Olfactory Mucosa Research Articles

Eosinophilic globules (syn. hyaline inclusions) and protein crystalloids in the nasal mucosa of rats treated with synthetic amorphous silica, an unspecific chitinase-like-protein-positive background lesion

In a 13-week inhalation toxicity study with three recovery periods (3, 6, and 12 months), Crl: WI rats were allocated to nine groups, each containing 25 animals per sex. Eight groups were treated daily by inhalation with the test items at concentrations of 0.5, 1.0, 2.5, or 5.0 mg/m3 (SAS 1 groups 2, 3, 4, or 5, respectively; SAS 2 groups 6, 7, 8, or 9, respectively). Controls (group 1) were treated with air only.In nasal cavities, the major lesions consisted of increased eosinophilic globules and chitinase-3-like-protein-positive crystalloids* in the nasal mucosa, mainly in nasal cavity levels 2–4 up to week 26 of recovery without any further injury in olfactory mucosa, mainly in SAS 1-treated animals. Eosinophilic globules in the rodent nasal cavity are common and increase with age; they represent a particular finding of the rodent nasal mucosa. The relevance of chitinase-3-like protein (Ym1 + Ym2) expression in the rodent nasal mucosa is unknown but is normal in control animals. Both findings developed without any indicator for inflammatory processes. The increase of these unspecific background findings is considered an indicator of minor irritative effects.Due to the clear lack of nasal tissue injury or concurrent changes (degeneration, necrosis, inflammatory infiltrate, dysplasia, and/or neoplasia) following repeated inhalation exposure to SAS, it is deemed that the eosinophilic globules (hyaline inclusions) combined with the formation of eosinophilic protein crystalloids in this study represent an adaptive response.

Evolution of nasal and olfactory infection characteristics of SARS-CoV-2 variants.

SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in nasal tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variant revealed that SARS-CoV-2 WA1 or Delta infect a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possessed broader cellular invasion capacity into the submucosa, while Omicron displayed enhanced nasal respiratory infection and longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon were more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa were accompanied by a decline of phagocytosis-related genes. Further, robust basal stem cell activation contributed to neuroepithelial regeneration and restored ACE2 expression postinfection. Together, our study characterized the nasal tropism of SARS-CoV-2 strains, immune clearance, and regeneration after infection. The shifting characteristics of viral infection at the airway portal provide insight into the variability of COVID-19 clinical features, particularly long COVID, and may suggest differing strategies for early local intervention.

ACKR3 in olfactory glia cells shapes the immune defense of the olfactory mucosa.

Barrier-forming olfactory glia cells, termed sustentacular cells, play important roles for immune defense of the olfactory mucosa, for example as entry sites for SARS-CoV-2 and subsequent development of inflammation-induced smell loss. Here we demonstrate that sustentacular cells express ACKR3, a chemokine receptor that functions both as a scavenger of the chemokine CXCL12 and as an activator of alternative signaling pathways. Differential gene expression analysis of bulk RNA sequencing data obtained from WT and ACKR3 conditional knockout mice revealed upregulation of genes involved in immune defense. To map the regulated genes to the different cell types of the olfactory mucosa, we employed biocomputational methods utilizing a single-cell reference atlas. Transcriptome analysis, PCR and immunofluorescence identified up-regulation of NF-κB-related genes, known to amplify inflammatory signaling and to facilitate leukocyte transmigration, in the gliogenic lineage. Accordingly, we found a marked increase in leukocyte-expressed genes and confirmed leukocyte infiltration into the olfactory mucosa. In addition, lack of ACKR3 led to enhanced expression and secretion of early mediators of immune defense by Bowman's glands. As a result, the number of apoptotic cells in the epithelium was decreased. In conclusion, our research underlines the importance of sustentacular cells in immune defense of the olfactory mucosa. Moreover, it identifies ACKR3, a druggable G protein-coupled receptor, as a promising target for modulation of inflammation-associated anosmia.

A Novel Approach for Mucosal and Bulbar Olfactory Ensheathing Cells Isolation Based on the Non-adherent Subculture Technique.

Olfactory ensheathing cells (OECs) are widely used in transplantation studies. The high purification of this unique cell type is valuable for medical applications. Although recent improvements in OECs isolation procedures opened a new era in this field, the high purification efficacy and viability rate are still of concern. The most widely used OECs isolation techniques can be broadly classified based on adherence properties, particularly in olfactory bulb-derived OEC isolation. Considering the invasive nature of harvesting OECs from human olfactory bulbs, a highly efficient purification of these cells from olfactory mucosa can benefit clinical trials. In this study, we isolated OECs from rats' olfactory bulbs and mucosa due to their differential adherence properties and compared them. Cell preparations were characterized by NGFR p75 and S100β antibodies, the specific markers for OECs, using immunocytochemistry and western blot analysis, respectively. OECs morphology and viability were monitored over time by microscopy and MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide) assay. We found that OECs could be purified from the olfactory mucosa using our suggested method as efficiently as the olfactory bulb. Both derived OECs showed high levels of NGFR p75 and S100β expression, although the S100β expression was higher in olfactory mucosa-derived OECs preparations (P<0.05). Moreover, there was no significant difference between the two sources in cell viability in our suggested protocol. Due to the non-invasive harvesting method, olfactory mucosa-derived OECs are preferred from a clinical point of view in transplantation studies.

Apoptosis and turnover disruption of olfactory sensory neurons in eosinophilic chronic rhinosinusitis.

Olfactory dysfunction (OD) is one of the important and difficult-to-treat symptoms of eosinophilic chronic rhinosinusitis (CRS), which is typically associated with type 2 inflammation where eosinophils (EOSs) function as both effectors and initiators. Eosinophilic infiltration in the olfactory mucosa (OM) is associated with severe OD, mucosal erosion, and more loss of olfactory sensory neurons (OSNs). Active EOS-derived cytokines, chemokines, and eosinophil granule proteins may lead to aggravation of inflammation, tissue damage, and impairment of the survival and regeneration of OSNs. Recent studies show that EOSs can lead to apoptosis of OSNs through axonal and neural body damage, turnover disorder of OSNs through the loss of immature OSNs and globose basal cells (GBCs), changed proliferative activity of horizontal basal cells (HBCs), and dysfunction of OSNs through the breakdown of neuroepithelial integrity and alteration of ion concentration in OSNs and mucin. In this review, we outline the current progress on the role of EOSs on OD in patients with eosinophilic CRS and the mechanism of EOS-associated injury of the OM and OSNs in experimental animal models with sinonasal inflammation. Further investigations on the molecular mechanisms of tissue eosinophilia-induced injury of OSNs are warranted to obtain new therapeutic targets and achieve better restoration of olfactory function.

Optical Anatomy and Microscopical for the Comparison of the Olfactory Mucosa of Puppies with Lambs

Dogs are animal that have been used by humans for a long time in many fields because of its characteristics, which have been used in fishing, guarding, insurance, drug detection and in the field of prevention and detection of crime, The present study was done to demonstrate the histological structure and the olfactory mucosa in both lambs and puppies, so two lambs and two puppies were included in this study. The puppies were killed by using xylazine 4ml and ketamine 8ml, the lambs were already slaughtered, the nasal cavity was arrived by using the wire sow and the olfactory mucosa was immediately obtained and immersed in 10% formalin for 24 hour, then the histological technique was applied to obtain slide of 5 µm thickness and stained by haematoxylin and Eosin, and examined under light microscopic. The present result, revealed that the olfactory epithelium of the puppies was taller than in lambs and the sensory hairs was crowded in both species, also the lamina properia of the lambs showed more mucus glands continues with surface.

A technological comparison of freeze-dried poly-ɛ-caprolactone (PCL) and poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with clozapine for nose-to-brain delivery

Clozapine (CZP) is an atypical antipsychotic, the most effective for the treatment of resistant schizophrenia. With the aim of overcome the limitations of current dosage forms (tablets or suspensions) and routes of administration (oral and intramuscular), this research project proposes nanocarrier systems for intranasal (IN) administration of CZP. In particular, the systems have been developed for the direct nose-to-brain (N2B) route, which allows the direct delivery of the drug to the central nervous system (CNS), bypassing the blood-brain barrier (BBB). This study focusses on the development and investigation of two distinct types of lyophilized polymeric nanoparticles (NPs) consisting of poly- ɛ -caprolactone (PCL) or poly (lactic-co-glycolic acid) (PLGA). The lyophilized formulations were cryoprotected with 5 % (w/v) d-maltose for PLGA-NPs and 25 % (w/v) d-maltose for PCL-NPs. The NPs showed a size of approximately 200 nm, suitable for reuptake from the olfactory nerve to reach the brain directly after the IN administration. In vitro release studies performed in artificial cerebrospinal fluid (aCSF) and simulated nasal fluid (SNF) showed that PLGA NPs released 100 % of the drug in 4 days in SNF, while in aCSF they released about 80 % in 20 days. On the contrary, PCL-NPs released the entire amount of drug within 24 h in both fluids. Furthermore, preliminary in vitro mucoadhesion studies were performed on NPs in liquid form (suspensions) to compare the possible variation in their mucoadhesive properties with respect to powdered NPs, which were tested directly on pig nasal mucosa tissue, where permeability was also assessed. CZP-loaded NPs showed some interactions with mucin in vitro, but no ex-vivo mucoadhesiveness was demonstrated. Furthermore, the results of permeation indicated that the use of CZP-PCL-NPs did not increase the effective permeation of CZP into the mucosa; however, a faster onset of permeation compared to free CZP and a controlled profile were demonstrated. Toxicity studies on primary human olfactory mucosa cells extracted from volunteer patients and on MDCKII cells were also done. Finally, stability studies were carried out under different conditions: suspensions stored at 4 and 25 °C for 3 months, in SNF at 35 °C and in aCSF at 37 °C; dry powders were instead stored at 25 °C with 50 % relative humidity for 8 months. All the results demonstrated a greater stability of NPs in powder form and the absence of aggregation phenomena under artificial physiological conditions.

Characterizing Immune Infiltration in Esthesioneuroblastoma Subtypes Through Gene Expression Deconvolution

BackgroundEsthesioneuroblastoma (ENB) is a rare cancer deriving from the olfactory mucosa. Among the basal or neural genomic subtypes, the basal subtype is associated with poorer survival, and poor differentiation, and higher levels of tumor-infiltrating immune cells (TIICs). The immune microenvironment of these ENB subtypes remains unclear. We used an established machine-learning algorithm on ENB transcriptomic profiles. MethodsThe authors characterized 22 immune cell populations using the CIBERSORTx deconvolutional machine-learning pipeline on RNAseq data from 18 ENB cases. The characterization aimed to elucidate differences in relative proportions and populations of TIICs between basal and neural ENB. ResultsNo differences in age, Hyams, Dulguerov, IDH2 mutation, or PD-L1 expression were seen between basal and neural subtypes of ENB (p>0.05). No difference in median overall survival was also appreciated (52.0±13.1 vs. 50.0±43.2 months, p=0.5). As a cohort, M2 macrophages were the most abundant subpopulation (14%) followed by naïve B-cells (13%) and CD4 memory resting T cells (12%). No gross differences on CD20, CD4, or CD8 cells/mm2 were apparent on gross histology (p>0.05). However, further analysis showed that activated CD4 memory T cells were significantly increased in the basal ENBs, whereas resting dendritic cells were increased in the neural ENB subtype. The TIIC profiles alone could not differentiate between basal and neural ENB but did suggest immunoprofile differences. ConclusionBasal and neural subtypes display distinct TIIC involvement, which may impact their difference in outcome. These findings provide the framework for further investigation in novel immunomodulation strategies for ENB.

SARS-CoV-2 induces inflammation and intracranial infection through the olfactory epithelium-olfactory bulb pathway in non-human primates

We examined the histopathological changes in the olfactory mucosa of cynomolgus and rhesus macaque models of SARS-CoV-2 infection. SARS-CoV-2 infection induced severe inflammatory changes in the olfactory mucosa. A major histocompatibility complex (MHC) class II molecule, HLA-DR was expressed in macrophage and supporting cells, and melanocytes were increased in olfactory mucosa. Supporting cells and olfactory neurons were infected, and SARS-CoV-2 N protein was detected in the axons of olfactory neurons and in olfactory bulbs. Viral RNA was detected in olfactory bulbs and brain tissues. The olfactory epithelium-olfactory bulb pathway may be important as a route for intracranial infection by SARS-CoV-2.

The possible effects of Zinc sulfate on adult rat olfactory mucosa. A histological study

The possible effects of Zinc sulfate on adult rat olfactory mucosa. A histological study

Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

Olfactory mucosa mesenchymal stem cells alleviate pulmonary fibrosis via the immunomodulation and reduction of inflammation

BackgroundPulmonary fibrosis (PF) is a progressive fibrosing interstitial pneumonia that leads to respiratory failure and other complications, which is ultimately fatal. Mesenchymal stem cells (MSCs) transplant is a promising strategy to solve this problem, while the procurement of MSCs from the patient for autotransplant remains a challenge.MethodsHere, we presented olfactory mucosa mesenchymal stem cells (OM-MSCs) from mouse turbinate and determined the preventing efficacy of allotransplant for PF. We demonstrated the antiinflammation and immunomodulatory effects of OM-MSCs. Flow cytometric analysis was used to verify the effect of OM-MSCs on monocyte-derived macrophage populations in the lung.ResultsAdministration of OM-MSCs reduces inflammation, attenuates the matrix metallopeptidase 13 (MMP13) expression level and restores the bleomycin (BLM)-induced pulmonary fibrosis by assessing the architecture of lung, collagen type I; (COL1A1), actin alpha 2, smooth muscle, aorta (ACTA2/α-SMA) and hydroxyproline. This therapeutic effect of OM-MSCs was related to the increase in the ratio of nonclassical monocytes to proinflammatory monocytes in the lung.ConclusionsThis study suggests that transplant of OM-MSCs represents an effective and safe treatment for PF.

Intranasal Nose-to-Brain Drug Delivery via the Olfactory Region in Mice: Two In-Depth Protocols for Region-Specific Intranasal Application of Antibodies and for Expression Analysis of Fc Receptors via In Situ Hybridization in the Nasal Mucosa.

A region-specific catheter-based intranasal administration method was successfully developed, established, and validated as reported previously. By using this method, drugs can be applicated specifically to the olfactory region. Thereby, intranasally administered drugs could be delivered via neuronal connections to the central nervous system. Here, we present a detailed protocol with a step-by-step procedure for nose-to-brain delivery via the olfactory mucosa.Fc receptors such as the neonatal Fc receptor (FcRn) and potentially Fcγ receptor IIb (FcγRIIb) are involved in the uptake and transport of antibodies via the olfactory nasal mucosa. To better characterize their expression levels and their role in CNS drug delivery via the nose, an in situ hybridization (ISH) protocol was adapted for nasal mucosa samples and described in abundant details.

Olfactory immunology: the missing piece in airway and CNS defence.

The olfactory mucosa is a component of the nasal airway that mediates the sense of smell. Recent studies point to an important role for the olfactory mucosa as a barrier to both respiratory pathogens and to neuroinvasive pathogens that hijack the olfactory nerve and invade the CNS. In particular, the COVID-19 pandemic has demonstrated that the olfactory mucosa is an integral part of a heterogeneous nasal mucosal barrier critical to upper airway immunity. However, our insufficient knowledge of olfactory mucosal immunity hinders attempts to protect this tissue from infection and other diseases. This Review summarizes the state of olfactory immunology by highlighting the unique immunologically relevant anatomy of the olfactory mucosa, describing what is known of olfactory immune cells, and considering the impact of common infectious diseases and inflammatory disorders at this site. We will offer our perspective on the future of the field and the many unresolved questions pertaining to olfactory immunity.

Human-derived air–liquid interface cultures decipher Alzheimer’s disease–SARS-CoV-2 crosstalk in the olfactory mucosa

BackgroundThe neurological effects of the coronavirus disease of 2019 (COVID-19) raise concerns about potential long-term consequences, such as an increased risk of Alzheimer's disease (AD). Neuroinflammation and other AD-associated pathologies are also suggested to increase the risk of serious SARS-CoV-2 infection. Anosmia is a common neurological symptom reported in COVID-19 and in early AD. The olfactory mucosa (OM) is important for the perception of smell and a proposed site of viral entry to the brain. However, little is known about SARS-CoV-2 infection at the OM of individuals with AD.MethodsTo address this gap, we established a 3D in vitro model of the OM from primary cells derived from cognitively healthy and AD individuals. We cultured the cells at the air–liquid interface (ALI) to study SARS-CoV-2 infection under controlled experimental conditions. Primary OM cells in ALI expressed angiotensin-converting enzyme 2 (ACE-2), neuropilin-1 (NRP-1), and several other known SARS-CoV-2 receptor and were highly vulnerable to infection. Infection was determined by secreted viral RNA content and confirmed with SARS-CoV-2 nucleocapsid protein (NP) in the infected cells by immunocytochemistry. Differential responses of healthy and AD individuals-derived OM cells to SARS-CoV-2 were determined by RNA sequencing.ResultsResults indicate that cells derived from cognitively healthy donors and individuals with AD do not differ in susceptibility to infection with the wild-type SARS-CoV-2 virus. However, transcriptomic signatures in cells from individuals with AD are highly distinct. Specifically, the cells from AD patients that were infected with the virus showed increased levels of oxidative stress, desensitized inflammation and immune responses, and alterations to genes associated with olfaction. These results imply that individuals with AD may be at a greater risk of experiencing severe outcomes from the infection, potentially driven by pre-existing neuroinflammation.ConclusionsThe study sheds light on the interplay between AD pathology and SARS-CoV-2 infection. Altered transcriptomic signatures in AD cells may contribute to unique symptoms and a more severe disease course, with a notable involvement of neuroinflammation. Furthermore, the research emphasizes the need for targeted interventions to enhance outcomes for AD patients with viral infection. The study is crucial to better comprehend the relationship between AD, COVID-19, and anosmia. It highlights the importance of ongoing research to develop more effective treatments for those at high risk of severe SARS-CoV-2 infection.Graphical

Antiviral Functions of Type I and Type III Interferons in the Olfactory Epithelium.

The olfactory neuroepithelium (OE) is one of the few neuronal tissues where environmental pathogens can gain direct access. Despite this vulnerable arrangement, little is known about the protective mechanisms in the OE to prevent viral infection and its antiviral responses. We systematically investigated acute responses in the olfactory mucosa upon exposure to vesicular stomatitis virus (VSV) via RNA-seq. VSVs were nasally inoculated into C57BL/6 mice. Olfactory mucosae were dissected for gene expression analysis at different time points after viral inoculation. Interferon functions were determined by comparing the viral load in interferon receptor knockout (Ifnar1-/- and Ifnlr1-/-) with wildtype OE. Antiviral responses were observed as early as 24 h after viral exposure in the olfactory mucosa. The rapidly upregulated transcripts observed included specific type I as well as type III interferons (Ifn) and interferon-stimulated genes. Genetic analyses demonstrated that both type I and type III IFN signaling are required for the suppression of viral replication in the olfactory mucosa. Exogenous IFN application effectively blocks viral replication in the OE. These findings reveal that the OE possesses an innate ability to suppress viral infection. Type I and type III IFNs have prominent roles in OE antiviral functions.

Spinal cord injury: Olfactory ensheathing cell-based therapeutic strategies.

Spinal cord injury (SCI) is a highly disabling neurological disorder that is difficult to treat due to its complex pathophysiology and nerve regeneration difficulties. Hence, effective SCI treatments are necessary. Olfactory ensheathing cells (OECs), glial cells derived from the olfactory bulb or mucosa, are ideal candidates for SCI treatment because of their neuroprotective and regenerative properties, ample supply, and convenience. Invitro, animal model, and human trial studies have reported discoveries on OEC transplantation; however, shortcomings have also been demonstrated. Recent studies have optimized various OEC transplantation strategies, including drug integration, biomaterials, and gene editing. This review aims to introduce OECs mechanisms in repairing SCI, summarize the research progress of OEC transplantation-optimized strategies, and provide novel research ideas for SCI treatment.

Olfactory Mucosal Cells of Individuals with Alzheimer’s Disease Display Alterations Similar to the Alzheimer Brain

AbstractBackgroundA sense of smell is orchestrated in the first place by the cells of the olfactory mucosa (OM), located in the rooftop of the nasal cavity. Impairments in olfaction are implicated in aging but also in Alzheimer’s disease (AD). Here, we describe the observed alterations in cultures of OM cells derived from individuals with late‐onset AD or amnestic form of mild cognitive impairment (MCI) when compared to OM cells derived from their cognitively healthy control subjects.MethodPrimary cultures of OM cells were harvested from biopsies of OM derived from cognitively healthy individuals and individuals with late‐onset AD or amnestic MCI and cultured in vitro. First, the cell types present in cultures of OM cells and genes differentially expressed between the OM cells derived from control subjects and individuals AD or MCI OM were assessed by single cell RNA‐sequencing (scRNA‐seq). Next, a total RNA‐seq was applied to a larger cohort of samples. The results of total and scRNA‐seq were further validated with various functional in vitro assays including inductively coupled plasma mass spectrometry (ICP‐MS) and measurements of intracellular ATP levels.ResultThe scRNA‐seq unveiled a total of five distinct cell populations in OM cell cultures. A total of 240 previously AD‐related genes were found to be differentially expressed between the AD and control OM cell cultures. Similarly, the MCI OM cells harbored 224 differentially expressed genes when compared to OM cells derived from their control subjects. Common for both AD and MCI OM cells were the alterations in genes related to mitochondrial function when compared to controls. Additionally, the ICP‐MS revealed increased levels of zinc, calcium and sodium in the AD OM cells in comparison to the OM cells derived from cognitively healthy subjects.ConclusionThe results implicate the role of olfactory mucosa as a site displaying early alterations related to Alzheimer’s disease.

Possible Protective Effects of Quercetin versus Quercetin Loaded Chitosan Nanoparticles on Zinc Sulfate Induced Olfactory Mucosa and bulb Injury in Adult Male Albino Rats; Behavioral, Biochemical, Histological and immunohistochemical study

Possible Protective Effects of Quercetin versus Quercetin Loaded Chitosan Nanoparticles on Zinc Sulfate Induced Olfactory Mucosa and bulb Injury in Adult Male Albino Rats; Behavioral, Biochemical, Histological and immunohistochemical study

The Effect of Sildenafil Citrate (Viagra®) on Nasal Olfactory Mucosa of Adult Male Albino Rat: caspase-3 and inducible nitric oxide synthase protein expressions.

The Effect of Sildenafil Citrate (Viagra®) on Nasal Olfactory Mucosa of Adult Male Albino Rat: caspase-3 and inducible nitric oxide synthase protein expressions.