Through The Back Door: Expiratory Accumulation of SARS-Cov-2 in the Olfactory Mucosa as Mechanism for CNS Penetration.

Introduction: SARS-CoV-2 is a respiratory virus supposed to enter the organism through aerosol or fomite transmission to the nose, eyes and oropharynx. It is responsible for various clinical symptoms, including hyposmia and other neurological ones. Current literature suggests the olfactory mucosa as a port of entry to the CNS, but how the virus reaches the olfactory groove is still unknown. Because the first neurological symptoms of invasion (hyposmia) do not correspond to first signs of infection, the hypothesis of direct contact through airborne droplets during primary infection and therefore during inspiration is not plausible. The aim of this study is to evaluate if a secondary spread to the olfactory groove in a retrograde manner during expiration could be more probable. Methods: Four three-dimensional virtual models were obtained from actual CT scans and used to simulate expiratory droplets. The volume mesh consists of 25 million of cells, the simulated condition is a steady expiration, driving a flow rate of 270 ml/s, for a duration of 0.6 seconds. The droplet diameter is of 5 µm. Results: The analysis of the simulations shows the virus to have a high probability to be deployed in the rhinopharynx, on the tail of medium and upper turbinates. The possibility for droplets to access the olfactory mucosa during the expiratory phase is lower than other nasal areas, but consistent. Discussion: The data obtained from these simulations demonstrates the virus can be deployed in the olfactory groove during expiration. Even if the total amount in a single act is scarce, it must be considered it is repeated tens of thousands of times a day, and the source of contamination continuously acts on a timescale of several days. The present results also imply CNS penetration of SARS-CoV-2 through olfactory mucosa might be considered a complication and, consequently, prevention strategies should be considered in diseased patients.

Respiratory syncytial virus tropism for olfactory sensory neurons in mice.

The olfactory mucosa, where the first step of odor detection occurs, is a privileged pathway for environmental toxicants and pathogens toward the central nervous system. Indeed, some pathogens can infect olfactory sensory neurons including their axons projecting to the olfactory bulb allowing them to bypass the blood-brain barrier and reach the central nervous system (CNS) through the so-called olfactory pathway. The respiratory syncytial virus (RSV) is a major respiratory tract pathogen but there is growing evidence that RSV may lead to CNS impairments. However, the mechanisms involved in RSV entering into the CNS have been poorly described. In this study, we wanted to explore the capacity of RSV to reach the CNS via the olfactory pathway and to better characterize RSV cellular tropism in the nasal cavity. We first explored the distribution of RSV infectious sites in the nasal cavity by in vivo bioluminescence imaging and a tissue clearing protocol combined with deep-tissue imaging and 3D image analyses. This whole tissue characterization was confirmed with immunohistochemistry and molecular biology approaches. Together, our results provide a novel 3D atlas of mouse nasal cavity anatomy and show that RSV can infect olfactory sensory neurons giving access to the central nervous system by entering the olfactory bulb. Cover Image for this issue: doi: 10.1111/jnc.14765.

Evaluation of the effect of cigarette smoking on the olfactory neuroepithelium of New Zealand white rabbit, using scanning electron microscope.

To detect ultra-structural changes of Rabbit's olfactory neuro-epithelium using scanning electron microscope after exposure to cigarette smoking. Sixty six rabbits (Pathogen free New Zealand white rabbits weighing 1-1.5 kg included in the study were randomly assigned into one of three groups: control group did not expose to cigarette smoking, study group 1 was exposed to cigarette smoking for 3 months and study group 2 was exposed to cigarette smoking 3 months and then stopped for 2 months. Olfactory neuro-epithelium from all rabbits were dissected and examined under Philips XL-30 scanning electron microscope. Changes that were found in the rabbits of study group 1 in comparison to control group were loss of microvilli of sustentacular cells (p = 0.016) and decreases in distribution of specialized cilia of olfactory receptor cells (p = 0.046). Also respiratory metaplasia was detected. These changes were reversible in study group 2. Cigarette smoking causes ultra-structural changes in olfactory neuro-epithelium which may explain why smell was affected in cigarette smokers. Most of these changes were reversible after 45 days of cessation of cigarette smoking to the rabbits.

"Mucosal maps" of the canine nasal cavity: Micro-computed tomography and histology.

Nasal turbinals, delicate and complex bones of the nasal cavity that support respiratory or olfactory mucosa (OM), are now easily studied using high resolution micro-computed tomography (µ-CT). Standard µ-CT currently lacks the capacity to identify OM or other mucosa types without additional radio-opaque staining techniques. However, even unstained mucosa is more radio-opaque than air, and thus mucosal thickness can be discerned. Here, we assess mucosal thickness of the nasal fossa using the cranium of a cadaveric adult dog that was µ-CT scanned with an isotropic resolution of 30 µm, and subsequently histologically sectioned and stained. After co-alignment of µ-CT slice planes to that of histology, mucosal thickness was estimated at four locations. Results based on either µ-CT or histology indicate olfactory mucosa is thicker on average compared with non-olfactory mucosa (non-OM). In addition, olfactory mucosa has a lesser degree of variability than the non-OM. Variability in the latter appears to relate mostly to the varying degree of vascularity of the lamina propria. Because of this, in structures with both specialized vascular respiratory mucosa and OM, such as the first ethmoturbinal (ET I), the range of thickness of OM and non-OM may overlap. Future work should assess the utility of diffusible iodine-based contrast enhanced CT techniques, which can differentiate epithelium from the lamina propria, to enhance our ability to differentiate mucosa types on more rostral ethmoturbinals. This is especially critical for structures such as ET I, which have mixed functional roles in many mammals.

Post-viral effects of COVID-19 in the olfactory system and their implications.

BACKGROUND: The mechanisms by which any upper respiratory virus, including SARS-CoV-2, impairs chemosensory function are not known. COVID-19 is frequently associated with olfactory dysfunction after viral infection, which provides a research opportunity to evaluate the natural course of this neurological finding. Clinical trials and prospective and histological studies of new-onset post-viral olfactory dysfunction have been limited by small sample sizes and a paucity of advanced neuroimaging data and neuropathological samples. Although data from neuropathological specimens are now available, neuroimaging of the olfactory system during the acute phase of infection is still rare due to infection control concerns and critical illness and represents a substantial gap in knowledge. RECENT DEVELOPMENTS: The active replication of SARS-CoV-2 within the brain parenchyma (ie, in neurons and glia) has not been proven. Nevertheless, post-viral olfactory dysfunction can be viewed as a focal neurological deficit in patients with COVID-19. Evidence is also sparse for a direct causal relation between SARS-CoV-2 infection and abnormal brain findings at autopsy, and for trans-synaptic spread of the virus from the olfactory epithelium to the olfactory bulb. Taken together, clinical, radiological, histological, ultrastructural, and molecular data implicate inflammation, with or without infection, in either the olfactory epithelium, the olfactory bulb, or both. This inflammation leads to persistent olfactory deficits in a subset of people who have recovered from COVID-19. Neuroimaging has revealed localised inflammation in intracranial olfactory structures. To date, histopathological, ultrastructural, and molecular evidence does not suggest that SARS-CoV-2 is an obligate neuropathogen. WHERE NEXT?: The prevalence of CNS and olfactory bulb pathosis in patients with COVID-19 is not known. We postulate that, in people who have recovered from COVID-19, a chronic, recrudescent, or permanent olfactory deficit could be prognostic for an increased likelihood of neurological sequelae or neurodegenerative disorders in the long term. An inflammatory stimulus from the nasal olfactory epithelium to the olfactory bulbs and connected brain regions might accelerate pathological processes and symptomatic progression of neurodegenerative disease. Persistent olfactory impairment with or without perceptual distortions (ie, parosmias or phantosmias) after SARS-CoV-2 infection could, therefore, serve as a marker to identify people with an increased long-term risk of neurological disease.

Olfactory Cleft Measurements and COVID-19-Related Anosmia.

OBJECTIVE: This study aimed to investigate the differences in olfactory cleft (OC) morphology in coronavirus disease 2019 (COVID-19) anosmia compared to control subjects and postviral anosmia related to infection other than severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). STUDY DESIGN: Prospective. SETTING: This study comprises 91 cases, including 24 cases with anosmia due to SARS-CoV-2, 38 patients with olfactory dysfunction (OD) due to viral infection other than SARS-CoV-2, and a control group of 29 normosmic cases. METHODS: All cases had paranasal sinus computed tomography (CT), and cases with OD had magnetic resonance imaging (MRI) dedicated to the olfactory nerve. The OC width and volumes were measured on CT, and T2-weighted signal intensity (SI), olfactory bulb volumes, and olfactory sulcus depths were assessed on MRI. RESULTS: This study showed 3 major findings: the right and left OC widths were significantly wider in anosmic patients due to SARS-CoV-2 (group 1) or OD due to non-SARS-CoV-2 viral infection (group 2) when compared to healthy controls. OC volumes were significantly higher in group 1 or 2 than in healthy controls, and T2 SI of OC area was higher in groups 1 and 2 than in healthy controls. There was no significant difference in olfactory bulb volumes and olfactory sulcus depths on MRI among groups 1 and 2. CONCLUSION: In this study, patients with COVID-19 anosmia had higher OC widths and volumes compared to control subjects. In addition, there was higher T2 SI of the olfactory bulb in COVID-19 anosmia compared to control subjects, suggesting underlying inflammatory changes. There was a significant negative correlation between these morphological findings and threshold discrimination identification scores. LEVEL OF EVIDENCE: Level 4.

Olfactory dysfunction in allergic rhinitis.

BACKGROUND: Olfactory dysfunction in patients with allergic rhinitis has long been thought to be secondary to coexisting chronic rhinosinusitis and polyposis with obstruction of airflow over the olfactory epithelium. Recent evidence suggests that the allergic inflammatory infiltrate may itself affect olfaction in the absence of mucosal hypertrophy. OBJECTIVE: We undertook a study to determine olfactory function in patients with allergic rhinitis in the presence and absence of chronic sinusitis. METHODS: Fifty-one subjects with symptoms of rhinitis who presented for allergy testing were administered the University of Pennsylvania Smell Identification Test. In addition each patient underwent computed tomography (CT) scanning of the sinuses. RESULTS: Eighty percent of subjects were allergic. Subjects with allergic rhinitis and no evidence of sinusitis scored on average in the 30th percentile (95% CI 20-40th percentile) on objective olfactory testing compared to age- and gender-specific norms. Half the allergic patients were classified as normosmic, while half had some degree of hyposmia. CONCLUSIONS: Our study demonstrates that even in the absence of mucosal disease on CT scan, a significant subset of patients with allergic rhinitis will exhibit hyposmia, mostly to a mild or moderate degree. The pathophysiology and potential treatments for olfactory loss in these patients should be further explored.

Optical coherence tomography for observation of the olfactory epithelium in mice.

OBJECTIVE: Optical coherence tomography (OCT) is an imaging tool that exploits the coherence of infrared light and is clinically utilized in the field of ophthalmology and dermatology. This study aimed to examine the feasibility of using OCT for diagnosing degeneration and regeneration of the olfactory epithelium in mice. METHODS: The olfactory and respiratory epithelia in excised nasal septa of adult mice were observed using OCT. Subsequently, histological assessments were performed with hematoxylin and eosin (H-E) staining. The thicknesses of the olfactory or respiratory epithelia were measured in both OCT images and H-E-stained paraffin sections. The ability of OCT to distinguish olfactory epithelia from respiratory epithelia in normal mice was compared with that of H-E staining. The feasibility of using OCT assessments for detecting changes in the thickness of olfactory epithelia was tested in a mouse model of the degeneration and regeneration of olfactory epithelia. RESULTS: OCT allowed visualization of the gross morphology of the olfactory and respiratory epithelium in normal mice, although it was limited in terms of visualizing cellular components. OCT-based measurements of epithelial thickness helped to distinguish olfactory epithelia from respiratory epithelia. Similar to H-E staining, OCT also clarified changes in the olfactory epithelium thickness after methimazole application. CONCLUSIONS: These findings indicate the utility of OCT for assessment of olfactory epithelial thickness and its potential for clinical evaluation of human olfactory epithelia.

In Vivo Inspection of the Olfactory Epithelium: Feasibility of Robotized Optical Biopsy.

Inspecting the olfactory cleft can be of high interest, as it is an open access to neurons, and thus an opportunity to collect in situ related data in a non-invasive way. Also, recent studies show a strong link between olfactory deficiency and neurodegenerative diseases such as Alzheimer and Parkinson diseases. However, no inspection of this area is possible today, as it is very difficult to access. Only robot-assisted interventions seem viable to provide the required dexterity. The feasibility of this approach is demonstrated in this article, which shows that the path complexity to the olfactory cleft can be managed with a concentric tube robot (CTR), a particular type of continuum robot. First, new anatomical data are elaborated, in particular for the olfactory cleft, that remains hardly characterized. 3D reconstructions are conducted on the database of 20 subjects, using CT scan images. Measurements are performed to describe the anatomy, including metrics with inter-subject variability. Then, the existence of collision-free passageways for CTR is shown using the 3D reconstructions. Among the 20 subjects, 19 can be inspected using only 3 different robot geometries. This constitutes an essential step towards a robotic device to inspect subjects for clinical purposes.

Microstructural network alterations of olfactory dysfunction in newly diagnosed Parkinson's disease.

Olfactory dysfunction is a robust and early sign for Parkinson's disease (PD). Previous studies have revealed its association with dementia and related neural changes in PD. Yet, how olfactory dysfunction affects white matter (WM) microstructure in newly diagnosed and untreated PD remains unclear. Here we comprehensively examined WM features using unbiased whole-brain analyses. 88 newly diagnosed PD patients without dementia (70 with hyposmia and 18 without hyposmia) and 33 healthy controls underwent clinical assessment and diffusion tensor imaging (DTI) scanning. Tract-based special statistics (TBSS), graph-theoretic methods and network-based statistics (NBS) were used to compare regional and network-related WM features between groups. TBSS analysis did not show any differences in fractional anisotropy and mean diffusivity between groups. Compared with controls, PD patients without hyposmia showed a significant decrease in global efficiency, whilst PD patients with hyposmia exhibited significantly reduced global and local efficiency and additionally a disrupted connection between the right medial orbitofrontal cortex and left rectus and had poorer frontal-related cognitive functioning. These results demonstrate that hyposmia-related WM changes in early PD only occur at the network level. The confined disconnectivity between the bilateral olfactory circuitry may serve as a biomarker for olfactory dysfunction in early PD.

Morphology of the nasal capsule of primates--with special reference to Daubentonia and Homo.

Primitive mammals are considered macrosmatic. They have very large and complicated nasal capsules, nasal cavities with extensive olfactory epithelia, and relatively large olfactory bulbs. The complicated structures of the nasal capsule follow a relatively conservative "bauplan," which is normally easy to see in earlier fetal stages; especially in altricial taxa it differentiates well into postnatal life. As anteriormost part of the chondrocranium, the nasal capsule is at first cartilaginous. Most of it ossifies endochondrally, but "appositional bone" ("Zuwachsknochen") is also common. Many fetal structures become resorbed. Together, all surviving bone structures form the ethmoid bone, but cartilages of the external nose and of the vomeronasal complex can persist throughout life. We describe in detail the anatomy of Daubentonia madagascariensis based on a fetal stage (41 mm HL) and an adult skull was analyzed by µCT. We found that the nasal capsule of this species is by far the most complicated one of all extant Primates. We also describe older fetuses of Homo sapiens (35 and 63 mm HL) as representative of a derived primate. The most significant feature of man--and probably of all anthropoids--is the complete loss of the recessus frontoturbinalis and its associated structures. It can be demonstrated that the evolutionary reductions within the primate nasal capsule mainly affect those structures associated with olfaction, whereas cartilages that are important for the biomechanics of the facial skull of the fetus persist.

Mapping the nasal airways: using histology to enhance CT-based three-dimensional reconstruction in Nycticebus.

Three-dimensional reconstructions of imaging data are an increasingly common approach for studying anatomical structure. However, certain aspects of anatomy, including microscopic structure and differentiating tissue types, continue to benefit from traditional histological analyses. We present here a detailed methodology for combining data from microCT and histological imaging to create 3D virtual reconstructions for visualization and further analyses. We used this approach to study the distribution of olfactory mucosa on ethmoturbinal I of an adult pygmy slow loris, Nycticebus pygmaeus. MicroCT imaging of the specimen was followed by processing, embedding, and sectioning for histological analysis. We identified corresponding features in the CT and histological data, and used these to reconstruct the plane of section in the CT volume. The CT volume was then digitally re-sliced, such that orthogonal sections of the CT image corresponded to histological sections. Histological images were annotated for the features of interest (in this case, the contour of soft tissue on ethmoturbinal I and the extent of olfactory mucosa), and annotations were transferred to binary masks in the CT volume. These masks were combined with density-based surface reconstructions of the skull to create an enhanced 3D virtual reconstruction, in which the bony surfaces are coded for mucosal function. We identified a series of issues that may be raised in this approach, for example, deformation related to histological processing, and we make recommendations for addressing these issues. This method provides an evidence-based approach to 3D visualization and analysis of microscopic features in an anatomic context.

Digital volume tomography (DVT) measurements of the olfactory cleft and olfactory fossa.

CONCLUSION: Preoperative imaging is important, because of the small size of the cribriform plate, different anatomical variants of the olfactory fossa in varied patients and unequal width of the cribriform plate in the anterior and posterior third. OBJECTIVE: Digital volume tomography (DVT) is a rather new imaging technique for the diagnosis of diseases of paranasal sinuses. This technology is dedicated to the evaluation of the distinctive structures of the anterior skull base due to the high resolution of the DVT. Based on the Keros classification this anatomic area was analyzed radiologically and also in relation to the uncinate process. METHODS: The investigation was performed on 111 patients. The Accu-I-tomo F17 was used. Patients with total nasal polyposis and patients who had undergone sinus surgery were excluded from the study. RESULTS: Keros type I was found in 11.25% of the patients, type II in 68.05% and type III in 20.7%. Significant asymmetry of the olfactory fossa was identified in nine patients (8.1%). The width of the olfactory cleft varied from 0 to 3.25 mm. No relation between Keros type and a particular onset of the uncinate process to orbit, skull base or middle turbinate could be detected.

Radiographic features of olfactory neuroblastoma.

Olfactory neuroblastomas are malignant, slowly growing neurogenic tumors originating from the olfactory mucosa of the nasal cavity. Fourteen patients with this tumor were evaluated and showed variable extension into adjacent structures such as the ethmoid and sphenoid sinuses, orbit, and anterior cranial cavity.