Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue
May 8th, 2018
Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.
The overall goal of these psPACT and mPACT methods is the fast clearing of whole CNS tissue without the need for an electrophoretic tissue clearing system. This method can help answer key questions in the human disease research model field, such as Alzheimer's disease or injury and degeneration and tumorigenesis. The main advantage of this technique is that the mouse CNS tissue is not damaged by the electrophoretic tissue clearing, and it's rapidly cleared by modified passive clearing.
We first had the idea for this method after using the original PACT and past method for brain clearing. Visual demonstration of this method is critical as psPACT and mPACT method are difficult to run because this method has multiple stages of sample preparation. Begin by preparing PACT cocktail solution by adding VA-044 powder to a solution of 4%PFA and 4%acrylamide at a final concentration of 0.25%After perfusing the tissues with PACT solution, completely immerse the whole brain and spinal cord into a 50-milliliter tube containing chilled PACT solution, and store at four degrees Celsius for 24 hours.
24 hours later, transfer the tissue to a 50-milliliter tube containing fresh, cold PACT solution, and connect the tube cap to a tissue gel hybridization system, connected to a nitrogen tank and set to 37 degrees Celsius. Then, turn on the vacuum and gas for 10 minutes to embed the sample in nitrogen gas. To polymerize the hydrogel, place the tube containing the sample in a shaking incubator set to 150 rpm and 37 degrees Celsius for three hours or until polymerization is complete.
Following the incubation, use blotting paper to remove the remaining polymerized hydrogel surrounding the tissues. Then, transfer the tissue to a 50-milliliter tube containing clearing solution. Place the sample in a shaking incubator set to 37 degrees Celsius and 150 rpm until the tissue has been cleared.
On average, it takes about 20 days for the mouse CNS to achieve full clarity. Begin psPACT with the CNS from a mouse perfused with 4%PFA that has been post-fixed in 4%PFA at four degrees Celsius for 24 hours. Wash the fixed tissue for one hour in 0.1 molar PBS.
After washing, transfer the tissue to A4P0 solution, and incubate at 37 degrees Celsius for 24 hours. The next day, after washing the tissue in PBS for five minutes, immerse the tissue in 0.25%VA-044 in PBS, and incubate at 37 degrees Celsius for six to 24 hours. Separating the acrylamide and subsequent VA-044 step avoids the need for removing remaining unpolymerized hydrogel monomers that surround the tissue, which can compromise final tissue integrity after achieving transparency with PACT.
Following the incubation, transfer the tissues to fresh, cold VA-044 solution. Then, embed the sample in nitrogen gas for 10 minutes. Transfer the tissue to clearing solution.
Then, incubate the sample in a shaking incubator set to 37 degrees Celsius and 150 rpm until the tissue has been cleared. On average, it takes approximately 17 days for mouse CNS to achieve full clarity. For mPACT, follow the protocol for psPACT until the clearing solution step.
Then, fully immerse the tissue clearing solution containing 0.5%alpha-thioglycerol, an un-browning agent, in addition to 8%SDS in 0.1 molar PBS. Place the sample in a shaking incubator set to 37 degrees Celsius and 150 rpm until the tissue has been cleared. On average, it takes only two weeks for the mouse CNS to achieve full clarity using mPACT.
In the present study, mouse brain samples treated via the mPACT protocol achieved optical transparency after 14 days, ahead of samples treated via the PACT and psPACT protocols. Rat whole CNS samples cleared by mPACT achieved maximal clearance in 20 days. Adult rat brains alone, as opposed to the entire CNS, were cleared with mPACT in a mere five days.
Once mastered, this technique can be done in two weeks with the mouse whole CNS if it is performed properly. After its development, this technique paved the way for researchers in the field of neuroscience and anatomy to explore pathophysiologic disease, such as tissue injury, developmental malformations, cancer, and neurodegenerative disease in mammalian CNS models.
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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
Executive Industry Relevance
Novel passive clearing methods like psPACT and mPACT address a critical bottleneck in neuroscience R&D by enabling rapid, high-fidelity optical transparency of whole CNS tissue without electrophoretic damage. This capability supports target validation and mechanistic de-risking in neurodegenerative disease models by preserving subcellular architecture while accelerating imaging timelines. The techniques enhance predictive confidence in preclinical studies of Alzheimer's, tumorigenesis, and injury models through improved visualization of neuropathological phenotypes.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses in intact CNS models by preserving subcellular structures and cell circuitry for target engagement studies.
- Operational Value: Reduces tissue processing artifacts associated with electrophoretic clearing, improving reproducibility of phenotypic screening outcomes.
- Predictive Value: Supports portfolio triage by providing clearer readouts of neurodegenerative progression in disease-relevant systems.
Screening & Assay Development
- Scientific Value: Generates standardized, transparent CNS preparations suitable for high-resolution imaging of subcellular biomarkers and pathway modulation.
- Operational Value: Eliminates need for specialized electrophoretic equipment, lowering barriers to assay implementation across discovery teams.
- Scalability: mPACT achieves mouse CNS clarity in ~14 days, enabling predictable timelines for compound screening campaigns.
Translational & Preclinical Research
- Translational Continuity: Facilitates seamless transition from discovery to preclinical validation by maintaining tissue integrity for longitudinal studies of tumorigenesis and developmental malformations.
- Mechanistic De-risking: Allows visualization of subcellular structures in pathological tissues, reducing ambiguity in target mechanism interpretation.
- Disease Model Fidelity: Supports consistent CNS clearing across rodent species (mouse/rat), enhancing cross-study comparability for safety pharmacology.
Pipeline & Workflow Integration
psPACT and mPACT fit within the early discovery to preclinical continuum, specifically supporting target validation through lead optimization by providing quantifiable, imaging-ready CNS models.
- Discovery Biology: Enables hypothesis testing via 3D mapping of protein expression and cell distribution in intact tissue, clarifying pathway involvement in disease models.
- Screening: Delivers reproducible, quantitative tissue preparations that support standardized evaluation of compound effects on CNS architecture.
- Analytics: Generates high-resolution imaging outputs that allow teams to compare structural changes across treatment conditions with improved signal-to-noise.
- Translational Research: Maintains proteomic and structural continuity from discovery through preclinical stages, supporting biomarker alignment in neurodegeneration and injury models.
- Enterprise Reuse: Establishes a reusable CNS clearing platform applicable across multiple projects studying neuropathology, reducing redundant method development.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity through intact tissue visualization.
- Operational Value: Enhances standardization and reproducibility by avoiding variable electrophoretic parameters and equipment dependencies.
- Strategic Value: Improves go/no-go decision efficiency by accelerating preclinical data generation for CNS-targeted candidates.
- Portfolio Impact: Enables risk-adjusted prioritization of neurodegeneration and oncology programs through faster, clearer phenotypic readouts.
Implementation Considerations
- Requires expertise in tissue histology, hydrogel chemistry, and incubation protocol execution to manage multi-stage sample preparation.
- Dependent on shaking incubators, nitrogen gas systems, and temperature-controlled environments for hydrogel polymerization and clearing phases.
- Necessitates cross-team standardization of solution preparation (e.g., VA-044, SDS concentrations) and timing controls to ensure inter-lab reproducibility.
- Adaptation to non-CNS tissues (e.g., kidney, spleen) requires validation of clearing times and structural preservation, as noted in source material.
- Practical limitation: Protocol success hinges on meticulous execution due to multiple preparation stages, with deviations risking incomplete clearing or tissue damage.
