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. 2016 Nov 1;9(11):1397-1403.
doi: 10.1242/dmm.026526. Epub 2016 Sep 16.

High- and ultrahigh-field magnetic resonance imaging of naïve, injured and scarred vocal fold mucosae in rats

Ayami Ohno Kishimoto  1 , Yo Kishimoto  1 , David L Young  1 , Jinjin Zhang  2 , Ian J Rowland  3 , Nathan V Welham  4
Affiliations

High- and ultrahigh-field magnetic resonance imaging of naïve, injured and scarred vocal fold mucosae in rats

Ayami Ohno Kishimoto et al. Dis Model Mech. .

Abstract

Subepithelial changes to the vocal fold mucosa, such as fibrosis, are difficult to identify using visual assessment of the tissue surface. Moreover, without suspicion of neoplasm, mucosal biopsy is not a viable clinical option, as it carries its own risk of iatrogenic injury and scar formation. Given these challenges, we assessed the ability of high- (4.7 T) and ultrahigh-field (9.4 T) magnetic resonance imaging to resolve key vocal fold subepithelial tissue structures in the rat, an important and widely used preclinical model in vocal fold biology. We conducted serial in vivo and ex vivo imaging, evaluated an array of acquisition sequences and contrast agents, and successfully resolved key anatomic features of naïve, acutely injured, and chronically scarred vocal fold mucosae on the ex vivo scans. Naïve lamina propria was hyperintense on T1-weighted imaging with gadobenate dimeglumine contrast enhancement, whereas chronic scar was characterized by reduced lamina propria T1 signal intensity and mucosal volume. Acutely injured mucosa was hypointense on T2-weighted imaging; lesion volume steadily increased, peaked at 5 days post-injury, and then decreased - consistent with the physiology of acute, followed by subacute, hemorrhage and associated changes in the magnetic state of hemoglobin and its degradation products. Intravenous administration of superparamagnetic iron oxide conferred no T2 contrast enhancement during the acute injury period. These findings confirm that magnetic resonance imaging can resolve anatomic substructures within naïve vocal fold mucosa, qualitative and quantitative features of acute injury, and the presence of chronic scar.

Keywords: Fibrosis; Hemorrhage; Larynx; MRI; Tissue repair; Voice; Wound healing.

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Conflict of interest statement

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
MRI of the naïve rat larynx, in vivo and ex vivo. (A) T1-weighted (T1W) serial axial images of the rat neck, acquired in vivo at 4.7 T using intravenous contrast enhancement. (B) Enlarged image of the region indicated by the dashed square in A. The red arrow indicates the larynx. (C) T1W axial image of the rat larynx, acquired ex vivo at 4.7 T using immersion contrast enhancement. (D) T1W axial image of the naïve rat larynx, acquired ex vivo at 9.4 T using immersion contrast enhancement. (E) Pseudocolored volume render of the rat larynx, generated with data from an ex vivo scan at 4.7 T using immersion contrast enhancement. Data represent n=5 animals per in vivo/ex vivo condition at 4.7 T (A-C,E) and n=2 animals at 9.4 T (D). Gd, gadobenate dimeglumine contrast agent; R, right; L, left.
Fig. 2.
Fig. 2.
Superparamagnetic iron oxide (SPIO) contrast enhancement of acute vocal fold injury. (A) T2- and T2*-weighted (T2W, T2*W) coronal images of the rat abdomen and neck, acquired in vivo at 4.7 T with and without intravenous SPIO contrast enhancement. Red asterisks indicate livers, red arrows indicate larynges. (B) T2W axial and coronal images of the rat larynx, 5 days following right-sided vocal fold mucosal injury. Images were acquired ex vivo at 4.7 T, with and without (pre-explant) intravenous SPIO contrast enhancement. Red arrows indicate hypointense mucosal lesions. (C) Pseudocolored volume renders of the vocal fold mucosal lesions shown in B. Lesions are red; thyroid (brown), cricoid (green) and arytenoid (cyan) cartilages are shown for anatomic orientation. (D) Effect of contrast enhancement on vocal fold mucosal lesion volume (mean±s.e.m.); n.s., no significant difference (P>0.01), calculated using a Student's t-test. (E) H&E-, Prussian Blue- and CD68-stained vocal fold coronal sections, 5 days following mucosal injury. Black arrows indicate blood (red) and hemosiderin (brown) in the H&E-stained sections and ferric iron (blue) in the Prussian Blue-stained sections; white arrows indicate CD68+ cells (green) in the immunosections (nuclei are counterstained blue). Scale bars: 100 µm. Data represent n=5 animals per experimental condition in A-E, with the exception of the injury+SPIO images and render in panels B and C; these data represent n=2/5 animals in which contrast enhancement was associated with larger lesion volumes. R, right; L, left.
Fig. 3.
Fig. 3.
Characterization of the acute vocal fold injury time course. (A) T2-weighted (T2W) coronal images of the rat larynx, 1-7 days following right-sided vocal fold mucosal injury. Images were acquired ex vivo at 4.7 T. Red arrows indicate hypointense mucosal lesions. (B) Pseudocolored volume renders of the vocal fold mucosal lesions shown in A. Lesions are red; thyroid (brown), cricoid (green) and arytenoid (cyan) cartilages are shown for anatomic orientation. (C) Change in vocal fold mucosal lesion volume, 1-7 days post-injury (mean±s.e.m.); *P<0.01 compared with day 1, calculated using one-way ANOVA. (D) T2*W coronal image of the rat larynx, 1 day following right-sided vocal fold mucosal injury. The image was acquired ex vivo at 4.7 T and is from the same 1 day post-injury larynx shown in A. The red arrow indicates a hypointense mucosal lesion. (E) H&E- and Prussian Blue-stained vocal fold coronal sections, 1-7 days following mucosal injury. Black arrows indicate ferric iron (blue). Scale bars: 100 µm. Data represent n=5 animals per experimental time point. R, right; L, left.
Fig. 4.
Fig. 4.
Characterization of vocal fold scar. (A) T1- and T2-weighted (T1W, T2W) axial images of the rat larynx, 2 months following right-sided vocal fold mucosal injury. Images were acquired ex vivo at 4.7 T; T1W images were acquired with (center) and without (left) immersion contrast enhancement. (B) T1W serial axial images of the rat larynx, 2 months following right-sided vocal fold mucosal injury. Images were acquired ex vivo at 4.7 T using immersion contrast enhancement. (C) Resliced serial coronal images of the larynx shown in B. (D) Enlarged image of the region indicated by the dashed square in C (left); Masson's Trichrome-stained section of the same larynx (right). Scale bar: 300 µm. Red arrows indicate hypointense scar tissue. Data represent n=5 animals. Gd, gadobenate dimeglumine contrast agent; R, right; L, left.
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