Development of an olfactory epithelial organoid culture system based on small molecule screening / 生物工程学报

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.

Alteração na representação de subpopulações de neurônios no epitélio olfatório de camundongos Ric8b cKO / Altered representation of neuronal sub-populations in the olfactory epithelium of Ric8b conditional knock-out mice

O objetivo desse trabalho foi identificar as consequências moleculares e funcionais da falta da proteína Ric8b no epitélio olfatório de camundongos. Para esse fim, comparamos o transcriptoma de epitélio olfatório de camundongos knock-out tecido específico para a proteína RIC8B (Ric8b cKO) com o dos seus irmãos tipo selvagem (WT). Identificamos muitos genes que apresentaram expressão reduzida no epitélio olfatório do camundongo Ric8b cKO, mas também vários genes que apresentaram a sua expressão aumentada. A maioria dos genes com expressão reduzida corresponde a genes normalmente expressos em neurônios olfatórios maduros, como por exemplo os genes de receptores olfatórios, o que é compatível com o fato já conhecido de que os camundongos Ric8b cKO apresentam um menor número desses neurônios. Inesperadamente, apesar de a maioria dos genes de receptores olfatórios ter a sua expressão diminuída no camundongo Ric8b cKO, observamos que um grupo destes genes de receptores teve a sua expressão aumentada. Os camundongos Ric8b cKO apresentaram também genes marcadores de outros tipos celulares que não neurônios canônicos com expressão aumentada no seu epitélio olfatório. Dentre eles, os mais significativamente alterados foram os genes marcadores de neurônios Trpc2+ tipo B (que expressam a guanilato ciclase solúvel Gucy1b2). Sabe-se que este tipo de neurônio é responsável pela sensibilidade a diferentes gases, e concordantemente, observamos que os camundongos Ric8b cKO apresentaram um aumento da sensibilidade a gás carbônico. Como o olfato apresenta um papel importante na regulação de ingestão alimentar, analisamos como os camundongos Ric8b cKO se comportam frente a diferentes dietas. Interessantemente, observamos que esses animais não apresentam preferência por alimento rico em gorduras quando comparado aos seus irmãos tipo selvagem. Nossos resultados sugerem, portanto, que a ausência da proteína RIC8B resulta na alteração de representatividade de neurônios canônicos e não canônicos no epitélio olfatório de camundongos, o que por sua vez leva a alterações funcionais e comportamentais

The objective of this work was to identify the molecular and functional consequences of the lack of the RIC8B protein in the main olfactory epithelium of mice. To this end, we compared the olfactory epithelium transcriptome of Ric8b tissue-specific knock-out mice (Ric8b cKO) with that of their wild-type littermates (WT). We identified many genes with differential expression, many of which were downregulated and also some which were upregulated in the olfactory epithelium of the Ric8b cKO mice. Most of the downregulated genes correspond to genes normally expressed in mature olfactory sensory neurons, such as olfactory receptor genes. This is compatible with the already known fact that the Ric8b cKO mice have less of this kind of neuron. Unexpectedly, even though most of the olfactory receptor genes were downregulated, we observed a subset of these genes that had their expression upregulated in the Ric8b cKO mice. The Ric8b cKO mice also showed upregulation for genes that are markers for cell types other than canonic neurons in their olfactory epithelium. Among these, the most significantly altered were the markers for neurons Trpc2+ type B (that express the soluble guanylate cyclase Gucy1b2). It is known that this kind of neuron is responsible for sensitivity to different gases. Accordingly, we observed that the Ric8b cKO mice presented a higher sensitivity to carbon dioxide. Since olfaction has an important role in food intake, we analyzed how the Ric8b cKO mice behaved with different diets. Interestingly, we observed that the Ric8b cKO mice lack preference for high fat diet when compared to their wild-type littermates. Our results indicate, therefore, that the lack of the RIC8B protein results in altered representativity of canonic and non-canonic neurons in the olfactory epithelium of mice, which then leads to altered function and behavior

Rabbit vomeronasal organ-derivered cells have mesenchymal profile and neuronal commitment / Las células derivadas del órgano vomeronasal del conejo tienen perfil mesenquimatoso y compromiso neuronal

SUMMARY: The vomeronasal organ (VNO) is an accessory organ involved on the olfactory pathway, that detects pheromones and emits signals in order to modulate social and reproductive behavior. The VNO stem cells replace neurons throughout life. The aim of this study was to isolate and characterize cells derived from the vomeronasal organ from New Zealand rabbits. Five male rabbits with 120 days were used for cell isolation and culture. Results: VNO-derived cells presented labelling for proliferation (PCNA), undifferentiated profile (Nanog), neuronal (GFAP), mesenchymal stem cells (CD73, CD90 and CD105 and Stro-1). Also, presence of cytoskeletal (Vimentin, b-tubulin and CK-18) and absence of hematopoietic markers (CD34, CD117 and CD45) both by immunofluorescence and flow cytometry. By PCR it was possible to verify the expression of some undifferentiated profile (Oct-4), neuronal (Nestin) and mesenchymal (CD73, CD105 and Vimentin) genes. Functionally, VNO-derived cells differentiate in vitro into adipocytes, osteocytes and chondrocytes, and presented no tumorigenic potential when injected to Balb/c nu/nu mice. In conclusion, the rabbit VNO-derived cells have a profile that could be supportive to VNO olfactory/neuroreceptor epithelium by delivering factors to epithelial turnover or even by differentiation into epithelial cells to replacement of commissural epithelium.

RESUMEN: El órgano vomeronasal (OVN) es un órgano accesorio de la vía olfatoria, que detecta feromonas y emite señales que afectan la modulación del comportamiento social y reproductivo. Las células madre OVN reemplazan las neuronas durante toda la vida. El objetivo de este estudio fue aislar y caracterizar células derivadas del órgano vomeronasal de conejos raza Nueva Zelanda. Para el aislamiento y el cultivo celular se utilizaron cinco conejos machos con una edad de 120 días. Las células del OVN presentaron etiquetado para la proliferación (PCNA), un perfil indiferenciado (Nanog), neuronal (GFAP), células madre mesenquimales (CD73, CD90 y CD105 y Stro-1). Además, se ob- servó presencia de citoesqueleto (Vimentina, β-tubulina y CK-18) y ausencia de marcadores hematopoyéticos (CD34, CD117 y CD45) tanto por inmunofluorescencia como por citometría de flujo. Me- diante PCR fue posible verificar la expresión de algunos genes de perfil indiferenciado (Oct-4), neuronal (Nestin) y mesenquimatoso (CD73, CD105 y Vimentin). Las células derivadas del OVN se diferencian in vitro en adipocitos, osteocitos y condrocitos, y no presentan un potencial tumorigénico al ser infiltrados en ratones Balb / c nu / nu. En conclusión, las células derivadas de OVN de conejo tienen un perfil que podría ser compatible con el epitelio olfatorio / neurorreceptor de OVN transmitiendo factores al recambio epitelial o incluso mediante la diferenciación en células epiteliales para reemplazar el epitelio comisural.

Neurogenesis and Regulation of Olfactory Epithelium

The olfactory epithelium is capable of structural and functional recovery after injury through neurogenesis. Neurogenesis occurs via stem cells in the olfactory epithelium. Horizontal basal cells and globose basal cells in the basal layer of the epithelium have the characteristics of stem cells and progenitor cells of olfactory neurons. In order for the horizontal basal cells and globose basal cells to differentiate into olfactory neurons, distinct transcriptional factors are required at each stage. These transcription factors inhibit or synergize with each other or cells at each differentiation stage, regulating olfactory neurogenesis. Recently, the regulation of neurogenesis and development through epigenetic controls that change gene expression without changing the gene sequence have been studied. Studies of olfactory epithelium have helped to elucidate complex neurological systems including spinal cord and brain. In particular, features of neurogenesis will lead to medical advances in the treatment of central nervous diseases, which until this time have been considered impossible.

Células-tronco derivadas do epitélio olfatório: perspectivas terapêuticas na medicina veterinária / Stem cells derived from olfactory epithelium: therapeutic perspectives in veterinary medicine

O epitélio olfatório (EO) é uma fonte promissora de células-tronco (CTEO) para o uso terapêutico na medicina veterinária e humana, especialmente em doenças correlacionadas com o sistema nervoso periférico (medula espinhal) e central (cérebro e tronco encefálico) , pois as CTEO possuem a capacidade de se diferenciar em células do sistema nervoso, tais como: neurônios, oligodendrócitos e astrócitos. Em humanos estas células são utilizadas em ensaios terapêuticos de doenças degenerativas como o Alzheimer e Parkinson. Em animais a casuística relativa das doenças neurodegenerativas crônicas ou agudas é baixa, devido à dificuldade de diagnóstico definitivo, desta forma o enfoque das pesquisas com terapia celular são em sua grande maioria em lesões mecânicas na medula espinhal. Devido à falta de padronização e seleção das melhores metodologias que permitam confrontação de estudos, esta revisão busca reunir as mais recentes publicações, descrevendo o potencial uso das células-tronco do epitélio olfatório em terapias celulares, discutindo os principais desafios e perspectivas futuras com enfoque na medicina veterinária.(AU)

The olfactory epithelium (OE) is a promising source of stem cells (OESC) for therapeutic use in veterinary and human medicine, especially in diseases correlated with the peripheral (spinal cord) and central (brain and brainstem) nervous system (CNS), because of its ability to differentiate into neurons, astrocytes and oligodendrocytes cells. In humans, OESC has been used primarily in therapeutic trials for degenerative diseases such as Alzheimer and Parkinson. In animals, the frequency of corresponding cases of chronic or acute neurodegenerative diseases is very low, because of the difficulty of a definitive diagnosis; thus, the focus of cell therapy research are mostly mechanical spinal cord injuries. Due to the lack of normalization and selection of the best methodologies for comparative studies, this review aims to analyze recent reports on the potential use of stem cells from the olfactory epithelium in cell therapies and to discuss the main challenges and future prospects in veterinary medicine.(AU)

El cultivo de precursores neuronales del epitelio olfatorio: Un modelo para estudiar la neurofisiopatología de la esquizofrenia / The culture of olfactory ephitelium neuronal precursors: A model for studying schizophrenia neurophysiopathology

Schizophrenia is a mental disorder characterized by delusions, hallucinations, disorganization in speech and thinking as well as alterations in social behavior, and affective flattening. Schizophrenic patients also have an olfactory deficit since prodromal stages of this disorder. The olfactory deficit could be present in schizophrenic patients due to anatomic and structural alteration of the Central Nervous System, or peripheral abnormalities in the olfactory epithelium. The major alterations of the Central Nervous System are diminished volumes of olfactory bulb and structures of primary olfactory cortex, hippocampus and amygdala. While, olfactory epithelium has functional abnormalities in cellular differentiation and electric response of sensory olfactory neurons, which suggest an impairment of the odor transduction. The cellular culture of olfactory epithelium has allowed isolating multipotential progenitor cells that have the ability to proliferate and differentiate in mature neurons and glia. This model could provide evidences on the causes that could explain the olfactory deficits in schizophrenia. Moreover, it will allow testing hypothesis on pathophysiological causes of this mental disorder in different of stages of the neurodevelopment. In addition, olfactory epithelial neuronal precursors constitute a novel model to detect genetic, proteomic and functional biomarkers that allow a biological diagnosis.

La esquizofrenia (EZ) es un trastorno psiquiátrico que se caracteriza por la presencia de delirios, alucinaciones, pensamiento desorganizado, lenguaje desestructurado, alteraciones del comportamiento social y aplanamiento afectivo, entre otros síntomas. Los pacientes con EZ también presentan un déficit en la capacidad olfatoria desde la fase prodrómica del trastorno. El déficit olfatorio en la EZ puede presentarse por alteraciones anatómico-estructurales del SNC o por anomalías a nivel periférico en el epitelio olfatorio. Las alteraciones principales del SNC son la disminución del volumen de los bulbos olfatorios, de estructuras de la corteza olfatoria primaria, del hipocampo y de la amígdala coronal. El epitelio olfatorio en los estadios tempranos de la EZ presenta anomalías funcionales en la diferenciación y en la respuesta biofísica de las neuronas sensoriales olfatorias, lo que sugiere que existe un desacoplamiento de la transducción olfatoria. El cultivo celular del epitelio olfatorio ha permitido aislar células progenitoras multipotenciales que poseen la capacidad de proliferar y diferenciarse en neuronas y glía. El estudio de este modelo podría aportar evidencia sobre las causas que explicarían el déficit olfatorio en la esquizofrenia y permitiría estudiar hipótesis que intenten explicar las causas de la fisiopatología de este trastorno en el neurodesarrollo así como detectar biomarcadores genéticos, proteómicos o funcionales que permitan un diagnóstico biológico.

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium / Estabelecimento de um protocolo para obtenção de linhagens de células-tronco neuronais a partir do epitélio olfatório de cães

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5 percent CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

Foi realizado um estudo morfológico e por cultivo celular a partir de células provenientes da mucosa olfatória de cães, como forma de estabelecer um protocolo de cultivo, como uma proposta para o tratamento de lesões traumáticas e nervosas degenerativas nestes animais e futuramente, para que tais resultados possam ser aplicados a espécie humana. Foram utilizados doze cães sem raça definida, a termo, oriundos de castrações do Centro de Controle de Zoonoses de São Paulo. O tecido da lâmina cribiforme do etmóide dos fetos foi coletado sob necropsia, em condições estéreis, 1 a 2 horas post mortem, por meio de incisão uterina e acesso da região fetal supracitada. Depois as células isoladas desse tecido foram adicionadas em médio DMEM/F-12 sob condições padrão (5 por cento CO2, >37ºC). As células obtidas a partir de biópsias do epitélio olfatório de cães apresentaram rápido crescimento após 24 horas de cultivo, demonstrando morfologia esférica, sendo encontradas flutuando ao redor do fragmento aderido à garrafa de cultura. Após 20 dias, foram verificados tipos celulares específicos, predominantemente elipsóides ou fusiformes, foram observadas in vitro. Sob avaliação por imunofluorescência indireta observaram-se células com expressão positiva para marcadores de precursores neuronais (GFAP, Neurofilamentos, oligodendrócitos e â-tubulina III). O índice de proliferação celular mostrou-se positivo para Ki67 com uma tendência de marcação de grupos celulares ao longo da região apical, enquanto a imunomarcação para PCNA mostrou-se predominantemente em grupos celulares residentes sobre a lâmina basal. A microscopia eletrônica de transmissão do epitélio olfatório de cães revelou células com citoplasma eletrodenso e mesma distribuição das células marcadas positivamente para PCNA. A atividade metabólica foi confirmada pela presença de eucromatina em muitas regiões celulares. Todos estes aspectos sustentam a hipotese sobre a presença de células progenitoras ...

Multipotency of cultured olfactory epithelium neural stem cells / 第二军医大学学报

Objective: To investigate the culture method for adult olfactory epithelium neural stem cells and their multipotency during in vitro differentiation. Methods: Olfactory epithelium was sequentially digested with neutral protease and collagenase I A, then the olfactory epithelium neural stem cells were enriched and amplified. Neurospheres were formed by culturing the cells with serum free medium containing bFGF and EGF. Differentiation of neurospheres was induced with medium containing serum; the differentiated cells were identified by immunocytochemistry. Results: Primary cultured olfactory epithelium neural stem cells proliferated and formed colonies. Cells in the colonies expressed the in vivo markers GBC2 and cytokeratin 5. Passaged cells proliferated and formed neurospheres when cultured with serum free medium containing bFGF and EGF. Medium containing serum induced the differentiation of neurospheres. TUJ1 positive neurons, P75NGFR positive olfactory ensheathing cells, GFAP positive astrocytes and NG2 positive oligodendrocyte progenitors were detected in the highly heterogeneous differentiated cells; besides, the cells also contained nestin positive neural stem cells. Conclusion: Cultured adult olfactory epithelium neural stem cells can proliferate and form neurospheres. The neurosphere cells have the multipotency to differentiate into neurons, astrocytes and oligodendrocyte progenitors.

Effect of Systemic Steroid on the Olfactory Epithelium Injured by 3-Methylindole in Mice

BACKGROUND AND OBJECTIVES: Various chemicals can affect the function of olfaction and steroids have been used for the treatment of olfactory dysfunction. In this study, we investigated the effect of chronic dexamethasone treatment on olfactory epithelium injured by 3-methylindole (3-MI). SUBJECTS AND METHODS: 0.75 mg/kg of dexamethasone and 0.15 mL of normal saline were administered to each of the 12 mice belonging to the experimental and control group respectively every other day from 1 week, before a single intraperitoneal administration of 175 mg/kg 3-MI, to 4 weeks after 3-MI injection. Three mice from each group were sacrificed every week, and olfactory epithelium was examined after H & E and immunohistochemical staining. RESULTS: On H & E staining, the height of the olfactory epithelium and the polarity of the cells showed no difference between the two groups. On olfactory marker protein (OMP) staining, the number of OMP-immunoreactive (IR) olfactory receptor cells was significantly increased in the experimental groups from 2 and 4 weeks after 3-MI injection compared with the control group. On proliferating cell nuclear antigen (PCNA) staining, PCNA-IR basal cells were significantly reduced in groups that received dexamethasone from 2 weeks to 3 weeks after injection compared with the control group. CONCLUSION: Dexamethasone shows no protective effect in early necrosis of olfactory epithelium by 3-MI, but showed positive effect on the regeneration of the olfactory receptor cells of olfactory epithelium.

The Properties of Glycoconjugates in the Rat Nasal Mucosa following Inhalation of Formaldehyde / 대한해부학회지

The present study was carried out to investigate the glycoconjugate properties of the nasal mucosa in the rat after inhalation of formaldehyde. Sprague-Dawley male rats were inhalated 30 ppm formaldehyde for 3 times with 3 hours exposure. The olfactory and respiratory mucosa in the nasal mucosa were taken from the animals on 3, 6,9 days and 2, 3, 4, 5 weeks after inhalation of formaldehyde. The properties of glycoconjugate of the olfactory and respiratory mucosa were investigated using nine biotinylated lectins (PSA, UEA I, PHA-L, BSL I, PNA, MAL I, DBA, BSL II or sWGA). In experimental groups, the degenerative changes of the olfactory epithelium were observed until 3 weeks after inhalation of formaldehyde, but the respiratory epithelium was no change. In control group, the olfactory cells in the olfactory epithelium reacted with PSA, UEA I, PNA, DBA, BSL II, sWGA, and the supporting cells reacted with PSA, PHA-L, PNA, MAL I, DBA, BSL II, sWGA, and Bowman's glands reacted with all the lectins. In experimental groups, the olfactory cells reacted with UEA I, DBA, and the supporting cells reacted with PHA-L, MAL I, DBA, UEA I, and the positive reaction of Bowman's glands was increased. In control group, the goblet cells in the respiratory epithelium reacted with UEA I, MAL I, and the ciliated columnar cells reacted with PSA, UEA I, PHA-L, BSL I, DBA, BSL II, sWGA, and the septal nasal glands reacted with all the lectins except UEA I. In experimental groups, the goblet cells reacted with UEA I, MAL I and PNA. Conclusively, the olfactory mucosa was shown a lot of changes in the properties of glycoconjugates following inhalation of formaldehyde, but respiratory mucosa was shown feeble change. These results suggest that there were different sugar residues of glycoconjugate in the olfactory and respiratory mucosa following inhalation of formaldehyde, respectively.

Dinâmica dos precursores celulares do epitélio olfatório de cães sem raça definida: um estudo imunohistoquímico e ultra-estrutural / The dynamic of precursor of the olfactory epithelium of mongrel dogs: an immunohistochemical and ultrastructural study

O epitélio olfatório apresenta um mecanismo de diferenciação em que células-tronco dão origem a células progenitoras amplificadoras, as quais expressam um gene pró-neural denominado Mammalian Achaete Scute Homolog 1 (Mash1). Estas células podem se diferenciar em receptores olfatórios. O epitélio olfatório de cães sem raça definida (3 machos de um ano e 2 fêmeas de três de idade) foi analisado por imunolocalização do antígeno nuclear de proliferação celular (PCNA) e por microscopia eletrônica de transmissão. Verificou-se marcação positiva para PCNA em células do epitélio olfatório, particularmente acima da linha da membrana basal. A ultra-estrutura do epitélio olfatório revelou células adjacentes à lâmina basal, cuja eletrodensidade assemelha-se àquelas presentes no epitélio de sustentação, reforçando a idéia da renovação das células de sustentação e dos neurônios olfatórios locais. O epitélio olfatório é composto células basais, comprometidas com sua renovação, caracterizadas através da intensa atividade mitótica, identificada pela reação positiva ao PCNA. Estes resultados sugerem que há reposição das células sustentaculares locais e do sistema através de mecanismos semelhantes.

Olfactory epithelium presents a mechanism of differentiation where stem cells give arise to amplifying progenitor cell which express Mammalian Achaete Scute Homolog 1 (Mash1). These cells can be differentiated into olfactory receptors. An immunolocalization study and ultrastructural analysis by transmission electron microscopy of olfactory epithelium of mongrel dogs were made using 3 males (one year old) and 2 females (three years old). Labeled cells with positive staining by Proliferating cell nuclear antigen (PCNA) were observed in specific areas of the olfactory epithelium, especially above the basal membrane. The ultrastructure revealed cells adjacent to the basal membrane with morphology resembling sustentacular cells, supporting the idea of renewal of sustentacular and olfactory sensorial cells. Olfactory epithelium contains basal cells committed to self-renewal, characterized by high metabolic activity, identified by positive reaction to PCNA. These results suggested the renewal of sustentacular and sensorial olfactory cells through the same pathway.

Effect of Systemic Steroid on the Olfactory Epithelium Injured by 3-Methylindole in Rats / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Various chemicals can affect the function of olfaction and steroids have been used for the treatment of the olfactory dysfunction. In this study, we investigated the effect of chronic dexamethasone treatment on olfactory epithelium injured by 3-methylindole (3-MI). MATERIALS AND METHOD: 0.75 mg/kg of dexamethasone were administered to 12 experimental rats every other day for 1-4 weeks, following a single intraperitoneal administration of 150 mg/kg 3-MI at the first week. Three rats from each group were sacrificed every week, and olfactory epithelium were examined after H & E and immunohistochemical staining. RESULTS: On H & E staining, the height of the olfactory epithelium and the polarity of the cells showed no difference. On protein gene product 9.5 staining, the number of immunoreactive olfactory receptor cells significantly increased in the experimental groups from 2 and 4 weeks after 3-MI injection compared with the control group. On proliferating cell nuclear antigen staining, immunoreactive basal cells were significantly reduced in groups that received dexamethasone from 2 weeks to 3 weeks after injection compared with the control group. CONCLUSION: Dexamethasone shows no protective effect in early necrosis of olfactory epithelium by 3-MI and showed positive effect on the regeneration of the olfactory receptor cells of olfactory epithelium.

Histologic Study on the Development of Olfactory Mucosa in Rats

BACKGROUND AND OBJECTIVES: There is evidence which suggests that mammals have functional olfactory systems at birth or shortly before birth. This study was performed to investigate perinatal development of the olfactory mucosa. MATERIALS AND METHODS: Sprague-Dawley rats at the 19th gestational day, and of the 1st, 3rd, 7th, 14th, 21st, and 28th postnatal day were sacrificed. The light microscopic investigation of the olfactory mucosa was conducted with hematoxylineosin stain, immunohistochemical stain for proliferating cell nuclear antigen (PCNA) and protein gene product (PGP) 9.5. RESULTS: Number of the cell layers, epithelial thickness, and density of the olfactory receptor cell peaked at the postnatal 14th day. The cells of the basal layer changed from globose cells to basal cell proper with age. The number of the basal cells to the receptor cells decreased with age. PCNA was positive both in the supporting and basal layers. PCNA positivity decreased with age in the supporting layer but stayed stationary in the basal layer. PGP 9.5 was strongly positive in the olfactory receptor cells, dendrites, and the nerve bundles but negative in the supporting and basal layers. CONCLUSION: The olfactory epithelium proliferated maximally at postnatal 14 day, and afterwards the olfactory mucosa tended to show their characteristic maturation with slowed neurogenesis.

Changes of the Olfactory Epithelium in Artificially Induced Hypothyroid Rats

BACKGROUND AND OBJECTIVES: This study was performed to investigate, through immunohistochemical analysis, the thyroid hormone's effect on the olfactory receptor neurons of adult rats. MATERIALS AND METHODS: Hypothyroidism was induced by adding reversible goitrogen propylthiouracil (PTU) to the rats' drinking water (30 mg/kg weight). Sprague-Dawly rats aged nine to ten weeks were divided into three groups : control, six weeks or PTU therapy, and 12 weeks of PTU therapy. Light microscopic investigation of the olfactory mucosa was conducted with an immunohistochemical stain to observe for proliferating cell nuclear antigen (PCNA) and protein genepeptide (PGP) 9.5. RESULTS: The rats in the experimental groups gained less weight compared with normal rats of the same age. Light microscopic examination revealed no statistically significant differences in the thicknesses of the olfactory epithelium and the numbers of cells among the three groups in H-E stains, but the ratio of PCNA(+) supporting cells decreased significantly with longer durations of PTU treatment. As PTU therapy continued, immunoreactivities to PGP 9.5 in olfactory receptor cells decreased remarkably. After 12 weeks of PTU treatment, no immunoreactivity was observed in the olfactory receptor cells. CONCLUSION: These results indicate that the thyroid hormone is essential for maturation of the olfactory receptor neuron.

Formaldehyde Gas Induced Morphological Changes in the Rat Olfactory Epithelium / 대한해부학회지

Experimental animals were inhalated 50 ppm formaldehyde gas for 3 times with one hour exposure and one hour rest. The olfactory mucosa were taken from the animals on 4, 7, 9, 11 days and 2-6 weeks after the inhalation. The characteristics of the various glycoproteins and the mitotic activity of the olfactory epithelial cells were investigated using lectins and 5'-bromodeoxyuridine (BrdU, 50 mg/kg) which injected one hour before sacrifice of the animals. The results were as follows; 1. In experimental animals, the degenerative changes of the olfactory epithelium such as atrophy and squamous metaplasia were observed until 2 weeks after formaldehyde gas inhalation. 2. In control animals, positive reactions appeared in the supporting cell to PNA, SBA, WGA, ECL, PHA-L and in the olfactory cells to PNA, SBA, WGA, UEA and in the proper basal cell to GS-I, SBA, WGA, PHA-L. In experimental groups, the positive reaction was increased in the supporting cells to SBA, ECL, PHA-L and in Bowman's gland to used lectins except ECL, GS-I. 3. The number of BrdU labelled cells in the olfactory epithelium was 14.8+/-1.2/mm in the control animals. The mitotic activities were decreased to 4.8+/-0.8 mm in 2 weeks and recovered in 3 weeks after the gas inhalation. 4. To find the stem cells of olfactory receptor cells, double labelling method was performed with lectins which were specific for proper basal cells (GS-I or PHA-L) and BrdU immunohistochemistry. The ratio of globose/proper basal cells in BrdU labelled cells with GS-I lectin positive reaction was 82%/18% in control group, 39.3%/60.7% in lesion group and 55.5%/44.5% in recovery group.

Ultrastructure of cell types of the olfactory epithelium in a catfish, Heteropneustesfossilis (Bloch).

Transmission electron microscopical study of olfactory epithelium of a mud-dwelling catfish, Heteropneustes fossilis (Bloch) shows receptor, supporting, goblet and basal cells. The receptor cells are of ciliated and microvillous type. Both ciliated and microvillous receptor cells are provided with olfactory knob. The dendrite of all the receptor cells bears many longitudinally arranged microtubules. Occurrence of the rod cell and its function is quite debatable. Specialized juctional complexes between the receptor and adjacent cells are clearly noted. The supporting cells are both ciliated and nonciliated. The ciliated supporting cells are responsible for water ventilation in the olfactory chamber as well as in the inter-lamellar spaces. This facilitates better perception of odours by the receptor cells. In addition to providing mechanical support to other cells, the nonciliated supporting cells also have a secretory function which is evident from the present study. The different stages of maturity of goblet cells are well documented. The presence of white cells in the olfactory epithelium is a very rare finding.

Regional Differences in Proliferative Activity of Nasal Epithelium in Rat

We investigated the active proliferation sites of epithelial cells in normal nasal mucosa by immunohistochemical staining of proliferating cell nuclear antigen (PCNA), the marker of S phase of cell cycle and active cell proliferation. The whole nasal mucosa of the ten normal Sprague-Dawley rats were processed for PCNA immunolabeling. In respiratory portion, distinctly positive reaction was seen mainly in the anterior aspect, that is, the nuclei of squamous and non-ciliated cuboidal/transitional epithelium. These types of epithelial cells are transformed to pseudostratified ciliated epithelium in the posterior direction where positive reaction became scanty. In olfactory epithelium, the nuclei immunoreactive for PCNA were distinct in some area, but absent in other adjacent areas, lacking of region-specific immunolabeling that was observed in respiratory mucosa. These results suggest that anterior portion of nasal cavity is the main proliferation zone of normal nasal respiratory epithelium as well as the main site of protective function. In contrast, the neurogenesis of the olfactory nerve cells is not site-specific, indicating that any region covered by olfactory mucosa may be the main proliferation zone.

EFFECT OF bFGF ON THE PROLIFERATION OF CULTURED OLFACTORY EPITHELIUM ENSHEATHING CELLS IN ADULT RAT / 解剖学报

Objective To investigate the effect of bFGF on the proliferation of olfactory epithelium ensheathing cells. Methods With BrdU incorporation method,the effect of bFGF on the proliferating rate of olfactory epithelium ensheathing cells was observed in the research. Results The olfactory epithelium ensheathing cells can proliferate without any proliferating factor.The bFGF(10?g/L)can enhance the proliferating rate in a moderate way.In this experiment BPE(bovine pituitary extract) can not enhance the stimulating effect of bFGF.Conclusion The bFGF(10?g/L)can stimulate the proliferating rate of the olfactory epithelium ensheathing cells in vitro.

STUDIES ON PURIFIACATION AND IDENTIFICATION OF THE OLFACTORY ENSHEATHING CELLS FROM BOTH ADULT MAN AND CANINE / 解剖学报

Objective To investigate the technical methods for culturing and purifying the olfactory ensheathing cells(OECs) from the adult canine and human olfactory epithclium.To establish a basis for future studying the transplantation of peripheral(OECs) to repair the spinal cord injury in human. Methods Purifying the OECs from the olfactory epithelium of adult canine and man according to their different attachment time with other types of cells.Culturing for 25 days,observed at 6d,10d and 25d,and immunostained with NGFRp75 antibody to identify the OECs. Results The number of cultured olfactory epithelium OECs from both adult canine and man were increased much more after 10 days of culture,and its sharp showed to be bi-polar or tripe-polar and are immunopositive to NGFRp75 antibody.The in vitro OECs of canine grew better than that in man's in the present conditions.Conclusion\ The method of different attachment time seems available in purifying olfactory ensheathing cells from both the adult canine and man olfactory epithelium.