Name: Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice (Video) | JoVE Journal
Uploaded: 2020-08-13T12:30:00.000+00:00
Duration: 5 min 27 s
Description: Learn in situ closed circuit perfusion of lower abdominal organs and hind limbs in mice for studying drug delivery and immune interactions. This method enables targeted research without traditional ex-vivo limitations.

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5 minutes

The study was supported by the NIH grant CA194058 to DS, Skaggs School of Pharmacy ADR seed grant program (DS); National Natural Science Foundation of China (Grant No. 31771093), the Project of International Collaboration of Jilin Province (No.201180414085GH), the Fundamental&#8194;Research&#8194;Funds&#8194;for&#8194;the&#8194;Central&#8194;Universities, the Program for JLU Science and Technology Innovative Research Team (2017TD-27, 2019TD-36).

All methods described here have been approved by the University of Colorado&#8217;s Institutional Animal Care and Use Committee (IACUC).
1. Pre-heat the perfusion system
<ol>
	<li>Prepare the perfusion system before surgery by starting a 37 &#176;C circulating water bath for all water-jacketed components (perfusate reservoir, moist chamber, and lid) as shown in a customized configuration in Figure 1A. Make sure the tubing is clean and replace if necessary. To limit perfusate volume, use a bubble trap within a moist chamber as the perfusate reservoir (Figure 1B-6).</li>
</ol>
2. Vascular catheterization
<ol>
	<li>Induce anesthesia in an 8-10 week old BALB/c mouse using an isoflurane veterinary anesthesia machine with 3-5% isoflurane and oxygen flow rate at 0.3 L/min. As an alternative, use ketamine/xylazine or any other type of intraperitoneal anesthesia. Evaluate the depth of anesthesia by 2 methods: toe-pinch and corneal reflex.</li>
	<li>Prewet a 4-0 silk suture with needle in double distilled water.</li>
	<li>Place the anesthetized mouse in a supine position on a Styrofoam board with the head facing the surgeon and immobilize forelimbs and hind limbs with tape. Wipe the abdomen with isopropyl alcohol and cut the abdomen along the midline in a &#34;T&#34; shape with scissors. Stop bleeding around the edge of the incision by electrocoagulation (cauterizing).</li>
	<li>Push the stomach, jejunum and colon to the right side of the abdomen to reveal the abdominal aorta, vena cava, and common iliac and iliolumbar arteries and veins.</li>
	<li>Under a dissection microscope, find and ligate the iliolumbar artery/vein in the male, and ovarian artery/vein and the iliolumbar artery/vein in the female using 4-0 silk sutures (Figure 2 yellow lines).</li>
	<li>Under a dissection microscope, loop two 4-0 silk sutures underneath the abdominal aorta and inferior vena cava (about 1 cm above iliac artery and vein, 1 mm apart, Figure 2), and make a loose knot in the suture closest to the iliac vessels (Figure 2, white dotted line). Alternatively, a 6-0 silk suture can be used for this knot.</li>
	<li>Under a dissection microscope, horizontally align and stretch both the inferior vena cava and the abdominal aorta with porte-aiguille. Use a 24 G winged shielded I.V. catheter to puncture the abdominal aorta, push the button to retract the needle core and insert the catheter about 5 mm into the vessel.
	<ol>
		<li>Repeat the same procedure with the inferior vena cava and tie up knots of both sutures around the catheterized vessels. 
		NOTE: The needle can easily puncture through the blood vessel; therefore, keep the vessels stretched and needle parallel with the vessel. Retract the needle core as soon as the needle penetrates about 1 mm into the vessel. The abdominal aorta is underneath the inferior vena cava and much thinner and more elastic due to being encased in connective tissue. Therefore, the aorta can &#8220;hold on&#8221; to the catheter, and should thus be catheterized before the vena cava to reduce the likelihood of the catheter slipping out.</li>
	</ol>
	</li>
	<li>Apply instant glue to immobilize the catheters to the erector spinae, replace the abdominal organs, and end the surgery while maintaining anesthesia. 
	NOTE: Organs that cannot be completely replaced into the abdominal cavity will need to be periodically moistened with perfusion medium during the perfusion process.</li>
</ol>
3. Set up the perfusion system
<ol>
	<li>Transfer the mouse into the water-jacketed moist chamber prewarmed to 37 &#176;C on a silicon pad and immobilize the catheter wings to the pad with 19 G needles.</li>
	<li>Fill the arterial catheter&#8217;s end (inlet) with prewarmed perfusion buffer (Ringer&#8217;s lactate solution supplemented with 5% BSA), and then connect the catheter end with the inlet perfusion tubing using a screw-on connector (Figure 1B, red arrow). 
	NOTE: Hold the connector with hemostatic forceps and immobilize the tubing with tape to avoid moving the catheters.</li>
	<li>Adjust the peristaltic flow rate to 0.6 mL/min and keep the perfusion outflow (Figure 1B, blue arrow) open-ended for 5-10 min to wash out the blood through the venous catheter. There will be some clots in the outlet catheter; flush out the clots with perfusate buffer before closing the perfusion circuit.</li>
	<li>Connect the venous catheter&#8217;s end with the outlet tubing using a screw-on connector to close the circuit (Figure 1B). At this point, perform CO2&#160;gas euthanasia and verify by chest puncture or any other method.</li>
	<li>Cover the moist chamber with the warmed lid. Check the level of perfusate periodically and add more if needed. Perfusion can be performed for up to 2 hours. 
	NOTE: 5 mL of perfusate will be needed to set up the closed perfusion system. If there is no leaking or edema, the volume of perfusate will decrease by less than 1 mL and additional buffer will not be needed. To avoid edema induced by peripheral circulating thrombus, perfusion buffer containing 0.002% heparin can be used in the first 10 minutes of perfusion, but should be changed to buffer without heparin to avoid the leaking at the edge of the incisions.</li>
	<li>If needed, add a reagent of choice into the perfusion reservoir or to the injection port at any time (Figure 1B-5). For example, 10 &#181;L of 10 mg/mL Hoechst33342 can be added into the perfusate to stain the cell nuclei 2 h before the end of perfusion, or 50 &#181;L of 1 mg/mL DyLight 649-lectin to stain the vascular endothelial cells 30 min before the end of perfusion. 
	NOTE: If attempting to stain bone marrow with Hoechst solution, mice will need to be pre-injected 30 minutes before surgery.</li>
	<li>After perfusion with fluorescent reagents, wash out with perfusion buffer for another 10 minutes to minimize the background fluorescence.</li>
</ol>
4. Analysis of perfused organs
<ol>
	<li>Collect organs including testis, prostate, bladder, femur, muscle, and skin (e.g., feet). Excise a piece of organ about 1 mm3 and flatten between two glass slides.
	<ol>
		<li>Study under inverted fluorescent confocal microscope using DAPI/Cy5 excitation and emission filters (excitation lasers : DAPI, 405 nm; Cy5,640 nm). Use at least 200x magnification objective with a 0.45 numerical aperture.</li>
		<li>Alternatively, fix the organs with 4% formaldehyde solution for 24 h and perform hematoxylin-eosin staining19.</li>
	</ol>
	</li>
	<li>To create a bone window for intact bone marrow observation, immobilize both ends of the femur or tibia and scrape away the cortical bone with the lateral edge of a 19 G needle to expose the periosteum; take care to keep a thin layer of residual bone. Place bone on a cover slip with the window facing the glass and image with inverted fluorescent confocal microscope using DAPI and Cy5 channels as described above. The cells and vascular network in the bone marrow cavity can be readily observed.</li>
</ol>

<ol><li>Ghaidan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ten+year+follow-up+of+lung+transplantations+using+initially+rejected+donor+lungs+after+reconditioning+using+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22Journal+of+Cardiothoracic+Surgery%22%5BJournal%5D)">Ten year follow-up of lung transplantations using initially rejected donor lungs after reconditioning using ex vivo lung perfusion.</a> Journal of Cardiothoracic Surgery. 14 (1), 125(2019).</li>
<li>Kabagambe, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+Ex+vivo+Hypothermic+and+Normothermic+Perfusion+for+Assessment+of+High-risk+Deceased+Donor+Human+Kidneys+for+Transplantation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Combined Ex vivo Hypothermic and Normothermic Perfusion for Assessment of High-risk Deceased Donor Human Kidneys for Transplantation.</a> Transplantation. 103 (2), 392-400 (2019).</li>
<li>Knaak, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technique+of+subnormothermic+ex+vivo+liver+perfusion+for+the+storage%2C+assessment%2C+and+repair+of+marginal+liver+grafts%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Technique of subnormothermic ex vivo liver perfusion for the storage, assessment, and repair of marginal liver grafts.</a> Journal of Visualized Experiments. (90), e51419(2014).</li>
<li>Hems, R., Ross, B. D., Berry, M. N., Krebs, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gluconeogenesis+in+the+perfused+rat+liver%5BTitle%5D)+AND+%22Biochemical+Journal%22%5BJournal%5D)">Gluconeogenesis in the perfused rat liver.</a> Biochemical Journal. 101 (2), 284-292 (1966).</li>
<li>Nielsen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vasopressin+increases+water+permeability+of+kidney+collecting+duct+by+inducing+translocation+of+aquaporin-CD+water+channels+to+plasma+membrane%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (4), 1013-1017 (1995).</li>
<li>Sutherland, F. J., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolated+blood+and+perfusion+fluid+perfused+heart%5BTitle%5D)+AND+%22Pharmacological+Research%22%5BJournal%5D)">The isolated blood and perfusion fluid perfused heart.</a> Pharmacological Research. 41 (6), 613-627 (2000).</li>
<li>Schreiter, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+ex+vivo+perfusion+system+emulating+in+vivo+conditions+in+noncirrhotic+and+cirrhotic+human+liver%5BTitle%5D)+AND+%22Journal+of+Pharmacology+and+Experimental+Therapeutics%22%5BJournal%5D)">An ex vivo perfusion system emulating in vivo conditions in noncirrhotic and cirrhotic human liver.</a> Journal of Pharmacology and Experimental Therapeutics. 342 (3), 730-741 (2012).</li>
<li>Sevick, E. M., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viscous+resistance+to+blood+flow+in+solid+tumors%3A+effect+of+hematocrit+on+intratumor+blood+viscosity%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Viscous resistance to blood flow in solid tumors: effect of hematocrit on intratumor blood viscosity.</a> Cancer Research. 49 (13), 3513-3519 (1989).</li>
<li>Duyverman, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+isolated+tumor+perfusion+model+in+mice%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">An isolated tumor perfusion model in mice.</a> Nature Protocols. 7 (4), 749-755 (2012).</li>
<li>Sears, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+perfusion+of+a+tumor-containing+colon+with+monoclonal+antibody%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">Ex vivo perfusion of a tumor-containing colon with monoclonal antibody.</a> J Surg Res. 31 (2), 145-150 (1981).</li>
<li>Duda, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Malignant+cells+facilitate+lung+metastasis+by+bringing+their+own+soil%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Malignant cells facilitate lung metastasis by bringing their own soil.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (50), 21677-21682 (2010).</li>
<li>Kristjansen, P. E., Boucher, Y., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dexamethasone+reduces+the+interstitial+fluid+pressure+in+a+human+colon+adenocarcinoma+xenograft%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Dexamethasone reduces the interstitial fluid pressure in a human colon adenocarcinoma xenograft.</a> Cancer Research. 53 (20), 4764-4766 (1993).</li>
<li>Sevick, E. M., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Geometric+resistance+to+blood+flow+in+solid+tumors+perfused+ex+vivo%3A+effects+of+tumor+size+and+perfusion+pressure%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Geometric resistance to blood flow in solid tumors perfused ex vivo: effects of tumor size and perfusion pressure.</a> Cancer Research. 49 (13), 3506-3512 (1989).</li>
<li>Zhang, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+Maintenance+of+Primary+Human+Multiple+Myeloma+Cells+through+the+Optimization+of+the+Osteoblastic+Niche%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Ex vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.</a> PLoS One. 10 (5), (2015).</li>
<li>Di Buduo, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modular+flow+chamber+for+engineering+bone+marrow+architecture+and+function%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Modular flow chamber for engineering bone marrow architecture and function.</a> Biomaterials. 146, 60-71 (2017).</li>
<li>Lokerse, W. J. M., Eggermont, A. M. M., Grull, H., Koning, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+evaluation+of+an+isolated+limb+infusion+model+for+investigation+of+drug+delivery+kinetics+to+solid+tumors+by+thermosensitive+liposomes+and+hyperthermia%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">Development and evaluation of an isolated limb infusion model for investigation of drug delivery kinetics to solid tumors by thermosensitive liposomes and hyperthermia.</a> Journal of Controlled Release. 270, 282-289 (2018).</li>
<li>Tietjen, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoparticle+targeting+to+the+endothelium+during+normothermic+machine+perfusion+of+human+kidneys%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Nanoparticle targeting to the endothelium during normothermic machine perfusion of human kidneys.</a> Science Translational Medicine. 9 (418), (2017).</li>
<li>Ternullo, S., de Weerd, L., Flaten, G. E., Holsaeter, A. M., Skalko-Basnet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolated+perfused+human+skin+flap+model%3A+A+missing+link+in+skin+penetration+studies%5BTitle%5D)+AND+%22European+Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">The isolated perfused human skin flap model: A missing link in skin penetration studies.</a> European Journal of Pharmaceutical Sciences. 96, 334-341 (2017).</li>
<li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Hematoxylin and eosin staining of tissue and cell sections.</a> Cold Spring Harbor Protocols. 2008, 4986(2008).</li>
<li>Hekman, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+Dual-Modality+Imaging+in+Renal+Cell+Carcinoma%3A+An+Ex+vivo+Kidney+Perfusion+Study%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">Targeted Dual-Modality Imaging in Renal Cell Carcinoma: An Ex vivo Kidney Perfusion Study.</a> Clinical Cancer Research. 22 (18), 4634-4642 (2016).</li>
<li>Graham, R. A., Brown, T. R., Meyer, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+ex+vivo+model+for+the+study+of+tumor+metabolism+by+nuclear+magnetic+resonance%3A+characterization+of+the+phosphorus-31+spectrum+of+the+isolated+perfused+Morris+hepatoma+7777%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">An ex vivo model for the study of tumor metabolism by nuclear magnetic resonance: characterization of the phosphorus-31 spectrum of the isolated perfused Morris hepatoma 7777.</a> Cancer Research. 51 (3), 841-849 (1991).</li>
</ol>

The authors have nothing to disclose.

The described circuit can be used to probe various research questions, for example the role of different serum components and tissue barriers in drug delivery, or immune and stem cell trafficking. Different drug delivery systems (e.g., liposomes and nanoparticles) can be added to the perfusate in order to understand the role of physiological and biochemical factors in delivery. The duration of perfusion can vary, depending on the tissue studied, scientific goals, and the composition of perfusate. We present here the resu...

Isolated organ perfusion was originally developed to study organ physiology for transplantation1,2,3, and enabled understanding of functions of the organs without interference from other body systems. For example, isolated kidney and heart perfusion was immensely useful in understanding basic principles of hemodynamics and effects of vasoactive agents, whereas liver perfusion was important to understanding the metabolic function, including drug metabolism in healthy and diseased tissue4,5,6,7. In addition, perfusion studies were critical in understanding viability and function of organs intended for transplantation. In Cancer Researchearch, isolated tumor perfusion has been described by several groups using mouse, rat, and freshly resected human tissues8,9. In some isolated tumor perfusion, the tumor was implanted in the ovary fat pad to force the growth of tumor supplying blood vessels from the mesentery artery10. The Jain group performed pioneering studies using isolated perfusion of colon adenocarcinomas to understand tumor hemodynamics and metastasis8,11,12,13. Other innovative engineered ex vivo setups include a 96-well plate-based perfusion device to culture the primary human multiple myeloma cells14 and a modular flow chamber for engineering bone marrow architecture and function research15.
In addition to physiology and pathology studies, organ perfusion has been used to study the basic principles of drug delivery. Thus, one group described isolated rat limb perfusion and studied accumulation of liposomes in implanted sarcomas16, whereas another group performed dissected human kidney perfusion to study the endothelial targeting of nanoparticles17. Ternullo et al. used an isolated perfused human skin flap as a close-to-in vivo skin drug penetration model18.
Despite these advancements in perfusion of large organs and tissues, there have been no reports on in situ perfusion models in mice that: a) bypass clearance organs such as liver, spleen and kidneys; b) include pelvic organs, skin, muscle, reproductive organs (in male), bladder, prostate and bone marrow. Due to the small size of these organs and the supplying vasculature, ex vivo cannulation and establishment of a perfusion circuit has not been feasible. The mouse is the most important animal model in cancer and immunology research, and drug delivery. The ability to perfuse small mouse organs would allow interesting questions regarding drug delivery to these organs, including to tumors implanted in the pelvis (bladder, prostate, ovary, bone marrow), to be answered, as well as studies of basic physiology and immunology of diseases of these organs. To address this deficiency, we developed an in situ perfusion circuit in mice that can potentially avoid tissue injury and is much better suited for functional research than isolated organ perfusion.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3.5x-90x stereo zoom microscope on boom stand with LED light</td><td>Amscope</td><td>SKU: SM-3BZ-80S</td><td></td></tr><tr><td>Carbon dioxide, USP</td><td>Airgas healthcare</td><td>19087-5283</td><td></td></tr><tr><td>Confocal microscope</td><td>NIKON</td><td>ECLIPSE Ti2</td><td></td></tr><tr><td>Disposable Sterile Cautery Pen with High Temp</td><td>FIAB</td><td>F7244</td><td></td></tr><tr><td>Moist chamber bubble trap (part 6 in Figure 1)</td><td>Harvard Apparatus</td><td>733692</td><td>Customized as the perfisate container; also enabled constant pressure perfusion</td></tr><tr><td>Moist chamber cover with quartz window (part 3 in Figure 1)</td><td>Harvard Apparatus</td><td>733524</td><td>keep the chamber&#039;s temperature</td></tr><tr><td>Moist chamber with metal tube heat exchanger</td><td>Harvard Apparatus</td><td>732901</td><td>Water-jacketed moist chamber with lid to maintain perfusate and mouse temperature</td></tr><tr><td>Olsen-Hegar needle holders with suture cutters</td><td>Fine Science Tools (FST)</td><td>125014</td><td></td></tr><tr><td>Oxygen compressed, USP</td><td>Airgas healthcare</td><td>C2649150AE06</td><td></td></tr><tr><td>Roller pump (part 4 in Figure 1)</td><td>Harvard Apparatus</td><td>730113</td><td>deliver perfusate to cannula in the moist chamber</td></tr><tr><td>SCP plugsys servo control F/Perfusion (part 1 in Figure 1)</td><td>Harvard Apparatus</td><td>732806</td><td>control the purfusion speed</td></tr><tr><td>Silicone pad</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Silicone tubing set (arrows in Figure 1)</td><td>Harvard Apparatus (TYGON)</td><td>733456</td><td></td></tr><tr><td>Student standard pattern forceps</td><td>Fine Science Tools (FST)</td><td>91100-12</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools (FST)</td><td>14001-14</td><td></td></tr><tr><td>Table for moist chamber</td><td>Harvard Apparatus</td><td>734198</td><td></td></tr><tr><td>Thermocirculator (part 2 in Figure 1)</td><td>Harvard Apparatus</td><td>724927</td><td>circulating water bath for all water-jacketed components</td></tr><tr><td>Three-way stopcock (part 5 in Figure 1)</td><td>Cole-Palmer</td><td>30600-02</td><td></td></tr><tr><td>Veterinary anesthesia machine</td><td>Highland</td><td>HME109</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>19-G BD PrecisionGlide needle</td><td>BD</td><td>305186</td><td>For immobilizing the Insyte Autoguard Winged needle and scratching the cortical bone</td></tr><tr><td>4-0 silk sutures</td><td>Keebomed-Hopemedical</td><td>427411</td><td></td></tr><tr><td>6-0 silk sutures</td><td>Keebomed-Hopemedical</td><td>427401</td><td></td></tr><tr><td>Filter (0.2 &amp;micro;m)</td><td>ThermoFisher</td><td>42225-CA</td><td>Filter for 5% BSA-RINGER&amp;rsquo;S</td></tr><tr><td>Permanent marker</td><td>Staedtler</td><td>342-9</td><td></td></tr><tr><td>Syringe (10 mL)</td><td>Fisher Scientific</td><td>14-823-2E</td><td></td></tr><tr><td>Syringe (60 mL)</td><td>BD</td><td>309653</td><td>Filter for 5% BSA-RINGER&amp;rsquo;S</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1% Evans blue ( w/v ) in phosphate-buffered saline (PBS, pH 7.5)</td><td>Sigma</td><td>314-13-6</td><td></td></tr><tr><td>10% buffered formalin</td><td>velleyvet</td><td>36692</td><td></td></tr><tr><td>BALB/c mice ( 8-10 weeks old )</td><td>Charles River</td><td></td><td></td></tr><tr><td>Baxter Viaflex lactate Ringer&#039;s solution</td><td>EMRN Medical Supplies Inc.</td><td>JB2324</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Thermo Fisher</td><td>11021-037</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Krazy Glue</td><td></td><td></td></tr><tr><td>DyLight-649-lectin</td><td>Vector Laboratories,Inc.</td><td>ZB1214</td><td></td></tr><tr><td>Ethanol (70% (vol/vol))</td><td>Pharmco</td><td>111000190</td><td></td></tr><tr><td>Hoechst33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Enterprises Limited</td><td>66794-017-25</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010023</td><td></td></tr></tbody></table>

establishing in situ closed circuit perfusion of lower abdominal organs and hind limbs in mice

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

We set up a closed circuit perfusion system through cannulation of the abdominal aorta and the inferior vena cava of 8-10 week old mice while keeping the volume of perfusion buffer less than 10 mL. Figure 3A shows confocal images after perfusing tissues with Ringer’s solution containing Hoechst 33342 and DyLight 649-lectin. Muscle, bone marrow, testis, bladder, prostate, and foot skin show efficient nuclear and vascular staining. Figure 3B shows hematoxylin-eosi...

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...

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JoVE Journal

Medicine

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of Göttingen minipigs in biomedical research.

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

Methods for Acute and Subacute Murine Hindlimb Ischemia

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The Mouse Isolated Perfused Kidney Technique

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

Hepatic insulin clearance is critical for regulating glucose homeostasis. This article describes a user-friendly hepatic perfusion procedure for directly evaluating the hepatic insulin clearance rate in situ in mice.

Assessing Insulin Clearance in Mice via In Situ Liver Perfusion

Learn in situ closed circuit perfusion of lower abdominal organs and hind limbs in mice for studying drug delivery and immune interactions. This method enables targeted research without traditional ex-vivo limitations.

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

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