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

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 transplantation^1,2,3, 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 tissue^4,5,6,7. 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 tissues^8,9. 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 metastasis^8,11,12,13. 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.

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 limit perfusate volume, use a bubble trap within a moist chamber as the perfusate reservoir (Figure 1B-6).

2. Vascular catheterization

1. 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.
2. Prewet a 4-0 silk suture with needle in double distilled water.
3. 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 "T" shape with scissors. Stop bleeding around the edge of the incision by electrocoagulation (cauterizing).
4. 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.
5. 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).
6. 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.
7. 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.
   1. 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 “hold on” to the catheter, and should thus be catheterized before the vena cava to reduce the likelihood of the catheter slipping out.
8. 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.

3. Set up the perfusion system

1. Transfer the mouse into the water-jacketed moist chamber prewarmed to 37 °C on a silicon pad and immobilize the catheter wings to the pad with 19 G needles.
2. Fill the arterial catheter’s end (inlet) with prewarmed perfusion buffer (Ringer’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.
3. 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.
4. Connect the venous catheter’s end with the outlet tubing using a screw-on connector to close the circuit (Figure 1B). At this point, perform CO₂ gas euthanasia and verify by chest puncture or any other method.
5. 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.
6. 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 µL 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 µL 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.
7. After perfusion with fluorescent reagents, wash out with perfusion buffer for another 10 minutes to minimize the background fluorescence.

4. Analysis of perfused organs

1. 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.
   1. 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.
   2. Alternatively, fix the organs with 4% formaldehyde solution for 24 h and perform hematoxylin-eosin staining¹⁹.
2. 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.

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...

Materials

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