Lipid Embolism in Obese Göttingen Minipigs

All studies were conducted in accordance with national regulations in Denmark, which are fully compliant with internationally accepted principles for the care and use of laboratory animals and with animal experimental licenses granted by the Danish Ministry of Justice.

In the 7-year period, the obese minipigs were housed either at the experimental animal facility at Copenhagen University (Taastrup, Denmark, Studies 1-4) or at CitoxLAB Denmark (Lille Skensved, Denmark, Studies 5-8). The typical study setup is described below and in Figure 1.

Female GMs (Ellegaard Göttingen Minipigs A/S, Dalmose, Denmark) were selected at approximately 5 to 6 months of age, and either they were ovariectomized at Ellegaard Göttingen Minipigs A/S or later after arrival to CitoxLAB. Denmark Ovariectomy was performed in order to avoid the large variation in food consumption in relation to the reproductive cycle.¹⁰ Obesity was induced by gradually offering increasing amounts of food approaching ad libitum levels after 9 to 10 months, aiming for a BW of approximately 80 to 85 kg at study start. In the induction period, the pigs were fed SDS minipig diet (Special Diet Services, Essex, United Kingdom), Porcine G+ (Scanbur, Denmark), or Altromin 9023 (Brogaarden, Denmark), but in all cases, the pigs were changed to Altromin 9023 at least 2 months before study start and continued on this diet during the studies. This diet contains 1.5% polyunsaturated fat corresponding to 44% of the total fat content. Domestic water was always available ad libitum. After the induction period, at an age of approximately 15 to 16 months, the pigs entered the first study. In some cases, they were terminated after the first study and in other cases, they were subjected to a suitable washout period and reused in a second study before termination (Table 1). In the study periods, the pigs were fed ad libitum, and daily food intake was monitored using an automated system (Mpigwin/MP2, Ellegaard Systems/Mbrose, Faaborg, Denmark). In general, the ad libitum fed pigs consumed 1 to 1.5 kg per day, which correspond to 2 to 3 times the recommended amount. In the interstudy period, the pigs were fed restrictedly with 200 to 250 g Altromin 9023 twice daily to limit further development of obesity, and generally the animals had a limited weight loss in this period. In all studies, the obese pigs were allowed ad libitum access to food at least 3 weeks before study start. The lean reference pigs were fed restrictedly with 250 g Altromin 9023 twice daily. Body weight was measured on a large animal scale twice weekly (TW) during the study periods.

Various anti-obesity drugs were tested in the model either as monotherapy or as combination therapy, as indicated in Table 1. All the animals were treated by subcutaneous (SC) injection in the skin behind the ears once daily, TW, or once weekly depending on the compound in question. Various vehicles were used: 8 mmol/L phosphate, 184 mmol/L propylene glycol, 58 mmol/L phenol, pH 7.4 or pH 8.15; 10 mmol/L phosphate, 2% glycerol, pH = 7.6; 50 mmol/L sodium phosphate, 70 mmol/L sodium chloride, 0.05% tween 80, pH 7.4; or 8 mmol/L phosphate, 18 mg/mL propylene glycol, pH 7.4 (see Supplemental Table 1 for details). The scheduled treatment period ranged from 53 to 140 days; in 3 cases, the unscheduled deaths lead to premature termination of the study as specified in Table 1.

In some studies, and in the lean and obese reference pigs just before euthanasia, the body composition was determined by dual-energy X-ray absorptiometry scanning (DEXA scanning; see Supplemental Table 2 for details) and a central-venous catheter for stress-free blood sampling during metabolic tests, and exposure profiles were implanted under general anesthesia as described in Supplemental Table 2.

In the pigs, where clinical signs were observed, a limited clinical examination was performed. Typically, body temperature, respiratory frequency and quality, capillary refill time, heart rate, and in some cases oxygenation were obtained.

Blood samples for hematology and selected clinical chemistry parameters were obtained in the prestudy period in study 5 (in the anesthesia for baseline DEXA scanning), and clinical chemistry was obtained in the lean and obese reference pigs just before euthanasia to evaluate obesity-related differences in the measured parameters. For hematology, EDTA stabilized blood was taken, and hematological parameters were measured on an ABX Pentra DX120SPS (Horiba ABXdiagnostics, France). For clinical chemistry, blood was taken in plain glass tubes or in EDTA tubes, and concentration of triglycerides, total cholesterol, and albumin in the resulting serum or plasma was analyzed on a Cobas 6000 (Roche, Switzerland) according to the manufacturer’s instructions. Samples were analyzed for C-reactive protein (CrP) using luminescence oxygen channeling immunoassay. Acceptor beads were conjugated with a monoclonal antibody (mAb) toward porcine CrP (AM26061Pu-N, OriGene Technologies GmbH, Germany), donor beads were coated with streptavidin, and a mAb toward human/mouse/porcine CrP (MAB1707, R&D systems, Minnesota) was biotinylated. Five microliter of the calibrators, controls, or ×50 diluted unknown samples in pig plasma were incubated 1 hour at room temperature (RT) with a 15 µL mixture of acceptor beads (79 µg/mL) and biotinylated mAb (15 nmol/L). Then 30 µL streptavidin-coated donor beads (66.67 µg/mL) was added and incubated for 30 minutes at RT. The plates were read in an Envision plate reader at 21°C to 22°C with a filter having a bandwidth of 520 to 645 nm after excitation by a 680 nm laser. The total measurement time per well was 210 milliseconds including a 70 milliseconds excitation time. The assay had a lower and a higher limit of quantification of 20 ng/mL and 1200 ng/mL, respectively, and a pig CrP reference serum (RS-5CrP) was used as calibrator.

Seven animals died unexpectedly, and 5 animals had to be immediately euthanized, these 12 animals are referred to as “unscheduled deaths” (Table 1). Three obese animals were euthanized via scheduled termination in the interstudy period after a clinical examination before enrollment in the second study, as the animals were judged not to be fit for a second study period. These animals are referred to as “scheduled termination” in Table 1. All animals had been obese for approximately 6 to 12 months at the time of death. Furthermore, 13 obese drug-naive or previously treated GMs without clinical observations and 5 lean, age-matched, drug-naive GMs were included, these animals are referred to as “obese reference minipigs” and “lean reference minipigs,” respectively. The obese reference minipigs had been obese for approximately 18 months.

The euthanasia was performed by an intravenous injection of the Zoletil mixture described in Supplemental Table 2 followed by exsanguination. A macroscopic examination was performed after opening the thoracic and abdominal cavities and by observing the appearance of the organs and tissues in situ. In studies 3, 4, and 5, 2 to 4 lung samples were fixed in neutral phosphate buffered 4% formaldehyde. In study 7, for the scheduled termination after study 7, and for the obese and lean reference animals, lung samples from left cranial, left caudal, right cranial, right medial, right caudal, and the accessory lobes were included. Tissues from kidney and liver were sampled in all animals. Furthermore, if abnormalities were found at necropsy in tissues/organs, these were sampled as well, which for many animals included samples of necrotic adipose tissue (retroperitoneal and/or intra-abdominal).

The fixed tissues were trimmed, embedded in paraffin, and approximately 3-µm thick sections were stained with hematoxylin and eosin. The tissue sections were evaluated by the study pathologist using a light microscope. The severity of vacuolation in lung and kidney was assigned one of the following 5 grades: minimal (very few), mild (few), moderate (moderate number), marked (many), and severe (extensive number).

For demonstration of lipid, formalin-fixed tissue was soaked in 30% glucose overnight, embedded in OCT compound (Optimal Cutting Temperature, VWR, Denmark), and frozen on dry ice. Cryosections were stained with Oil Red O (Sigma-Aldrich, Denmark), counterstained with hematoxylin, and mounted with Faramount (Dako-Agilent, Denmark). Oil Red O staining was performed on lung tissue from 4 unscheduled deaths, 3 scheduled terminations, and 1 obese and 2 lean reference GMs.

The composition of lipid in the lipid emboli was evaluated by acetone treatment of cryosections from lung tissue. The sections were treated with 99.9% acetone (VWR, Denmark) for 30 minutes at either 4°C, RT, or 37°C followed by Oil Red O staining.

Immunohistochemistry (IHC) was performed to demonstrate macrophages with an antibody toward CD68 (rabbit antimouse CD68, Abcam, United Kingdom), lymph vessels with an antibody toward LYVE1 (rabbit antimouse LYVE1, Abcam), and blood vessels with an antibody toward von Willebrand factor (VWF; rabbit antihuman VWF, Abcam). In short, deparaffinized sections were blocked with peroxidase-blocking solution (Dako-Agilent, Denmark) followed by blocking in 3% bovine serum albumin (BSA) in tris-buffered saline (TBS; Ampliqon, Denmark). The sections were incubated 30 minutes with primary antibody diluted 1:250 (anti-CD68), 1:2500 (anti-LYVE1), or 1:8000 (anti-VWF) in TBS, 3% BSA, followed by incubation with BrightVision-HRP (Immunologic, VWR, Denmark) for 30 minutes and diaminobenzidine (DAB) + substrate chromogen system (Dako-Agilent, Denmark) for 5 minutes. Slides were washed in TBS, 0.01% Tween 20 between incubations. Finally, slides were counterstained with hematoxylin and mounted in Pertex. All incubations were performed at RT. Double staining, IHC anti-CD68/Oil Red O, was performed as described above except that cryosections were used, and incubation with DAB + chromogen was followed by Oil Red O staining.

The tissue was processed for transmission electron microscopy (TEM) as described previously.¹¹ In short, the formalin-fixed tissues were refixed in cooled 0.5% glutaraldehyde and 2% paraformaldehyde and postfixed in 2% OsO₄ in cacodylate buffer (Ampliqon, Denmark) for 1 hour at RT followed by a wash in cacodylate buffer and staining with 0.5% uranyle acetate (Sigma-Aldrich, Denmark) before processing to epon (Ax-Lab, Denmark) blocks using increasing gradients of ethanol and increasing concentrations of epon in propylene oxide (Sigma-Aldrich, Denmark). The presence of lipid was verified in 1 µm semithin sections stained with 1% toluidine blue (Merck, Denmark), before preparing sections for the electron microscope. Ultrathin sections (∼70 nm) cut with a diamond knife (Diatome 3 mm) collected at copper grids, mesh 200, were examined in a Tecnai G2 Bio TWIN FP 5018/41 transmission electron microscope (FEI, the Netherlands). Images were obtained with a Veleta CCD Camera (Olympus-SIS, Germany) using Microscope User Interface version 4.6.4 TEM Imaging & Analysis version 4.7 SP3 software.

Body weight, fat percentages, and blood parameters from the lean and obese reference groups were compared using Student t test. In addition, triglycerides, total cholesterol, and albumin concentrations from obese pigs with pulmonary embolism of moderate, severe, or marked degree were compared to the plasma values from obese pigs without pulmonary embolism (unscheduled death and obese reference pigs) using Student t test. All statistical analyses were done in GraphPad Prism version 8.0.0 for Windows, GraphPad Software (San Diego, California; www.graphpad.com).

Prestudy DEXA scanning data from study 5 have been used as an example of typical BW and fat percentage range in this model, and in addition, BW and fat percentage from the lean and obese reference pigs are given to show the level of obesity in the model (Table 2). As can be seen, a typical starting BW is around 90 kg (in this case ranging from 68 to 97 kg), and fat percentage is around 47% (here ranging from 39% to 50%). The BW and the fat percentage in the obese controls are significantly increased, approximately 100% compared to the lean controls.

The most prominent clinical signs requiring euthanasia included varying degrees of respiratory distress, cyanosis, prolonged capillary refill time, and/or anorexia. Low oxygen tension (Spo ₂) was measured by pulse oximetry in some of the animals, and in 3 cases vomiting was observed. Individual details are given in Table 3.

Prestudy samples were included in one study (study 5), data from the lean and obese reference pigs together with values from the unscheduled deaths and scheduled termination, where blood sampling was performed before death/euthanasia, are given in Table 2. Obese pigs had significantly higher triglyceride levels, whereas total cholesterol, CrP, and albumin were not significantly different between the lean and obese reference groups.

The obese pigs with clinical signs did not display a uniform pattern. The statistical analysis comparing values from pigs with embolism of moderate, severe, or marked degree to those from obese pigs with no or minimal embolism revealed a trend for higher triglyceride concentrations (0.60 ± 0.24 vs 0.97 ± 0.47 mM, P = .06) and significantly higher albumin concentrations (46.4 ± 3.4 vs 50.0 ± 3.6, P < .05) in the pigs with lipid embolism, whereas no differences were found in the cholesterol concentrations (2.5 ± 0.5 vs 2.0 ± 0.4 mM, P = ns). In addition, 4 animals with clear respiratory signs had low platelet counts compared to the prestudy samples in apparently healthy drug-naive obese pigs (Table 2), and some had high numbers of white blood cells and/or neutrophils. Note that blood samples only were taken in 8 of 16 animals with clinical observations, and not all parameters were measured in all the pigs.

The gross appearance of the lungs did not reveal any findings except for occasional small focal red areas. In general, no macroscopic findings were recorded for kidney and liver, whereas white or yellowish masses, up to 10 cm in diameter, were found in the retroperitoneal and/or visceral adipose tissue of 19 examined animals at the macroscopic examination; 11 of the 12 unscheduled deaths, 3 scheduled terminations, and 5 obese reference minipigs.

On histopathological examination, marked to severe thickening of the alveolar septa throughout the lung was observed in 10 of the 12 unscheduled deaths. The thickening of the alveolar septa was due to variable sized vacuoles (Figure 2A) involving most lobes, but with a tendency to be most severe in the caudal lobes. Oil Red O staining of formalin-fixed cryosections demonstrated that the vacuoles contained lipid (Figure 2B) and that the lipid was disseminated in the alveolar septa (Figure 2C). In general, there was no inflammation associated with the lipid changes, whereas in some animals, the lipid changes were accompanied by focal alveolar edema and hemorrhage (Figure 2D) consistent with the focal red areas observed macroscopically in these animals. Intra-alveolar aggregation of vacuolated macrophages was seen in variable degrees (Figure 2E) and Oil Red O staining of formalin-fixed cryosections demonstrated that the vacuoles contained lipid (Figure 2F). However, the finding was not associated with the vacuoles in the alveolar septa, and aggregation of vacuolated alveolar macrophages was also observed in 2 of the 5 lean age-matched GMs although the vacuoles were smaller. Table 3 gives an overview of the individual animals where the severity grades refer to the most affected lung samples.

In the kidney, the main microscopic finding was variable-sized vacuoles in glomeruli (Figure 3A), this was observed in 9 of the 12 unscheduled deaths. The glomerular capillaries were dilated due to lipid and sometimes erythrocytes were seen to be displaced in the capillaries. Oil red O staining of formalin-fixed cryosections confirmed that the vacuoles contained lipid (Figure 3B). There was no accompanying inflammation, hemorrhage, or necrosis. In the liver, macrovesicular vacuoles in the hepatocytes were observed in a few animals. Lipid emboli were observed in hepatic vessels in one unscheduled death and one scheduled termination (Figure 3C). Furthermore, multinucleated giant cells were observed in hepatic vessels of 2 acutely death animals with lipid embolism (Figure 3D).

Immunohistochemistry was performed to examine the presence of lipid in blood vessels and to determine whether lipid could be found in lymph vessels as well. Staining was absent adjacent to the vacuoles with the lymphatic marker LYVE1, although lymph vessels could easily be detected in the lungs (Figure 4A). In contrast, weak staining surrounding the vacuoles could be detected with an antibody against VWF indicating that the lipid emboli were present in the blood capillaries but not in the lymph vessels (Figure 4B). Transmission electron microscopy verified that the lipid in the alveolar septa of the lung was located in the blood capillaries (Figure 4C), although lipid droplets occasionally were observed in intravascular macrophages.

The composition of the lipid in the blood vessels was studied by acetone treatment of the cryosection from lung tissue at either 4°C, RT, or 37°C followed by Oil Red O staining. The lipid emboli were extracted from the tissue at all 3 incubation temperatures indicating that the lipid mainly consisted of triglycerides as phospholipids are not dissolved by acetone.¹²

Lipid embolism was not observed in any of the 5 lean GMs, whereas varying degree of lipid embolism was seen in the lung of 7 of the 13 examined obese reference GMs and in the 3 GMs with scheduled termination after study 7 (Tables 3 and 4). One of the obese reference GM demonstrated marked lipid embolism in the lung and one animal had moderate lipid embolism, whereas the remaining 5 animals only had minimal changes. The 3 GMs with scheduled termination had mild to marked lipid embolism in lung, and moderate lipid embolism was observed in the kidney in 2 of the animals.

Microscopic examination revealed that the macroscopic changes found in the retroperitoneal and/or visceral adipose tissue consisted of lipogranulomatous inflammation (lipogranulomatous steatitis; Figure 5A). The lipogranulomatous inflammation was characterized by cholesterol clefts surrounded by multinucleated giant cells (Figure 5B) and was observed in all examined animals with lipid embolism including the obese reference GM with minimal pulmonary lipid embolism (Table 4). A high number of macrophages could be detected by IHC using an anti-CD68 antibody (Figure 5C). Small lipid droplets were often observed in the macrophages (Figure 5D). The changes were accompanied by extensive fibrosis.