Tongue fat infiltration in obese versus lean Zucker rats

Abstract

Study objectives: Obesity is the most important risk factor for obstructive sleep apnea (OSA), and the effects of obesity may be mediated by tongue fat. Our objective was to examine the effects of obesity on upper airway structures in obese (OBZ) and non-obese (NBZ) Zucker rats.

Design: Animal study.

Setting: Academic Medical Center.

Participants: OBZ (638.2 ± 39 g; 14.9 ± 1.1 w) and age-matched NBZ Zucker (442.6 ± 37 g, 15.1 ± 1.5 w) rats.

Interventions: TONGUE FAT AND VOLUME AND WERE ASSESSED USING: in vivo magnetic resonance spectroscopy (MRS), magnetic resonance imaging including Dixon imaging for tongue fat volume, ex vivo biochemistry (fat quantification; triglyceride (mg)/tissue (g), and histology (Oil Red O stain).

Measurements and results: MRS: overall OBZ tongue fat/water ratio was 2.9 times greater than NBZ (P < 0.002) with the anterior OBZ tongue up to 3.3 times greater than NBZ (P < 0.002). Biochemistry: Triglyceride (TG) in the tongue was 4.4 times greater in OBZ versus NBZ (P < 0.0006). TG was greater in OBZ tongue (3.57 ± 1.7 mg/g) than OBZ masseter muscle (0.28 ± 0.1; P < 0.0001) but tongue and masseter TG were not different in NBZ rats (0.82 ± 0.3 versus 0.28 ± 0.1 mg/g, P = 0.67). Dixon fat volume was significantly increased in OBZ (56 ± 15 mm3) versus NBZ (34 ± 5 mm3, P < 0.004). Histology demonstrated a greater degree of intracellular muscle fat and extramuscular fat infiltration in OBZ versus NBZ rats.

Conclusions: Genetically obese rats had a large degree of fat infiltration in the tongue compared to both skeletal muscle and tongue tissues of the non-obese age-matched littermates. The significant fat increase and sequestration in the obese tongue may play a role in altered tongue neuromuscular function, tongue stiffness or metabolic function.

Keywords: Dixon imaging; MRI; Zucker rats; obesity; obstructive sleep apnea; spectroscopy; tongue; upper airway.

Figure 1

Location of voxels in representative obese Zucker rat. The exact location of three voxels (black squares) that were studied using the spectroscopy protocol in nine magnetic resonance imaging (MRI) slices from a single obese rat are shown. The first column shows the location of the first magnetic resonance spectroscopy (MRS) tongue voxel (Voxel 1) in the axial, coronal, and sagittal views (rows labeled, far right); the second column shows the location of the second tongue voxel (Voxel 2); and the third column shows the location of the masseter muscle voxel (Voxel 3). The posterior tongue voxel was placed proximal to the hyoid bone. The second (anterior) tongue voxel was placed 2-4 mm anterior to the posterior voxel in the middle part of the tongue. The third voxel was placed in the masseter muscle, lateral to the mandible bone. Annotations on the third column of images show: Mst, masseter muscle; Ton, tongue; Np, nasopharyngeal airway; Br, brain. Calibration bar (second column) = 10 mm and applies to all images. In the top row the oil (marked “O”) and water phantom (marked “W”) show the cross section of the tubular glass phantoms as round dots outside the rat but within the images. These phantoms were used to calibrate the Dixon method for analysis of fat content in the Dixon spin-echo magnetic resonance images (images shown above are T1 weighted, scout images not Dixon protocol images; see supplemental material for details on the imaging protocols and analyses).

Figure 2

MR H¹ spectra of tongue muscle in non-obese Zucker (NBZ) and obese Zucker (OBZ) rats. The H¹ spectra obtained during nonwater suppressed acquisition, in panels A (NBZ) and C (OBZ) show a single spectral peak at 4.7 ppm, that is the resonant frequency of water for the 200 MHz imaging system in this experiment. The water-suppressed point-resolved multiecho spectroscopy (PRESS) acquisition H¹ spectra in panels B (NBZ) and D (OBZ) are, respectively, from the same tongue voxels from which the nonsuppressed water spectra (A,C) were obtained. Color-coded curves are used in the figures to differentiate the original spectral data (blue lines) from the computer-generated curve-fitted data in each panel. The red lines denote the estimated area under the water spectra and lipid peaks (A,C), whereas in B and D two polynomial equations were combined (red and green lines) to better estimate the area under the lipid peak. The black lines represent the difference in calculated area from the original (blue line) data. In panel D: MRS, the blue line original spectra; Δ analysis, the black line as described above; typical spectral peaks (Tau, taurine; CR, creatinine). H¹ denotes the water peak; Lipid denotes the lipid peak; and E1 and E2 are peaks of the computer-generated curves that combine to estimate the lipid peak.

Figure 3

Sagittal magnetic resonance imaging MRI of obese rat tongue compared to postmortem histological slice. Panel A shows the midsagittal image of an obese rat (same rat from which magnetic resonance spectroscopy (MRS) spectra are shown in Figure 2). The rat in both images is oriented with the nose (anterior) toward the top of the image and the posterior tongue and larynx toward the bottom of the frame. In panel A, the location of the two voxels (2 × 2 × 2 mm) used for MRS, are drawn to scale, marked, 1 and 2 and highlighted using black squares. These locations depicted on the original scout image were aligned from the scanner data during the MRS protocol. Location 1 is superimposed to scale in the approximate location, on the histological slice in panel B. Note that these are thin slices (showing only one face of the three-dimensional voxel) and fat content was dispersed throughout the tissue, not just in the single sagittal slice. On panel A, the Cut line denotes the approximate location below which the histological slice in panel B, was obtained (the anterior portion of the tongue is not shown in this histology slice). Note that left and right panels have individual but different scale bars; in the lower left (10.0 mm, A) and upper right (2.0 mm, B).

Figure 4

Histological panels (A-F) with Oil Red O staining at increasing magnifications (top to bottom). Fat distribution in midsagittal sections of tongue tissue of lean and obese Zucker rats (NBZ and OBZ) is shown. NBZ rats are on the left (A,C,E) and OBZ rats are on the right (B,D,F). Neutral lipids are stained red. Hematoxylin counterstain shows cell membranes and organelles in blue. There is much more intracellular and extracellular fat in OBZ slides than NBZ. LM, longitudinal muscle fibers; TM, transverse muscle fibers; G, glands near posterior base of tongue; LP, lamina propria; F, filiform papillae of the dorsal mucosa. In A and B, calibration bars are 2 mm (40x). Full arrows (with “tails”) show neutral lipids accumulated between longitudinal muscle fibers (LM). In panels C and D, the same region as A and B are shown in high magnification (400x); calibration bars are 50 μm. Full arrows show large accumulated lipids, and arrowheads show lipid droplets lined on the edge of muscle fibers. Panels E and F are confocal microscopy images with differential interference contrast image overlay to show intracellular fat. Arrowheads show different sizes of lipid droplets within muscle fibers. Calibration bars are 20 μm.