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* These authors contributed equally
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,....
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
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 hematoxyli.......
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.......
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).
....Name | Company | Catalog Number | Comments |
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 |
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