Non-invasive diagnostic method to objectively measure olfaction and diagnose smell disorders by molecularly targeted fluorescent imaging agent

Abstract

The sense of smell (olfaction) is one of the most important senses for animals including humans. Despite significant advances in the understanding mechanism of olfaction, currently, there are no objective non-invasive methods that can identify loss of smell. Covid-19-related loss of smell has highlighted the need to develop methods that can identify loss of olfaction. Voltage-gated sodium channel 1.7 (Na_V1.7) plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. We have identified several conditions such as chronic inflammation and viral infections such as Covid-19 that lead to loss of smell correlate with downregulation of Na_V1.7 expression at transcript and protein levels in the olfactory epithelium. Leveraging this knowledge, we have developed a novel fluorescent probe Tsp1a-IR800 that targets Na_V1.7. Using fluorescence imaging we can objectively measure the loss of sense of smell in live animals non-invasively. We also demonstrate that our non-invasive method is semiquantitative because the loss of fluorescence intensity correlates with the level of smell loss. Our results indicate, that our probe Tsp1a-IR800, can objectively diagnose anosmia in animal and human subjects using infrared fluorescence. We believe this method to non-invasively diagnose loss of smell objectively is a significant advancement in relation to current methods that rely on highly subjective behavioral studies and can aid in studying olfaction loss and the development of therapeutic interventions.

Figure 1.

Histological slides of the olfactory bulb and olfactory epithelium of normosmic mice, mice with olfactory ablation, and a mouse infected with COVID-19. (A) H&E stain of normosmic mouse. (B) Na_V1.7 IHC slide of normosmic mouse. (C) IHC slide of the mouse after olfactory ablation. (D) IHC slide of olfactory tissue with IgG isotype primary antibody. (E) IHC slides of a mouse with SARS-CoV-2 infection. (F) Quantification of Na_V1.7 expression in the olfactory epithelium of 3 mice. (G) Quantification of Na_V1.7 expression in olfactory bulb of 3 mice. (** P ≤ 0.01; *** P ≤ 0.001; ON – olfactory nerve bundles; OSN – olfactory sensory neurons; ONL – olfactory nerve layer)

Figure 2.

SCN9A gene expression in the olfactory epithelium of hamsters and humans infected with SARS-CoV-2. A) UMAP plots of SCN9A gene expression in different cell types of olfactory epithelium in mock and SARS-CoV-2 infected hamsters at 1, 3, 10 dpi. B) IHC slides of Na_V1.7 expression in mock and infected hamster’s olfactory epithelium C) Violin plots of SCN9A gene expression in olfactory epithelium bulk tissues in mock and SARS-CoV-2 infected hamsters at 1, 3, 10 dpi. D) SCN9A gene expression in human OE tissues in control and SARS-CoV-2 infected cadavers. E) IHC slides of Na_V1.7 expression in control and infected human olfactory epithelium

Figure 3.. Tsp1a-IR800 accumulation in the ROEB in normosmic mice and mice with olfactory ablation.

Chemical synthesis of Tsp1a-IR800. The IR800 fluorophore with an attached azido group reacts with an alkyne group on Tsp1a to yield the fluorescent imaging agent. (B-C) Epifluorescence images and fluorescent intensity quantification of animals injected with PBS, Tsp1a-IR800, and Tsp1a-IR800/Tsp1a blocking formulation, respectively. Images were taken 30 min after tail vein injection. (D-E) Epifluorescence images and fluorescence intensity quantification of normosmic control animals (injected with Tsp1a-IR800 or Tsp1a-IR800/Tsp1a blocking formulation) and mice with prior olfactory ablation with methimazole (injected with Tsp1a-IR800.) Images were taken 30 min after tail vein injection. (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** P ≤ 0.0001).

Figure 4.

Fluorescent confocal microscopy images of the olfactory epithelium of animals injected with PBS, Tsp1a-IR800, and Tsp1a-IR800/Tsp1a blocking formulation, respectively. (ON – olfactory nerve (bundles), OSN – olfactory sensory neurons.)

Figure 5.

Buried food test. (A) Schematic illustrating mouse cage with a cookie buried in the upper right corner of the cage. (B) Bars show time (seconds) spent for mice treated with PBS (n=5) or methimazole (n=5) to find the buried food. (C) Correlation of Tsp1a-IR800 radiant efficiency at ROEB and time in buried food test. (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.)

Figure 6.

Imaging of olfactory epithelium in non-human primates. (A) Images were taken using Quest system (approved for clinical use) of the olfactory bulb, muscle, olfactory epithelium, and the brain of the NHP after intravenous injection of TSP1a-IR800 (B) Quantification of the near infra-red fluorescence intensity using authorized Quest software. (C) Fluorescent confocal microscopy images of the olfactory bulb, muscle, olfactory epithelium and brain tissues of the same NHPs (D) Schematic depiction of the potential use of the TSP1a-IR800 in the physician’s office setting using the Quest or other vendor NIR fluorescence imaging systems. (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.)