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Elimination of olfactory sensory neurons by zinc sulfate inoculation prevents SARS-CoV-2 infection of the brain in K18-hACE2 transgenic mice.

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Affiliations

  • 1. Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul, 05029, South Korea.
      Authors
    • Lee JH 1
    • Yoo ES 1
    • Kim NW 1
    • Shim WY 1
    • Jeong HB 1
    • Kim DH 1
    • Park YJ 1
    • Seo SM 1
    • Choi YK 1
    (9 authors)
  • 2. Laboratory of Veterinary Toxicology, College of Veterinary Medicine, Research Institute for Veterinary Science, Seoul National University, Seoul, 08826, South Korea.
      Authors
    • Yun JW 2
    (1 author)
  • 3. Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.
      Authors
    • Park JW 3
    (1 author)
  • 4. Laboratory of Avian Diseases, College of Veterinary Medicine, Research Institute for Veterinary Science, Seoul National University, Seoul, 08826, South Korea.
      Authors
    • Choi KS 4
    (1 author)
  • 5. Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam, 13488, South Korea.
      Authors
    • Lee HY 5
    (1 author)
    • Show all (7)

Scientific Reports, 13 Nov 2024, 14(1):27863
https://doi.org/10.1038/s41598-024-78538-5 PMID: 39537718 PMCID: PMC11561319

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Abstract 

Coronavirus disease-2019 (COVID-19), attributed to the severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), has posed global health challenges since it first emerged in 2019, and its impact continues to persist. The neurotropic nature of SARS-CoV-2 remains undisclosed, though researchers are proposing hypotheses on how the virus is transmitted to the central nervous system. One of the prevailing hypotheses is that SARS-CoV-2 travels through the olfactory nerve system via the olfactory epithelium (OE). Using a K18-human angiotensin converting-enzyme 2 (hACE2) transgenic mouse model with impaired olfactory sensory neurons (OSNs) induced by zinc sulfate, we examined the role of the olfactory nerve in the brain invasion by SARS-CoV-2. Mice lacking OSNs exhibited reduced levels of viral transmission to the brain, leading to significantly improved outcomes following SARS-CoV-2 infection. Moreover, a positive correlation was observed between viral persistence in the OE and brain infection. These results indicate that early inhibition of the olfactory nerve pathway effectively prevents viral invasion of the brain in K18-hACE2 mice. Our study underscores the significance of the olfactory nerve pathway in the transmission of SARS-CoV-2 to the brain.

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Logo of scirepAboutEditorial BoardFor AuthorsScientific Reports
Sci Rep. 2024; 14: 27863.
Published online 2024 Nov 13. https://doi.org/10.1038/s41598-024-78538-5
PMCID: PMC11561319
PMID: 39537718

Elimination of olfactory sensory neurons by zinc sulfate inoculation prevents SARS-CoV-2 infection of the brain in K18-hACE2 transgenic mice

Ji-Hun Lee,1 Eun-Seon Yoo,1 Na-Won Kim,1 Won-Yong Shim,1 Han-Bi Jeong,1 Dong-Hyun Kim,1 Young-Jun Park,1 Sun-Min Seo,1 Jun-Won Yun,2 Jun Won Park,3 Kang-Seuk Choi,4 Ho-Young Lee,5 Jun-Young Seo,6 Ki Taek Nam,6 Je Kyung Seong,corresponding author7,8 and Yang-Kyu Choicorresponding author1,9
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Associated Data

Supplementary Materials
Data Availability Statement

Abstract

Coronavirus disease-2019 (COVID-19), attributed to the severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), has posed global health challenges since it first emerged in 2019, and its impact continues to persist. The neurotropic nature of SARS-CoV-2 remains undisclosed, though researchers are proposing hypotheses on how the virus is transmitted to the central nervous system. One of the prevailing hypotheses is that SARS-CoV-2 travels through the olfactory nerve system via the olfactory epithelium (OE). Using a K18-human angiotensin converting-enzyme 2 (hACE2) transgenic mouse model with impaired olfactory sensory neurons (OSNs) induced by zinc sulfate, we examined the role of the olfactory nerve in the brain invasion by SARS-CoV-2. Mice lacking OSNs exhibited reduced levels of viral transmission to the brain, leading to significantly improved outcomes following SARS-CoV-2 infection. Moreover, a positive correlation was observed between viral persistence in the OE and brain infection. These results indicate that early inhibition of the olfactory nerve pathway effectively prevents viral invasion of the brain in K18-hACE2 mice. Our study underscores the significance of the olfactory nerve pathway in the transmission of SARS-CoV-2 to the brain.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-78538-5.

Keywords: SARS-CoV-2, Olfactory epithelium, Olfactory sensory neurons, Brain, Zinc sulfate, K18-hACE2
Subject terms: Virology, Infection

Introduction

A novel coronavirus denominated as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in late December 2019, and is the causative agent of the coronavirus disease-2019 (COVID-19)1. SARS-CoV-2 is the third member of the Betacoronavirus genus, which caused deadly respiratory illnesses in humans following SARS-CoV in 2002 and Middle East Respiratory Syndrome Coronavirus outbreaks in 20122. COVID-19 was declared a pandemic by the World Health Organization in 2020 and still persists as a leading global health concern3. As of March 2024, a count of more than 700 million COVID-19 patients has been confirmed, and 7 million deaths have been reported4.

SARS-CoV-2 gains access to a cell by binding its spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell membrane5. The ACE2 receptor is distributed throughout various organs in the human body6,7, making them susceptible to SARS-CoV-2 infection8. For example, respiratory diseases are the most common clinical manifestations exhibited in patients with COVID-19 infection, owing to a high level of ACE2 expression in human lungs9,10. Additionally, multiple organ failures in patients with COVID-19 can also be explained by ACE2 distribution in the affected organs11,12.

Patients with COVID-19 may also exhibit neurological manifestations, including anosmia, ageusia, headache, and stroke13,14. Although the neurological symptoms of COVID-19 are now believed to be largely dependent on the host’s immune response to viral particles, the neurotropism of the virus remains an unresolved issue that requires further investigation concerning central nervous system (CNS) infection of SARS-CoV-215–17. Recent studies have suggested several possible neuroinvasive mechanisms of SARS-CoV-2: (1) the virus crosses the blood-brain-barrier18,19, (2) the virus is transported to the CNS through axons from the peripheral nervous system20,21, (3) the virus produces and triggers inflammatory mediators to damage the nervous system22,23, (4) the virus invades the olfactory epithelium (OE) and is subsequently transported to the olfactory bulb24,25.

In this regard, we directed our attention toward understanding the role of the OE in the brain invasion of SARS-CoV-2 in K18-hACE2 transgenic mice. We utilized the K18-hACE2 mouse model, which, while not fully appropriate for directly reflecting human conditions, is still suitable for COVID-19 research in investigating viral pathogenesis26. We aimed to explore the potential consequences of SARS-CoV-2 infection in the brains of mice with disrupted OE. We established a mouse model lacking olfactory sensory neurons (OSNs) by inoculating K18-hACE2 transgenic mice with zinc sulfate solution to reveal the intricate involvement of the OE in the neuroinvasive process of SARS-CoV-2 infection.

Materials and methods

Ethics statement

All procedures in this study were approved by the Institutional Animal Care and Use Committee of Konkuk University (approval number: KU23061), in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011). All research was conducted in accord with the ARRIVE guidelines.

Cell lines

Vero E6 cells (CRL-1586; ATCC, Manassas, VA, USA) were used for viral growth. Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco) supplemented with 2% fetal bovine serum (Gibco) and 1% antibiotic-antimycotic solution (Gibco). The cells were maintained at 37 [degree celsius] with 5% CO2.

SARS-CoV-2 propagation and titration

The viral stock of SARS-CoV-2 (NCCP 43326) was provided by the Korea Disease Control and Prevention Agency (KDCA). For viral propagation, Vero E6 cells in DMEM were inoculated with the parental virus and cultured until 80% of the cells exhibited cytopathic effects. The supernatant containing the virus was then harvested through centrifugation at 3,000 rpm for 10 min with subsequent filtration using 0.22 μm syringe filters. Viral titration was assessed using a tissue culture infectious dose 50% (TCID50) assay.

Mice and experimental design

Eight-week-old, female K18-hACE2 transgenic mice with a C57BL/6J background were purchased from Jackson Laboratory. Mice were randomly divided into three groups: normal control, virus-infected, and zinc sulfate. All mice received pretreatment for three consecutive days before viral infection. For pretreatment, the mice were lightly anesthetized with 5% isoflurane, and subsequently administered with either 20 µl of 0.9% normal saline (normal control and virus-infected group) or 3% zinc sulfate (Sigma-Aldrich) solution (zinc sulfate group) into each naris.

Following pretreatment, SARS-CoV-2 infection was conducted in two groups (virus-infected and zinc sulfate groups). All procedures concerning SARS-CoV-2 were conducted in the Biosafety Level 3 (BSL3) facilities at Konkuk University. Both groups of mice underwent deep anesthesia with an intraperitoneal injection of 10 ml/kg zoletil (60 mg/kg)-xylazine (5 mg/kg) solution. Each mouse was then infected via intranasal inoculation with 1 × 105 TCID50 SARS-CoV-2 in 40 µl phosphate-buffered saline (PBS). After viral infection, the zinc sulfate group received 10 µl of 3% zinc sulfate solution into each naris every other day from one day post-infection (DPI).

The body weight, temperature, activity level, and survival rate of the mice were monitored daily for a full-period observation of 16 days. The activity level of the mice was scored according to the criteria outlined in Table 1. Necropsy was performed at 2, 5, and 16 DPI. The numbers of mice used in the study are listed as follows: the normal control group, n = 8 for full-period observation; virus-infected group, n = 5 for 2 DPI, n = 5 for 5 DPI, and n = 7 for full-period observation; zinc sulfate group, n = 5 for 2 DPI, n = 5 for 5 DPI, and n = 13 for full-period observation. All surviving mice scheduled for necropsy were anesthetized with 5% isoflurane and euthanized by exsanguination of the abdominal aorta. Organs, including the lungs, brains, spleens, and facial bones, were collected.

Table 1

Criteria for scoring activity level.

Activity score (%)Criteria
100Healthy
75Healthy, but with ruffled fur
50Less movement, lethargy, ruffled fur, and hunched posture
25Moribund, eye discharge, and inability to maintain physical balance
0Death

Viral RNA extraction and reverse transcriptase qPCR (RT-qPCR)

The infected tissues were weighed and emulsified in PBS at 100 times the tissue volume using three-way stopcocks locked with a syringe at each end. The lysed tissues were centrifuged for 5 min at 3,000 rpm, and the supernatants were collected. Approximately 200 µl of each supernatant was mixed with 100 µl MagNA Pure 96 External Lysis Buffer (Roche). Total RNA was extracted from the mixtures using a MagNA Pure 96 system (Roche). The RNA concentration of the extracts was estimated using a NanoDrop. Reverse transcription was performed with a BIO-RAD T100 thermal cycler (Bio-Rad) using Maxime RT PreMix (Random Primer, iNtRon Biotechnology) under the following conditions; 60 min at 45 [degree celsius] for cDNA synthesis and 5 min at 94 [degree celsius] for RTase inactivation. qPCR was performed using AccuPower® 2X GreenStar™ qPCR Master Mix (Bioneer). The primers were designed to target the ORF1ab gene of SARS-CoV-2: ORF1ab-F: 5′-CCCTGTGGGTTTTACACTTAAAAA-3′; ORF1ab-R: 5′-GATTGTGCATCAGCTGACTG-3′. qPCR was performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with the thermal cycling settings outlined as follows: initial denaturation at 95 [degree celsius] for 3 min; 40 cycles of denaturation at 95 [degree celsius] for 10 s, and annealing/elongation at 51 [degree celsius] for 30 s. Melting curve analysis was performed at the end of the cycling procedure.

Histopathological analysis

Harvested tissues were fixed in 10% neutral phosphate-buffered formalin for 24 h and routinely processed. Facial bones were decalcified in 20% ethylenediaminetetraacetic acid (EDTA) solution and replaced daily for three weeks before processing. The processed tissues were embedded in paraffin blocks, sectioned into 4-µm thick sections, and placed onto glass slides. Sections for histopathological analysis were stained with hematoxylin and eosin (H&E) using a Gemini AS Automated Slide Stainer (Epredia). The slides were scanned under a light microscope to examine histopathological changes in the tissues. Images were captured and analyzed using the Olympus cellSens imaging software (Olympus).

To quantify the degree of pneumonia in the lung tissues, three parameters were evaluated: interstitial pneumonia, perivascular edema, and bronchiolitis. Each parameter was assigned a score from 0 to 3 or 5 according to the severity of the lesion, as previously described27. The total score was calculated by summing all three scores. For perivascular cuffing in the cerebral tissues, the layers of leukocytic infiltrates around the vessels were counted and scored. The scoring criteria for perivascular cuffing are shown in Table 2. The OE thickness of the nasal cavity was assessed in two separate sections: T2 and T3 sections. A T2 section was obtained at the location of the incisive papilla. A T3 section was obtained at the level of the second palatal ridge. The measurements were conducted at three specific points on the most dorsal area of the OE in both the T2 and T3 sections. The average of the three measured values was calculated.

Table 2

Criteria for scoring perivascular cuffing.

Perivascular cuffing scoreCriteria
0No leukocyte around the vessels
1One layer of leukocytes around the vessels
2Two layers of leukocytes around the vessels
3Three layers of leukocytes around the vessels
4> Three layers of leukocytes around the vessels, with more than five sites of vessels affected

Immunohistochemistry and immunofluorescence

The 4 μm-thick sections were cleared and dehydrated using a set of xylene and a graded series of ethanol concentrations. Antigen retrieval was performed by heating the slides immersed in sodium citrate buffer (0.01 M, pH 6.0) in a commercial cooker for 10 min. Nonspecific binding was blocked by the sequential application of 3.0% hydrogen peroxide in methanol for 10 min and 10% goat serum for 1 h. For immunohistochemistry, the slides were incubated with SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (Sino Biological, 1:500) for 2 h at room temperature. After washing with PBS, the sections were incubated with a biotinylated secondary antibody (PK-6101, Vector Laboratories) for 30 min, followed by 30 min of incubation with ABC reagent (PK-6101, Vector Laboratories). Staining was performed with the 3,3′-diaminobenzidine (DAB) substrate (SK-4100, Vector Laboratories) and counterstained with hematoxylin. Finally, the slides were mounted with a mounting medium and scanned under a light microscope.

For immunofluorescence, the sections were incubated with primary antibodies for 2 h at room temperature. After washing with PBS, the secondary antibodies were added for 45 min. Double immunostaining for the two antigens from identical hosts was performed using the SuperBoost tyramide signal amplification kit (B40922, Thermo Fisher Scientific). Antibody elution was achieved by incubating slides in 0.5% sodium dodecyl sulfate (SDS) buffer (pH 2) for 30 min at 50 [degree celsius]. The antibodies used in this study were olfactory marker protein (OMP, Abcam, Cambridge, UK; 1:1000), cytokeratin 8 (CK8, Abcam, Cambridge, UK; 1:1000), cytokeratin 5 (CK5, Abcam, Cambridge, UK; 1:500), and ACE2 (Abcam, Cambridge, UK; 1:500). The sections were then incubated with a secondary antibody (Abcam, 1:500) labeled with a fluorescent marker for 1 h. All sections were mounted with 4′,6-diamidino-2-phenylindole (DAPI) and scanned with a fluorescent microscope (Olympus). Images were obtained and merged using the Olympus cellSens imaging software (Olympus).

Quantification and statistical analysis

Statistical analyses were performed using GraphPad Prism software (GraphPad Prism 9.0). All data were presented as the mean with the standard error of the mean (SEM). The normality of the samples was assessed using the Shapiro-Wilk test. The Brown-Forsythe test was employed to test for equal variance among the samples. If the samples passed the normality test, one-way analysis of variance (ANOVA) or Welch ANOVA was conducted to compare data across groups, depending on the results of the Brown-Forsythe test. If the samples did not pass the normality test, the Kruskal-Wallis test was used for data comparison. Survival rates were analyzed using Kaplan-Meier survival curves and compared using the log-rank test. Statistical significance was attributed to data with p-values less than 0.05 (*), 0.01 (**), and 0.001 (***).

Results

Zinc sulfate inoculation improved clinical outcomes following a lethal dose of SARS-CoV-2 infection

To investigate the effect of zinc sulfate inoculation on lethal doses of SARS-CoV-2, two groups of 8-week-old female hACE2 transgenic mice were treated with 0.9% normal saline or 3% zinc sulfate (Fig. 1A). After 3 days of treatment, both groups were infected with 1 × 105 TCID50 of SARS-CoV-2. The clinical outcomes of both groups were compared with those of the normal control group, which was not subjected to viral infection. The virus-infected group exhibited a decline in overall clinical signs, including body weight (Fig. 1B), temperature (Fig. 1C), and activity levels (Fig. 1D), starting from 5 DPI. The survival rate of the virus-infected group also began to decrease at 6 DPI, reaching 0% by 9 DPI (Fig. 1E). In contrast, the zinc sulfate group exhibited an increase in body weight for the initial 5 days following virus infection (Fig. 1B). This was assumed to be indicative of weight loss due to pretreatment with zinc sulfate before viral infection. Subsequently, the activity levels of the zinc sulfate group also decreased from 6 DPI at a rate comparable to that of the virus-infected group (Fig. 1D). However, the zinc sulfate group exhibited gradual recovery from 10 DPI. At 16 DPI, the survival rate of the zinc sulfate group was 61.5%, which was significantly higher than that of the virus-infected group (Fig. 1E). Spleen-to-body weight ratio of the zinc sulfate group also displayed a significant difference compared to the virus-infected group, indicating ameliorated disease progression regarding SARS-CoV-2 infection (Fig. 1F).

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Study design and clinical outcomes in response to SARS-CoV-2 infection. (A) Schematic illustration of the experimental model used in our studies. Eight-week-old female hACE2 transgenic mice were treated with either 0.9% normal saline or 3% zinc sulfate. Each group received either intranasal inoculation of 0.9% normal saline (normal control; n = 8, and virus-infected group; n = 7) or 3% zinc sulfate (zinc sulfate group; n = 13). Following three consecutive days of inoculation, viral infection was performed, followed by treatment every other day. Clinical findings including body weight (B), body temperature (C), activity level (D), and survival rate (E) monitored for 16 DPI. (F) Spleen-to-body weight ratio of each group, normalized to the normal control group. Data are presented as mean ± SEM. p-values are indicated above the plots: *p < 0.05; **p < 0.01; ***p < 0.001. Survival rates were compared with the log-rank test.

Histopathological changes in the lungs were unaffected by zinc sulfate inoculation

Pulmonary lesions resulting from SARS-CoV-2 infection were compared between groups on dates of sacrifice during the observational period by H&E staining (Fig. 2A). The evaluation parameters include inflammation, perivascular edema, and bronchiolitis in the lung tissues. Compared to the normal control group, the other two groups displayed a higher degree of inflammation, characterized by aggregates of mononuclear inflammatory cells within the lung parenchyma. Perivascular edema, with enlarged perivascular spaces containing infiltrations of inflammatory cells, was also observed in both groups. Bronchiolitis was only present in the virus-infected group, although with a minor severity score. Overall, the extent of pneumonia was quantified using pathological scoring, which revealed no notable differences between the two groups (Fig. 2B). The lung-to-body weight ratios of both groups were significantly higher than the normal control group, indicating indistinct levels of lung lesions due to SARS-CoV-2 infection (Fig. 2C).

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Comparison of histopathological findings in lung tissues collected at dates of sacrifice during the full-period observation. (A) Representative histopathological image of H&E-stained lungs from each group. Mononuclear cell infiltrates (red heads), and perivascular edema (yellow heads) are presented in both the virus-infected and zinc sulfate groups. Upper; scale bars, 500 μm, middle; scale bars, 200 μm, lower; scale bars, 100 μm. (B) Histopathological scores of lungs for each group (normal control group; n = 8, virus-infected group; n = 7, zinc sulfate group; n = 13). Histopathological changes include inflammation, perivascular edema, and bronchiolitis. Each parameter was scored on a scale from 0 to 5, with a cumulative total score calculated. Data are presented as mean ± SEM. (C) Lung-to-body weight ratio of each group, normalized to the normal control group. Data are presented as mean ± SEM. p-values are indicated above the plots: ***p < 0.001.

Cerebral tissues developed milder lesions in mice inoculated with zinc sulfate

We then compared the level of inflammation in the cerebral tissues in response to SARS-CoV-2 infection on dates of sacrifice during the observational period by H&E staining. The degree of perivascular cuffing, characterized by mononuclear inflammatory cell infiltration around the capillaries in the cerebral tissues, was analyzed. A comparison of the degree of perivascular cuffing in the cerebra indicated that the zinc sulfate group exhibited fewer signs of perivascular inflammation in the cerebra than the virus-infected group (Fig. 3A–B). These results suggest that zinc sulfate inoculation affects viral replication and accumulation in cerebral tissues.

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Comparison of histopathological findings in brain tissues collected at dates of sacrifice during the full-period observation. (A) Representative histopathological image of H&E-stained brains from each group. Perivascular mononuclear cell infiltrates (red heads) were notably present in the virus-infected group. Scale bars, 200 μm (upper), 50 μm (lower). (B) Histopathological scores of perivascular cuffing found in cerebral tissues (normal control group; n = 8, virus-infected group; n = 6, zinc sulfate group; n = 12). Scores range from 0 to 4 by the number of layers of monocytic infiltrates surrounding the vessels. Data are presented as mean ± SEM. p-values are indicated above the plots: **p < 0.01.

Zinc sulfate inoculation resulted in lower SARS-CoV-2 viral loads in brain tissues

The degree of SARS-CoV-2 infection in groups at 5 DPI was compared by quantifying the viral loads in tissues collected from the mice using RT-qPCR. Viral titers in the lungs did not exhibit any notable differences between the virus-infected and zinc sulfate groups (Fig. 4A). However, significant variations in viral loads were observed in brain tissues between the two groups (Fig. 4B-D). Specifically, certain individuals in the zinc sulfate group displayed no viral loads in cerebral and cerebellar tissues. As a result, zinc sulfate inoculation led to less accumulation of SARS-CoV-2 viral RNA in brain tissues, with no significant difference observed in lung tissues.

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Comparison of SARS-CoV-2 viral loads per mg of different tissues at 5 DPI. SARS-CoV-2 viral RNA copies in the lungs (A), cerebra (B), cerebella (C), and olfactory bulbs (D) of each group (normal control group; n = 5, virus-infected group; n = 5, zinc sulfate group; n = 5). Significant differences in SARS-COV-2 viral loads between virus-infected and zinc sulfate groups were observed in cerebra, cerebella, and olfactory bulbs. Viral quantification was performed by RT-qPCR. Data are presented as mean ± SEM. p-values are indicated above the plots: *p < 0.05.

Zinc sulfate inoculation induced morphological alterations in the OE and lamina propria of nasal mucosa

The morphological alterations in the nasal cavity collected at 5 DPI were assessed by H&E staining to determine the effects of zinc sulfate inoculation and SARS-CoV-2 infection on the OE. In the virus-infected group, we observed a decrease in OE thickness in both the T2 and T3 sections (Fig. 5B and D). Statistical significance was observed in both sections. OE thickness was reduced to 52% in the T2 section and 73.7% in the T3 section compared to those in the normal control group. Also, the zinc sulfate group displayed a significant reduction in OE thickness in both the T2 and T3 sections (Fig. 5C and D). The thickness of OE decreased to 24.8% in the T2 section and 14.7% in the T3 section when compared to the normal control group. These values also show statistical differences compared to those of the virus-infected group. In addition, a considerable portion of nerve bundles within the lamina propria were depleted in the zinc sulfate group, resulting in a looser lamina propria compared with that in the other two groups (Fig. 5A-C). In summary, both the virus-infected and zinc sulfate groups displayed OE atrophy in the nasal cavities, with the zinc sulfate group demonstrating more severe atrophy. The OE also exhibited microscopic differences (Fig. 5E). The OE is normally lined with microvilli and possesses an abundance of cytoplasmic content. In comparison, the OE of the zinc sulfate group exhibited a loss of microvilli and cytoplasmic deficiency.

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Morphological alterations in the OE and lamina propria of nasal mucosa collected at 5 DPI. Representative histopathological images of H&E-stained T2 (upper) and T3 (lower) sections of the OE and lamina propria from the normal control group (A), virus-infected group (B), and zinc sulfate group (C). Nerve bundles (red asterisks) are presented in both normal control and virus-infected groups. (D) Bar graphs comparing the thickness of the OE at T2 and T3 sections from each group, normalized to the normal control group (normal control group; n = 5, virus-infected group; n = 5, zinc sulfate group; n = 5). Data are presented as mean ± SEM. p-values are indicated above the plots: *p < 0.05; **p < 0.01 ***p < 0.001. (E) Representative histopathological images of H&E-stained OE from each group, at T3 sections of the nasal cavities. Scale bars, 50 μm (A-C), 25 μm (E).

Zinc sulfate inoculation induced a complete reduction of OMP-expressing cells in the nasal mucosa

Antigen immunodetection was conducted with nasal bone samples collected at 5 DPI to examine the cellular changes in the OE. First, the OMP antigen in the OE was detected to visualize the OSNs (Fig. 6 and Fig. S1). Compared with the normal control group (Fig. 6A), the virus-infected group displayed a minimal degree of impairment in the OSNs (Fig. 6B). This is believed to be attributable to SARS-CoV-2 infection. In contrast, the zinc sulfate group displayed a dramatic reduction in OMP-expressing cells in both the OE and lamina propria (Fig. 6C). This indicates that zinc sulfate inoculation resulted in the removal of OSNs and nerve bundles from the nasal cavities.

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Immunofluorescence staining of different antigens in the nasal mucosa collected at 5 DPI. Representative immunofluorescence images of the OE from the normal control group (A, D), virus-infected group (B, E), and zinc sulfate group (C, F). Each antigen is visualized as follows; OMP (red), CK5 (red), and CK8 (green). Nuclei are stained with DAPI (blue). The cilia of intact OSNs at the top of the OE were detectable as red in the normal control group (A). The zinc sulfate group exhibited a significant reduction in OMP-expressing cells in the OE and lamina propria compared to the other two groups, while CK8-expressing cells showed changes in size, particularly in cell width (C, F). Scale bars, 50 μm.

Detection of CK8 in the OE revealed that the number of sustentacular cells was maintained in both the virus-infected and zinc sulfate groups (Fig. 6B and C). However, the sustentacular cells in both the virus-infected and zinc sulfate groups exhibited reduced cellular height and loss of microvilli, with this change more prominent in the zinc sulfate group (Fig. 6C). Additionally, both virus-infected and zinc sulfate groups displayed an increase in CK5-expressing cells (Fig. 6D and F), suggesting that the horizontal basal cells which express CK5 were augmented as part of a recovery mechanism following both SARS-CoV-2 infection and zinc sulfate inoculation28. Also, the removal of the OSNs in the OE of the zinc sulfate group caused a loosening of the cellular structure, resulting in alterations in the cellular morphology of the OE.

Persistent SARS-CoV-2 infection within the OE is positively linked to the incidence of brain infection

To explore the viral pathway by which SARS-CoV-2 reaches the brain through the OE, we assessed the presence of the virus in the organs of mice sacrificed at 2 and 5 DPI. Examination of SARS-CoV-2 in T2 sections of the nasal cavity indicated that viral infection in the OE occurred at 2 DPI in both the virus-infected and zinc sulfate groups (Fig. 7A and Fig. S2). In contrast, at 5 DPI, the virus was not detected in the OE of the zinc sulfate group, whereas the virus-infected group exhibited viral antigens in the OE. These findings may be related to viral detection in the cerebral cortex at 2 and 5 DPI (Fig. 7B). In cases where the viral presence persisted until 5 DPI, it was likely to be detected in the CNS. However, if, as observed in the zinc sulfate group, the virus was no longer detected at 5 DPI, it was not detected in the brain tissues. In addition, OE destruction by zinc sulfate inoculation inhibited SARS-CoV-2 invasion into cerebral tissues, while the virus remained detectable in lung tissues (Fig. 7C). Therefore, it can be concluded that the persistence of SARS-CoV-2 in the OE can influence the potential for viral invasion of the brain.

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Detection of SARS-CoV-2 antigen across various tissues at different DPI. Representative immunohistochemical images of the OE (A), brain (B), and lungs (C) of each group at 2 (upper) and 5 (lower) DPI. The presence of the SARS-CoV-2 antigen is compared between groups. The SARS-CoV-2 antigen was stained with the DAB reagent (brown). The slides were counterstained with hematoxylin (purple). Scale bars, 50 μm.

Discussion

SARS-CoV-2 is the most recently identified neurotropic betacoronavirus in humans, and not much of its pathological mechanism has been elucidated thus far29,30. Prior research has primarily focused on understanding the viral mechanisms responsible for viral invasion of the brain. Even though neurological symptoms are attributed primarily to the host’s immune activation rather than the virus’s direct neurotropic properties, studying the potential of SARS-CoV-2 for neuroinvasion is still vital. Currently, the direct invasion by SARS-CoV-2 through the OE is considered the most plausible and influential hypothesis. In this context, our objective was to explore the precise mechanisms by which the OE is related to the neuroinvasive traits of SARS-CoV-2. To achieve this, we used zinc sulfate to disrupt OSNs in K18-hACE2 transgenic mice and examined whether SARS-CoV-2 was able to invade brain tissue. To provide a clearer comparison of mortality in the affected mice based on viral neuroinvasiveness, we utilized the Wuhan strain (NCCP 43326) rather than the Omicron variants, which exhibit less virulence in these animals31.

The OE comprises various cell types, including sustentacular cells and OSNs32. Sustentacular cells are columnar cells attached to the basal lamina of the OE. They are also referred to as supporting cells, as they offer structural and metabolic assistance to the OSNs they enclose33. OSNs, being bipolar neurons, receive olfactory signals from odorants34. Their dendritic portions are projected to the apical surface of the OE, while the axons are gathered to form nerve bundles within the lamina propria beneath the OE35. The olfactory nerves pass through the cribriform plate and establish a connection to the outermost layer of the olfactory bulb. The dendrites of OSNs accept the olfactory signal, which is then transported to the olfactory bulb following the olfactory nerve pathway36.

Several neurotropic coronaviruses use this olfactory nerve pathway to invade the central nervous system, as reported in previously published research34,37–40. Likewise, following intranasal inoculation, SARS-CoV-2 is likely transported to the olfactory bulbs following the olfactory nerve pathway after entering the olfactory mucosa. This mechanism was postulated based on accumulating evidence from studies verifying the presence of SARS-CoV-2 in the olfactory nervous tract41,42. Given that SARS-CoV-2 uses the ACE2 receptor for cell entry, the olfactory nerve pathway exploited by the virus appears to involve cells expressing hACE2 within the OE of K18-hACE2 transgenic mice. However, the OSNs of K18-hACE2 mice are not the main entry point for SARS-CoV-2 because they do not express hACE2 (Fig. S3). Among the epithelial cells of the OE, hACE2 is predominantly expressed in non-neuronal cells, including sustentacular cells, making them the primary entrance for SARS-CoV-243–45. Consequently, the virus predominantly infects non-neuronal cells within the OE via the hACE2 receptor, with the presumption that it subsequently travels along the route of OSNs, finally reaching the olfactory bulb46.

In this study, we present additional evidence regarding the neuroinvasive pathway of SARS-CoV-2 through the olfactory nervous system, as illustrated in Fig. 8. The persistent presence of the virus within the OE appeared to be crucial for the infection of the brain by SARS-CoV-2, as it facilitated viral infection of OSNs. This contrasts with the observations made in the zinc sulfate group. Intranasal inoculation with zinc sulfate solution led to OE damage, particularly the removal of OSNs, while leaving sustentacular cells relatively unaffected. As confirmed by our results, viral infection within the OE proceeded normally; however, transmission of the virus to the brain did not occur in mice lacking OSNs. Restricting subsequent viral transport to OSNs was vital for defending against brain infection by SARS-CoV-2. This finding implies that brain invasion by intranasally introduced SARS-CoV-2 is mediated by the presence of OSNs despite lack of hACE2 in these cells, leading to the conclusion that the virus travels along the olfactory nerve pathway to reach the brain.

An external file that holds a picture, illustration, etc. Object name is 41598_2024_78538_Fig8_HTML.jpg

Schematic diagram illustrating the impact of OSN removal on the transmission of SARS-CoV-2 to the brain. The mouse OE consists of various cell types, including OSNs, sustentacular cells, and horizontal basal cells, as represented in the diagram. Upon intranasal infection, SARS-CoV-2 enters the OE through cells expressing hACE2, primarily the sustentacular cells. Subsequently, the virus is transmitted via the olfactory nervous system to reach the olfactory bulb. When the mouse is subjected to zinc sulfate inoculation, significant alterations occur in the nasal mucosa due to OSN removal. In the OE, most OSNs become eliminated, leading to morphological changes in sustentacular cells and horizontal basal cells. Nerve bundles within the lamina propria are also removed. When the mouse lacking OSNs is infected with SARS-CoV-2, the virus is detected only in the sustentacular cells, and no further transmission into the brain occurs.

From these results, we may carefully speculate the significance of the viral route through the olfactory nervous system in terms of brain invasion by SARS-CoV-2. In the zinc sulfate group, the viral route via the OE was believed to be obstructed via damage to the OSNs through inoculation with zinc sulfate solution. The general amelioration of the pathological conditions resulting from SARS-CoV-2 infection observed in the zinc sulfate group implies that brain invasion by the virus was noticeably hindered. This is further supported by previous research reporting a correlation between viral infection in brain tissues and survival in K18-hACE2 transgenic mice47. Therefore, early inhibition of the OE route resulted in a reduction in mortality associated with SARS-CoV-2 infection, indicating that brain invasion through the OE plays a significant role in the neuroinvasive mechanisms of SARS-CoV-2.

In summary, the K18-hACE2 transgenic mouse model, in which OSNs were removed through zinc sulfate inoculation, exhibited resistance to a lethal dose of SARS-CoV-2 infection. Our study demonstrates that the OE, specifically OSNs, facilitate viral transmission to the CNS and that this pathway is a major contributor to the neuroinvasion of SARS-CoV-2 in K18-hACE2 transgenic mice. Additionally, early intervention to prevent brain infection through this crucial pathway can halt the deadly progression of the disease, providing insights into the development of novel treatment strategies for SARS-CoV-2-related neurological diseases. However, it is important to note that the findings in K18-hACE2 mice may not be directly generalizable to human cases. Also, the mechanisms by which SARS-CoV-2 gains access to the olfactory nervous system following entry into the OE via non-neuronal cells need to be explored. Further investigation is necessary to deepen our understanding of the specific mechanisms of the cells in the OE concerning SARS-CoV-2 transmission as various speculations can be made. For example, potential interactions between sustentacular cells and OSNs should be considered48,49. With growing interest in the research field of the olfactory system, we anticipate that our data will contribute to elucidation of SARS-CoV-2 pathogenesis.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Acknowledgements

All authors thank Ji-Hoon Han at Koatech Inc. for providing the graphical summary.

Author contributions

Conceptualization: J.-H.L., E.-S.Y., N.-W.K., W.-Y.S., H.-B.J., D.-H.K., Y.-J.P., S.-M.S.; Data curation: J.-H.L.; Formal analysis: J.-H.L., J.-W.Y., J.-W.P., K.-S.C., H.-Y.L., J.-Y.S., K.-T.N., J.-K.S., Y.-K.C.; Funding acquisition: J.-K.S., Y.-K.C.; Investigation: J.-H.L., E.-S.Y., N.-W.K., W.-Y.S., H.-B.J., D.-H.K., Y.-J.P., S.-M.S.; Methodology: J.-H.L., E.-S.Y., N.-W.K., W.-Y.S., H.-B.J., D.-H.K., Y.-J.P., S.-M.S., J.-W.Y., J.-W.P., K.-S.C., H.-Y.L., J.-Y.S., K.-T.N.; Project administration: J.-H.L., J.-K.S., Y.-K.C.; Resources: J.-H.L., Y.-K.C.; Supervision: J.-K.S., Y.-K.C.; Writing – original draft: J.-H.L., Y.-K.C.; Writing – review & editing: J.-H.L., E.-S.Y., N.-W.K., W.-Y.S., H.-B.J., D.-H.K., Y.-J.P., S.-M.S., J.-W.Y., J.-W.P., K.-S.C., H.-Y.L., J.-Y.S., K.-T.N., J.-K.S., Y.-K.C.

Funding

This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Korean government (MSIT) (Nos. 2021M3H9A1030260 and 2021M3H9A1097269).

Data availability

Data cannot be shared openly but are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Acknowledgments.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Je Kyung Seong, rk.ca.uns@esuomuns.

Yang-Kyu Choi, rk.ca.kuknok@cuykgnay.

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Bio &amp; Medical Technology Development Program of the National Research Foundation (NRF), funded by the Korean government (MSIT) (2)