Pulmonary hypertension (PH) is a progressive and challenging disease to treat clinically despite advances in medical therapy. PH patients experience severe respiratory and cardiac deficits due to pulmonary vasculature derangements and the subsequent right ventricular (RV) strain and failure. Combining durable mechanical circulatory support1,2 with low-impedance artificial lungs3 may enable long-term mechanical support that effectively meets PH patients’ unique physiologic needs. However, development of effective mechanical cardiopulmonary support devices for PH-derived RV failure requires a large animal model that recapitulates the hemodynamic derangements and pathophysiology of advanced PH with RV hypertrophy (PH-RVH).
Here, we present a comprehensive guide to left pulmonary artery (LPA) ligation combined with progressive main pulmonary artery (MPA) banding model in adult sheep. Our reported experience suggests this method is a safe and effective approach to establish this disease model. The combination of LPA ligation and MPA banding synergistically induces PH-RVH by first eliminating PA blood flow to the left lung, and then progressively stenosing the MPA over a 2-month period in a controlled fashion. The model will facilitate the research and development of mechanical circulatory and respiratory support devices for patients with PH-RVH.
We used Dorset-cross wethers between 60 and 70 kg (Noble Life Sciences, Woodbine, MD) for model development and refinement. The Institutional Animal Care and Use Committee at Vanderbilt University Medical Center approved the protocol. The described procedures were conducted in accordance with the U.S. National Research Council’s Guide for the Care and Use of Laboratory Animals, 8th edition. A comprehensive list of supplies, medications, and equipment is provided in the Tables S1–S7 (see Supplemental Digital Content 1, http://links.lww.com/ASAIO/A505).
Detailed procedural steps are listed under Supplementary Method 1 (see http://links.lww.com/ASAIO/A505) by each stage of the experimental preparation: 1) preoperative preparation; 2) operative procedure; 3) postoperative recovery; and 4) development of PH-RVH. The preoperative care of sheep is similar to that of most other large animal experiments with respect to sedation and analgesics (see Supplementary Table S4, http://links.lww.com/ASAIO/A505). However, as sheeps are ruminants, there are some specific considerations, such as extended nil per os period and use of an orogastric tube for gastric drainage. The operative procedure requires meticulous dissection and execution to minimize surgical trauma and to expedite recovery. The entire procedure is performed through a mini thoracotomy (Figure 1A). The surgical steps include MPA occluder placement (Figure 1C), insertion of a pressure sensor in the RV outflow tract (RVOT, Figure 1D), and LPA ligation (Figure 1E). Thereafter, both RVOT pressure tubing and occluder’s actuating tube are tunneled out the left dorsum and each connected to a separate subcutaneous port (Figure 1F). Postoperative recovery entails standard care measures for appropriate analgesia and operative site management (see Supplementary Table S4, http://links.lww.com/ASAIO/A505).
Development of PH-RVH is initiated soon after the immediate postoperative recovery period in a programmatic, measured process to develop progressive PH-RVH without undue risk of acute physiologic decompensation (Figure 2). Every 2–4 days, the occluder cuff is inflated by injecting 3% hypertonic saline through its port (Figure 2B, and 2C), while monitoring for changes in heart rate, respiratory rate, and RV and cuff pressures (Figure 3, see Supplementary Figure 1, http://links.lww.com/ASAIO/A505). The frequency and volume of cuff inflation can be titrated according to the animal’s physiologic response and tolerance to band tightening. Furthermore, blood may be drawn periodically from the RV subcutaneous port for health assessments and labs, such as complete blood counts, liver function tests, and cardiac biomarkers. The chronic MPA banding procedure is continued throughout a period of at least 60 days to gradually increase RV pressure and achieve RV remodeling. Standard humane endpoint criteria are enforced throughout this period to minimize unexpected pain and distress of the animal (see Supplementary Table 7, http://links.lww.com/ASAIO/A505). Thereafter, the sheeps can be used for various therapeutic and medical device interventions for treating PH-RVH.
We performed the described survival surgery on four sheeps. All four animals survived to the intended nonsurvival experiment. The mean duration between the survival and nonsurvival surgeries was 66 ± 2 days without major complication or elective study termination. Hemodynamic parameters were recorded at Week 1 (i.e., immediately following LPA ligation and MPA banding) and throughout the PH-RVH development phase.
We used the Student’s paired t-test to compare hemodynamic and physiologic parameters between Week 1 and Week 9 of PH development. The mean respiratory rate significantly increased between Week 1 and 9 (Figure 3B, p = 0.024), while mean heart rate did not (Figure 3C, p = 0.86). The RV systolic pressure (RVSP) was elevated in all animals at Week 1 at 45.1 ± 7.0 mmHg, and increased to 68.9 ± 34.2 mmHg at Week 9 (Figure 3A); however, the observed increase in RVSP from Week 1 to Week 9 was not significant (p = 0.22), likely due to the small sample size and variation in RVSP between animals. Nevertheless, respiratory rate, heart rate, and RVSP values were generally above the normal physiologic range across all animals and time points (Figure 3, dotted lines).
Although there have been reports of PA banding in small animal models,4,5 this approach has been rarely explored in large animal models to induce chronic PH and RVH.6,7 Prior sheep PH models have used PA bead embolization, but bead injection can trigger a severe inflammatory response.6,7 Here, we describe a technique to induce progressive PH and RV pressure overload with clearly established dose–response relationships, while permitting partial reversibility. The entire surgical procedure is performed through a mini-thoracotomy incision. This surgical approach facilitates a rapid postoperative recovery and transition to the PH-RVH development phase of the study with minimal morbidity. The LPA ligation serves to increase the starting RV pressure of the PH-RVH model and has been done in previous models.8–11 In our work, the ligation alone achieved an RVSP of 45.1 ± 7.0 mmHg at Week 1 (Figure 3A). The adjustable silicone occluder allows for adjustment of PA occlusion through the subcutaneous port to further induce PH-RVH as tolerated by the animal. Measuring cuff and RV pressures enables controlled titration of occlusion that may reduce unexpected mortality compared with prior sheep PH models.6,7 Further analyses, including echocardiography, necropsy, and histology can be performed to confirm RV hypertrophy and ventricular remodeling (see Supplementary Figure 2, http://links.lww.com/ASAIO/A505; Supplementary Method 2, http://links.lww.com/ASAIO/A505). The controlled nature of this model facilitates complex investigations into RV remodeling in response to increased RV pressures as well as interventions to ameliorate the deleterious effects of PH-RV overload.
The presented model significantly increased respiratory rate but did not significantly increase RVSP between Week 1 and 9. Week 1 RVSP readings were obtained after LPA ligation and were markedly elevated in all animals (RVSP = 45.1 ± 7.0 mmHg). It is important to note that this change in RVSP did not achieve statistical significance likely due to the small sample size and large variation in RVSP between animals. This is an early report of our model, and there will be improvements in the technique that will reduce variability between animals. Despite these initial concerns, the model still achieved RVSP measurements that were well above the normal physiologic range for all animals presented (Figure 3A), especially in the case for Animal 4, which achieved RVSP greater than 120 mmHg. Importantly, the cuff banding can be tailored to achieve higher or lower levels of pulmonary blood pressures according to investigator’s needs.
A potential obstacle in the model is MPA cuff leakage. Although heavy-duty occluders were used in the presented model, the MPA cuff still leaked in one of the four cases (Animal 4, Supplementary Figure 1, http://links.lww.com/ASAIO/A505). We did not reoperate on the animal as it was still achieving high RV systolic pressure. Testing the cuff integrity before surgeries could minimize these potential risks. If leakage were to occur, investigators will need to weigh their options and decide on continuing the model as is, reoperate on the sheep to replace the cuff, or to electively terminate the experiment.
We describe a well-tolerated and reproducible model of PH-RVH using LPA ligation and progressive PA banding in a large animal model. The minimally invasive surgical approach and the controlled nature of progressive PA banding allow for an effective disease model while reducing mortality. This large animal disease model provides an ideal pathophysiologic platform for biomedical engineering investigations into medical and device therapies for PH-RVH.
The histology slides in Supplementary Files were imaged using the Digital Histology Shared Resource at Vanderbilt University Medical Center (www.mc.vanderbilt.edu/dhsr).
Jose Diaz, MD, Phil Williams, Jamie M. Adcock, LVMT, Mary S. Fultz, LVMT, Franz Baudenbacher, PhD
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