unit using both RNA sequencing and Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Both the transcriptome and proteome are obtained and compared, demonstrating that the isolated brain microvessel is a robust model for the NVU and can be used to generate highly informative datasets in both physiological and disease contexts.
ArticleNumber 100684
Author Richard, Vincent
Yerko, Volodymyr
Davoli, Maria Antonietta
Aouabed, Zahia
Almeida, Daniel
Leonova-Erko, Elena
Wakid, Marina
Rahimian, Reza
Mechawar, Naguib
Turecki, Gustavo
Borchers, Christoph
Zahedi, René
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Keywords RNA sequencing
Microvessels
LC-MS/MS
Neurovascular unit
Postmortem
Blood-Brain Barrier
Language English
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Snippet The neurovascular unit, comprised of vascular cell types that collectively regulate cerebral blood flow to meet the needs of coupled neurons, is paramount for...
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SubjectTerms Blood-Brain Barrier
LC-MS/MS
Microvessels
Neurovascular unit
Postmortem
RNA sequencing
Study Protocol
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Title Universal method for the isolation of microvessels from frozen brain tissue: A proof-of-concept multiomic investigation of the neurovasculature
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