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 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.
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.
All methods described here have been approved by the University of Colorado’s Institutional Animal Care and Use Committee (IACUC).
1. Pre-heat the perfusion system
2. Vascular catheterization
3. Set up the perfusion system
4. Analysis of perfused organs
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-eosin staining of organs after 3 hours of normothermic perfusion.
Figure 1. Simplified perfusion setup. (A) The system includes (1) a pressure/flow rate controller, (2) a circulating heated water bath, (3) a heated moist chamber with heated cover and custom Styrofoam spacer, and (4) a peristaltic pump. (B) The perfusion circuit shows inlet (red lines) and outlet (blue lines), (5) the injection port and (6) the customized perfusion reservoir. Please click here to view a larger version of this figure.
Figure 2. Location of ligated and cannulated blood vessels in male and female mice. In order to outline the blood vessels, 5 µL/kg of 1% Evans Blue dye was added to perfusion medium 30 min before the end of the perfusion. In female mice, both iliolumbar and ovarian arteries and veins are ligated. In male mice, iliolumbar artery and vein are ligated. Yellow and white lines show approximate position of ligation knots; yellow arrows show actual sutures and knots. Both venous and arterial catheters are inserted. Intestines were removed for demonstration purposes. Please click here to view a larger version of this figure.
Figure 3. Confocal and H&E images show successful perfusion of organs for 3 h with no apparent tissue injury. Confocal scale bar: 20 µm for bone marrow and 100 µm for other organs. H&E scale bar: 100 µm. Please click here to view a larger version of this figure.
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 results of perfusion up to 2 h using a basic perfusion media consisting of Ringer’s solution with lactate and albumin. It must be noted that the goal of the present work was to establish the perfusion circuit rather than developing and optimizing perfusion medium and conditions to support optimal organ/tissue function and oxygenation. The addition of nutrients, vitamins, hormones, and blood cells have been extensively investigated in the previous literature, and numerous perfusion media and oxygenation protocols have been described in dozens of research publications in human and animal tissues7,9,10,11,12,13,16,17,20,21. Refinements of the perfusate composition as well as oxygenation can enable long-term maintenance of tissue metabolism at body temperature. Thus, some groups used red blood cells and blood oxygenation, which greatly improves viability of sensitive tissues17. Perfusion controls are also highly customizable; if necessary, a gas manifold to control oxygenation and pressure manometer can be added. In addition, a larger perfusate chamber can be used, and the physiological parameters such as pressure, temperature, and flow rate can be controlled by a computer.
There are several limitations in the perfusion circuit. The uterus and ovaries in female mice could not be perfused due to the ligation of ovarian artery and vein. Also, we observed that perfusion of testis was incomplete, possibly due to alternative blood supply not included in the perfusion circuit.
With sufficient practice, the cannulation procedure can be performed within 20-30 min with a success rate of over 80%. The success rate highly depends on the ability to cannulate the aorta and vena cava without puncturing the vessel as discussed in Step 2.7. It is important in step 2 to minimize injury to tissues and small blood vessels in the surgical area because of potential leaks and loss of perfusate. In step 2.5, one must always puncture the artery first, and take precaution so that the catheter will not be pulled out. In step 3, when connecting the catheters’ end to the tubing, make sure to hold the catheter tightly using porte-aiguille to avoid moving the needle. Blood pressure is directly proportional to flow rate; therefore, the flow rate of perfusate must be lower than 0.6 mL/min to maintain physiological pressure. Lastly, it is preferred to maintain the heartbeat until the perfusion circuit is closed, as this will improve the perfusion of microvasculature.
The authors have nothing to disclose.
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 Research Funds for the Central Universities, the Program for JLU Science and Technology Innovative Research Team (2017TD-27, 2019TD-36).
Equipment | |||
3.5x-90x stereo zoom microscope on boom stand with LED light | Amscope | SKU: SM-3BZ-80S | |
Carbon dioxide, USP | Airgas healthcare | 19087-5283 | |
Confocal microscope | NIKON | ECLIPSE Ti2 | |
Disposable Sterile Cautery Pen with High Temp | FIAB | F7244 | |
Moist chamber bubble trap (part 6 in Figure 1) | Harvard Apparatus | 733692 | Customized as the perfisate container; also enabled constant pressure perfusion |
Moist chamber cover with quartz window (part 3 in Figure 1) | Harvard Apparatus | 733524 | keep the chamber's temperature |
Moist chamber with metal tube heat exchanger | Harvard Apparatus | 732901 | Water-jacketed moist chamber with lid to maintain perfusate and mouse temperature |
Olsen-Hegar needle holders with suture cutters | Fine Science Tools (FST) | 125014 | |
Oxygen compressed, USP | Airgas healthcare | C2649150AE06 | |
Roller pump (part 4 in Figure 1) | Harvard Apparatus | 730113 | deliver perfusate to cannula in the moist chamber |
SCP plugsys servo control F/Perfusion (part 1 in Figure 1) | Harvard Apparatus | 732806 | control the purfusion speed |
Silicone pad | Harvard Apparatus | ||
Silicone tubing set (arrows in Figure 1) | Harvard Apparatus (TYGON) | 733456 | |
Student standard pattern forceps | Fine Science Tools (FST) | 91100-12 | |
Surgical Scissors | Fine Science Tools (FST) | 14001-14 | |
Table for moist chamber | Harvard Apparatus | 734198 | |
Thermocirculator (part 2 in Figure 1) | Harvard Apparatus | 724927 | circulating water bath for all water-jacketed components |
Three-way stopcock (part 5 in Figure 1) | Cole-Palmer | 30600-02 | |
Veterinary anesthesia machine | Highland | HME109 | |
Materials | |||
19-G BD PrecisionGlide needle | BD | 305186 | For immobilizing the Insyte Autoguard Winged needle and scratching the cortical bone |
4-0 silk sutures | Keebomed-Hopemedical | 427411 | |
6-0 silk sutures | Keebomed-Hopemedical | 427401 | |
Filter (0.2 µm) | ThermoFisher | 42225-CA | Filter for 5% BSA-RINGER’S |
Permanent marker | Staedtler | 342-9 | |
Syringe (10 mL) | Fisher Scientific | 14-823-2E | |
Syringe (60 mL) | BD | 309653 | Filter for 5% BSA-RINGER’S |
Reagents | |||
1% Evans blue ( w/v ) in phosphate-buffered saline (PBS, pH 7.5) | Sigma | 314-13-6 | |
10% buffered formalin | velleyvet | 36692 | |
BALB/c mice ( 8-10 weeks old ) | Charles River | ||
Baxter Viaflex lactate Ringer's solution | EMRN Medical Supplies Inc. | JB2324 | |
Bovine serum albumin | Thermo Fisher | 11021-037 | |
Cyanoacrylate glue | Krazy Glue | ||
DyLight-649-lectin | Vector Laboratories,Inc. | ZB1214 | |
Ethanol (70% (vol/vol)) | Pharmco | 111000190 | |
Hoechst33342 | Life Technologies | H3570 | |
Isoflurane | Piramal Enterprises Limited | 66794-017-25 | |
Phosphate buffered saline | Gibco | 10010023 |