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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+year+follow-up+of+lung+transplantations+using+initially+rejected+donor+lungs+after+reconditioning+using+ex+vivo+lung+perfusion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+Ex+vivo+Hypothermic+and+Normothermic+Perfusion+for+Assessment+of+High-risk+Deceased+Donor+Human+Kidneys+for+Transplantation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+of+subnormothermic+ex+vivo+liver+perfusion+for+the+storage,+assessment,+and+repair+of+marginal+liver+grafts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gluconeogenesis+in+the+perfused+rat+liver.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasopressin+increases+water+permeability+of+kidney+collecting+duct+by+inducing+translocation+of+aquaporin-CD+water+channels+to+plasma+membrane.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+blood+and+perfusion+fluid+perfused+heart.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+perfusion+system+emulating+in+vivo+conditions+in+noncirrhotic+and+cirrhotic+human+liver.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscous+resistance+to+blood+flow+in+solid+tumors:+effect+of+hematocrit+on+intratumor+blood+viscosity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+tumor+perfusion+model+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+perfusion+of+a+tumor-containing+colon+with+monoclonal+antibody.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+cells+facilitate+lung+metastasis+by+bringing+their+own+soil.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+reduces+the+interstitial+fluid+pressure+in+a+human+colon+adenocarcinoma+xenograft.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+resistance+to+blood+flow+in+solid+tumors+perfused+ex+vivo:+effects+of+tumor+size+and+perfusion+pressure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+Maintenance+of+Primary+Human+Multiple+Myeloma+Cells+through+the+Optimization+of+the+Osteoblastic+Niche.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+flow+chamber+for+engineering+bone+marrow+architecture+and+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+an+isolated+limb+infusion+model+for+investigation+of+drug+delivery+kinetics+to+solid+tumors+by+thermosensitive+liposomes+and+hyperthermia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+targeting+to+the+endothelium+during+normothermic+machine+perfusion+of+human+kidneys.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+perfused+human+skin+flap+model:+A+missing+link+in+skin+penetration+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Dual-Modality+Imaging+in+Renal+Cell+Carcinoma:+An+Ex+vivo+Kidney+Perfusion+Study.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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 border="0" cellpadding="0" cellspacing="0" style="width:1073px;" width="1071">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.5x-90x stereo zoom microscope on boom stand with LED light</td>
			<td>Amscope</td>
			<td>SKU: SM-3BZ-80S</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Carbon dioxide, USP</td>
			<td>Airgas healthcare</td>
			<td style="width:135px;">19087-5283</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Confocal microscope</td>
			<td style="width:191px;">NIKON</td>
			<td style="width:135px;">ECLIPSE Ti2</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Disposable Sterile Cautery Pen with High Temp</td>
			<td style="width:191px;">FIAB</td>
			<td style="width:135px;">F7244</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Moist chamber bubble trap (part 6 in Figure 1)</td>
			<td style="width:191px;">Harvard Apparatus</td>
			<td style="width:135px;">733692</td>
			<td style="width:313px;">Customized as the perfisate container; also enabled constant pressure perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Moist chamber cover with quartz window (part 3 in Figure 1)</td>
			<td style="width:191px;">Harvard Apparatus</td>
			<td style="width:135px;">733524</td>
			<td style="width:313px;">keep the chamber&#39;s temperature</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Moist chamber with metal tube heat exchanger</td>
			<td style="width:191px;">Harvard Apparatus</td>
			<td style="width:135px;">732901</td>
			<td style="width:313px;">Water-jacketed moist chamber with lid to maintain perfusate and mouse temperature</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:55px;width:435px;">Olsen-Hegar needle holders with suture cutters</td>
			<td style="width:191px;">Fine Science Tools (FST)</td>
			<td style="width:135px;">125014</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:435px;">Oxygen compressed, USP</td>
			<td>Airgas healthcare</td>
			<td style="width:135px;">C2649150AE06</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Roller pump (part 4 in Figure 1)</td>
			<td style="width:191px;">Harvard Apparatus</td>
			<td style="width:135px;">730113</td>
			<td style="width:313px;">deliver perfusate to cannula in the moist chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SCP plugsys servo control F/Perfusion (part 1 in Figure 1)</td>
			<td style="width:191px;">Harvard Apparatus</td>
			<td style="width:135px;">732806</td>
			<td style="width:313px;">control the purfusion speed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone pad</td>
			<td style="width:191px;">Harvard Apparatus</td>
			<td style="width:135px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Silicone tubing set (arrows in Figure 1)</td>
			<td style="width:191px;">Harvard Apparatus (TYGON)</td>
			<td style="width:135px;">733456</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student standard pattern forceps</td>
			<td style="width:191px;">Fine Science Tools (FST)</td>
			<td style="width:135px;">91100-12</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors</td>
			<td style="width:191px;">Fine Science Tools (FST)</td>
			<td style="width:135px;">14001-14</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table for moist chamber</td>
			<td style="width:191px;">Harvard Apparatus</td>
			<td style="width:135px;">734198</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Thermocirculator (part 2 in Figure 1)</td>
			<td style="width:191px;">Harvard Apparatus</td>
			<td style="width:135px;">724927</td>
			<td style="width:313px;">circulating water bath for all water-jacketed components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Three-way stopcock (part 5 in Figure 1)</td>
			<td style="width:191px;">Cole-Palmer</td>
			<td style="width:135px;">30600-02</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Veterinary anesthesia machine</td>
			<td style="width:191px;">Highland</td>
			<td style="width:135px;">HME109</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">19-G BD PrecisionGlide needle</td>
			<td style="width:191px;">BD</td>
			<td style="width:135px;">305186</td>
			<td style="width:313px;">For immobilizing the Insyte Autoguard Winged needle and scratching the cortical bone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 silk sutures</td>
			<td style="width:191px;">Keebomed-Hopemedical</td>
			<td style="width:135px;">427411</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-0 silk sutures</td>
			<td style="width:191px;">Keebomed-Hopemedical</td>
			<td style="width:135px;">427401</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Filter (0.2 &#181;m)</td>
			<td style="width:191px;">ThermoFisher</td>
			<td>42225-CA</td>
			<td style="width:313px;">Filter for 5% BSA-RINGER&#8217;S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permanent marker</td>
			<td style="width:191px;">Staedtler</td>
			<td style="width:135px;">342-9</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe (10 mL)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">14-823-2E</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe (60 mL)</td>
			<td style="width:191px;">BD</td>
			<td style="width:135px;">309653</td>
			<td style="width:313px;">Filter for 5% BSA-RINGER&#8217;S</td>
		</tr>
		<tr height="21">
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:435px;">1% Evans blue ( w/v ) in phosphate-buffered saline (PBS, pH 7.5)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">314-13-6</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% buffered formalin</td>
			<td>velleyvet</td>
			<td style="width:135px;">36692</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BALB/c mice ( 8-10 weeks old )</td>
			<td style="width:191px;">Charles River</td>
			<td style="width:135px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Baxter Viaflex lactate Ringer&#39;s solution</td>
			<td style="width:191px;">EMRN Medical Supplies Inc.</td>
			<td style="width:135px;">JB2324</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Bovine serum albumin</td>
			<td>Thermo Fisher</td>
			<td>11021-037</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyanoacrylate glue</td>
			<td style="width:191px;">Krazy Glue</td>
			<td style="width:135px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DyLight-649-lectin</td>
			<td style="width:191px;">Vector Laboratories,Inc.</td>
			<td style="width:135px;">ZB1214</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol (70% (vol/vol))</td>
			<td style="width:191px;">Pharmco</td>
			<td style="width:135px;">111000190</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Hoechst33342</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:135px;">H3570</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Isoflurane</td>
			<td>Piramal Enterprises Limited</td>
			<td style="width:135px;">66794-017-25</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Phosphate buffered saline</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:135px;">10010023</td>
			<td style="width:313px;"></td>
		</tr>
	</tbody>
</table>

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&#8217;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 hematoxyli...

Watch this Scientific Journal Video about Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice at JoVE.com

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

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 ...

establishing-situ-closed-circuit-perfusion-lower-abdominal-organs

Research

JoVE Journal

Medicine

5.9K Views.  The First Hospital of Jilin University. 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.

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

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been widely used to measure aortic dimensions in mouse models of aortic aneurysms. Aortic aneurysms are defined as permanent dilations of the aorta, which occur most frequently in the ascending and abdominal regions. Sequential measurements of aortic dimensions by ultrasound are the principal approach for assessing the development and progression of aortic aneurysms in vivo. Although many reported studies used ultrasound imaging to measure aortic diameters as a primary endpoint, there are confounding factors, such as probe position and cardiac cycle, that may impact the accuracy of data acquisition, analysis, and interpretation. The purpose of this protocol is to provide a practical guide on the use of ultrasound to measure the aortic diameter in a reliable and reproducible manner. This protocol introduces the preparation of mice and instruments, the acquisition of appropriate ultrasound images, and data analysis.

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been ...

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) generated by recording both pressure and volume during cardiac catheterization are vital when assessing both systolic and diastolic cardiac function. Left and right heart function are closely related, reflected in ventricular interdependence. Thus, recording biventricular function in the same animal is important to get a complete assessment of cardiac function. In this protocol, a closed chest approach to cardiac catheterization consistent with the way catheterization is performed in patients is adopted in mice. While challenging, the closed chest strategy is a more physiological approach, because opening the chest results in major changes in preload and afterload that create artifacts, most notably a fall in systemic blood pressure. While high-resolution echocardiography is used to assess rodents, cardiac catheterization is invaluable, particularly when assessing diastolic pressures in both ventricles.
Described here is a procedure to perform invasive, closed chest, sequential left and right ventricular pressure-volume (PV) loops in the same animal. PV loops are acquired using&#160;admittance technology with a mouse pressure-volume catheter and pressure-volume system acquisition. The procedure is described, beginning with the neck dissection, which is required to access the right jugular vein and the right carotid artery, to the insertion and positioning of the catheter, and finally the data acquisition. Then, the criteria required to ensure the acquisition of high-quality PV loops are discussed. Finally, the analysis of the left and right ventricular PV loops and the different hemodynamic parameters available to quantify systolic and diastolic ventricular function are briefly described.

Presented here is a protocol to assess biventricular heart function in mice by generating pressure-volume (PV) loops from the right and left ventricle in the same animal using closed chest catheterization. The focus is on the technical aspect of surgery and data acquisition.

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) ...