Name: novel passive clearing methods for the rapid production of optical transparency in whole cns tissue
Uploaded: 2018-05-08T14:00:00
Description: Watch this Scientific Journal Video about Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue at JoVE.com

This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University. In addition, this work was supported by a grant from the National Research Foundation of Korea (NRF-2017R1D1A1B03030315).

All procedures have been approved by the appropriate research ethics committee at Yonsei University College of Medicine. All experimental animals are sacrificed in accordance with the guidelines of the laboratory animal care committee at Yonsei University College of Medicine.
1. Preparation of Reagents
Caution: Paraformaldehyde (PFA), acrylamide and sodium dodecyl sulfate (SDS) are toxic irritants and thus should be handled in a fume hood with appropriate personal pro...

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While the passive, non-electrophoretic extraction methods employed in PACT significantly improved the consistency achieved with previous tissue clearing methods such as CLARITY2,3,4,7,8, the technique still bears several shortcomings, the most pressing of which is the length of time required to achieve maximal tissue clarity12. In the c...

A fundamental objective of scientific and clinical inquiry involves attaining a complete understanding of organ structure and function; however, the exceedingly complex nature of mammalian organs often serves as a barrier to fully achieving this aim1. CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tisssue-hYdrogel)2,3,4, which involves building an acrylamide-based hydrogel hybrid from intact tissues, achieves optical clearance of a variety of organs, including the brain, liver, and spleen, while preserving their structural integrity5. CLARITY has thus enabled not only visualization but also the opportunity to finely dissect complex cellular networks and tissue morphologies without the need for sectioning.
In order to achieve tissue clearance, CLARITY employs electrophoretic methods to remove the lipid content of the sample at hand. While CLARITY has been noted for producing physically stable tissue-hydrogel hybrids, studies have shown that its use of electrophoretic tissue clearing (ETC) methods yields variable results in terms of tissue quality, including browning, epitope damage, and protein loss5,6. To address these issues, modified protocols such as PACT (PAssive Clarity Technique), which replaces the ETC treatment with a passive, ionic-detergent based delipidation technique, have been developed7,8,9. Despite achieving a greater consistency in results, however, PACT requires more time to obtain maximal clearance. Furthermore, none of these techniques have yet been applied to the whole CNS form, or in larger rodent models such as rats and guinea pigs.
The present study seeks to address these limitations by proposing novel methodologies, psPACT (process-separate PACT) and mPACT (modified PACT), for facilitating the fast clearance of the whole CNS and internal organs in both mouse and rat models10. Specifically, psPACT processes tissues in 4% acrylamide and 0.25% VA-044 in two separate steps during hydrogel formation; mPACT essentially involves the same steps, but supplements the SDS-based clearing solution with 0.5% &#945;-thioglycerol as a key reagent. Both techniques harness the endogenous systemic and cerebrospinal circulatory systems to significantly reduce the time needed to produce optical clearance. As a proof of principle, we demonstrate the use of confocal microscopy to analyze blood vessel patterns in the cleared tissues10.

<table>
	<tbody>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Affymetrix, Inc.</td>
			<td>75819</td>
			<td>Clearing solution</td>
		</tr>
		<tr>
			<td>Nycodenz</td>
			<td>Axia-Shield</td>
			<td>1002424</td>
			<td>nRIMS solution</td>
		</tr>
		<tr>
			<td>40% Acrylamide Solution</td>
			<td>Bio Rad Laboratories, Inc.</td>
			<td>161-0140</td>
			<td>Polymerization (A4P0)</td>
		</tr>
		<tr>
			<td>2,2&#180;-Azobis[2-(2-imidazolin-2-yl)propane] Dihydrochloride</td>
			<td>Wako Pure Chemical Industries, Ltd.</td>
			<td>017-19362</td>
			<td>Polymerization (VA-044)</td>
		</tr>
		<tr>
			<td>1-Thioglycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M1753-100ML</td>
			<td>Clearing solution (mPACT)</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Georgiachem</td>
			<td>9005-64-5</td>
			<td>nRIMS solution</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ML</td>
			<td>Immuno Staining</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Bovogen</td>
			<td>BSA100</td>
			<td>Immuno Staining</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck Millipore</td>
			<td>375095</td>
			<td>Perfusion (PBS)</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-25G</td>
			<td>nRIMS solution</td>
		</tr>
		<tr>
			<td>PECAM-CD31 antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-28188</td>
			<td>Immuno Staining</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-IgG Cy3 fluorescent conjugate</td>
			<td>Jackson ImmunoResearch Inc.</td>
			<td>111-165-003</td>
			<td>Immuno Staining</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Tech &#38; Innovation</td>
			<td>BPP-9004</td>
			<td>Perfusion, Polymerization</td>
		</tr>
		<tr>
			<td>20X Phosphate Buffered Saline (pH 7.4)</td>
			<td>Tech &#38; Innovation</td>
			<td>BPB-9121</td>
			<td>Perfusion, Buffer</td>
		</tr>
		<tr>
			<td>10 mL stripette</td>
			<td>Coatar</td>
			<td>4488</td>
			<td>Solution transfer</td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>Clearing tube</td>
		</tr>
		<tr>
			<td>35 mm Cell culture dish</td>
			<td>SPL</td>
			<td>20035</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Confocal dish</td>
			<td>SPL</td>
			<td>211350</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Korea vaccine Co., Ltd</td>
			<td>26G 1/2</td>
			<td>Anesthetize&#160;</td>
		</tr>
		<tr>
			<td>50 mL syringe</td>
			<td>Korea vaccine Co., Ltd</td>
			<td>21G1 1/4</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A3553</td>
			<td>Polymerization (A4P0)</td>
		</tr>
		<tr>
			<td>Whatman 3MM paper</td>
			<td>Sigma-Aldrich</td>
			<td>Z270849</td>
			<td>Blotting paper for gel removal</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM780</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>ZEN lite Software</td>
			<td>Zeiss</td>
			<td>ZEN 2012</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Longerpump</td>
			<td>BT100-1F</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>EasyGel</td>
			<td>Lifecanvas Technologies</td>
			<td>EasyGel</td>
			<td>Tissue gel hybridization system</td>
		</tr>
	</tbody>
</table>

novel passive clearing methods for the rapid production of optical transparency in whole cns tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Generation of a transparent model of the whole CNS using optimized passive clearing techniques
Optical clearance of mouse and rat whole CNS tissues was rapidly achieved using various passive clearing techniques (Figure 1). A schematic of tissue clearing over time is shown in Figure 2A. Unlike the original PACT method, psPACT (process-separate PACT) invo...

Watch this Scientific Journal Video about Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue at JoVE.com

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

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.

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

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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