Supplementing Immunostaining with Complementary Methods for a Holistic Understanding of the Mechanisms Behind COVID-19-Induced Anosmia

Introduction 

The exact mechanisms behind COVID-19-induced anosmia, or the complete loss of smell, remain unclear due to conflicting findings that fail to establish a direct and consistent link between viral presence, cellular damage, and the subsequent disruption of olfactory function and inflammation within the olfactory system. Anosmia affects nearly 19% of COVID-19 patients, lasting an average of two months after recovery, significantly reducing their quality of life [1]. Fortunately, prior research on SARS-CoV, the closest known relative of SARS-CoV-2, provides scientists with a valuable foundation for understanding the development and progression of COVID-19-related symptoms. SARS-CoV relies on the angiotensin-converting enzyme 2 (ACE2) receptor for entry into human cells [2]. Similarly, ACE2 has been identified as a receptor for the spike proteins on the surface of SARS-CoV-2, establishing a critical starting point for scientists to investigate the infection pathway within the olfactory epithelium [3].

Despite this foundation, the precise mechanisms underlying COVID-19-induced anosmia remain unclear, with multiple competing hypotheses proposed. Through inhalation, odorants from the air enter the nose, reaching a tissue called the olfactory epithelium (OE). One leading theory suggests that SARS-CoV-2 may directly impact the OE by infecting sustentacular cells, which express ACE2 receptors [4]. The infection of these cells may impair their supportive role to olfactory sensory neurons, potentially disrupting olfactory function and resulting in anosmia [4]. Alternatively, some researchers have proposed that SARS-CoV-2 may directly infect the olfactory sensory neurons themselves, bypassing sustentacular cells to induce a loss of smell [5]. Yet another hypothesis points to direct viral infiltration into the brain through the olfactory bulb (OB), leading to disruptions in olfactory processing [6]. While these theories are not mutually exclusive, they collectively showcase the complexity of anosmia as a COVID-19 symptom and emphasize the need for further research to delineate the precise pathophysiological processes involved. 

This review examines a key challenge in current research on the mechanisms underlying COVID-19-induced anosmia and SARS-CoV-2 infection patterns, initially focusing on the findings from two of the four studies reviewed. The varying conclusions regarding the specific cell types infected by SARS-CoV-2, as reported by Bryche et al. (2020) and Zhang et al. (2021), can be attributed to the limited scope of their methodologies, which primarily rely on histological staining and immunohistochemistry after intranasal administration of the virus in golden Syrian hamsters. Bryche et al. (2020) concluded that sustentacular cells within the olfactory epithelium are the primary targets of SARS-CoV-2, finding no evidence of infection in olfactory sensory neurons. This contradicts the findings of Zhang et al. (2021), which identified olfactory sensory neurons as also being infected by the virus. These articles both attribute anosmia to a single cell type or a narrow component of the olfactory system, despite the complex, multi-cellular nature of olfactory dysfunction. Notably, Zhang et al. (2021) included tissue samples from both male and female hamsters and employed RT-qPCR (quantitative reverse transcription polymerase chain reaction) to measure the expression levels of key chemokines and cytokines, or secreted proteins that coordinate immune responses, providing a more comprehensive dataset compared to Bryche et al. However, these two studies largely overlook or dismiss the potential infection or inflammation of the OB, a crucial region for odor processing. In contrast, the other two reviewed studies offer a more comprehensive approach, providing valuable insights into the potential role of the OB in COVID-19-related anosmia. Ueha et al. (2022) utilized oral inoculation of SARS-CoV-2, exploring its potential to induce nasal viral infection, which may subsequently spread to the brain, a methodology distinct from the nasal administration approach used in other studies. Similarly, de Melo et al. (2021) expanded their investigation by incorporating human patient data alongside hamster models and employing additional food-finding experiments to confirm the presence of anosmia in infected hamsters, a factor often neglected in other studies that rely solely on viral detection. However, while these innovative methodologies enhance our understanding of SARS-CoV-2's impact on the olfactory system and provide new insights into the potential role of the OB in anosmia among COVID-19 patients, further research is necessary to validate these findings through replication and the use of larger sample sizes to ensure their reliability. 

Discussion 

Methodological Limitations of Immunostaining in COVID-19-Induced Anosmia 

A significant challenge in understanding COVID-19-induced anosmia lies in the methodological shortfalls of existing studies, as demonstrated by Bryche et al. (2020) and Zhang et al. (2021). While Zhang et al. (2021) address sex biases by utilizing tissue samples from both male and female hamsters in their staining methods, they still reveal inconsistencies in understanding the virus’s infection mechanism. Although both studies agree that SARS-CoV-2

infects sustentacular cells, Zhang et al. (2021) also report infection of olfactory sensory neurons, adding complexity to the infection profile. Furthermore, Zhang et al. (2021) incorporated chemokine and cytokine profiling into their experiment, confirming a pro-inflammatory status, which Bryche et al. (2020) did not assess. However, neither study considers the OB as a potential contributor to olfactory dysfunction, a critical oversight when attempting to comprehensively address the underlying causes of COVID-19-induced anosmia. 

Although Bryche et al. (2020) and Zhang et al. (2021) both employed immunostaining on tissue samples from hamsters nasally inoculated with SARS-CoV-2 to identify infection sites, their differing conclusions indicate the limits of relying exclusively on these methods. Bryche et al. (2020) utilized an antibody targeting the nucleocapsid protein of SARS-CoV-2 and detected its presence in sustentacular cells of the OE, suggesting that these cells are the primary targets of the virus. They also emphasize that olfactory sensory neurons (OSNs) are not directly infected by SARS-CoV-2, as there is an absence of viral antigen in these cells [7]. This starkly contrasts with the findings of Zhang et al. (2021), who identified direct infection of both immature olfactory sensory neurons (iOSNs) and mature olfactory sensory neurons (mOSNs), alongside sustentacular cells, through detection of the nucleocapsid protein. A critical distinction lies in the sex biases inherent in the two studies. Bryche et al. (2020) conducted their analysis using only female hamsters, potentially skewing their findings. In contrast, Zhang et al. (2021) addressed this limitation by utilizing tissue samples from both male and female hamsters, providing a more comprehensive view of SARS-CoV-2 infection patterns. This difference highlights the importance of considering sex as a factor in research, particularly when relying on small sample sizes. Both studies were constrained by limited hamster cohorts, and given the difficulty of significantly increasing sample sizes, alternative methodologies are needed to complement immunofluorescence and immunohistochemistry techniques, ensuring more consistent and reliable results. 

Despite the differing results observed by Bryche et al. (2020) and Zhang et al. (2021), both studies share the fact that they failed to provide conclusive evidence of SARS-CoV-2 infection in the OB, accentuating, once again, the limitations in their methodologies. Bryche et al. (2020), despite staining brain tissue collected from golden Syrian hamsters, found no indication of direct viral infection in the OB. Similarly, Zhang et al. (2021) did not detect nucleocapsid protein in OB tissue from their own hamsters. Zhang et al. (2021) determined the expression levels of chemokine- and cytokine-encoding genes in an attempt to offer a broader view of the infection mechanism. However, despite these advancements, Zhang et al. (2021) still rely on the same methodology as Bryche et al. (2020) in examining brain tissue, which may not be sufficient to conclusively rule out the OB as an affected region. While Bryche et al. (2020) and Zhang et al. (2021) at least acknowledge the OB as a potential site of SARS-CoV-2 infection in the context of COVID-19-induced anosmia, they do not offer substantial evidence to support this hypothesis [7, 8]. The use of nearly identical methodologies for analyzing both nasal and brain tissues in the studies by Bryche et al. (2020) and Zhang et al. (2021) raises concerns about the validity of their conclusion that SARS-CoV-2 does not infect the OB when considering their data alone. This is particularly noteworthy given that the OB is largely surrounded by the blood-brain barrier, a protective multicellular structure that controls the passage of substances from the bloodstream into brain tissue, which may influence the virus's ability to invade this region differently than it does inside the nose (Bryche et al., 2020). To fully understand the methods underlying COVID-19-induced anosmia, supplementary experiments that are specific to the OB are essential to either substantiate or refute its involvement in olfactory dysfunction.

In summary, while studies by Bryche et al. (2020) and Zhang et al. (2021) collectively provide valuable insights into the infection patterns of SARS-CoV-2 in the OE, they all share significant methodological limitations that hinder a clear understanding of COVID-19-induced anosmia. The inconsistent findings across these studies, particularly regarding the types of infected cells and the lack of evidence for OB involvement, stress the inadequacy of relying solely on immunostaining methods. Moreover, the small sample sizes (12 or fewer) and sex biases present in these studies further compromise the robustness of their conclusions. These methodological shortcomings underscore the urgent need for more comprehensive and multi-experimental approaches, including those that specifically target the OB, in order to clarify the precise mechanisms behind olfactory dysfunction in COVID-19 and inform future research on potential therapeutic interventions. 

Role of Oral Inoculation, Behavioral Assays, and Additional Patient Data in Enhancing Understanding of COVID-19-Induced Anosmia Mechanisms Related to the Olfactory Bulb  

Though prior research has identified potential sites of SARS-CoV-2 infection, inconsistencies in findings, largely due to methodological limitations, have created ambiguity regarding the virus's pathogenesis and the mechanisms underlying COVID-19-induced anosmia. Ueha et al. (2022) and de Melo et al. (2021) contribute to the understanding of the OB’s role in this olfaction loss by incorporating innovative approaches to immunohistochemistry and histological staining, offering a more comprehensive understanding of COVID-19-induced anosmia. Additionally, both studies highlight brain infection and inflammation as potential mechanisms contributing to anosmia. However, to validate these findings, future research must replicate these methods with larger sample sizes to ensure dependable and generalizable results.

Both Ueha et al. (2022) and de Melo et al. (2021) employ innovative methodologies that build upon the conventional immunohistochemistry and histological staining techniques, allowing for a more comprehensive investigation into the effects of SARS-CoV-2 on the olfactory system. Ueha et al. (2022) administered the SARS-CoV-2 virus to their hamsters through oral inoculation to better model the actual spread of the disease in the real world. They were able to then utilize RT-qPCR to confirm infection in the lungs and immunohistochemistry to confirm viral presence in parts of the OE [9]. De Melo et al. (2021), however, took their methodology further by incorporating a critical behavioral assay that directly assessed anosmia in their hamster models, ensuring that the infection led to measurable olfactory dysfunction. A sucrose preference test showed that infected hamsters had no preference for sucrose-supplemented water, whereas uninfected hamsters preferred it [10]. Additionally, infected hamsters took longer or failed to find hidden food compared to their non-infected counterparts; however, both groups were able to locate visible food [10]. These results demonstrate that infected hamsters exhibited impaired ability to find food due to olfactory dysfunction rather than visual or motor deficits [10]. Additionally, de Melo et al. (2021) enhanced the study’s relevance to humans by including patient nasal tissue samples, comparing the viral infection patterns in humans with those observed in hamsters. This added layer of analysis provided important insight into how SARS-CoV-2 impacts olfactory function across species. In contrast, while Ueha et al. (2022) focused primarily on the mechanistic details of the infection process without directly assessing anosmia in their hamster model, de Melo et al.’s (2021) inclusion of behavioral testing ensures that the observed viral effects are indeed linked to olfactory dysfunction, which makes their findings more robust and directly applicable to the study of COVID-19-induced anosmia in humans. 

Despite differing methods, a key area of investigation in both Ueha et al. (2022) and de Melo et al. (2021) was the impact of SARS-CoV-2 on the OB, which was absent in prior studies. Both articles provide compelling evidence that the virus does affect this region, though their findings differ in terms of localization and the cellular mechanisms involved. Ueha et al. (2022) reported evidence of SARS-CoV-2 infection in the olfactory nerve bundles that connect the olfactory mucosa to the OB, highlighting the potential for viral propagation through the olfactory sensory pathway. On the other hand, de Melo et al. (2021) identified SARS-CoV-2 in olfactory nerve bundles near the neuroepithelium. Their observation that viral nucleoprotein co-localized with olfactory marker protein (OMP)-positive sensory neuron axons suggests that the virus may infect olfactory sensory neurons (OSNs) and travel backward along their axons toward the OB. Moreover, de Melo et al. (2021) detected SARS-CoV-2 nucleoprotein at the junction between the olfactory nerve and the OB, indicating potential infection of cells with neuronal or glial morphology. Together, these studies highlight the olfactory nerve as a key route for viral spread. However, the findings of de Melo et al. (2021) provide more detailed evidence for retrograde neuronal infection, lending further support to the idea that the OB also plays an important role in COVID-19-induced anosmia. 

To sum up, Ueha et al. (2022) and de Melo et al. (2021) contribute valuable insights into the pathogenesis of COVID-19-induced anosmia by addressing distinct methodological and scientific gaps in previous research. The incorporation of behavioral assays and human tissue samples by de Melo et al. (2021) enhances the clinical relevance of their findings, while the work of Ueha et al. (2022), which emphasizes the oral route leading to viral spread in the olfactory pathway, adds an important real-world application. Both studies contribute to the growing body of evidence that suggests SARS-CoV-2 affects the olfactory system, including the OB, through direct infection of sensory neurons and retrograde transmission along the olfactory nerve. However, additional research, including larger sample sizes and PET scan imaging, is required to fully determine the role of the OB in the broader context of COVID-19-related anosmia [10]. 

Conclusion

In conclusion, the investigation into the mechanisms underlying COVID-19-induced anosmia has revealed significant advancements, but substantial gaps in understanding remain. Early studies, such as those by Bryche et al. (2020) and Zhang et al. (2021), provide useful insights into infection location but are hindered by methodological limitations like small sample sizes, sex biases, and an overreliance on histological techniques, which led to inconsistent findings about which cells are infected by SARS-CoV-2 and whether the OB is involved. These studies primarily focus on specific cell types located in the nose, but fail to explore the OB's role in anosmia. In contrast, Ueha et al. (2022) and de Melo et al. (2021) employ more comprehensive methods. Ueha et al.’s (2022) oral inoculation in hamsters provides new mechanistic insights into viral spread via the olfactory sensory pathway, while de Melo et al. (2021) confirm anosmia through behavioral assays and show SARS-CoV-2 infection in sensory neurons, suggesting retrograde transmission to the OB. Their findings strengthen the evidence that the OB plays a critical role in anosmia, a key area overlooked in the prior research. Despite these advances, both Ueha et al. (2022) and de Melo et al. (2021) would benefit from larger sample sizes and replication of their methods. Further research is needed to fully explore the OB's involvement in COVID-19-related anosmia, including in-depth imaging techniques for direct evidence of viral spread in the brain. Studies should also address sex biases by including both male and female samples and broader species variations. Ultimately, while recent studies provide important insights, more rigorous research is needed to clarify the mechanisms underlying COVID-19-induced anosmia and guide effective treatment strategies. Although interventions such as nasal injections of platelet-rich plasma and olfactory training have been employed to reverse long-term smell loss, their outcomes have varying degrees of success [11]. A deeper understanding of the condition is essential for developing targeted therapies and improving patient outcomes.

Author’s Note

This literature review was initially written as part of a UWP 104E assignment; however, the topic aligns closely with my own research interests and work in the Gong Lab, which focused on identifying immune cells in the olfactory bulb of mice infected with SARS-CoV-2 and examining the possible connections to Alzheimer’s disease. I chose to examine COVID-19-induced anosmia due to the significant scientific debate surrounding its underlying mechanisms and the broader implications for understanding viral impacts on the nervous system. The intended audience for this piece is primarily specialists and researchers in the fields of neuroscience, virology, and sensory biology. Through this review, I aim to highlight the current uncertainty and conflicting findings regarding how SARS-CoV-2 leads to anosmia and which structures in the olfactory pathway are involved, particularly the olfactory bulb. In particular, I emphasize the limitations of relying solely on immunohistochemistry data and advocate for the integration of additional experimental approaches to achieve a more precise and comprehensive understanding of this symptom. 

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