Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Yinyu Yuan; Jacquelyn  R. Dayton; Marie-Lena Freese; Bryce  G. Dorflinger; Lillian Cruz-Orengo

doi:10.3791/60291

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

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10:35 min

January 12, 2020

DOI: 10.3791/60291-v

Yinyu Yuan*¹, Jacquelyn R. Dayton*¹, Marie-Lena Freese², Bryce G. Dorflinger¹, Lillian Cruz-Orengo¹

¹Department of Anatomy, Physiology and Cell Biology,University of California Davis, ²University of Veterinary Medicine Hannover Foundation

Overview

This protocol describes a method to isolate microvessels from various regions of the central nervous system (CNS) in both lissencephalic and gyrencephalic vertebrates. By enabling the comparison of microvasculature across different CNS regions and individuals, this technique facilitates deeper insights into vascular structures. The protocol can be completed within a day without the need for ultracentrifugation or enzymatic dissociation.

Key Study Components

Area of Science

Neuroscience
Microvasculature Studies
Central Nervous System Research

Background

The isolation of CNS microvessels is crucial for understanding regional differences in vascularization.
Microvascular studies can provide insights into neurological disorders and vascular health.
The study addresses challenges in removing complex tissues while ensuring a clean specimen for analysis.
Comparative analysis across species can enhance our understanding of vertebrate CNS structures.

Purpose of Study

To establish a reliable method for isolating microvessels from various CNS regions.
To compare microvasculature in lissencephalic versus gyrencephalic vertebrates.
To simplify the isolation process by avoiding traditional techniques like ultracentrifugation.

Methods Used

The method involves dissection and homogenization of CNS tissue, followed by centrifugation for microvessel purification.
Both small and large vertebrate specimens are utilized, with specific protocols for each size.
Manual techniques such as careful tissue removal and homogenization are emphasized to avoid damaging microvessels.
Fresh MV-1 and MV-2 solutions are critical for maintaining tissue viability throughout the process.
Filtration through nylon net filters aids in the purification of isolated microvessels.

Main Results

The outlined process effectively isolates microvessels, providing a robust platform for future comparative studies.
Challenges in tissue removal, particularly of the meninges and choroid plexus, are addressed with specific recommendations.
This method is versatile, allowing for isolation from both small and large vertebrate species.

Conclusions

This study demonstrates an efficient technique for microvessel isolation that can enhance research on CNS vascular networks.
The ability to compare microvasculature across different vertebrates opens avenues for understanding vascular adaptations.
Overall, the protocol contributes to our knowledge of CNS morphology and functionality, particularly in relation to microvascular health.

Frequently Asked Questions

What are the advantages of this microvessel isolation technique?

This technique allows for quick isolation within a day and eliminates the need for ultracentrifugation, making it more user-friendly and efficient.

How is the biological model prepared for microvessel isolation?

The model involves careful dissection of CNS tissue from vertebrate specimens, ensuring that meninges and choroid plexus are fully removed.

What outcomes are expected from this isolation method?

Successful isolation of microvessels will enable comparative analyses of vascular structures, enhancing understanding of their roles in the CNS.

Can this method be adapted for use with other species?

Yes, the method can be adapted for various vertebrate species, with specific considerations for size and tissue complexity.

What limitations should be considered when using this protocol?

Key limitations include the potential difficulty in removing complex layers of tissue and the need for precision during dissection to achieve clean specimens.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

This protocol is significant because it allows for the comparison of microvasculature between different central nervous system regions, as well as between different individuals. This microvessel isolation technique can be completed within a single day, and it eliminates the need for ultracentrifugation and enzymatic dissociation. Removing the meninges and the choroid plexus can be difficult.

Be sure to work slowly and carefully to ensure the complete removal of each tissue. For CNS tissue dissection from a small lissencephalic vertebrate specimen, place the harvested brain into a 15 milliliter conical tube containing MV-1 solution on ice, and use forceps to retrieve the pituitary from the sella turcica of the skull. Place the pituitary in a 1.7 milliliter microcentrifuge tube of MV-1 solution on ice, and remove the skin and muscle to expose the vertebral column.

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