The Microvillar and Solitary Chemosensory Cells as the Novel Targets of Infection of SARS-CoV-2 in Syrian Golden Hamsters.

Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019, suffer from respiratory and non-respiratory symptoms. Among these symptoms, the loss of smell has attracted considerable attention. The objectives of this study were to determine which cells are infected, what happens in the olfactory system after viral infection, and how these pathologic changes contribute to olfactory loss. For this purpose, Syrian golden hamsters were used. First, we verified the olfactory structures in the nasal cavity of Syrian golden hamsters, namely the main olfactory epithelium, the vomeronasal organ, and their cellular components. Second, we found angiotensin-converting enzyme 2 expression, a receptor protein of SARS-CoV-2, in both structures and infections of supporting, microvillar, and solitary chemosensory cells. Third, we observed pathological changes in the infected epithelium, including reduced thickness of the mucus layer, detached epithelia, indistinct layers of epithelia, infiltration of inflammatory cells, and apoptotic cells in the overall layers. We concluded that a structurally and functionally altered microenvironment influences olfactory function. We observed the regeneration of the damaged epithelium, and found multilayers of basal cells, indicating that they were activated and proliferating to reconstitute the injured epithelium.

Transient Effects of Cyclophosphamide on Basal Cell Proliferation of Olfactory Epithelia.

Cancer is often treated with broad-spectrum cytotoxic drugs that not only eradicate cancerous cells but also have detrimental side effects. One of these side effects, disruption of the olfactory system, impedes a patient's ability to smell, perceive flavor, and ultimately may interfere with their nutritional intake and recovery from cancer. Recent studies reported that the chemotherapy drug, cyclophosphamide (CYP), can damage gustatory epithelia and disrupt cell proliferation in olfactory epithelia. In this study, we asked if CYP altered globose and horizontal basal cell proliferation in the murine main olfactory epithelium (MOE) and vomeronasal organ (VNO). We used antibodies for Ki67, a marker strictly associated with cell proliferation, and Keratin 5, a marker for the cytoskeleton of horizontal basal cells. Our results revealed a significant CYP-induced decrease in the number of proliferative cells in both epithelia, especially globose basal cells in the MOE, within the first 1-2 days postinjection. Recovery of cell renewal was apparent 6 days after injection. The immunohistochemical markers showed significantly higher levels of globose and horizontal basal cell proliferation in CYP-injected mice at 14 and 30 days postinjection compared with control mice. The prolonged proliferative activation of globose and horizontal basal cells suggests that, besides altering proliferation of olfactory epithelia, the epithelial substrate needed for successful cell renewal may be adversely affected by CYP.

Cyclophosphamide has Long-Term Effects on Proliferation in Olfactory Epithelia.

Chemotherapy patients often experience chemosensory changes during and after drug therapy. The chemotherapy drug, cyclophosphamide (CYP), has known cytotoxic effects on sensory and proliferating cells of the taste system. Like the taste system, cells in the olfactory epithelia undergo continuous renewal. Therefore, we asked if a single injection of 75 mg/kg CYP would affect cell proliferation in the anterior dorsomedial region of the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) from 0 to 125 days after injection. Both epithelia showed a decrease in Ki67-labeled cells compared to controls at day 1 and no Ki67+ cells at day 2 postinjection. In the sensory layer of the MOE, cell proliferation began to recover 4 days after CYP injection and by 6 days, the rate of proliferation was significantly greater than controls. Ki67+ cells peaked 30 days postinjection, then declined to control levels at day 45. Similar temporal sequences of initial CYP-induced suppression of cell proliferation followed by elevated rates peaking 30-45 days postinjection were seen in the sustentacular layer of the MOE and all 3 areas (sensory, sustentacular, marginal) of the VNO. CYP affected proliferation in the sensory layer of the MOE more than the sustentacular layer and all 3 areas of the VNO. These findings suggest that chemotherapy involving CYP is capable of affecting cell renewal of the olfactory system and likely contributes to clinical loss of function during and after chemotherapy.

Main Olfactory and Vomeronasal Epithelium Are Differently Affected in Niemann-Pick Disease Type C1.

INTRODUCTION: Olfactory impairment is one of the earliest symptoms in neurodegenerative disorders that has also been documented in Niemann-Pick disease type C1 (NPC1). NPC1 is a very rare, neurovisceral lipid storage disorder, characterized by a deficiency of Npc1 gene function that leads to progressive neurodegeneration. Here, we compared the pathologic effect of defective Npc1 gene on the vomeronasal neuroepithelium (VNE) with that of the olfactory epithelium (OE) in an NPC1 mouse model. METHODS: Proliferation in the VNE and OE was assessed by applying a bromodeoxyuridine (BrdU) protocol. We further compared the immunoreactivities of anti-olfactory marker protein (OMP), and the lysosomal marker cathepsin-D in both epithelia. To investigate if degenerative effects of both olfactory systems can be prevented or reversed, some animals were treated with a combination of miglustat/allopregnanolone/2-hydroxypropyl-cyclodextrin (HPßCD), or a monotherapy with HPßCD alone. RESULTS: Using BrdU to label dividing cells of the VNE, we detected a proliferation increase of 215% ± 12% in Npc1-/- mice, and 270% ± 10% in combination- treated Npc1-/- animals. The monotherapy with HPßCD led to an increase of 261% ± 10.5% compared to sham-treated Npc1-/- mice. Similar to the OE, we assessed the high regenerative potential of vomeronasal progenitor cells. OMP reactivity in the VNE of Npc1-/- mice was not affected, in contrast to that observed in the OE. Concomitantly, cathepsin-D reactivity in the VNE was virtually absent. Conclusion: Vomeronasal receptor neurons are less susceptible against NPC1 pathology than olfactory receptor neurons. Compared to control mice, however, the VNE of Npc1-/- mice displays an increased neuroregenerative potential, indicating compensatory cell renewal.

Patent nasopalatine ducts: an update of the literature and a series of new cases.

PURPOSE: The objective of this review is to present an update and summary of clinical findings of cases with a patent nasopalatine duct (NPD) reported in the literature from 1881 to 2016. METHODS: Previous articles and reviews about patent NPDs were studied and copies of all original publications were obtained for data verification. Furthermore, a literature search was conducted. In addition, the study sample was complemented with four cases recently seen in our institution. RESULTS: Ten out of 67 published cases were to be excluded for this analysis due to misinterpretation or misreporting in previous articles. Overall, 57 cases with NPD patency could be analyzed. Males outnumbered females in a ratio of 2:1. The mean age (when this information was available) was 34.1 ± 17.6 years (range 6-69 years). NPDs were located bilaterally (60%), unilaterally (20%) or centrally (20%). Complete or partial patency was reported in 73.9 and 26.1%, respectively. 74.1% of patients presented a variety of clinical signs and symptoms. The ability of the patient to produce a squeaky or whistling sound was the most frequent clinical finding (23.8%). CONCLUSIONS: Caution must be exercised when reading review articles about NPD patency since wrong data have been copied in several subsequent publications. Since epidemiological data are missing with regard to patent NPDs, age and gender predilections are not warranted. Bilateral occurrence and full patency were prevailing features in the evaluated case reports of patent NPDs.

Pathology and behaviour in feline medicine: investigating the link between vomeronasalitis and aggression.

OBJECTIVES: The aim of the study was to investigate if the feline vomeronasal organ (VNO) can be affected by inflammatory lesions and if these changes are associated with behavioural alterations. METHODS: VNOs from 20 cats were sampled during necropsy, submitted for routine tissue processing and stained with haematoxylin and eosin for histopathological evaluation. For the 20 cats, data on the presence of aggressive behaviours towards cats or humans were collected by questionnaire survey at the point of death. Inflammatory lesions were classified depending on the duration of the process as acute or chronic, both in vomeronasal sensory epithelium (VNSE) and in non-sensory epithelium (NSE). Fisher's exact test was used to compare VNO inflammation with behavioural data. RESULTS: The VNSE was inflamed in 11/20 VNOs (55%) while the NSE was inflamed in 13/20 (65%). Overall, the VNO was affected by inflammation in 14/20 (70%) cats, and all the lesions were classified as chronic. Five out of 20 cats (25%) had documented intraspecific aggressive behaviours and 8/20 (40%) had shown aggression towards humans. Fisher's exact test showed a statistically significant correlation between inflammation of the VNSE and intraspecific aggression (P = 0.038). No statistically correlations were observed between VNSE inflammation and aggression towards humans and between NSE inflammation and aggression towards cats or humans. CONCLUSIONS AND RELEVANCE: Our results show, for the first time, the existence of vomeronasalitis in animals and its possible association with intraspecific aggressive behaviours. The inflammatory microenvironment could impair VNSE functionality, causing intraspecific communication alterations, probably through a reduction in chemical communication action and perception. Owing to the pivotal role of the VNO in the social life of cats and other species, this report provides a rationale to further investigate this disease in relation to a variety of behavioural disorders.

Aerosol and nasal transmission of chronic wasting disease in cervidized mice.

Little is known regarding the potential risk posed by aerosolized prions. Chronic wasting disease (CWD) is transmitted horizontally, almost surely by mucosal exposure, and CWD prions are present in saliva and urine of infected animals. However, whether CWD may be transmissible by the aerosol or nasal route is not known. To address this question, FVB mice transgenetically expressing the normal cervid PrP(C) protein [Tg(cerPrP) mice] were exposed to CWD prions by either nose-only aerosol exposure or by drop-wise instillation into the nostrils. Mice were monitored for signs of disease for up to 755 days post-inoculation (p.i.) and by examination of tissues for lesions and PrP(CWD) after necropsy. In particular, nasal mucosa, vomeronasal organ, lungs, lymphoid tissue and the brain were assessed for PrP(CWD) by Western blotting and immunohistochemistry. Six of seven aerosol-exposed Tg(cerPrP) mice developed clinical signs of neurological dysfunction mandating euthanasia between 411 and 749 days p.i. In all these mice, CWD infection was confirmed by detection of spongiform lesions and PrP(CWD) in the brain. Two of nine intranasally inoculated Tg(cerPrP) mice also developed transmissible spongiform encephalopathy associated with PrP(CWD) between 417 and 755 days p.i. No evidence of PrP(CWD) was detected in CWD-inoculated Tg(cerPrP) mice examined at pre-terminal time points. These results demonstrate that CWD can be transmitted by aerosol (as well as nasal) exposure and suggest that exposure via the respiratory system merits consideration for prion disease transmission and biosafety.

The olfactory system is affected by steroid aerosol treatment in mice.

Asthma needs continuous treatment often for years. In humans, some drugs are administered via aerosol, therefore they come in contact with both respiratory and olfactory mucosa. We explored the possibility that antiasthma corticosteroid treatment could influence the olfactory function by passage through the nose. A group of mice was exposed twice daily for 42 days to fluticasone propionate aerosol and was compared with a control group. Olfactory behavior, respiratory mechanics, histology, and immunoreactivity in the olfactory system were assessed. Fluticasone-treated mice were slower in retrieving a piece of hidden food, but both groups were similarly fast when the food was visible. When a clearly detectable odor was present in the environment, all mice behaved in a similar way. Respiratory mechanics indices were similar in all mice except for the viscose resistance, which was reduced in fluticasone-treated mice. Olfactory mucosa of fluticasone-treated mice was thicker than that of controls. Slight but consistent differences in staining were present for Olfactory Marker Protein but not for other proteins. A mild impairment of olfactory function is present in mice chronically treated with fluticasone aerosol, apparently accompanied by slight modifications of the olfactory receptor cells, and suggests monitoring of olfactory function modifications in long-term steroid users.

A rare case of jugular foramen schwannoma arising from Jacobson's nerve.

The jugular foramen (JF) region is a complex area of the cranial base where venous structures such as the jugular bulb and the inferior petrosal sinuses are strictly related to the lower cranial nerves IX, X and XI. The most common tumours include glomus jugulare, schwannomas of the mixed cranial nerves (IX-XI) and meningiomas. Schwannomas involving the jugular foramen are rare neoplasms and in most of the cases are thought to originate from the X cranial nerve. We report a case of a schwannoma of the JF diagnosed at an early stage, allowing radiological and surgical evidence to support its origin from the tympanic branch of the IX cranial nerve. To our knowledge this is the first case reported in the literature of such a tumour.

The vomeronasal chemosensory system as a route of neuroinvasion by herpes simplex virus.

We have investigated the potential of neurotropic microbes to invade the central nervous system (CNS) via the peripheral nervous system. Herpes simplex virus type 1 (HSV-1) strain KH6 and herpes simplex virus type 2 (HSV-2) strain 186 were found to infect chemosensory neurons in the vomeronasal organ (the pheromone detector) following intranasal inoculation of mice. HSV-1 strain KH6 infection was further transmitted to the accessory olfactory bulb (first relay), the medial amygdala (second relay), and the bed nucleus of the stria terminalis and the ventromedial hypothalamus (third relay). HSV-1 strain KH6 also targeted the olfactory and trigeminal systems. HSV-2 strain 186 predominantly attacked the brainstem including the trigeminal system. While both viruses did not induce apoptosis in infected chemosensory neurons, they did in infected brain tissue. These results suggest that neurotropic viruses can invade the brain by infecting vomeronasal chemosensory neurons and that the restrained induction of apoptosis in the infected neurons may facilitate viral transmission to the CNS.

Enlarged vomeronasal organ in a child: imaging findings.

The vomeronasal organ is a special sensory organ that exists in both animals and humans. It is located on the sides of nasal septum and although it involutes with age, occasionally it may be seen in humans. We present the imaging findings in a child with an enlarged nasal septum whose features we believe are compatible with a vomeronasal organ.

Widespread defects in the primary olfactory pathway caused by loss of Mash1 function.

MASH1, a basic helix-loop-helix transcription factor, is widely expressed by neuronal progenitors in the CNS and PNS, suggesting that it plays a role in the development of many neural regions. However, in mice lacking a functional Mash1 gene, major alterations have been reported in only a few neuronal populations; among these is a generalized loss of olfactory receptor neurons of the olfactory epithelium. Here, we use a transgenic reporter mouse line, in which the cell bodies and growing axons of subsets of central and peripheral neurons are marked by expression of a tau-lacZ reporter gene (the Tattler-4 allele), to look both more broadly and deeply at defects in the nervous system of Mash1-/- mice. In addition to the expected lack of olfactory receptor neurons in the main olfactory epithelium, developing Mash1-/-;Tattler-4+/- mice exhibited reductions in neuronal cell number in the vomeronasal organ and in the olfactory bulb; the morphology of the rostral migratory stream, which gives rise to olfactory bulb interneurons, was also abnormal. Further examination of cell proliferation, cell death, and cell type-specific markers in Mash1-/- animals uncovered parallels between the main olfactory epithelium and the vomeronasal organ in the regulation of sensory neuron development. Interestingly, this analysis also revealed that, in the olfactory epithelium of Mash1-/- animals, there is an overproduction of proliferating cells that co-express markers of both neuronal progenitors and supporting cells. This finding suggests that olfactory receptor neurons and olfactory epithelium supporting cells may share a common progenitor, and that expression of Mash1 may be an important factor in determining whether these progenitors ultimately generate neurons or glia.

Aberrant sensory innervation of the olfactory bulb in neuropilin-2 mutant mice.

The mammalian olfactory system consists of two anatomically segregated structures, the main olfactory system and the vomeronasal system, which each detect distinct types of chemical stimuli in the environment. During development, sensory neurons establish precise axonal connections with their respective targets within the olfactory bulb. The specificity of the odorant or vomeronasal receptor expressed by the sensory neuron is crucial in this process, yet it is less clear which of the more conventional axon guidance molecules are involved. Here, we show that neuropilin-2, a coreceptor for some of the class 3 semaphorins, is expressed in subpopulations of olfactory and vomeronasal sensory neurons. We generated a knock-out mutation in the neuropilin-2 gene by gene targeting in embryonic stem cells. Neuropilin-2 mutant mice exhibit profound and distinct effects on target innervation within the olfactory bulb. In the main olfactory system, axons of olfactory sensory neurons penetrate into the deeper layers of the main olfactory bulb. In the vomeronasal system, axonal fasciculation within the vomeronasal nerve is affected; some axons are misrouted and innervate glomeruli in an ectopic domain of the accessory olfactory bulb.

Colchicine-induced cell death and proliferation in the olfactory epithelium and vomeronasal organ of the mouse.

The cytotoxic agent colchicine induced apoptotic cell death and subsequent regeneration in the mouse olfactory epithelium and vomeronasal organ. The TUNEL method revealed the presence of many apoptotic bodies in the middle to basal region of the septal olfactory epithelium and vomeronasal organ near the boundary of the respiratory epithelium at 1 day after a single i.p. injection of colchicine (4 mg/kg b.w.). In some regions of the third and the fourth nasal turbinates, massive apoptosis was observed in the olfactory epithelium. Electron micrographs of the septum showed that immature olfactory cells and globose basal cells were killed by the colchicine and had been phagocytized by the supporting cells and macrophages. In the vomeronasal organ, immature sensory cells and precursors died in response to the colchicine. In response to cell death, active proliferation of precursor cells (globose basal cells) and subsequent regeneration of olfactory cells occurred in the olfactory epithelium and vomeronasal organ. Incorporation of the mitotic tracer BrdU by precursor cells reached its peak at 4 days after colchicine treatment in the vomeronasal organ, and at 6 to 7 days in the olfactory epithelium; however, in some regions in the third and the fourth nasal turbinates, where many olfactory cells and globose basal cells had died by colchicine effect, the regeneration did not occur even in 1 month, forming the epithelium of only supporting cells and horizontal basal cells. In the next month, these regions became normal olfactory epithelium. This suggests that the globose basal cells in the surrounding normal olfactory epithelium might invade these regions to give rise to the olfactory cells.

The effect of ammonia on olfactory epithelium and vomeronasal organ neuroepithelium of rabbits. A histological and histochemical study.

In recent studies, the vomeronasal organ (VNO), although vestigial and with unknown function in humans, was reported to be present in almost every person examined. In rabbits, it is a well-developed organ, one lying on each side of the nasal septum. Histologically it was found to contain neuroepithelium which is considered an accessory olfactory system taking charge of an olfactory discrimination different from that of olfactory epithelium. Experimental removal of the VNO in male animals reduces reproductive performance and aggression. In this study, the effects of prolonged exposure to ammonia vapor on the histological pattern and enzymatic activity of the olfactory epithelium and the VNO neuroepithelium of 30 adult male rabbits were investigated and compared with a control group. In the exposed animals, the supporting cells in both types of epithelia showed hyperplasia which was more marked in the olfactory epithelium. Manifestations of cytotoxicity were found more in the bipolar cells of the olfactory epithelium than in the VNO neuroepithelium. The enzymatic activity in the exposed group supported the histological results. The presence of the VNO neuroepithelium of the rabbit in a narrow duct with a minute nasal orifice could have some protective effects on the cells.

Cell death, phagocytosis, and neurogenesis in mouse olfactory epithelium and vomeronasal organ after colchicine treatment.

The cytotoxic agent colchicine induced apoptotic cell death in the mouse olfactory epithelium and vomeronasal organ. The terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) method revealed the presence of many apoptotic bodies in the middle to basal region of the septal olfactory epithelium and vomeronasal organ near the boundary of the respiratory epithelium at 1 day after intraperitoneal (i.p.) injection of colchicine (4 mg/kg). In some areas of the nasal turbinates, massive apoptosis was observed in the olfactory epithelium. Electron micrographs showed that immature olfactory cells and globose basal cells were killed by the colchicine and had been phagocytized by the supporting cells and macrophages. In some areas of the nasal turbinates, mature olfactory cells were also killed, and thus only the supporting cells and horizontal basal cells remained in the epithelium. Bromodeoxyuridine (BrdU) labeling showed that regeneration occurred in the septum and vomeronasal organ at 4-6 days after colchicine treatment; however, there were no regenerated olfactory cells in some areas of the turbinates up to 30 days after colchicine treatment.