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

Abstract

Despite the recent advances in understanding the mechanisms of olfaction, no tools are currently available to noninvasively identify loss of smell. Because of the substantial increase in patients presenting with coronavirus disease 2019-related loss of smell, the pandemic has highlighted the urgent need to develop quantitative methods. Methods: Our group investigated the use of a novel fluorescent probe named Tsp1a-IR800_P as a tool to diagnose loss of smell. Tsp1a-IR800_P targets sodium channel 1.7, which plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. Results: Intuitively, we have identified that conditions leading to loss of smell, including chronic inflammation and coronavirus disease 2019, correlate with the downregulation of sodium channel 1.7 expression in the olfactory epithelium, both at the transcript and at the protein levels. We demonstrated that lower Tsp1a-IR800_P fluorescence emissions significantly correlate with loss of smell in live animals-thus representing a potential tool for its semiquantitative assessment. Currently available methods rely on delayed subjective behavioral studies. Conclusion: This method could aid in significantly improving preclinical and clinical studies by providing a way to objectively diagnose loss of smell and therefore aid the development of therapeutic interventions.

Keywords: COVID-19; anosmia; fluorescence imaging; olfaction; optical; smell.

Graphical abstract

FIGURE 1.

Histologic slides of olfactory bulb and olfactory epithelium of normosmic mice. (A) Hematoxylin and eosin staining. (B) Na_V1.7 immunohistochemistry. H&E = hematoxylin and eosin; IHC = immunohistochemistry; ON = olfactory nerve bundles; ONL = olfactory nerve layer.

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 Na_V1.7 expression in olfactory epithelium of 3 mouse groups. (E) Quantification of Na_V1.7 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.

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.

Tsp1a-IR800_P 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-IR800_P, and Tsp1a-IR800_P/Tsp1a blocking formulation. (C and D) Epifluorescence images and fluorescence intensity quantification of normosmic animals injected with Tsp1a-IR800_P (control) and Tsp1a-IR800_P/Tsp1a (blocking) and mice with prior olfactory ablation using methimazole injected with Tsp1a-IR800_P. All images were taken 30 min after tail vein injection. *P ≤ 0.05. ***P ≤ 0.001. Olf. abl. = olfactory ablation.

FIGURE 5.

Fluorescent confocal microscopy images of olfactory epithelium of animals injected with PBS, Tsp1a-IR800_P, and Tsp1a-IR800_P/Tsp1a-Pra0 blocking formulation. Blue fluorescence indicates nucleus of cells, and red fluorescence indicates infrared fluorescence coming from Na_V1.7 of olfactory nerve bundles. ON = olfactory nerve (bundles).

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-IR800_P 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.

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-IR800_P. (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-IR800_P 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.