Research ArticleBasic Science Investigation
Open Access

Noninvasive Diagnostic Method to Objectively Measure Olfaction and Diagnose Smell Disorders by a Molecularly Targeted Fluorescence Imaging Agent

Dauren Adilbay, Junior Gonzales, Marianna Zazhytska, Paula Demetrio de Souza Franca, Sheryl Roberts, Tara D. Viray, Raik Artschwager, Snehal Patel, Albana Kodra, Jonathan B. Overdevest, Chun Yuen Chow, Glenn F. King, Sanjay K. Jain, Alvaro A. Ordonez, Laurence S. Carroll, Stavros Lomvardas, Thomas Reiner and Nagavarakishore Pillarsetty
Journal of Nuclear Medicine August 2024, 65 (8) 1293-1300; DOI: https://doi.org/10.2967/jnumed.123.266123

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Figures

  • Figure
  • FIGURE 1.
    FIGURE 1.

    Histologic slides of olfactory bulb and olfactory epithelium of normosmic mice. (A) Hematoxylin and eosin staining. (B) NaV1.7 immunohistochemistry. H&E = hematoxylin and eosin; IHC = immunohistochemistry; ON = olfactory nerve bundles; ONL = olfactory nerve layer.

  • FIGURE 2.
    FIGURE 2.

    Histologic slides of olfactory bulb and olfactory epithelium of mice with olfactory ablation using methimazole and mouse infected with COVID-19. (A) Immunohistochemistry slide of mouse after olfactory ablation. (B) Immunohistochemistry slide of olfactory tissue with IgG isotype primary antibody, as control for possible unspecific binding. (C) Immunohistochemistry slides of mouse with SARS-CoV-2 infection. (D) Quantification of NaV1.7 expression in olfactory epithelium of 3 mouse groups. (E) Quantification of NaV1.7 expression in olfactory bulb of 3 mouse groups. ** P ≤ 0.01. *** P ≤ 0.001. IHC = immunohistochemistry; ON = olfactory nerve bundles; ONL = olfactory nerve layer.

  • FIGURE 3.
    FIGURE 3.

    SCN9A gene expression in olfactory epithelium of hamsters and humans infected with SARS-CoV-2. (A) Uniform manifold approximation and projection for dimension reduction plots of SCN9A gene expression in different cell types of olfactory epithelium in mock and SARS-CoV-2–infected hamsters at 1, 3, and 10 d after infection. (B) Violin plots of SCN9A gene expression in olfactory epithelium bulk tissues in mock and SARS-CoV-2–infected hamsters at 1, 3, 10 d after infection. (C) SCN9A gene expression in human olfactory epithelium tissues in control and SARS-CoV-2–infected cadavers. DPI = days after infection; GBC = glucose basal cells; HBC = horizontal basal cells; INP = immediate neuronal precursors; MV2 = microvillus cells 2; OSN = olfactory sensory neurons; SUS = sustentacular cells.

  • FIGURE 4.
    FIGURE 4.

    Tsp1a-IR800P accumulation in ROEB in normosmic mice and mice with olfactory ablation. (A and B) Fluorescence quantification and epifluorescence images of animals injected with PBS, Tsp1a-IR800P, and Tsp1a-IR800P/Tsp1a blocking formulation. (C and D) Epifluorescence images and fluorescence intensity quantification of normosmic animals injected with Tsp1a-IR800P (control) and Tsp1a-IR800P/Tsp1a (blocking) and mice with prior olfactory ablation using methimazole injected with Tsp1a-IR800P. All images were taken 30 min after tail vein injection. *P ≤ 0.05. ***P ≤ 0.001. Olf. abl. = olfactory ablation.

  • FIGURE 5.
    FIGURE 5.

    Fluorescent confocal microscopy images of olfactory epithelium of animals injected with PBS, Tsp1a-IR800P, and Tsp1a-IR800P/Tsp1a-Pra0 blocking formulation. Blue fluorescence indicates nucleus of cells, and red fluorescence indicates infrared fluorescence coming from NaV1.7 of olfactory nerve bundles. ON = olfactory nerve (bundles).

  • FIGURE 6.
    FIGURE 6.

    Buried-food test. (A) Schematic of experimental design illustrating mouse cage with cookie buried in upper right corner. (B) Average time (seconds) spent per mouse treated with PBS (n = 5) or methimazole (n = 5) to find buried food. Graph indicates that healthy mice found buried food much more quickly than ones treated with methimazole (P < 0.001), suggesting presence of olfactory dysfunction in the latter. (C) Correlation of Tsp1a-IR800P radiant efficiency at ROEB and time on buried-food test demonstrating that the more quickly mice find buried food, the brighter is fluorescence detected from olfactory nerve region.

  • FIGURE 7.
    FIGURE 7.

    Imaging olfactory epithelium of NHPs. (A) Images were taken using Quest system (approved for clinical use) of olfactory bulb, muscle, olfactory epithelium, and brain of 4 NHPs, after intravenous injection of Tsp1a-IR800P. (B) Quantification of near-fluorescence intensity demonstrates that signal from olfactory epithelium is significantly higher than that from surrounding tissues. (C) Fluorescent confocal microscopy images of olfactory bulb, muscle, olfactory epithelium, and brain tissues of same NHPs. (D) Schematic depiction of potential use of Tsp1a-IR800P in physician’s office setting using Quest or other vendor NIR fluorescence imaging systems. ****P ≤ 0.0001. a.u. = arbitrary units; olf. bulb = olfactory bulb; olf. epi = olfactory epithelium.

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