Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Jiwon Woo; Eunice Yoojin Lee; Hyo-Suk Park; Jeong Yoon Park; Yong Eun Cho

doi:10.3791/57123

Neuartige passiv Clearing Methoden für die schnelle Produktion von optischen Transparenz im gesam...

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Neuartige passiv Clearing Methoden für die schnelle Produktion von optischen Transparenz im gesamten ZNS-Gewebe

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06:14 min

May 8, 2018

DOI: 10.3791/57123-v

Jiwon Woo^1,2,3, Eunice Yoojin Lee⁴, Hyo-Suk Park^1,3, Jeong Yoon Park^1,3, Yong Eun Cho^1,2,3

¹Department of Neurosurgery, Gangnam Severance Hospital,Yonsei University College of Medicine, ²Brain Korea 21 PLUS Project for Medical Science,Yonsei University, ³The Spine and Spinal Cord Institute, Biomedical Center, Gangnam Severance Hospital,Yonsei University College of Medicine, ⁴Columbia University College of Physicians and Surgeons

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Overview

This study introduces two innovative methodologies, psPACT and mPACT, to enhance optical transparency and facilitate microscopic analysis of intact rodent central nervous system (CNS) vasculature. The techniques aim to quickly clear whole CNS tissue without damaging it, addressing key questions related to human disease models such as Alzheimer's disease and tumorigenesis.

Key Study Components

Area of Science

Neuroscience
Tissue clearing techniques
Microscopic imaging

Background

The study builds on previous methods, highlighting the limitations of electrophoretic tissue clearing.
Emphasizes the need for rapid and effective clearing processes for rodent CNS tissue.
Offers solutions for both basic research and clinical implications in neuroscience.

Purpose of Study

To develop and validate psPACT and mPACT as efficient protocols for CNS tissue clearing.
To expand the capabilities for researchers studying neuropathological conditions.
To facilitate better visualization of tissue vasculature in the CNS.

Methods Used

The methods involve a multi-stage protocol for preparing and clearing CNS tissue using PACT solutions.
Key biological models include mouse and rat CNS samples, with a focus on achieving optical transparency.
psPACT generally takes about 20 days for full clarity, while mPACT can achieve this in approximately 14 days.
Specialized solutions and incubation environments are employed for polymerization and clearing processes.

Main Results

mPACT offers a significant reduction in tissue clearing time compared to traditional methods.
Samples treated with mPACT displayed optical transparency in a shorter timeframe, suggesting enhanced efficiency for research applications.
The techniques prove effective in facilitating the study of various neurological conditions.

Conclusions

This study enables rapid and efficient clearing of CNS tissue, improving research capabilities in the field of neuroscience.
The novel methodologies serve as valuable tools for exploring disease mechanisms and tissue injury.
Implications for understanding neurodegenerative diseases and enhancing pathological studies via improved imaging techniques are substantial.

Frequently Asked Questions

What advantages do psPACT and mPACT provide?

Both methods allow for fast clearing of CNS tissue while preserving its integrity, making them valuable for microscopic analyses.

How are the biological models implemented in this study?

The study uses mouse and rat CNS samples adeptly processed through specific protocols to achieve clarity.

What data outcomes can be expected using these methods?

Results include enhanced visibility of tissue vasculature and improved understanding of disease mechanisms related to the CNS.

How can the methods be adapted for other research purposes?

Researchers may modify the concentration of clearing solutions or the timing of incubations to tailor protocols for different tissues.

What are the key limitations of the psPACT and mPACT protocols?

The methods require meticulous execution and may be challenging due to multiple sample preparation stages.

Can these techniques be applied to other species or tissue types?

While primarily developed for rodent CNS tissue, further validation may enable adaptations for other mammals or specific tissues.

Hier stellen wir zwei neue Methoden, PsPACT und mPACT zur Erreichung maximaler optischer Transparenz und anschließende mikroskopische Analyse des Gewebes Gefäßsystem in das intakte Nagetier ganze CNS.

Das übergeordnete Ziel dieser psPACT- und mPACT-Methoden ist die schnelle Reinigung des gesamten ZNS-Gewebes, ohne dass ein elektrophoretisches Gewebereinigungssystem erforderlich ist. Diese Methode kann helfen, zentrale Fragen im Modellfeld der Humankrankheitsforschung zu beantworten, wie z. B. die Alzheimer-Krankheit oder Verletzungen und Degeneration sowie die Tumorgenese. Der Hauptvorteil dieser Technik besteht darin, dass das ZNS-Gewebe der Maus durch die elektrophoretische Gewebereinigung nicht geschädigt wird und durch die modifizierte passive Reinigung schnell entfernt wird.

Wir hatten die Idee zu dieser Methode, nachdem wir die ursprüngliche PACT- und frühere Methode zur Gehirnreinigung verwendet hatten. Die visuelle Demonstration dieser Methode ist von entscheidender Bedeutung, da psPACT und mPACT-Methoden schwierig durchzuführen sind, da diese Methode mehrere Phasen der Probenvorbereitung umfasst. Beginnen Sie mit der Zubereitung der PACT-Cocktaillösung, indem Sie VA-044-Pulver zu einer Lösung aus 4 % PFA und 4 % Acrylamid in einer Endkonzentration von 0,25 % hinzufügen. Nachdem Sie das Gewebe mit PACT-Lösung durchblutet haben, tauchen Sie das gesamte Gehirn und das Rückenmark vollständig in ein 50-Milliliter-Röhrchen mit gekühlter PACT-Lösung und lagern Sie es 24 Stunden lang bei vier Grad Celsius.

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