Name: establishing in situ closed circuit perfusion of lower abdominal organs and hind limbs in mice
Uploaded: 2020-08-13T12:30:00
Description: Watch this Scientific Journal Video about Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice at JoVE.com

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 li...

<ol>
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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. 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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 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">
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	</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...

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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 ...

This protocol can be used to answer questions about drug delivery and immune interactions within the organs included in our circuit. This technique allows for the perfusion of organs that are not possible to perfuse by traditional ex-vivo methods, while avoiding the main clearance organs. Before beginning the surgery, set a circulating water bath and all of the water-jacketed components, to 37 degrees Celsius.Confirm that the tubing is cleaned, and use a bubble trap within a moist chamber as the perfusate reservoir to limit the perfusate volume. To perform the catheterization procedure, confirm a lack of response to pedal reflex, in an eight to 10 week old male anesthetized bulb see mouse. And place the mouse in the supine position on a styrofoam board, with the head at six o'clock.Immobilize the animals limbs with tape, and wipe the abdomen with isopropyl alcohol. Make a T-shaped incision in the abdomen, using electro coagulation to stop any bleeding around the edge of the incision. 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.Under a dissection microscope, locate and ligate the ovarian and iliolumbar arteries and veins with 4-O silk sutures. Loop two 4-O silk sutures under the abdominal aorta and inferior vena cava about one centimeter above the iliac artery and vein, one millimeter apart. Then make a loose nod in the suture closest to the iliac vessels.Use a needle holder to horizontally align and stretch both the inferior vena cava and the abdominal aorta. And, keeping the vessels stretched, use a 24-gauge winged shield IV catheter to puncture the abdominal aorta. As soon as the needle penetrates about one millimeter into the vessel, depress the button to retract the needle core, and insert the catheter about five millimeters into the vessel.Insert a catheter five millimeters into the inferior vena cava, in the same manner. And tie both sutures around the catheterized vessels. Then apply instant glue to immobilize the catheters to the erector spin a, and replace the abdominal organs.To set up the perfusion system, place the mouse on a silicon pad in the water-jacketed moist chamber, and immobilize the catheter wings to the pad with 19-gauge needles. Fill the arterial catheter inlet, with pre-warmed perfusion buffer. Use hemostatic forceps, and a screw-on connector to attach the catheter inlet to the tubing, and immobilize the inlet perfusion tubing with tape.Adjust the peristaltic flow rate to 0.6 milliliters per minute, and keep the perfusion outflow open-ended for five to 10 minutes, to wash the blood out through the venous catheter. Some clots will be present in the outlet catheter, flush out the clots with perfusate buffer, and use a screw on connector to attach to the end of the venous catheter to the outlet tubing to close the circuit. Then cover the moist chamber with a pre-warmed lid for up to two hours of perfusion, periodically checking on the level of perfusate.Here, confocal images after perfusion with ringer solution containing Hoechst 33342 and DyLight-649 lectin, are shown. The muscle, bone marrow, testes, bladder, prostate and foot skin typically demonstrate an efficient nuclear and vascular staining. Hematoxylin and eosin staining of these tissues after three hours of normal thermic perfusion, reveals a successful flushing of the organs, with no apparent tissue injury.This model can be used to understand drug uptake pathways, in the physiological irrelevant environment. For example, to understand the role of serum components in drug accumulation. We're using the system, to understand the mechanism of liposome accumulation in the skin.

Research

JoVE Journal

Medicine

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