1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine
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Chen, W. C., Park, S. H., Hoffman, C., Philip, C., Robinson, L., West, J., et al. Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice. J. Vis. Exp. (71), e50023, doi:10.3791/50023 (2013).
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 artery5-13. 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 echocardiography5,14-17 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.
2. Right Heart Catheterization
3. Collection of Blood and Spleen Samples
4. Collection of Lung and Heart Samples
5. Analysis of Pressure Curves Generated from the Right Heart Catheter
The recorded right ventricular pressure data are analyzed using LabChart 7 software with no knowledge of the group identity of each recording. More than 20 curves are selected randomly and the difference between maximum and minimum ventricular pressure for each curve measured (ΔP). The average ΔP is calculated to give the right ventricular systolic pressure.
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 animals, the standard deviation of the ΔP, and the heart rate needs to be calculated. Both values are expected to be not significantly different between treatment groups. Furthermore, the expectation is that the ΔP values and the heart rate do not show a drift across the recording time. For example, slowly increasing ΔP values would indicate hypoxic pulmonary vasoconstriction 18-20. Decreasing heart rate would indicate that the level of anaesthesia is too high leading to the death of the animal. In our laboratory different people perform the procedure. Across all groups of mice we routinely achieve 90-95 % success rates in obtaining right ventricular pressure data.
While learning the procedure, it is best to start with older female mice (25-30 g and more) that have a larger jugular vein and tend to have less subcutaneous fat deposits than male mice. The best way to ascertain the placement of the catheter is to euthanize the animal as soon as the catheter is secured inside of the vein. Dissect the animal starting from the heart to directly visualize the placement of the catheter. This will greatly facilitate problem solving.
The first outcome for obtaining BAL fluid is the correct placement of the tracheal cannula. This is indicated by the inflation of the lungs when Hanks buffer is instilled, and deflation of the lungs when the buffer is removed. The expected recovery is 70-80 % of the instilled volume in control animals, less (down to 50 %) in animals that have lung inflammation. Foam on top of the recovered BAL fluid indicates the presence of surfactant. Blood contamination of the recovered BAL fluid either occurs because of the presence of significant lung inflammation, or because instillation and recovery of the wash fluid was too fast. It is of note that some acute measures of injury within the lungs can be induced by the BAL procedure. If the experimental design is to understand these acute injury parameters and examination of BAL is also important, then one lung lobe needs to be tied off and removed prior to conducting BAL.
In the procedure shown here, we harvest lung tissues for histology without standardized inflation and perfusion of the lungs. In cases where exact histological morphometry is the major read-out, the lungs should be inflated via the trachea, and perfusion should be performed via the pulmonary artery, all under standardized pressure.
In the protocol shown, the heart is dissected to provide the Fulton index as measure of right heart hypertrophy (weight of right ventricle / weight of left ventricle and septum). In addition, new methods are being developed that are based on histological evaluation of the right heart. Emerging histological measures of right heart hypertrophy are a) the number of nuclei within a defined area of randomly selected right heart, and b) counts of vessels, and fibrotic index per area of heart.
In our laboratory, the correct recovery of lung draining mediastinal and tracheobronchial lymph nodes (Figure 5) is routinely verified by flow cytometry because these tissues are very small in control mice. However, depending on the microbiome in the housing facility and the age of the animals, the size of the lymph nodes in unchallenged, control mice may be sufficiently large for only visual confirmation. When verifying by flow cytometry, lymph node cells need to be distinguished from thymocytes. By accident, the thymus can easily be sampled together with the lymph node because the thymus and the lymph nodes are located close to each other and are contained within the rib cage. The distinction is that the thymus is located close to the breast-bone and the lymph node close to the spine. Further challenging is that the thymus is very big, containing many more cells than the small lymph nodes. Thymocytes can easily be distinguished from lymph node cells by using flow cytometry and CD3, CD4, and CD8 markers. Lymph nodes have characteristically CD3+ cells, the majority of which are either positive for CD4 or CD8. In contrast, the majority of thymocytes are positive for all three markers (CD3+, CD4+, CD8+). Further in depth information on the localization of mouse lymph nodes can be found in the manuscript by van den Broeck et al.21.
Consistent timing of each main procedural step (heart catheterization, recovery of blood and spleen, harvest of BAL, lung and heart tissues) is critical for the generation of data that allows for robust comparison between treatment groups. Therefore, it is recommended that at least two, and best three, investigators collaborate to accomplish the experiment. Furthermore, it is critical that the investigators perform the procedure without knowledge of the group identity of the animals. Therefore, the identification of the data has to be made in a manner that obscures the group identity. Finally, it is also important that the order in which the animals are analyzed is randomized. For example, a simple identifier is the date of the experiment and consecutive numbering of the animals. If there are for example 4 groups of animals (A-D), assign identification and study the animals in the following order: date_1: Aa, date_2: Ba, date_3: Ca, date_4: Da, date_5: Ab, date_6: Bb, date_7: Cb, and so on.
This experimental flow allows for an in-depth, simultaneous study of the molecular mechanisms that control lung inflammation and right heart function. The simultaneous data and sample collection achieves better insights into molecular networks, and a reduction in the number of animals needed to obtain novel biological knowledge.
|Type of tube / beaker||Solution (volume)||Utilization|
|Processing of blood|
|Eppendorf, 1.5 ml||Blood for serum|
|EDTA coated 2.0 ml||Blood for plasma|
|Eppendorf, 0.5 ml||Freeze serum or plasma|
|Processing of BAL|
|Polypropylene, round bottom, 4.5 ml||Collect bronchoalveolar lavage|
|Polypropylene, round bottom, 4.5 ml||Freeze BAL supernatant|
|96-well plate, skirted||BAL cells|
|Processing of tissue|
|Polypropylene, 50 ml||Formaldehyde, 7.5 ml||Fix lung tissue|
|Eppendorf, 1.5 ml||Snap freeze lung lobe|
|Eppendorf, 1.5 ml||Snap freeze right heart|
|Eppendorf, 1.5 ml||Snap freeze left heart|
|24-well plate||Hanks buffer, 0.5-1 ml||Quick-storage of lung lobes for later isolation of single cells|
|24-well plate||Hanks buffer, 0.5-1 ml||Quick storage of spleens for later isolation of single cells|
|24-well plate||Hanks buffer, 0.5-1 ml||Quick storage of lymph nodes for later isolation of single cells|
|For the procedures|
|Glass beaker, 300 ml||Autoclaved water||Moisturizing and cleaning catheter|
|Polypropylene tube, 50 ml||Hanks buffer, 50 ml||Perform BAL|
|Polypropylene tube, 50 ml||Hanks buffer, 50 ml||Wash tracheal cannula|
|Polypropylene tube, 50 ml||Disinfectant solution||Cleaning instruments|
|Polypropylene tube, 50 ml||70 % ethanol||Cleaning instruments|
|Polypropylene tube, 50 ml||Tap water||Cleaning instruments|
Table 1. Preparation and organization of tubes and solutions.
|Scissors||rounded or straight 5.5 inch, round or sharp ends||Area A|
|Scissors||rounded 4.0 inches, sharp ends|
|Forceps||4.5 inch, bent, with flat, grasping ends|
|Forceps||4.0 inch, bent, with flat, grasping ends|
|Scissors||Slim Blades/Angled- 9 cm||Area B|
|Micro-scissors||Vannas Spring Scissors - 2 mm Blades|
|Forceps||forceps (Dumon #5 Fine)|
|Forceps||4.0 inch, bent, with flat, grasping ends|
|Forceps||4.0 inch, straight, with flat, grasping ends|
|Suture||Braided Silk Suture 6-0|
|Scissors||rounded or straight 5.5 inch, round or sharp ends||Area C|
|Scissors||rounded 4.0 inches, sharp ends|
|Forceps||4.5 inch, bent, with flat, grasping ends|
|Forceps||4.0 inch, bent, with flat, grasping ends|
|Suture||Braided Silk Suture 4-0|
Table 2. Organization of instruments, suture, catheter, tracheal cannula.
Figure 1. Schematic sketch of the work-areas. A) Work-area A for recording, weighing, collection of blood and spleen tissue and temporary holding space for the animals. B) Work-area B for right heart catheterization. C) Work-area C for harvest of BAL, lungs, lung lymph node, and heart tissues.
Figure 2. Instruments used for the procedure. A) Instruments for work-area A; B) instruments for right heart catheterization (work-area B); C) instruments for work-area C. D) Tracheal cannulas. The ruler shows the size of the instruments.
Figure 3. Right heart catheter. Right heart catheter shown with marks and tape (A), or held by hand (B). The pencil points to the marks made on the catheter (A). Insertion of the catheter (C-E): cleaning of the right jugular vein (C); sutured vein held with forceps, catheter is being advanced towards a hole previously made into the vein (D); catheter following insertion into the vein (E). Arrows point to the catheter (D, E).
Figure 4. Sample trace of pressure changes in the right heart. The traces show pressure changes (mmHg) over time (min : sec); the software displays each of the curves at two different speeds. The traces of a control mouse (A); and a mouse with inflammation induced pulmonary arterial remodeling and hypertension (B) are shown. Curves used for the measurement of the right systolic ventricular pressure are highlighted. Note the clear pressure plateaus in the highlighted curves. The arrow points to a curve that has a spike indicating a technical error. These spiky types of curves cannot be used for measurement of right ventricular systolic pressures. Click here to view larger figure.
Figure 5. Location of a mediastinal / tracheobronchial lymph node relative to rib cage and spine. The arrow points to the lymph node.
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 can be studied simultaneously. Therefore, the procedure is suitable for small, targeted experiments 12 and large scale screens performed in a wide range of settings, from a small laboratory to a large pharmaceutical study.
The combination of heart physiology measurements and tissue harvest opens the possibility to perform analysis designed to detect biological networks that control or synchronize heart, lung and immune function, utilizing transgenic and KO mouse resources. An important advantage of the simultaneous procedure flow is the reduction in the number of animals needed per study. Another application of this procedural workflow is the possibility to study other or additional organs from the same animal as needed.
The procedure outlined here generates right heart pressure data very rapidly, within 5-10 min of anaesthesia induction. This makes it possible to study the animals while they are breathing room air spontaneously. A limitation of the approach is that it is invasive: the animals are anaesthetized, they are placed onto their backs, and a catheter is passed via the jugular vein through the atrium into the right ventricle. The limitation is also the advantage of this procedure because the data represent direct measurements of pressure changes over time in the right ventricle. Further technical improvements, particularly relying on miniaturizing the pressure catheters, may allow to place permanent, indwelling catheters in mice that allow for the direct recording of the pulmonary artery pressure over several weeks time, as is being performed in rats 22-24 and other larger animals. If insertion of a heart-catheter through the jugular vein in close-chested, spontaneously breathing animals is not possible, an alternative recording of right ventricular systolic pressure changes can be made in ventilated animals by opening of the thorax and placing the pressure catheter through an incision directly into the right ventricle 25. Non-invasive measurements of right heart function can be made using echocardiography. This procedure can either be performed prior to right heart catheterization, or mice can be examined by echocardiography for multiple times to study disease progression prior to the invasive measurements5,14-17.
Another application of this procedure is to obtain additional data. As an example, using a catheter that can measure flows and pressure, right heart output could be simultaneously recorded together with right heart pressure changes. The procedure outlined here can also be used to further understand the physiologic, cellular and molecular changes that are triggers of pulmonary hypertension other than the immune response, for example, genetic mutations 8,10, cigarette smoke exposure 26, or microbial infections 27-29.
No conflicts of interest declared.
This work was funded by the National Institutes of Health 1R21HL092370-01 (GG), 1R01 HL095764-01 (GG); R01HL082694 (JW); American Heart Association, Founders affiliate (0855943D, GG); Stony Wold - Herbert Fund, New York (SHP).
|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|
|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|
|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|
|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|
|Amplifier, PowerLab 4/30||ADInstrument||Model ML866|
|Catheter, pressure F1.4||Millar Instruments, Inc||840-6719|
|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|
|Pressure Control Unit||Millar Instruments, Inc||PCU-2000|
|Software, Labchart-Pro V.7||AD Instruments|