Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Anika M.S. Hartz; Julia A. Schulz; Brent S. Sokola; Stephanie E. Edelmann; Andrew N. Shen; Ralf G. Rempe; Yu Zhong; Nader El Seblani; Bjoern Bauer

doi:10.3791/57346

Isolement des capillaires cérébraux du tissu de cerveau humain frais

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Neuroscience

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JoVE Journal Neuroscience

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Isolement des capillaires cérébraux du tissu de cerveau humain frais

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06:35 min

September 12, 2018

DOI: 10.3791/57346-v

Anika M.S. Hartz¹, Julia A. Schulz², Brent S. Sokola², Stephanie E. Edelmann¹, Andrew N. Shen¹, Ralf G. Rempe², Yu Zhong¹, Nader El Seblani³, Bjoern Bauer²

¹Sanders-Brown Center on Aging, Department of Pharmacology and Nutritional Sciences,University of Kentucky, ²Department of Pharmaceutical Sciences, College of Pharmacy,University of Kentucky, ³Department of Neuroscience,University of Kentucky

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Overview

This study introduces a preclinical model using isolated brain capillaries from human brain tissue to investigate blood-brain barrier (BBB) function under both normal and pathological conditions. An optimized protocol for isolating these capillaries is detailed, enabling insights into BBB dysfunction and related signaling pathways.

Key Study Components

Area of Science

Neuroscience
Blood-Brain Barrier Research
Cellular Biology

Background

Blood-brain barrier integrity is critical for central nervous system function.
Understanding BBB dysfunction is pivotal for therapeutic development.
Existing models often lack the physiological relevance of human tissue.
This protocol leverages fresh human brain tissue for precise research applications.

Purpose of Study

To provide a reliable method for isolating human brain capillaries.
To study intact, physiologically active human capillaries.
To facilitate research into signaling pathways involved in BBB function.

Methods Used

The model involves isolating capillaries from fresh human brain tissue using a stepwise protocol.
Capillaries are separated from other brain debris using density gradient centrifugation.
Microscopic examination helps assess capillary purity post-isolation.
Capillaries can be stored for future experiments or processed immediately.
Safety measures for handling human tissue are emphasized due to infection risks.

Main Results

The method successfully isolates pure populations of human brain capillaries.
Insights on BBB modeling can further research into pharmacological effects and signaling pathways.
Isolation quality directly impacts the utility of capillaries in experimentation.
Potential applications include molecular, proteomic, and cellomic studies.

Conclusions

This study demonstrates a practical method for studying blood-brain barrier functionality in human tissue.
The approach enhances research capacity in understanding BBB-related mechanisms in health and disease.
Findings support advanced exploration of therapeutic avenues targeting barrier dysfunction.

Frequently Asked Questions

What are the advantages of using human brain capillaries?

Human brain capillaries provide a more relevant model for studying blood-brain barrier physiology and pathology compared to other species or in vitro systems.

How is the isolation protocol implemented?

The protocol involves careful dissection, density gradient centrifugation, and filtration to ensure high purity and yield of brain capillaries from tissue samples.

What types of experiments can isolated capillaries be used for?

Isolated capillaries can be used in pharmacological assays, cellular signaling studies, and various molecular analyses to investigate blood-brain barrier mechanisms.

What are key considerations during the isolation process?

Care must be taken to avoid capillary loss during handling, and the proper density of Ficoll is crucial for effective separation.

What safety precautions are necessary when working with human tissue?

Laboratory safety protocols include wearing protective gear, handling within a BSL-2 environment, and following guidelines for potential pathogen exposure.

Capillaires cérébraux isolés du tissu de cerveau humain peuvent servir comme un modèle préclinique pour étudier la fonction de barrière dans des conditions physiologiques et physiopathologiques. Nous présentons ici un protocole optimisé pour isoler les capillaires du cerveau du tissu de cerveau humain frais.

Cette méthode peut aider à répondre à des questions clés dans le domaine de la recherche sur la barrière hémato-encéphalique, telles que le dysfonctionnement de la barrière hémato-encéphalique, les fuites de barrière et les voies de signalisation spécifiques à la barrière hémato-encéphalique humaine. Le principal avantage de cette technique est qu’elle permet d’étudier des capillaires cérébraux humains intacts et physiologiquement actifs qui ont été isolés de tissu cérébral humain frais. Pour commencer cette procédure, documentez le poids du tissu cérébral humain, qui doit être d’environ 10 grammes.

Au microscope, retirez soigneusement toutes les méninges à l’aide d’une pince et coupez la substance blanche à l’aide d’un scalpel. Ensuite, coupez soigneusement le tissu cérébral et hachez-le avec un scalpel pendant environ cinq minutes. Transférez le tissu cérébral dans le broyeur de tissus et ajoutez 30 millilitres de tampon d’isolement.

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Neurosciences numéro 139 Neuroscience système neurovasculaire barrière hémato - encéphalique capillaires du cerveau les cellules endothéliales tissus cérébraux humains