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A Mouse Model of Endometriosis with Nanoparticle Labeling for In Vivo Photoacoustic Imaging

  • Endometriosis: Methodologies
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Abstract

Endometriosis is a condition of the female reproductive tract characterized by endometrium-like tissue growing outside the uterus. Though it is a common cause of pelvic pain and infertility, there is currently no reliable noninvasive method to diagnose the presence of endometriosis without surgery, and the pathophysiological mechanisms that lead to the occurrence of symptoms require further inquiry. Due to patient heterogeneity and delayed diagnosis, animal models are commonly used to study the development of endometriosis, but these are costly due to the large number of animals needed to test various treatments and experimental conditions at multiple endpoints. Here, we describe a method for synthesis of multimodal imaging gold-fluorescein isothiocyanate (FITC) nanoparticles with preclinical application via induction of nanoparticle-labeled endometriosis-like lesions in mice. Labeling donor endometrial tissue fragments with gold-FITC nanoparticles prior to induction of endometriosis in recipients enables in vivo detection of the gold-labeled lesions with photoacoustic imaging. The same imaging method can be used to visualize embryos noninvasively in pregnant mice. Furthermore, the conjugated FITC dye on the gold nanoparticles allows easy isolation of labeled lesion tissue under a fluorescence dissection microscope. After dissection, the presence of gold-FITC nanoparticles and endometrium-like histology of lesions can be verified through fluorescence imaging, gold enhancement, and immunostaining. This method for in vivo imaging of endometriosis-like lesions and fluorescence-guided dissection will permit new experimental possibilities for the longitudinal study of endometriosis development and progression as well as endometriosis-related infertility.

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The data that support the findings of this study are available from the article and Supplementary Information files.

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Acknowledgements

Research reported in this publication was supported in part by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Numbers R01HD084478, R01HD101243, and R01HD102170 to J.W.J., F31HD101207 to R.M.M. and T32HD087166; MSU AgBio Research, and Michigan State University. M.N. would like to thank Hyeonjoo Woo for assistance on TEM analysis. T.K. acknowledges funding from the Departmental Start-Up Grant, Michigan State University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Contributions

R.M.M., M.N., T.H.K., T.K., and J.W.J. designed the experiments and analyzed data; R.M.M., M.N., T.H.K., S.J.C., and K.H. performed experiments; R.M.M., M.N., T.K., and J.W.J. wrote the manuscript.

Corresponding authors

Correspondence to Taeho Kim or Jae-Wook Jeong.

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All protocols relating to mice were overseen and approved by the Institutional Animal Care and Use Committee at Michigan State University under PROTO201900328.

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Supplementary file1 Suppl. Video 1 (Related to Figure 5A) Video reconstruction from in vivo PA imaging of a control endometriosis mouse (no nanoparticle) showing PA signal detection for gold (yellow), Hb (blue), and HbO2 (red) four weeks after induction. PA signals were co-registered with mouse anatomy by B-mode ultrasound (grey). The viewing angle is transverse to the length of the mouse body, and the scanning direction is from cephalic to caudal. Scale bar = 5 mm. (WMV 400 KB)

Supplementary file2 Suppl. Video 2 (Related to Figure 5A) Video reconstruction from in vivo PA imaging of a mouse with gold-FITC nanoparticle-labeled lesions showing PA signal detection for gold (yellow), Hb (blue), and HbO2 (red) four weeks after induction. PA signals were co-registered with mouse anatomy by B-mode ultrasound (grey). The viewing angle is transverse to the length of the mouse body, and the scanning direction is from cephalic to caudal. Scale bar = 5 mm. (WMV 564 KB)

Supplementary file3 Suppl. Video 3 (Related to Figure 5D) Video reconstruction from in vivo PA imaging of a non-pregnant wild-type mouse showing PA signal detection of Hb (blue) and HbO2 (red) combined with ultrasound-derived anatomical structures (grey). The viewing angle is transverse to the length of the mouse body, and the scanning direction is from cephalic to caudal. Scale bar = 5 mm. (MP4 681 KB)

Supplementary file4 Suppl. Video 4 (Related to Figure 5D) Video reconstruction from in vivo PA imaging of a gestation day (GD) 7.5 wild-type mouse showing PA signal detection of Hb (blue) and HbO2 (red) combined with ultrasound-derived anatomical structures (grey). The viewing angle is transverse to the length of the mouse body, and the scanning direction is from cephalic to caudal. Scale bar = 5 mm. (MP4 739 KB)

Supplementary file5 Suppl. Video 5 (Related to Figure 5D) Video reconstruction from in vivo PA imaging of a gestation day (GD) 8.5 wild-type mouse showing PA signal detection of Hb (blue) and HbO2 (red) combined with ultrasound-derived anatomical structures (grey). The viewing angle is transverse to the length of the mouse body, and the scanning direction is from cephalic to caudal. Scale bar = 5 mm. (MP4 649 KB)

Supplementary file6 Suppl. Video 6 (Related to Figure 5D) Video reconstruction from in vivo PA imaging of a gestation day (GD) 9.5 wild-type mouse showing PA signal detection of Hb (blue) and HbO2 (red) combined with ultrasound-derived anatomical structures (grey). The viewing angle is transverse to the length of the mouse body, and the scanning direction is from cephalic to caudal. Scale bar = 5 mm. (MP4 672 KB)

Supplementary file7 Suppl. Video 7 (Related to Figure 5D) Video reconstruction from in vivo PA imaging of a gestation day (GD) 11.5 wild-type mouse showing PA signal detection of Hb (blue) and HbO2 (red) combined with ultrasound-derived anatomical structures (grey). The viewing angle is transverse to the length of the mouse body, and the scanning direction is from cephalic to caudal. Scale bar = 5 mm. (MP4 833 KB)

ESM 8

Supplementary file8 Suppl. Figure 1 (Related to Fig. 5A) In vivo imaging of endometriosis-like lesions based on oxygen saturation (SO2). (A) Representative in vivo photoacoustic (PA) images from control endometriosis mice (no nanoparticle; left) and mice with gold-FITC nanoparticle-labeled lesions (right) showing PA signal detection for SO2 (blue-red) four weeks after induction. The orange segmented circle (left panel) indicates a region containing an unlabeled lesion identified after dissection. The orange segmented circle (right panel) indicates lesion identified by PA signal and confirmed by dissection. (B) Mean intensities of SO2 PA signals from each group. We plotted PA signals from ROIs drawn at the endometriotic lesions of at least three mice. The graphs represent the mean ± SEM (control n=4, gold-FITC nanoparticle n=3; ns, p>0.05). Scale bar = 5 mm. (PNG 349 kb)

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Marquardt, R.M., Nafiujjaman, M., Kim, T.H. et al. A Mouse Model of Endometriosis with Nanoparticle Labeling for In Vivo Photoacoustic Imaging. Reprod. Sci. 29, 2947–2959 (2022). https://doi.org/10.1007/s43032-022-00980-5

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