Olfactory epithelium, which detects and transmits odor signals, is critical for the function of olfactory system. Olfactory epithelium is able to recover spontaneously after injury under normal circumstances, but this ability is dampened in certain diseases or senility, which causes olfactory dysfunction. The olfactory epithelium consists of basal cells, sustentacular cells and olfactory sensory neurons. In order to develop an olfactory epithelial organoid containing multiple olfactory cell types in vitro, we used three-dimensional culture model and small molecules screening. This organoid system consists of horizontal basal-like cells, globose basal-like cells, sustentacular-like cells and olfactory sensory neurons-like cells. Through statistical analysis of clone diameter, immunofluorescence staining and qPCR detection of the expression level of related marker genes. We identified a series of growth factors and small molecule compounds that affected the proliferation, composition and gene expression of the organoids. CHIR-99021, an activator of Wnt signaling pathway, increased the colony formation and proliferation rate of olfactory epithelial organoids and the expression level of marker genes of olfactory sensory neurons-like cells. In addition, each factor in the culture system increased the proportion of c-Kit-positive globose basal-like cell colonies in organoids. Moreover, EGF and vitamin C were both beneficial to the expression of horizontal basal-like cell marker genes in organoids. The established olfactory epithelial organoid system mimicked the process of olfactory epithelial stem cells differentiating into various olfactory epithelial cell types, thus providing a research model for studying olfactory epithelial tissue regeneration, the pathological mechanism of olfactory dysfunction and drug screening for olfactory dysfunction treatment.
Humans ; Olfactory Mucosa/metabolism* ; Epithelial Cells ; Organoids/metabolism* ; Olfaction Disorders/metabolism*

OBJECTIVE: To study the expression characteristics of NeuN (neuron-specific nuclear protein/neuronal nuclei) in the development of mice olfactory bulb and epithelium. METHOD: The expression of NeuN in the heads of E9.5, E11.5, E14.5, E17.5, newborn and adult mice were examined by immunofluorescence method. RESULT: NeuN was expressed in mature neural cells at the margin area of olfactory bulb of new natal mice while the layers of olfactory bulb could not be distinguished. Mature neural cells expressing NeuN aggregated in the granule cells layer, when layers of olfactory bulb of adult mice were clearly formed. The expression of NeuN could not be observed in bipolar olfactory receptor neurons (ORN) in olfactory epithelium. CONCLUSION It is usually believed that NeuN is expressed in most mature neurons. However, during the development of olfactory bulb and epithelium, NeuN is expressed only in mature neural cells migrating from margin area to the inner granule cells layer of olfactory bulb. It is proposed that the migration of granule cells in olfactory bulb might be related to the formation of smell pattern.
Animals ; Animals, Newborn ; Female ; Mice ; Nerve Tissue Proteins ; metabolism ; Neurons ; metabolism ; Nuclear Proteins ; metabolism ; Olfactory Bulb ; embryology ; growth & development ; metabolism ; Olfactory Mucosa ; embryology ; growth & development ; metabolism ; Pregnancy

Sialic acid residues are constant constituents of the glycoproteins of the airways in all species. Sialoglycoproteins are the main acidic glycoprotein and their functions are to mediate cell adherence, to control the viscoelasticity of mucus and to serve as receptor sites for the binding of exogenous macromolecules. The purpose of this study was to investigate the differences in the distribution of sialoglycoproteins as a terminal sugar and in the composition of the penultimate sugar according to aging in the murine nasal respiratory and olfactory mucosa. Nasal cavities of mice (BALB/c) were fixed by intracardiac perfusion with 2.0% glutaraldehyde and embedded in Epon 812. First, the serial sections were stained with Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA). Then, the adjacent sections were stained with DBA and PNA before and after neuraminidase digestion in all experimental groups. Apical cell surfaces of olfactory mucosa and cilia on a few ciliated cells in the mucosa of the septum and nasal floor were labelled with MAA, but cell surfaces of respiratory mucosa, Bowman's glands and goblet cells were not labelled with MAA, irrespective of aging. Apical cell surfaces of both olfactory and respiratory mucosa and Bowman's glands were stained with SNA, however, goblet cells were not labelled with SNA. After neuraminidase digestion to remove terminal sialic acid residues of sialoglycoproteins, only cell surfaces of respiratory mucosa were labelled with PNA, but goblet cells, cell surfaces of olfactory mucosa and Bowman's glands were not labelled with PNA. Cell surfaces and Bowman's glands of olfactory mucosa were labelled with DBA after neuraminidase digestion, but cell surfaces of respiratory mucosa and goblet cells were not labelled with DBA. Our results indicate that there were different carbohydrate structures of sialoglycoconjugates in olfactory and respiratory mucosa, and it was not influenced by aging.
Aging/metabolism* ; Animal ; Carbohydrates/analysis* ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa/chemistry* ; Olfactory Mucosa/chemistry* ; Sialoglycoproteins/analysis*

OBJECTIVE: To study the expression of PCNA and LNGFr in olfactory epithelium of patients suffering from dysosmia caused by chronic sinusitis, and the function of LNGFr. METHOD: Forty-six patients undergoing FESS were chosen. Before operation, their olfactory functions were examined with CCCRC. According to their CCCRC scores, they were divided into three groups. Group A: Patients with chronic sinusitis and dysosmia 25 cases; Group B: Patients with chronic sinusitis and a normal olfactory function 10 cases; Group C: Patients with deviation of nasal septum and a normal olfactory function 11 cases. The expressions of PCNA and LNGFr were measured in olfactory mucosas of the three groups by immunohistochemistry. RESULT: In basal cells, the expression of PCNA and LNGFr in group A was higher than that in group B (P < 0.01). and in group C (P < 0.01). There was negative correlation between positive cells of PCNA and CCCRC score in basal cells of group A (r = -0.7441, P < 0.01); There was negative correlation between integral optical density of LNGFr and CCCRC score in basal cells of group A (r = -0.4407, P < 0.05). There was positive correlation between positive cells of PCNA and integral optical density of LNGFr in basal cells of group A (r = 0.5317, P < 0.01). CONCLUSION Basal cells proliferated dramatically in patients suffering from dysosmia caused by chronic sinusitis. The proliferating capacity of basal cells was related to up-regulation of LNGFr expression.
Chronic Disease ; Humans ; Immunohistochemistry ; Nerve Tissue Proteins ; metabolism ; Olfaction Disorders ; metabolism ; Olfactory Mucosa ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; Receptors, Nerve Growth Factor ; metabolism ; Sinusitis ; complications

Objective: To investigate the effects of dopamine on olfactory function and inflammatory injury of olfactory bulb in mice with allergic rhinitis (AR). Methods: AR mouse model was established by using ovalbumin (OVA), and the mice were divided into two groups: olfactory dysfunction (OD) group and without OD group through buried food pellet test (BFPT). The OD mice were randomly divided into 2 groups, and OVA combined with dopamine (3, 6, 9 and 12 days, respectively) or OVA combined with an equal amount of PBS (the same treatment time) was administered nasally. The olfactory function of mice was evaluated by BFPT. The number of eosinophils and goblet cells in the nasal mucosa were detected by HE and PAS staining. Western blotting, immunohistochemistry or immunofluorescence were used to detect the expression of olfactory marker protein (OMP) in olfactory epithelium, the important rate-limiting enzyme tyrosine hydroxylase (TH) of dopamine, and the marker proteins glial fibrillary acidic protein (GFAP) and CD11b of glial cell in the olfactory bulb. TUNEL staining was used to detect the damage of the olfactory bulb. SPSS 26.0 software was used for statistical analysis. Results: AR mice with OD had AR pathological characteristics. Compared with AR mice without OD, the expression of OMP in olfactory epithelium of AR mice with OD was reduced (F=26.09, P<0.05), the expression of GFAP and CD11b in the olfactory bulb was increased (F value was 38.95 and 71.71, respectively, both P<0.05), and the expression of TH in the olfactory bulb was decreased (F=77.00, P<0.05). Nasal administration of dopamine could shorten the time of food globule detection in mice to a certain extent, down-regulate the expression of GFAP and CD11b in the olfactory bulb (F value was 6.55 and 46.11, respectively, both P<0.05), and reduce the number of apoptotic cells in the olfactory bulb (F=25.64, P<0.05). But dopamine had no significant effect on the number of eosinophils and goblet cells in nasal mucosa (F value was 36.26 and 19.38, respectively, both P>0.05), and had no significant effect on the expression of OMP in the olfactory epithelium (F=55.27, P>0.05). Conclusion: Dopamine can improve olfactory function in mice with AR to a certain extent, possibly because of inhibiting the activation of glial cells in olfactory bulb and reducing the apoptotic injury of olfactory bulb cells.
Animals ; Disease Models, Animal ; Dopamine ; Humans ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa/metabolism* ; Olfactory Bulb/pathology* ; Ovalbumin ; Rhinitis, Allergic/metabolism*

OBJECTIVESTo observe olfactory ensheathing glia (OEG) survival and repair in vivo for spinal cord injury after OEG transplantation.

METHODSThe OEG was cultured with the olfactory bulb of Wistar neonate rats. The spinal cords contusion was made in group A, B, and C with the New York University impactor, then complete transection was performed in the contusion area in group A. OEG labeled by Hoechst was transplanted in group A and B. In group C, DMEM were injected. In group D, laminectomies were done without cord contusion and transection. The functional recovery of the spinal cord injury [Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale scores] and changes of body weight were observed. The tissue sections were done 24 weeks postoperatively. HE staining, neurofibril (NF) immunohistochemical staining, and silver staining were performed respectively to observe the pathologic changes and axon regeneration. The survival of OEG labeled by Hoechst was observed under the fluorescence microscope.

RESULTSLocomotive behaviour improved 4 weeks postoperatively. The BBB locomotion scores of group A and B were significantly higher than that of group C in all periods (from 4 weeks to 24 weeks) (P < 0.01). Sixteen weeks after operation, the BBB locomotion scores became stable and showed no change. HE staining showed that the area of spinal cord injury was disorder and the number of nerve cell was more in group A and B. In group C, there was the obvious cavum and few wring nerve fiber in the area of spinal cord injury. The nerve fibers innervated to the injuried area in group A and B were more than that of group C, but less than that of group D. A great number of OEG labeled by Hoechst were observed around spinal injuried area under fluorescence microscope. After operation, the body weight reduced in every group. The body weight of group D had recovered after 2 weeks and gradully increased. After 4 weeks, the body weight in group A, B, and C decreased to the minimum and were significantly less than that of group D (P < 0.01). After this, body weight in group A and B increased and was significantly more than that of group C (P < 0.05).

CONCLUSIONSOEG transplantation can promote the axons regeneration and the recovery of locomotion function in experimental spinal cord injuries.

Animals ; Animals, Newborn ; Axons ; physiology ; Cells, Cultured ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Nerve Regeneration ; Neuroglia ; cytology ; transplantation ; Olfactory Bulb ; cytology ; transplantation ; Olfactory Mucosa ; cytology ; transplantation ; Rats ; Rats, Wistar ; Spinal Cord ; physiopathology ; surgery ; Spinal Cord Injuries ; surgery

OBJECTIVE: To study the expression of apoptosis related genes, Bcl-2, bax, and iNOS in the olfactory epithelium of mice infected with influenza virus, and to discuss how they regulate apoptosis of the olfactory sensory neurons. METHOD: The expression levels of apoptosis related genes were detected with semi-quantity RT-PCR. RESULT: (1) The expression levels of Bcl-2 mRNA remain relatively constant after virus inoculation; (2) The expression levels of bax mRNA increased massively, and decreased gradually; (3) The expression levels of iNOS mRNA increased significantly in a short period, and then fell down to the undetectable level gradually. CONCLUSION The apoptosis related genes, Bcl-2, bax, and iNOS may play important roles in regulation of apoptosis of olfactory sensory neurons of mice infected with influenza virus.
Animals ; Mice ; Mice, Inbred BALB C ; Nitric Oxide Synthase Type II ; metabolism ; Olfactory Mucosa ; metabolism ; Orthomyxoviridae ; Orthomyxoviridae Infections ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate unusual pathological features of olfactory neuroblastoma (ONB) and its correlation with the clinical prognosis.

METHODSTotally 40 cases of ONB were studied using histology and immunohistochemistry techniques. All the cases of ONB were graded according to Hyams Grading system.

RESULTSONB consisted of small round tumor cells growing in nests or lobules separated by fibrovascular septa. Characteristically, there were neurofibrillary intercellular matrices and Homer-Wright or Flexner-Wintersteiner rosette formation. The unusual structures including epithelial components such as mucous or squamous cell nests which were found in 45.0% (18/40), and 15.0% (6/40) respectively. In addition, 3 cases showed an in-situ form with invasion of tumor into olfactory epithelium, and there was exogenous papillary proliferation seen in 2 cases. Log-rank survival analysis demonstrated that Hyams Grading had no statistical correlation with the prognosis. The presence of necrosis was correlated with a poor prognosis (P = 0.016) while the presence of mucous cells was correlated with a good prognosis (P = 0.011).

CONCLUSIONSUnusual pathological structures including epithelial structures, in-situ invasion of tumor tissue into the involving olfactory epithelium and exogenous papillary proliferation can be found in ONB, suggesting that ONB may originate from the undifferentiated basal cells of olfactory epithelium, through bipotential differentiation. The presence of tumor necrosis in ONB is a poor prognostic indicator while the presence of mucous cells suggests a good prognosis.

Adolescent ; Adult ; Aged ; Child ; Chromogranin A ; metabolism ; Esthesioneuroblastoma, Olfactory ; metabolism ; pathology ; Female ; Follow-Up Studies ; Humans ; Lung Neoplasms ; secondary ; Lymphatic Metastasis ; Male ; Nasal Cavity ; Necrosis ; Neoplasm Invasiveness ; Neoplasm Recurrence, Local ; Nose Neoplasms ; metabolism ; pathology ; Olfactory Mucosa ; pathology ; Phosphopyruvate Hydratase ; metabolism ; Survival Rate ; Synaptophysin ; metabolism ; Young Adult

To explore whether hypoxic condition could promote the olfactory mucosa mesenchymal stem cells (OM-MSCs) to differentiate into neurons with the olfactory ensheathing cells (OECs) supernatant and the potential mechanisms.
 Methods: The OM-MSCs and OECs were isolated and cultured, and they were identified by flow cytometry and immunofluorescence. The OM-MSCs were divided into three groups: a 3%O2+ HIF-1α inhibitors (lificiguat: YC-1) + OECs supernatant group (Group A) , a 3%O2 + OECs supernatant group (Group B) and a 21%O2 + OECs supernatant group (Control group). The neurons, which were differentiated from OM-MSCs, were assessed by immunofluorescence test. The mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), βIII-tubulin and glial fibrillary acidic portein (GFAP) were detected by quantitative polymerase chain reaction (Q-PCR) and Western blot. The potassium channels were analyzed by patch clamp.
 Results: The neurons differentiated from OM-MSCs expressed the most amount of βIII-tubulin, and the result of Q-PCR showed that HIF-1α expression in the Group B was significantly higher than that in the other groups (all P<0.05). Western blot result showed that the βIII-tubulin protein expression was significantly higher and GFAP protein expression was obviously decreased in the Group B (both P<0.05). The patch clamp test confirmed that the potassium channels in the neurons were activated.
 Conclusion: Hypoxic condition can significantly increase the neuronal differentiation of OM-MSCs by the OECs supernatant and decrease the production of neuroglia cells, which is associated with the activation of HIF-1 signal pathway.
Blotting, Western ; Cell Differentiation ; physiology ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; pharmacology ; Flow Cytometry ; Glial Fibrillary Acidic Protein ; metabolism ; Hypoxia ; physiopathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Indazoles ; pharmacology ; Mesenchymal Stem Cells ; physiology ; Neurogenesis ; physiology ; Neuroglia ; metabolism ; physiology ; Neurons ; physiology ; Olfactory Mucosa ; Potassium Channels ; Signal Transduction ; Tubulin ; metabolism