The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.
The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance – the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW – provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.
In this protocol we demonstrate an isolated, ventilated, perfused mouse lung preparation that has previously been used to quantify the impact of pulmonary emboli and chronic hypoxia on pulsatile pulmonary vascular pressure-flow relationships (Tuchscherer, Webster, & Chesler, 2006; Tuchscherer et al., 2007). In brief, the mouse lungs are surgically isolated from surrounding tissues, placed in a heated chamber (IL-1; Harvard Apparatus, Holliston, MA) and ventilated (Ventilatory Control Module (VCM)-R with timer counter module (TCM); Harvard Apparatus). The lung vasculature is perfused with heated RPMI 1640 cell culture medium with 3.5% Ficoll using a syringe pump (Cole-Parmer, Vernon Hills, IL) to generate the steady flow waveforms or a high-frequency oscillatory pump (Bose -Electro Force, Eden Prairie, MN) in parallel with the syringe pump to create pulsatile pulmonary vascular flow waveforms. Pressure transducers (P75, Harvard Apparatus) measure the instantaneous pulmonary artery pressure (PAP) and left atrial pressure (LAP). Instantaneous flow rate (Q) is measured with an in-line flow meter (Transonic Systems, Inc., Ithaca, NY). Pulsatile pressure-flow relationships are derived from these measurements, which provide insight into pulmonary vascular physiology and pathology and right ventricular afterload.
1. Equipment:
2. Preparing the IL-1 system
3. Solutions
4. Ventilating a Mouse
5. Perfusing a Ventilated Mouse
5.1. Access the right ventricle to inject heparin
5.2. Cannulate the main pulmonary artery
5.3. Cannulate the left atrium
5.4. Begin perfusion
6. Measuring Pulsatile and Steady Pulmonary Pressure-Flow Relationships
7. Representative Results:
Representative Steady Results:
In the isolated lung set-up the experimenter has the ability to independently control not only the pulmonary flow Q but also the airway pressure Pair and left atrial pressure LAP. This is advantageous because Q, Pair and LAP influence the pulmonary vasculature and the resulting pulmonary artery pressure PAP. Another advantage is that results obtained are independent of sympathetic nervous system tone 1, volume status, and anesthesia 2.
PAP changes induced by stair-step changes in Q for fixed Pair and either fixed or varying LAP are shown in Figure 1. Note that in the isolated lung preparation, the LAP cannula is typically connected to tubing that directs the perfusate into a waste container. With this tubing in place, LAP is linearly dependent on Q due to Poiseuille flow. However, the height of the outlet and waste container can be manually adjusted to provide a constant, non-zero LAP or the tubing can be removed to allow zero LAP that is independent of Q.
Representative Pulsatile Results:
While arbitrary pulsatile flow waveforms can be generated with this system, we typically generate flow of the form Q = 3 + 2 sin (2fπt) ml/min at frequencies of f = 1, 2, 5, 10, 15 and 20 Hz to assess the linearized impedance of the pulmonary vasculature (Figure 2: top panel). From the resulting PAP, LAP and Q measurements, pulmonary vascular impedance magnitude (Z) and phase (θ) are calculated by first decomposing one full sinusoidal cycle of ΔP = PAP-LAP and Q at each imposed sinusoidal flow rate frequency into a series of sinusoidal harmonics using a Fourier transform. The ratio of the pressure transform to flow transform yields the pulmonary vascualar impedance, PVZ = FFT (ΔP)/FFT(Q), which has magnitude Z and phase θ. Input impedance Z0, characteristic impedance ZC, and index of wave reflection RW, are calculated from the impedance magnitude. In particular, Z0 is calculated from Z at the 0th harmonic (f = 0 Hz) averaged over all frequencies, ZC is calculated as the average of Z between the first minimum (5 Hz) and 20 Hz, and RW is calculated as (Z0-ZC)/(Z0+ZC) 3.
Figure 1. Steady flow waveforms (top row) and resulting pressures (second row: PAP, Pair, LAP) as a function of time with different combinations of LAP and Pair. The bottom row shows PAP vs. Q. In (A) and (B), LAP increases and decreases with Q because the outflow tubing was in place. This tubing was removed for (C) so that LAP is constant and independent of Q. In (D) and (E) the height of the outlet tubing was adjusted manually so that LAP is higher but independent of Q. Pair was either at end-inspiratory (A, C, D) or end-expiratory (B, E) pressure.
Figure 2. Pulsatile flow waveform Q (top panel) and resulting pressures (bottom panel: PAP, LAP and Pair) as a function of time. From these pulsatile pulmonary pressure-flow relationships, PVZ can be calculated, which reflects the total right ventricular afterload.
Critical steps in the surgery
It is critical that care is taken when cutting the rib cage away from the lungs. The lungs must be fully exposed and uninhibited by surrounding tissues during inflation but not damaged during the process of isolation. The use of a flat object like the back end of the forceps can be used to hold the lungs away from the chest wall so that there is a clear path for scissors to cut. Another critical step is the placement of the suture around the pulmonary artery and the aorta. Using a blunted straight tweezers will reduce the risk of puncturing the pulmonary artery. The final critical step during the surgery is the placement of the cannulas. If the cannulas are too high above the plane of the great vessels as they exit the heart, the cannulas can pull on either the pulmonary artery or pulmonary veins. If the cannula in the left atrium is too low, it can block flow into the left lung. As a consequence, more flow goes to the right lung, increasing PAP and hastening the development of edema in the right lung.
Critical steps during data collection
Steady flow data collection should be performed quickly, especially for high flow rates, so that the exposure of the lung vasculature to high fluid shear stresses is minimized. In our experience, high shear stresses lead to lung edema. Also, rapid increases in shear stress can cause lung edema. For steady flow conditions, an increase in flow of 6 ml/min/min does not cause edema. Steady flow rates over 5 ml/min can be obtained without edema in certain conditions. We have perfused lungs of control and chronically hypoxic mice with steady flow rates as high as 10 ml/min successfully.
Frequency limitations
The highest frequency tested by us is typically 20 Hz because we use a flow waveform Q = 3 + 2 sin (2fπt) ml/min. The pump we describe here can generate oscillations at higher frequencies (at least 50 Hz), however the trade-off is decreased stroke length, i.e., change in Q. A more physiological flow waveform in which the magnitude of flow oscillation decreases with increasing frequency could likely be simulated with this system. Alternatively, a custom perfusion pump could be used with the same surgical isolation and ventilation procedures described here. The frequency range of the pressure transducers (P75, Harvard Apparatus, Holliston, MA) is reported as 0-100 Hz. The actual frequency response of the transducers is dependent on the stiffness and size of the tubing used to connect the transducers to the PA and LA cannulas. Using metal tubing instead of polyethylene tubing would increase the response of the system. However it is not possible to use completely rigid tubing because flexibility in cannula location and placement are needed during surgery. Nevertheless, higher frequency response transducers and/or more rigid tubing would increase the signal-to-noise in the pressure measurements and enable PVZ to be obtained at higher frequencies.
Applications
This isolated lung preparation has been used to investigate the effect of pulmonary embolism 4 as well as chronic hypoxia 5 on pulsatile pulmonary pressure-flow relationships. It also was used to investigate the effects of vasoactive agents in the pulmonary circulation 6 and to quantify the proximal and distal pulmonary vascular effects of acute rho kinase inhibition 7. This technique can be used to quantify pulmonary vascular physiology in inbred or outbred strains of mice or genetically engineered mice 8. The interpretation of the pressure-flow data obtained with this isolated lung preparation is not complicated by differences in heart rate or cardiac output between strains of mice. It is important to note that the impedance spectra obtained in an isolated, ventilated perfused lung in response to a non-physiological waveform should not be directly compared to those obtained in an in vivo preparation in response to a normal heart beat. Also, in vivo, ventilation is by negative, not positive, pressure and the viscosity of blood is approximately 4-fold the viscosity of RPMI with Ficoll.
Significance
Using the isolated, ventilated, perfused mouse lung preparation, we have been able to show that smooth muscle cell de-activation by acute rho-kinase inhibition has no direct effect on the compliance of the large, conduit arteries that significantly impact RV afterload 7. The clinical importance of proximal artery compliance has been increasingly recognized 9-11. Additionally, decreased main pulmonary artery compliance has been shown to be an excellent predictor of mortality in pulmonary arterial hypertension 10,11. The main cause of death from pulmonary hypertension is right ventricular failure; however increased mean pulmonary artery pressure alone is not sufficient to cause failure 12. A more effective measure of total right ventricular afterload is PVZ, which depends on both proximal compliance and distal resistance and is calculated from pulsatile pulmonary pressure-flow relationships such as can be obtained in mouse lungs with this protocol.
The authors have nothing to disclose.
This research was supported by the National Institutes of Health grant R01HL086939 (NCC).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
1 ml syringe | Fisher Sci | 14-829-10F | ||
10 ml syringe | Fisher Sci | 14-823-2A | ||
60 ml syringe | Fisher Sci | 13-689-8 | ||
RPMI with GLN 6/PK | Fisher Sci | MT10040CV | ||
Bottle Top Filters | Fisher Sci | 09-761-57 | ||
Ficoll PM 70 | Sigma-Aldrich | F2878-100g | ||
Heparin | Sigma-Aldrich | |||
Y27632 | Sigma-Aldrich | Y0503 | ||
Angled Ball Iris scissors | Fine Science Tools | 14109-09 | ||
Vannas Spring Scissors – 4mm Blades | Fine Science Tools | 15018-10 | ||
Fine Iris Scissors – straight | Fine Science Tools | 14106-09 | ||
Dumont #5/45 Forceps | Fine Science Tools | 11251-35 | ||
Dumont Medical Biology Forceps | Fine Science Tools | 11254-20 | ||
Lauda E100 ECO-line 003 | VWR | Comparable to Lauda-Brinkmann E-103, 62400-922 | ||
IL-1 Isolated perfused mouse lung system | Harvard Apparatus | 739904 | ||
Blood Pressure Transducer P75 for PLUGSYS Module | Harvard Apparatus | 730020 | ||
TS410 Flow Modules | Transonic | TS410 | ||
ME 4 PXN Precision PXN Inline Flowsensors | Transonic | ME 4 PXN | ||
Cole-Parmer Multi-Syringe Pumps | Cole-Parmer | EW-74900-20 | ||
Nembutal 50MG/ML 20ML Vial | Amatheon |