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/pubmed?term=((Structural+changes+at+synapses+after+delayed+perfusion+fixation+in+different+regions+of+the+mouse+brain%5BTitle%5D)+AND+%22Journal+of+Comparative+Neurology%22%5BJournal%5D)">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/pubmed?term=((Whole+animal+perfusion+fixation+for+rodents%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">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/pubmed?term=((Perfusion+fixation+in+brain+banking%3A+a+systematic+review%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">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/pubmed?term=((Fixation%2C+fine+structure%2C+and+immunostaining+for+neuropeptides%3A+perfusion+versus+immersion+of+the+neuroendocrine+hypothalamus%5BTitle%5D)+AND+%22Journal+of+Histochemistry+and+Cytochemistry%22%5BJournal%5D)">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/pubmed?term=((Use+of+perfusion+fixation+for+improved+neuropathologic+examination%5BTitle%5D)+AND+%22Archives+of+Pathology+and+Laboratory+Medicine%22%5BJournal%5D)">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/pubmed?term=((Neuronal+alpha-synucleinopathy+with+severe+movement+disorder+in+mice+expressing+A53T+human+alpha-synuclein%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Parkinson%27s+disease+alpha-synuclein+transgenic+mice+develop+neuronal+mitochondrial+degeneration+and+cell+death%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((alpha-Synuclein+is+phosphorylated+in+synucleinopathy+lesions%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Parkinson%27s+disease%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">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/pubmed?term=((Phosphorylation+of+Ser-129+is+the+dominant+pathological+modification+of+alpha-synuclein+in+familial+and+sporadic+Lewy+body+disease%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Hyperphosphorylation+and+insolubility+of+alpha-synuclein+in+transgenic+mouse+oligodendrocytes%5BTitle%5D)+AND+%22EMBO+Reports%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+arterial+blood+pressure+in+different+strains+of+mice%5BTitle%5D)+AND+%22American+Journal+of+Hypertension%22%5BJournal%5D)">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><tbody><tr><td>2-Methylbutane (Certified ACS), Fisher Chemical</td><td>Fisher Scientific</td><td>03551-4</td><td></td></tr><tr><td>Andwin Scientific Tissue-Tek O.C.T Compound</td><td>Fisher Scientific</td><td>NC1029572</td><td></td></tr><tr><td>Artman Instruments 4.5&quot; Straight Castroveijo Spring Action Scissors</td><td>Amazon</td><td>B0752XHK2X</td><td>&quot;fine scissors&quot;</td></tr><tr><td>BD General Use and PrecisionGlide Hypodermic Needles, 18 G</td><td>Fisher Scientific</td><td>14-826-5D</td><td></td></tr><tr><td>BD General Use and PrecisionGlide Hypodermic Needles, 22 G</td><td>Fisher Scientific</td><td>14-826B</td><td></td></tr><tr><td>Corning PES Syringe Filters</td><td>Fisher Scientific</td><td>09-754-29</td><td></td></tr><tr><td>Cytiva HyClone Phosphate Buffered Saline (PBS), 10x</td><td>Fisher Scientific</td><td>SH30258</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Fisher BioReagents Bovine Serum Albumin (BSA) Protease-free Powder</td><td>Fisher Scientific</td><td>BP9703100</td><td></td></tr><tr><td>Fisherbrand Curved Medium Point General Purpose Forceps</td><td>Fisher Scientific</td><td>16-100-110</td><td>&quot;curved fenestrated forceps&quot;</td></tr><tr><td>Fisherbrand Curved Very Fine Precision Tip Forceps</td><td>Fisher Scientific</td><td>16-100-123</td><td>&quot;curved fine forceps&quot;</td></tr><tr><td>Fisherbrand Disposable Graduated Transfer Pipettes</td><td>Fisher Scientific</td><td>13-711-9AM</td><td></td></tr><tr><td>Fisherbrand High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td><td>Fisher Scientific</td><td>12-000-122</td><td>&quot;straight fine forceps&quot;</td></tr><tr><td>Fisherbrand Micro Spatulas with Rounded Ends</td><td>Fisher Scientific</td><td>21-401-5</td><td></td></tr><tr><td>Fisherbrand Porcelain Buchner Funnels with Fixed Perforated Plates</td><td>Fisher Scientific</td><td>FB966J</td><td></td></tr><tr><td>Fisherbrand Premium Cover Glass, 24 x 50</td><td>Fisher Scientific</td><td>12-548-5M</td><td></td></tr><tr><td>Fisherbrand Premium Tissue Forceps 1X2 Teeth 5 in. German Steel</td><td>Fisher Scientific</td><td>13-820-074</td><td>&quot;skin forceps&quot;</td></tr><tr><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>&quot;dissecting scissors&quot;</td></tr><tr><td>Fisherbrand Sterile Syringes for Single Use</td><td>Fisher Scientific</td><td>14-955-464</td><td></td></tr><tr><td>Fisherbrand Straight Locking Hemostats</td><td>Fisher Scientific</td><td>16-100-115</td><td></td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Fisherbrand Vinyl Tubing and Connector Kits, 1/4 in.</td><td>Fisher Scientific</td><td>14-174-1C</td><td></td></tr><tr><td>Fisherbrand Wet-Strengthened Qualitative Filter Paper Circles</td><td>Fisher Scientific</td><td>09-790-12F</td><td></td></tr><tr><td>Fisherbrand Y Connector with 1/4 in. ID - Polypropylene - QC</td><td>Fisher Scientific</td><td>01-000-686</td><td></td></tr><tr><td>Garvey Economy Single Edge Cutter Blade</td><td>Amazon</td><td>B001GXFAEQ</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 488</td><td>ThermoFisher Scientific</td><td>35552</td><td></td></tr><tr><td>Ideal Clamp Stant High-Pressure Clamps</td><td>Fisher Scientific</td><td>14-198-5A</td><td>&quot;hose clamps&quot;</td></tr><tr><td>IMEB, Inc Sakura Accu-Edge Low Profile Microtome Blades, Dispoisable</td><td>Fisher Scientific</td><td>NC9822467</td><td></td></tr><tr><td>Kawasumi 25 Gauge Standard Winged Blood Collection Set</td><td>Fisher Scientific</td><td>22-010-137</td><td>&quot;butterfly needle&quot;</td></tr><tr><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>&quot;buffer bottles&quot;</td></tr><tr><td>PAP pen</td><td>abcam</td><td>ab2601</td><td></td></tr><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></tr><tr><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></tr><tr><td>Triton X-100</td><td>Us Biological</td><td>T8655</td><td></td></tr><tr><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 α-synuclein in the midbrain and lumbar spinal cord of a 15-month-old mouse and a 7 month-old-mouse expressing human A53T α-synuclein (Figure 6).
The A53T mutation is overrepresented in patients with autosomal dominant Parkinson's disease (PD). Furthermore, human α-synuclein with the A53T mutation can recapitulate many of the features of human PD when expressed in mice6,7. Phosphorylation of α-synuclein at the Serine 129 residue has been shown in vivo and in vitro to induce α-synuclein aggregation8. Lewey bodies are the classical histologic finding present in patients with PD or Lewey body dementia9. A majority of the α-synuclein present in Lewey bodies is phosphorylated at Serine 12910,11. As a result, the accumulation of phosphorylated α-synuclein is used as a marker of the histologic severity of PD pathology. The present study finds that phosphorylated α-synuclein accumulates at a significantly higher level in 15.5-month-old symptomatic mice relative to 7-month-old asymptomatic mice expressing human A53T α-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="Midbrain, lumbar spine sections with α-syn-A53T staining, immunofluorescence, microscopy, PS129, DAPI." class="xfigimg" src="/files/ftp_upload/63386/63386fig06.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Label for phosphorylated α-synuclein in midbrain and lumbar spinal cord tissues from a mouse model of Parkinson'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 α-synuclein that induces Parkinson's-like pathology. Scale bars = 100 µm (upper panels) and 50 µm (lower panels). Abbreviation: α syn-A53T = A53T mutant of α-synuclein; DAPI = 4',6-diamidino-2-phenylindole; PS129 = phosphorylated Serine 129 of α-synuclein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.7K Views.  State University of New York Upstate Medical University. Dit protocol biedt een eenvoudige methode van fixatieve perfusie bij muizen die de histologische studie van CZS-weefsels mogelijk maakt zonder aanzienlijke investeringen. Het belangrijkste voordeel van de hier gepresenteerde zwaartekrachtperfusiemethode is dat het profusieapparaat kan worden gebouwd met gemakkelijk beschikbare componenten. De procedure wordt gedemonstreerd door Arnav Rana, een MD-PhD-student van het laboratorium van Dr.Xin Jie Chen.Om te beginnen, met behulp van een sc...

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

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Developmental Biology

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Immunology and Infection

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Medicine

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

Mouse Brain Histone Modification Quantification Using Flow Cytometry

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Author Spotlight: Enhancements in Gene Expression Regulation Research

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

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

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