The authors thank Xiaowen Wang, Liam Coyne, and Jason Grullon for their assistance in developing this protocol. This work was supported by the NIH grants AG061204 and AG063499.

<ol>
	<li>Tao-Cheng, J. H., Gallant, P. E., Brightman, M. W., Dosemeci, A., Reese, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+at+synapses+after+delayed+perfusion+fixation+in+different+regions+of+the+mouse+brain.">Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain.</a> Journal of Comparative Neurology. 501 (5), 731-740 (2007).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments: JoVE. (65), e3564 (2012).</li><li>McFadden, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+fixation+in+brain+banking:+a+systematic+review.">Perfusion fixation in brain banking: a systematic review.</a> Acta Neuropathologica Communications. 7 (1), 146 (2019).</li><li>Lamberts, R., Goldsmith, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation,+fine+structure,+and+immunostaining+for+neuropeptides:+perfusion+versus+immersion+of+the+neuroendocrine+hypothalamus.">Fixation, fine structure, and immunostaining for neuropeptides: perfusion versus immersion of the neuroendocrine hypothalamus.</a> Journal of Histochemistry and Cytochemistry. 34 (3), 389-398 (1986).</li><li>Adickes, E. D., Folkerth, R. D., Sims, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+perfusion+fixation+for+improved+neuropathologic+examination.">Use of perfusion fixation for improved neuropathologic examination.</a> Archives of Pathology and Laboratory Medicine. 121 (11), 1199-1206 (1997).</li><li>Giasson, B. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+alpha-synucleinopathy+with+severe+movement+disorder+in+mice+expressing+A53T+human+alpha-synuclein.">Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein.</a> Neuron. 34 (4), 521-533 (2002).</li><li>Martin, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease+alpha-synuclein+transgenic+mice+develop+neuronal+mitochondrial+degeneration+and+cell+death.">Parkinson's disease alpha-synuclein transgenic mice develop neuronal mitochondrial degeneration and cell death.</a> Journal of Neuroscience. 26 (1), 41-50 (2006).</li><li>Fujiwara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Synuclein+is+phosphorylated+in+synucleinopathy+lesions.">alpha-Synuclein is phosphorylated in synucleinopathy lesions.</a> Nature Cell Biology. 4 (2), 160-164 (2002).</li><li>Kalia, L. V., Lang, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease.">Parkinson's disease.</a> Lancet. 386 (9996), 896-912 (2015).</li><li>Anderson, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Ser-129+is+the+dominant+pathological+modification+of+alpha-synuclein+in+familial+and+sporadic+Lewy+body+disease.">Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease.</a> Journal of Biological Chemistry. 281 (40), 29739-29752 (2006).</li><li>Kahle, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperphosphorylation+and+insolubility+of+alpha-synuclein+in+transgenic+mouse+oligodendrocytes.">Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes.</a> EMBO Reports. 3 (6), 583-588 (2002).</li><li>Mattson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arterial+blood+pressure+in+different+strains+of+mice.">Comparison of arterial blood pressure in different strains of mice.</a> American Journal of Hypertension. 14, 405-408 (2001).</li></ol>

The authors declare no competing financial interests.

This work describes the critical steps to performing transcardial perfusion. When constructing the perfusion apparatus (protocol section 1), it is important to use tubing that is flexible enough to be completely occluded by a hemostat. Some stiff tubing may not be sufficiently occluded by a hemostat and may still allow PFA to leak into the main line during the initial PBS perfusion. When preparing the 4% PFA solution, it is important to ensure that the pH is physiologic (7.4). As preparation of the PFA solution involves heating it to 65 &#176;C, the solution must be cooled back down to 25 &#176;C prior to measuring the pH as this is the temperature at which pH is calibrated on the meter.
When making the initial incision into the abdomen, care must be taken to avoid laceration of the abdominal organs (protocol section 2). When dissecting superiorly towards the diaphragm, it is important to avoid laceration of the liver as it is common for the liver to be adherent with the anterior abdominal wall. To overcome this, the liver is carefully and bluntly dissected away from the anterior wall before continuing an incision towards the diaphragm. When entering the thoracic cavity through the diaphragm, it is important to avoid laceration of the heart, great vessels, and the lung. To avoid this, the scissor tip is kept superficially and at an acute angle with the ribcage.
The initial dissection to expose the heart takes approximately 2 min from the initial incision. It is expected that during this time, some air has entered the tip of the butterfly needle. The introduction of this air into the circulation of the mouse will yield poor-quality perfusion. Therefore, it is critical that the main line is opened and PBS is flushed through the needle immediately prior to cannulation of the heart to remove air bubbles. Ideally, the heart is cannulated while a small PBS trickle flows through the needle tip to ensure the complete absence of air when puncturing the LV.
When the needle enters the LV, it must not go so deep as to introduce the needle tip into the right ventricle (RV). Placement of the needle in either the RV or beyond the mitral valve will result in immediate &#34;inflation&#34; of the lungs when perfusion is started. This is undesirable, and the needle must be withdrawn slightly to ensure LV placement. If the needle is placed properly, the lungs will remain flat throughout perfusion. When perfusion is initiated, it is sometimes observed that a clear liquid is emerging from the open mouth of the animal. This is usually due to a perfusion pressure that is too high or due to misplacement of the needle within the heart. The authors speculate that elevated perfusion pressures result in extravasation of the perfusate from the arteriolar capillary bed and retrograde flow of PBS via the bronchial tree into the esophagus and oral cavity.
The perfusion pressure must be lowered by either decreasing the level of PBS in the PBS bottle or by lowering the height of the PBS bottle. Alternatively, if the needle is placed too deeply into the left ventricle, it may travel through the mitral valve and deliver perfusate to the left atrium. This may result in retrograde flow through the pulmonary veins and extravasation of perfusate into the arterioles, as described above. Thorough clearance of blood from the circulatory system with PBS is especially important to avoid fixative-induced cross-linking of blood components resulting in vessel occlusion upon subsequent fixative perfusion. Clearance is effectively assessed by a color change of the liver and PBS flow from an incision in the ventral tail base. Blood clearance is generally complete by 3 min of perfusion with PBS; however, if visual signs of clearance occur at shorter times, then fixative is introduced sooner than 3 min. Longer clearance times are not recommended as delayed fixative perfusion leads to artifacts in CNS fine structure1.
When PFA is being administered, it is important to monitor the level of PFA solution in the PFA bottle. Fill up the PFA bottle if the level of PFA drops to less than 4 cm above the mouth of the PFA bottle. After perfusion has been completed, the perfusion apparatus must be thoroughly rinsed with distilled water. This is important as residual PFA in the main line will contaminate&#160;the initial PBS perfusion with PFA and result in poor-quality perfusion. Finally, 25 G butterfly needles are generally recommended for average-sized adult mice in the 20-30 g range. However, larger or smaller mice may require slightly larger or smaller gauge needles in addition to the adjustment of the fixative bottles to provide optimal flow rates.
For CNS dissection and OCT embedding (protocol section 3), it is common for spinal cord tissues to not completely sink in 30% sucrose. These tissues are therefore left in sucrose for 2 days and then embedded in OCT, regardless of whether they sink or not. When freezing down tissues in OCT, it is possible that certain tissues may crack when placed in cooled 2-methylbutane. This is more common with the brain and usually occurs when too much OCT is placed on the tissue. To avoid this, place only enough OCT to cover the tissue surfaces prior to immediate freezing. In some protocols, cracking is less common despite complete immersion in OCT. This is usually due to a slower freezing method such as when using dry-ice-cooled 2-methylbutane or placing the cryomold on a block of dry ice directly. Liquid-nitrogen-cooled 2-methylbutane is preferred in this work as the rate of freezing is substantially more rapid and may better preserve tissue morphology than slower freezing methods.
When cryosectioning the tissue (protocol section 4), it is important to avoid multiple freeze-thaw cycles. Therefore, it is optimal to cut all sections from a single OCT block to obtain a specific brain region for analysis instead of thawing and refreezing selected areas. If this is not viable, after obtaining a few sections, users may refreeze and store the OCT blocks in the -80 &#176;C deep freezer 1-2 more times for future use.
The major benefits of this method over more traditional pump or air pressure delivery of perfusate are as follows: (1) low cost and accessibility of the perfusion apparatus. (2) Users do not need to manually maintain pressure in the perfusion apparatus throughout the perfusion. (3) Lower and more consistent perfusion pressure than other low-cost alternatives for perfusion such as via syringe delivery. Using Bernoulli&#39;s equation, it is calculated that the gravity-fed perfusion apparatus constructed here will maintain a perfusion pressure of approximately 73 mm Hg when the perfusate bottles are placed at 1 m of elevation relative to the needle. Given that this is significantly below the systolic blood pressure of these animals, this perfusion pressure is likely sufficiently low to avoid vascular rupture12.
The authors have thus far successfully used this perfusion system to detect the presence of phosphorylated &#945;-synuclein in a mouse model of Parkinson&#39;s disease. During this time, significant limitations with this perfusion method have not been encountered that are not present with a pump perfusion delivery method. The major limitation of this technique is the time-consuming nature of perfusion versus drop fixation. This technique is preferable to drop fixation as perfusion results in the deeper penetration of fixative to the CNS structures. A second limitation of this technique is that it requires some surgical skill to perform, as the heart must be cannulated quickly following entrance into the thoracic cavity. However, with experience, trained users can routinely canulate the heart within 1 min of the initial incision into the abdomen.

Histologic characterization of pathology in the mouse central nervous system (CNS) is a routine technique used in studies of neurodegeneration. As neuronal tissues rapidly degrade after death, it is common practice to deliver a chemical fixative such as paraformaldehyde to the CNS tissues to preserve their morphology1,2. Chemical fixation can be performed either through whole-body perfusion with a fixative solution or through the isolation of tissues and their immersion in a fixative solution (termed &#34;drop fixation&#34;). Perfusion is the preferred method of fixative delivery, as drop fixation may not allow rapid penetration of the fixative solution into deep CNS structures3,4,5. Furthermore, as it is difficult to remove the unfixed spinal cord from the vertebral column, delivery of the fixative solution via perfusion allows for the in situ preservation of spinal cord microscopic and gross anatomy and stiffens the tissue to minimize damage during removal.
Delivery of the buffer and fixative solutions necessary for fixation is commonly performed using commercially available pumps or air pressure. Gravity delivery of perfusate may serve as an alternative to pump delivery for the following reasons: (1) Pump or air pressure delivery may in some cases require a user to manually maintain pressure in the system throughout the perfusion. Gravity delivery of perfusate can be maintained without user intervention. (2) A gravity-delivered perfusate apparatus can be constructed at low cost to the user by obtaining materials available&#160;from standard scientific vendors. This work describes how to construct a simple gravity perfusion device using wash bottles and vinyl tubing. Using a mouse model of Parkinson&#39;s disease, this work demonstrates the efficacy of this system in perfusing the brain and spinal cord tissues prior to their isolation for frozen sectioning. This work comprehensively describes all steps, techniques, and materials needed to dissect the fixed tissue out of the animal, rapidly freeze/embed and cryosection the tissue, and detect the presence of phosphorylated &#945;-synuclein in both the brain and spinal cord via indirect immunofluorescence microscopy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Methylbutane (Certified ACS), Fisher Chemical</td>
			<td>Fisher Scientific</td>
			<td>03551-4</td>
			<td></td>
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			<td>Andwin Scientific Tissue-Tek O.C.T Compound</td>
			<td>Fisher Scientific</td>
			<td>NC1029572</td>
			<td></td>
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			<td>Artman Instruments 4.5&#34; Straight Castroveijo Spring Action Scissors</td>
			<td>Amazon</td>
			<td>B0752XHK2X</td>
			<td>&#34;fine scissors&#34;</td>
		</tr>
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			<td>BD General Use and PrecisionGlide Hypodermic Needles, 18 G</td>
			<td>Fisher Scientific</td>
			<td>14-826-5D</td>
			<td></td>
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			<td>BD General Use and PrecisionGlide Hypodermic Needles, 22 G</td>
			<td>Fisher Scientific</td>
			<td>14-826B</td>
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			<td>Corning PES Syringe Filters</td>
			<td>Fisher Scientific</td>
			<td>09-754-29</td>
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			<td>Cytiva HyClone Phosphate Buffered Saline (PBS), 10x</td>
			<td>Fisher Scientific</td>
			<td>SH30258</td>
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			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
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			<td>Fisher BioReagents Bovine Serum Albumin (BSA) Protease-free Powder</td>
			<td>Fisher Scientific</td>
			<td>BP9703100</td>
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			<td>Fisherbrand Curved Medium Point General Purpose Forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-110</td>
			<td>&#34;curved fenestrated forceps&#34;</td>
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			<td>Fisherbrand Curved Very Fine Precision Tip Forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-123</td>
			<td>&#34;curved fine forceps&#34;</td>
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			<td>Fisherbrand Disposable Graduated Transfer Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
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			<td>Fisherbrand High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-122</td>
			<td>&#34;straight fine forceps&#34;</td>
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			<td>Fisherbrand Micro Spatulas with Rounded Ends</td>
			<td>Fisher Scientific</td>
			<td>21-401-5</td>
			<td></td>
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			<td>Fisherbrand Porcelain Buchner Funnels with Fixed Perforated Plates</td>
			<td>Fisher Scientific</td>
			<td>FB966J</td>
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			<td>Fisherbrand Premium Cover Glass, 24 x 50</td>
			<td>Fisher Scientific</td>
			<td>12-548-5M</td>
			<td></td>
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			<td>Fisherbrand Premium Tissue Forceps 1X2 Teeth 5 in. German Steel</td>
			<td>Fisher Scientific</td>
			<td>13-820-074</td>
			<td>&#34;skin forceps&#34;</td>
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			<td>Fisherbrand Reusable Heavy-Wall Filter Flasks</td>
			<td>Fisher Scientific</td>
			<td>FB3002000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Standard Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-20</td>
			<td>&#34;dissecting scissors&#34;</td>
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			<td>Fisherbrand Sterile Syringes for Single Use</td>
			<td>Fisher Scientific</td>
			<td>14-955-464</td>
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			<td>Fisherbrand Straight Locking Hemostats</td>
			<td>Fisher Scientific</td>
			<td>16-100-115</td>
			<td></td>
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			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
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			<td>Fisherbrand Vinyl Tubing and Connector Kits, 1/4 in.</td>
			<td>Fisher Scientific</td>
			<td>14-174-1C</td>
			<td></td>
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			<td>Fisherbrand Wet-Strengthened Qualitative Filter Paper Circles</td>
			<td>Fisher Scientific</td>
			<td>09-790-12F</td>
			<td></td>
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			<td>Fisherbrand Y Connector with 1/4 in. ID - Polypropylene - QC</td>
			<td>Fisher Scientific</td>
			<td>01-000-686</td>
			<td></td>
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			<td>Garvey Economy Single Edge Cutter Blade</td>
			<td>Amazon</td>
			<td>B001GXFAEQ</td>
			<td></td>
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		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 488</td>
			<td>ThermoFisher Scientific</td>
			<td>35552</td>
			<td></td>
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			<td>Ideal Clamp Stant High-Pressure Clamps</td>
			<td>Fisher Scientific</td>
			<td>14-198-5A</td>
			<td>&#34;hose clamps&#34;</td>
		</tr>
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			<td>IMEB, Inc Sakura Accu-Edge Low Profile Microtome Blades, Dispoisable</td>
			<td>Fisher Scientific</td>
			<td>NC9822467</td>
			<td></td>
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			<td>Kawasumi 25 Gauge Standard Winged Blood Collection Set</td>
			<td>Fisher Scientific</td>
			<td>22-010-137</td>
			<td>&#34;butterfly needle&#34;</td>
		</tr>
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			<td>Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Probes ProLong Diamond Antifade Mountant with DAPI</td>
			<td>Fisher Scientific</td>
			<td>P36962</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Narrow-Mouth Right-to-Know LDPE Wash Bottles</td>
			<td>ThermoFisher Scientific</td>
			<td>2425-0506</td>
			<td>&#34;buffer bottles&#34;</td>
		</tr>
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			<td>PAP pen</td>
			<td>abcam</td>
			<td>ab2601</td>
			<td></td>
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		<tr>
			<td>Paraformaldehyde Granular</td>
			<td>Electron Microscopy Systems</td>
			<td>19210</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Glass Drying Dishes, 34.9 x 24.9 x 6 cm</td>
			<td>Fisher Scientific</td>
			<td>15-242D</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Anti-Alpha-synuclein (phospho S129) antibody [EP1536Y]</td>
			<td>abcam</td>
			<td>ab51253</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465-2.5kg</td>
			<td></td>
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			<td>Stainless Steel Drinking Cup 18-oz</td>
			<td>amazon</td>
			<td>B0039PPO9U</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose for Molecular Biology CAS: 57-50-1</td>
			<td>Us Biological</td>
			<td>S8010</td>
			<td></td>
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		<tr>
			<td>Triton X-100</td>
			<td>Us Biological</td>
			<td>T8655</td>
			<td></td>
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			<td>Vetone Fluriso Isoflurane USP</td>
			<td>MWI Animal Health</td>
			<td>502017</td>
			<td></td>
		</tr>
	</tbody>
</table>

a gravity-fed transcardial perfusion method for histologic analysis of the mouse central nervous system

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To maintain the morphology of these delicate tissues, it is routine to administer a chemical fixative such as paraformaldehyde via cannulation of the heart in anesthetized animals (transcardial perfusion). Transcardial perfusion of the mouse heart has traditionally relied on the use of peristaltic pumps or air pressure to deliver both the saline and fixative solutions necessary for this process. As an easily accessible alternative to these methods, this work demonstrates the use of a gravity-fed method of perfusate delivery that uses materials available in most hardware stores. 
To validate this new perfusion method, this work demonstrates all the subsequent steps necessary for the sensitive detection of phosphorylated &#945;-synuclein in both the brain and spinal cord. Included in these steps are the dissection of the fixed brain and spinal cord tissues, rapid freezing/embedding and cryosectioning of the tissues, and immunofluorescent staining. As this method results in whole-body delivery of the fixative, it may also be used to prepare other non-neuronal tissues for histologic analysis.

High-quality perfusion is indicated by the absence of blood in the liver, spinal cord, and deep CNS structures (Figure 4C and Figure 5G,H). Retained blood below the dura mater (for example, within the venous sinuses) or between the dura mater and the skull has not been problematic as this blood is not within the brain parenchyma. The blood seen in Figure 4A is located between the skull and the dura matter and is therefore not problematic or suggestive of poor-quality perfusion. The fresh brain and spinal cord are quite soft and easily damaged during handling. Adequately fixed tissues, by comparison, are firm. To assess the quality of tissue and the preservation of morphology of tissues by this perfusion method, this work demonstrates the detection of phosphorylated &#945;-synuclein in the midbrain and lumbar spinal cord of a 15-month-old mouse and a 7 month-old-mouse expressing human A53T &#945;-synuclein (Figure 6).
The A53T mutation is overrepresented in patients with autosomal dominant Parkinson&#39;s disease (PD). Furthermore, human &#945;-synuclein with the A53T mutation can recapitulate many of the features of human PD when expressed in mice6,7. Phosphorylation of &#945;-synuclein at the Serine 129 residue has been shown in vivo and in vitro to induce &#945;-synuclein aggregation8. Lewey bodies are the classical histologic finding present in patients with PD or Lewey body dementia9. A majority of the &#945;-synuclein present in Lewey bodies is phosphorylated at Serine 12910,11. As a result, the accumulation of phosphorylated &#945;-synuclein is used as a marker of the histologic severity of PD pathology. The present study finds that phosphorylated &#945;-synuclein accumulates at a significantly higher level in 15.5-month-old symptomatic mice relative to 7-month-old asymptomatic mice expressing human A53T &#945;-synuclein. This is consistent with reports describing an enrichment of cytopathology in the anterior horns of the spinal cord and the midbrain of these mice.6 From this, it is concluded that the simplified perfusion method described here provides high-quality fixation of CNS tissues for downstream histologic characterization.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63386/63386fig06.jpg" /> 
Figure 6: Label for phosphorylated &#945;-synuclein in midbrain and lumbar spinal cord tissues from a mouse model of Parkinson&#39;s Disease. The aged (15.5-month-old) end-stage paralyzed mouse is compared with the 7-month-old healthy mouse. Both mice express a misfolding prone mutant variant (A53T) of human &#945;-synuclein that induces Parkinson&#39;s-like pathology. Scale bars = 100 &#181;m (upper panels) and 50 &#181;m (lower panels). Abbreviation: &#945; syn-A53T = A53T mutant of &#945;-synuclein; DAPI = 4&#39;,6-diamidino-2-phenylindole; PS129 = phosphorylated Serine 129 of &#945;-synuclein. <a href="https://www.jove.com/files/ftp_upload/63386/63386fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System at JoVE.com

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

A convenient gravity-fed perfusion method for histological analysis of the mouse central nervous system is presented. Immunofluorescent detection of phosphorylated &#945;-synuclein is demonstrated in a mouse model of Parkinson's disease. This work also comprehensively describes the transcardial perfusion, dissection, tissue freezing/embedding, and frozen sectioning steps.

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To ...

This protocol provides a simple method of fixative perfusion in mice that will allow the histologic study of CNS tissues without significant investment. The main advantage of the gravity perfusion method presented here is that the profusion apparatus can be constructed with easily available components. Demonstrating the procedure will be Arnav Rana, an MD-PhD student from Dr.Xin Jie Chen's laboratory.To begin, using a sharp razor blade, trim the inner straws from two 500-milliliter wash bottles by cutting them flush to the inner side of the screw on the cap. Cut a four-by-four-centimeter square hole in the bottom of each buffer bottle, then cut another small hole in the bottom of each bottle to allow the passage of a bent stainless steel micro spatula. Next, create an S-shaped bend in the micros spatulas so that they may be used as hooks for hanging the buffer bottles.Insert the bent micro spatulas into the appropriate openings in the buffer bottles. Next, tightly connect two 25-centimeter-long tubes with the outer bottle straw outlets and join the ends of these tubes together with the Y connector, then join the 2-meter-long tubing to the free end of the Y connector. After removing the plunger from a 1-milliliter syringe, cut the syringe approximately 6 centimeters away from the tip by first scoring the plastic with a razor blade and then sharply snapping the syringe plastic.Insert the cut face of the syringe into the free end of the plastic tubing to a sufficient depth and ensure a tight seal. Next, label one buffer bottle as PFA and the other as PBS. Measure approximately 1/3 of the length away from the bottle opening and draw a line around the bottle circumference at this point to denote the appropriate filling level of the perfusate solutions.After straightening a large paperclip, create a loop that can fit around the circumference of the tubing and place this loop around the tubing, just proximal to the syringe, then place an appropriately-sized Styrofoam block in the glass tray, place the ends of the paperclip into the Styrofoam block to affix the tubing and using a paper towel, raise the front end of the glass tray by approximately 2 centimeters. After rinsing a 1-liter beaker with distilled water, fill it with approximately 800 milliliters of 18 milliohms molecular-biology-grade water. Heat the beaker in a microwave for three minutes or until the water temperature reaches 65 degrees Celsius, then place it on a heating or a stirring plate kept in a fume hood.Next, rinse a stirring rod with distilled water and place it in the beaker. Start the stirrer and turn the hot plate up to medium heat, ensuring that the water temperature does not go above 70 degrees Celsius. After wearing a surgical mask, gloves, and lab coat, measure 40 grams of PFA powder and add it into the heated water, then using a transfer pipette, add a few drops of 5-molar sodium hydroxide to the solution.Allow the powder to dissolve fully. If the powder has not fully dissolved, add more sodium hydroxide as needed. Once all the PFA has nearly dissolved, stop the stirring and heating and immediately add 100 milliliters of 10x PBS, then use the 18 milliohms molecular-biology-grade water to top up the beaker to the 1-liter mark.Cover the beaker with plastic wrap and place it in a minus 20-degrees Celsius freezer until the solution reaches room temperature. After calibrating a pH meter using appropriate standards, measure the pH of the solution while the beaker is on a stirring plate. Add hydrochloric acid until the pH reaches 7.4.If the pH is too low, add 5-molar sodium hydroxide to increase the pH. Next, connect a vacuum flask to vacuum and place a clean ceramic Buchner funnel with filter paper in the flask. After turning the vacuum on, wet the filter paper using a transfer pipette filled with the 4%PFA solution, then slowly pour the solution onto the filter paper until all the solution has been filtered.After filtering, store the solution in a light-protected container at 4 degrees Celsius until use. In the fume hood, place a Styrofoam dissecting block in a glass tray. Ensure that the dissecting block has five to six short needles to restrain the mouse during surgery and two long needles to support the perfusion tubing.Next, rinse the perfusion apparatus with distilled water. After all the water has drained, hang the profusion bottles one meter above the animal to be perfused, then prepare non-sterile 1x PBS using 10x PBS diluted with molecular-biology-grade-water. Clamp the main line of the perfusion apparatus using a hemostat, then clamp the PFA line with another hemostat.After filling the buffer container with PBS, place the syringe end of the perfusion apparatus in a beaker to collect waste buffer and remove the hemostat occluding the main line. While the PBS flows through the line, vigorously tap on the walls of the tube to remove trapped air. Once all the air has been removed from the buffer and main lines, occlude the flow by placing a hemostat on the main line.Next, remove the hemostat from the PFA line, allowing the PBS to flow in a retrograde manner up to the PFA bottle while tapping out any bubbles in the PFA line. Continue to allow the PBS into the PFA line until it can be seen just above the opening of the bottle, then occlude the PFA line with a hemostat to stop the flow of PBS into the PFA bottle. Next, connect the butterfly infusion needle to the perfusion syringe and open the main-line hemostat to flush PBS through the line and remove bubbles from the perfusion syringe.After the bubbles have been removed, close the main-line hemostat. Ensure that the PBS bottle is now approximately 1/3 full of PBS. If necessary, flush PBS through the main line or fill more PBS into the buffer bottle until 1/3 of its full capacity.Once all bubbles have been removed from the PBS, PFA, and main lines, fill the PFA bottle with 4%PFA solution at room temperature up to the black mark on the bottle. Next, prepare for non-survival surgery by cleaning the necessary instruments first with water and then with 70%ethanol. After securing an anesthetized C57 black 6J mouse to the Styrofoam block, begin the surgery by lifting the abdominal skin with skin forceps and cutting through the abdominal wall with large scissors.Continue the abdominal cut superiorly towards the liver, then detach the liver from the anterior abdominal wall and continue the initial incision superiorly towards the diaphragm. When the incision reaches the diaphragm, using fine-dissecting scissors, make a hole in the diaphragm and cut through the ribs on the mouse's right side approximately halfway to the right axilla, then make a similar, but longer incision through the left side of the diaphragm almost all the way to the left axilla. Reflect the chest wall and pin it to the Styrofoam block.After identifying the left ventricle, using fine-curved forceps, hold the heart steady with light pressure, then open the main-line hemostat to allow the PBS to flow through the needle and immediately pierce the left ventricle with the needle. Ensure that the needle is inserted no more than 0.5 centimeters into the ventricle. Next, rest the butterfly tubing on an X made in the Styrofoam with two large 18-gauge needles.Identify the inferior vena cava as it exits the liver and transect it using fine-dissecting scissors. Continue PBS perfusion until the liquid flowing out of the inferior vena cava is blood-free, then working quickly, occlude the PBS line with a hemostat and open the PFA line. Allow the PFA to perfuse for a few minutes until the tail starts to curl.Once the tail starts to curl, begin a 50-minute timer. After 50 minutes, occlude the main line with a hemostat and withdraw the needle from the left ventricle. Next, place the mouse in a labeled container of PFA solution at 4 degrees Celsius overnight.High-quality perfusion is indicated by the absence of blood in the liver, spinal cord and deep central nervous system structures. The blood observed in the brain is located between the skull and the dura mater and is therefore not problematic or suggestive of poor-quality perfusion. Detection of alpha-synuclein following this perfusion method shows that phosphorylated alpha-synuclein accumulates significantly higher in 15.5-month-old symptomatic mice relative to seven-month-old asymptomatic mice expressing human A53T alpha-synuclein.This result is consistent with reports describing an enrichment of cytopathology in the anterior horns of the spinal cord and the midbrain of these mice. It is important that no abdominal organs are cut when accessing the thoracic cavity. Furthermore, the perfusion needle must not be placed too deeply within the left ventricle.

a-gravity-fed-transcardial-perfusion-method-for-histologic-analysis

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

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