The Adult Sprague-Dawley Sugen-Hypoxia Rat Is Still “the One:” A Model of Group 1 Pulmonary Hypertension: Reply to Le Cras and Abman

From the Authors:

Kojonazarov and colleagues recently reported severe emphysema in the SU5416/hypoxia (SuHx) rat model of pulmonary hypertension (PH) (1). The authors found that adult male Wistar Kyoto (WKY) rats had an increased air-to-tissue ratio as judged by nongated in vivo microcomputed tomography, and an increased mean linear intercept (MLI) as a surrogate for emphysema (1, 2). Le Cras and Abman now respond to Kojonazarov and colleagues’ report by underlining the “important role of the developmental timing of disrupted VEGF signaling.” They cite earlier studies conducted on ovine fetuses that showed that VEGF inhibition caused vascular remodeling, a reduction in vascular and airway growth, and neonatal PH at birth (3).

Although the VEGFR inhibitor SU5416 is known to induce emphysema in rats housed in room air at Denver altitude (1,609 m altitude) (4, 5), we contended in our response letter (6) that, at least in male Sprague-Dawley (SD) rats, the combination of VEGFR inhibition and hypoxia does not lead to any biologically relevant emphysema or other significant parenchymal lung disease (7) but rather to pulmonary arterial hypertension (PAH) due to severe angioproliferative remodeling (7, 8). A similar degree of PH without apparent alveolar simplification was seen when VEGF blockade was administered in utero to fetal or neonatal sheep (3). In utero, Po₂ is approximately 19 mm Hg in the fetal pulmonary artery and 34 mm Hg in the umbilical vein (maximum systemic oxygenation), which represent hypoxemic/hypoxic values for newborns after postnatal cardiopulmonary adaptation. Thus, it may not be surprising that VEGF blockade in utero causes severe PH in fetal or neonatal sheep. The lack of significant alveolar simplification in this fetal model (with low systemic arterial Po₂) is consistent with earlier observations in adult hypoxic rats when VEGFR blockade did not cause emphysema in the setting of hypoxia (7).

As discussed by the authors, the WKY rat strain (Janvier Labs) (1, 2) may be more prone to emphysema after SuHx exposure than other strains (6, 9). In contrast, only a mild increase in MLI (+18%) was seen in adult male SuHx-SD rats obtained from Harlan (6), and no emphysema was found in male SD rats obtained from Charles River (6, 8). Of note, even in WKY rats, emphysema is not a universal finding after exposure to SuHx (10).

Although differences among rat strains may account for some of the discrepancies related to the observed extent of emphysema, there is yet another explanation. Importantly, different methodologies were used to assess the degree of emphysema. Kojonazarov and colleagues (1, 2) used nongated in vivo chest micro–computed tomography scans—a method that is fundamentally different from ex vivo MLI measurements. To perform a quantitative three-dimensional (3D) analysis of the lung parenchyma in SuHx-SD rats (8), we used scanning electron microscopy. Even this comprehensive, high-resolution 3D imaging method did not reveal any significant emphysema in the adult SuHx-SD rat model (Figure 1).

Figure 1.

Scanning electron microscopy does not reveal any significant emphysema in the adult Sprague-Dawley Sugen-hypoxia (SuHx) rat model of pulmonary arterial hypertension. (A) Scanning electron micrographs illustrate the alveolar architecture of adult rats subdivided into three experimental groups. The study design is described in Reference 8. Sprague-Dawley rats (6–8 wk old, ∼180–200 g) were purchased from Charles River and divided into three age-matched groups according to the experimental design: 1) control normoxia (ConNx); 2) control hypoxia (ConHx; i.e., rats injected once subcutaneously with vehicle [DMSO; vol/vol] and then exposed to chronic hypoxia [Fi_O2 = 0.1, CO₂ < 10.000 ppm] for 3 wk, followed by a 6-wk period in room air [Fi_O2 = 0.21]); and 3) SuHx (i.e., rats injected with the VEGFR inhibitor SU5416 [Sigma, 20 mg/kg/dose subcutaneously dissolved in DMSO] and subsequently exposed to chronic hypoxia [3 wk], followed by 6 wk of room air). The rat lungs were perfused in vivo by injecting a total of 50 ml of normal saline into the beating right ventricle. After perfusion, the heart and lungs were taken out en bloc. The left lung lobe was ligated and snap-frozen in liquid nitrogen, and the right lobes were tracheally inflated with 10% formalin at a standardized pressure of 25 cm H₂O for at least 5 minutes and fixed. The lungs were freeze-dried and sputtered with gold in an argon atmosphere and examined using a Philips ESEM XL-30 scanning electron microscope at 15 keV and 21 μA. Scale bars: top, 500μm; middle, 100μm; bottom, 50μm. (B) A morphometric analysis showed that the mean alveolar diameters did not differ significantly among the three groups (ConNx [45.8 ± 1.6 μm], ConHx [50.8 ± 2.0 μm], and SuHx [50.0 ± 2.5 μm] animals), in both nonparametric (Kruskal-Wallis/Benjamini-Krieger-Yekutieli) and parametric (ANOVA/Bonferroni post hoc) statistical tests and multiple comparisons. Mean alveolar diameters were determined by morphometric image analysis (Scandium, Olympus Soft Imaging Solutions) of scanning electron micrographs of different groups (n = 6 rats per group), with more than 100–150 measured data points per animal. Mean ± SEM; n = 6 adult male Sprague-Dawley rats per group. All animal experiments were conducted with the approval of the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (#15/2022, #13/1328; LAVES). n.s. = not significant.

Whether or not adult SuHx-exposed SD rats develop biologically relevant emphysema is an important question because the presence of significant parenchymal lung disease would invalidate this as a model of human PAH (group 1 PH). Based on our literature search and analysis of our own SuHx rat studies (6), now including 3D scanning electron microscopy (Figure 1), we conclude that there is little evidence of biologically relevant emphysema when SuHx is used to model PAH in adult male SD rats (4). At most, there may be mild enlargement of distal intraalveolar spaces in this rat substrain, depending on the method used for tissue fixation and the timing of the lung harvest.

Thus, we conclude that exposure of adult rats to SuHx still provides one of the best models to study PAH (8), and one that lacks a significant emphysema-like lung phenotype, at least in the SD strain (Figure 1) (8).