Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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,....

Protocol

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

  1. Prepare the perfusion system before surgery by starting a 37 °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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
Equipment
3.5x-90x stereo zoom microscope on boom stand with LED lightAmscopeSKU: SM-3BZ-80S
Carbon dioxide, USPAirgas healthcare19087-5283
Confocal microscopeNIKONECLIPSE Ti2
Disposable Sterile Cautery Pen with High TempFIABF7244
Moist chamber bubble trap (part 6 in Figure 1)Harvard Apparatus733692Customized as the perfisate container; also enabled constant pressure perfusion
Moist chamber cover with quartz window (part 3 in Figure 1)Harvard Apparatus733524keep the chamber's temperature
Moist chamber with metal tube heat exchangerHarvard Apparatus732901Water-jacketed moist chamber with lid to maintain perfusate and mouse temperature
Olsen-Hegar needle holders with suture cuttersFine Science Tools (FST)125014
Oxygen compressed, USPAirgas healthcareC2649150AE06
Roller pump (part 4 in Figure 1)Harvard Apparatus730113deliver perfusate to cannula in the moist chamber
SCP plugsys servo control F/Perfusion (part 1 in Figure 1)Harvard Apparatus732806control the purfusion speed
Silicone padHarvard Apparatus
Silicone tubing set (arrows in Figure 1)Harvard Apparatus (TYGON)733456
Student standard pattern forcepsFine Science Tools (FST)91100-12
Surgical ScissorsFine Science Tools (FST)14001-14
Table for moist chamberHarvard Apparatus734198
Thermocirculator (part 2 in Figure 1)Harvard Apparatus724927circulating water bath for all water-jacketed components
Three-way stopcock (part 5 in Figure 1)Cole-Palmer30600-02
Veterinary anesthesia machineHighlandHME109
Materials
19-G BD PrecisionGlide needleBD305186For immobilizing the Insyte Autoguard Winged needle and scratching the cortical bone
4-0 silk suturesKeebomed-Hopemedical427411
6-0 silk suturesKeebomed-Hopemedical427401
Filter (0.2 µm)ThermoFisher42225-CAFilter for 5% BSA-RINGER’S
Permanent markerStaedtler342-9
Syringe (10 mL)Fisher Scientific14-823-2E
Syringe (60 mL)BD309653Filter for 5% BSA-RINGER’S
Reagents
1% Evans blue ( w/v ) in phosphate-buffered saline (PBS, pH 7.5)Sigma314-13-6
10% buffered formalinvelleyvet36692
BALB/c mice ( 8-10 weeks old )Charles River
Baxter Viaflex lactate Ringer's solutionEMRN Medical Supplies Inc.JB2324
Bovine serum albuminThermo Fisher11021-037
Cyanoacrylate glueKrazy Glue
DyLight-649-lectinVector Laboratories,Inc.ZB1214
Ethanol (70% (vol/vol))Pharmco111000190
Hoechst33342Life TechnologiesH3570
IsofluranePiramal Enterprises Limited66794-017-25
Phosphate buffered salineGibco10010023

References

  1. Ghaidan, H., et al. Ten year follow-up of lung transplantations using initially rejected donor lungs after reconditioning using ex vivo lung perfusion. Journal of Cardiothoracic Surgery. 14 (1), 125 (2019).
  2. Kabagambe, S. K., et al.

Explore More Articles

In SituClosed Circuit PerfusionLower Abdominal OrgansHind LimbsMiceDrug DeliveryImmune InteractionsCatheterizationAbdominal AortaInferior Vena CavaIliac Arteries And VeinsPerfusion SystemWater jacketed Moist Chamber

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved