Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

Summary

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

Abstract

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes ¹.

Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension ². Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases ³. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention ⁴. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.

Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery^5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.

The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography^5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

Protocol

1. Preparation

1. Prepare the following solutions and tubes (Table 1) as follows:
   1. Hanks solution, no calcium, magnesium or indicator with Penicillin (100 U/ml) / Streptomycin (100 mg/ml).
   2. Phosphate buffered saline (PBS), 1x, no calcium, no magnesium.
   3. Ethanol, 70 %, make 500 ml.
   4. Buffered formaldehyde, 7-10 % with PBS, make 500 ml.
   5. Anaesthesia solutions:
      1. Avertin. Carefully add 5 ml of 2-Methyl-2-butanol to 5 g of 2,2,2-Tribromoethanol. Once dissolved, keep stock solution at room temperature in the dark. Take 0.25 ml of the stock solution and dilute it with 10 ml of 1xPBS in g....

Results

The primary outcome for obtaining right heart pressure curves is achieved by the correct position of the right heart catheter. The shape of the pressure time curves is critical because the correct placement of the catheter inside of the right ventricle will result in pressure plateaus (Figure 4). Spiky curves, instead, indicate a catheter that is moved by the breathing or heart beat motion against the wall of the right ventricle. To detect potential problems with the stage of survival of the anima.......

Discussion

The experimental flow outlined here allows for rapid and simultaneous measurement of right ventricular systolic pressure and harvest of samples for the analysis of the responses in the lungs, heart and the immune system in mice. The procedure combines heart physiology measurements, micro-dissection and subsequent tissue harvest for live cell studies, histological analysis, or omics-analysis of the tissues. The complete procedure takes less than 20 min per mouse. Because of the work-area-organized workflow, 2-3 animals ca.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
2-Methyl-2-butanol	Sigma-Aldrich	152463
2,2,2-Tribrom–thanol	Sigma-Aldrich	T48402
disinfectant soap (Coverage Spray TB plus Steris)	Fisher Scientific	1629-08
Ethyl Alcohol, 200 Proof, Absolute, Anhydrous ACS/USP Grade	PHARMCO-AAPER	111000200	Dilute to 70 % with distilled water
Formaldehyde solution	Sigma-Aldrich	F1635-500ML	Dilute to a 7-10 % formaldehyde concentration at a PBS concentration of 1x using PBS stock solution and water
Hanks solution, no calcium, magnesium	Fisher Scientific	21-022-CV
O.C.T	Tissue-Tek	4583
Penicillin (10,000 U/ml) / Streptomycin (10,000 mg/ml) solution	Thermo Scientific	SV30010
Phosphate buffered saline (PBS), no calcium, no magnesium, 1x and 10x solutions	Fisher Scientific
Sodium pentobarbital 26%	Fort Dodge Animal Health	NDC 0856-0471-01
Labware
Plates 12, 24, 96 well	Falcon
Transfer Pipet	Fisher Scientific	13-711-9BM
Tube, EDTA coated	Sarstedt	2013-08
Tubes 0.65 ml and 1.7 ml micro-centrifuge	VWR
Tubes 12 x 75 mm polypropylene	Fisher Scientific	14-956-1D
Tubes, various sizes, polypropylene	Fisher Scientific
Instruments
Forceps, Dumon #5 Fine	Fine Science Tools	11254-20
Forceps, extra fine graefe -0.5 mm tips curved	Fine Science Tools	11152-10
Forceps, extra fine graefe -0.5 mm tips straight	Fine Science Tools	11150-10
Cannula 18 ga, 19 ga	BD	Precision Glide Needles	Cut to optimal length, blunted and outside rasped to create a rough outside surface.
Scissors, Dissector scissors-slim blades 9 cm	Fine Science Tools	14081-09
Suture for BAL, braided silk suture, 4-0	Fine Science Tools	SP116
Suture for right heart catheterization, braided silk suture, 6-0	Teleflex medical	18020-60
Syringe, 1 ml	BD	309659
Equipment
Amplifier, PowerLab 4/30	ADInstrument	Model ML866
Catheter, pressure F1.4	Millar Instruments, Inc	840-6719
Dissecting Microscope	Variscope
Forceps, Vannas spring scissors-2 mm blades	Fine Science Tools	15000-00
Halogen Illuminated Desk Magnifier	Fisher Scientific	11-990-56
Laptop computer	Asus	Model number A52F i5 processor; 15 inch
Light Source	Amscope	HL-250-A
Pressure Control Unit	Millar Instruments, Inc	PCU-2000
Software, Labchart-Pro V.7	AD Instruments