Uptake of BrdU in Olfactory and Respiratory Epithelium of Rabbits with Experimental Sinusitis

Sinusitis was induced in six rabbits in order to evaluate its influence on the proliferation of cells in the olfactory epithelium compared with the respiratory epithelium during conservative antibiotic therapy. Then 1% ofloxacin was injected into the paranasal sinuses. Three normal rabbits were not administered any treatment and served as controls. The rabbits were sacrificed under intravenous anesthesia and the olfactory and respiratory mucosa was excised 24 hours after intravenous administration with the labeling reagent 5-bromo-2'-deoxyuridine (BrdU). The extent of cell proliferation in these tissues was estimated by immunohistochemical staining with BrdU-specific antibody. The uptake of BrdU was significantly increased (p=0.0099) in the respiratory mucosa, but not in the olfactory mucosa. Furthermore, in olfactory epithelium, 79.2% and 16.7% of all BrdU-positive cells were olfactory and basal, respectively. Thus, turnover of epithelial cells due to sinusitis was not as accelerated in the olfactory mucosa as in the respiratory mucosa during antibiotic treatment.     

Olfactory disturbances caused by the anti-cancer drug tegafur

Olfactory disturbances induced by the anticancer drug tegafur were studied in separate clinical and experimental investigations. Five patients with olfactory dysfunction after tegafur were studied and were found to have normal endoscopic findings of the olfactory cleft mucosa. The average period for drug administration was 22 months. Recovery from the olfactory disturbance was poor and biopsy of the olfactory mucosa revealed severely degenerated epithelium. In experimental studies in a guinea pig animal model, effects of oral tegafur on mitotic cells in the olfactory epithelium were examined using bromodeoxyuridine (BrdU) uptake as index. At the conclusion of 3 weeks' treatment, no pronounced morphological changes were seen, but the number of BrdU-incorporating cells decreased in proportion to the dose of tegafur used. Following long-term administration of tegafur 18 months, mitotic cells reacting to BrdU or proliferating cell nuclear antigen had virtually disappeared, indicating persistent inhibition of mitotic cell activity. Morphological changes present included decreased olfactory cell numbers, loss of cells in areas just above basal cells and degeneration of the mucous layer.     

Human olfactory biopsy. The influence of age and receptor distribution.

Thirty-six mucosal specimens were obtained with a biopsy instrument from the upper nasal septum of 12 human autopsy cases before the en bloc removal of the entire olfactory area. Examination of these 36 specimens with transmission electron microscopy demonstrated olfactory epithelium in only 17. A significant negative correlation (r = -.728) was noted between the age of the subject and the probability of obtaining olfactory epithelium, supporting the idea that the olfactory mucosa is gradually replaced by respiratory epithelium with aging. Using the en bloc specimens, the distribution of olfactory epithelium was reconstructed from light microscopic examination of silver-stained sections. Multiple patches of respiratory epithelium were observed over the upper portion of the nasal septum and superior turbinates, ie, the presumptive olfactory area. On transmission electron microscopic examination, frequent respiratory metaplasia was also suggested. Within the area of respiratory metaplasia, supporting cell-like and microvillar cell-like structures often were found; these structures may be remnants of olfactory epithelium. The sampling of olfactory tissue with a biopsy procedure is hampered by the irregular and patchy distribution of olfactory epithelium. The invasion of respiratory epithelial patches into the olfactory mucosa seems to be characteristic of the human olfactory epithelium and may increase as a function of age. Thus, conclusions about the structure of the olfactory mucosa in an individual patient must be based on several tissue samples.     

Evaluation of turnover of olfactory epithelium in mice by using anti-BrdU monoclonal antibody

Among nerve cells of vertebrates, the olfactory elements are uncommon in their capacity to turnover and to be replaced after injury. An autoradiographical and morphological observation has shown that degenerated olfactory nerve cells are reconstituted by a new population of neurons which originate from basal cells. However, an autoradiographic method requires a special isotope institute and it takes a long time for the final specimen to observe. Recently, a rapid technique without the radioisotope has been alternatively developed in which a thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU), is incorporated into replicating DNA and subsequently localized using a specific monoclonal antibody. In the present study, cell dynamics of olfactory mucosa in mice were investigated by means of immunohistological technique. The results were as follows. 1. The labelled elements were concentrated at the basal layer of the epithelium, which were observed 5 hrs after the first injection of BrdU. 2. At 15 days after administration of BrdU, the labelled elements were located in the mid-layer of the epithelium, where can be recognized as the compartment of nerve cells. 3. After 30 days, the labelled cells disappeared from the epithelium. It indicates that the period of turnover in the olfactory epithelium of mice is within 30 days. 4. Fifteen days after axotomy of the olfactory nerves, two stained patterns which were numerously or sparsely labelled regions were observed. The former is considered that immature neurons predominantly exist, and the latter is the area which mature neurons abundantly locate. It is considered that this immunohistological approach is useful for the observation of the turnover of the olfactory epithelium.     

Immunohistopathology of variations of human olfactory mucosa

Summary The characteristics of the human olfactory mucosa were studied immunohistologically. Regular, tonal distribution of the supporting cells, multilayered olfactory receptor cells and basal cells was commonly found in the olfactory mucosa of the human fetus. In contrast, most of the olfactory mucosa in the adult varied to some extent. In the relatively thick, slightly degenerated olfactory mucosa, olfactory marker protein positive receptor cells were arranged irregularly. The most common evidence for variation was the decrease or disappearance of the olfactory receptor cells. Serous-type lactorferrin-containing glandular acini were characteristically found beneath degenerated epithelium. Islands of respiratory epithelium were also seen. The ductules of the Bowman's glands were distended and the openings of these ductules were wide. There was invagination or epithelial cell processes into the glandular lumina. These findings suggest that the epithelial cells of Bowman's glands play an important role in the regeneration of the human olfactory mucosa. Offprint requests to: T. Nakashima     

The olfactory and respiratory epithelium in rhesus and squirrel monkeys studied with freeze-fracture technique

Olfactory and respiratory epithelia was prepared for thin-section and freeze-fracture replicas, and were investigated using electron microscopy. Three cell types were identified in the olfactory epithelium: olfactory receptor neurons (ORN), sustentacular cells (SC) and basal cells (BC); and respiratory epithelial cells (RC) in the surrounding mucosa. ORN and RC were provided with kinocilia. The cilia displayed their characteristic 5-7 rows of proximally situated 'necklaces', seen in freeze-fracture replicas. ORN and SC as well as RC were connected with tight junctions classified as being 'tight', whereas the RC displayed intermediate junctions. No nexuses (gap junctions) were found. It is concluded that the monkey olfactory/respiratory epithelium exhibit the same characteristics as seen in other vertebrates when using thin section/freeze-fracture technique.     

The dynamics of precursor cells in the olfactory epithelium of juvenile and adult guinea pigs

The dynamics of precursor cells in the olfactory epithelium of juvenile and adult guinea pigs were examined by immunohistochemical double staining using bromodeoxyuridine (BrdU), the neural cell adhesion molecule (N-CAM) and the protein gene product 9.5 (PGP9.5). Expression of apoptotic cells in the olfactory epithelium with the use of the TdT-mediated dUTP biotin nick end labeling (TUNEL) method was also observed. BrdU was given to healthy guinea pigs at the ages of 2 weeks and 6 months old. Tissue specimens were serially collected 1 h to 28 days after administration. BrdU-labeled cells were seen above the basal cell layer after 1 h and migrated to the middle layer of the olfactory epithelium, after 1 day in juveniles and 5 days in adults with expression of N-CAM. PGP9.5 was observed in BrdU-labeled cells after 5 days in juvenile guinea pigs and 7 days in adult. At 14 days after administration, BrdU-labeled cells in the epithelium appeared to decrease. However, a few of these cells were recognized above the basal cell layer after 28 days. The number and location of TUNEL-positive cells did not significantly differ between the juvenile and adult olfactory epithelium. Therefore, we conclude that the division speed from stem cells in juveniles is faster than that in adults, and apoptosis is unaffected by aging in the normal olfactory epithelium.     

电刺激嗅神经诱发电位实验研究

目的 探索一种不受主观因素影响,能客观反映嗅觉功能的实验方法。方法 采用２０只家兔,刺激电极分别置于筛板和鼻甲溴区粘膜及呼吸区粘膜;记录电极置于头皮表面近嗅球部,给予０．２～４．０ｍＡ电刺激,记录产生的诱发电位。结果 电极置于筛板和鼻甲嗅区粘膜可记录到一组“负－正－负”三相的诱发电位波,依次命名为Ｎ１、Ｐ１、Ｎ２,当刺激强度为２．０ｍＡ时,筛板粘膜诱发电位的潜伏期分别为１６．２７、２５．３６、４９     

The effects of zinc on the olfactory neuroepithelium and olfactory bulbs of the Sprague-Dawley rat after oral administration of zinc-gluconate trihydrate

BACKGROUND: The most frequent causes of upper respiratory infections are human rhinoviruses. The nasopharyngeal area, which includes the respiratory epithelium, mucosa, and the olfactory neuroepithelium (ONe), is a first-line of defense against airborne viruses and allergens, some of which manage to penetrate the nasal mucosa and invade the tissues of the nasal respiratory epithelium. Biochemical evidence from several studies suggests that zinc is an effective cold treatment and that over-the-counter (OTC) zinc-gluconate compounds may provide the high pharmacologic doses of zinc needed to act as an effective means of treating and reducing the duration and severity of symptoms of the common cold. METHODS: A series of male Sprague-Dawley rats were fed an oral preparation of zinc-gluconate trihydrate or received the equivalent through drinking water to investigate the potential cytotoxic and/or neurotoxic insult to the olfactory receptor cells and other tissue in the ONe and afferent neuronal pathways. RESULTS: Coronal sections of the rat ONe and corresponding olfactory bulbs showed consistent cellular and tissue damage of increasing severity that correlated with the duration of treatment with the zinc compound when compared with the control group animals. CONCLUSION: The results of this analysis indicate that the repeated oral administration of such zinc-containing compounds have neurotoxic effects on the ONe and to the mitral cells in the olfactory bulbs of treated rats. These findings point toward the need for increased investigation into the potential deleterious effects of zinc-containing compounds to humans as well.     

Effect of perchloroethylene inhalation on nasal mucosa in mice

An experimental group of 16 male pure-bred mice was exposed to perchloroethylene gas at 300ppm for 6h daily for 5 days. Histopathological study of the nasal mucosa was performed sequentially 1, 2, 4, and 7 days after exposure. Erosion of the olfactory epithelium and dilatation of Bowman's glands were observed from 1 to 7 days after exposure. Atrophy of the olfactory nerves was observed from 4 to 7 days after exposure. At 4 days after exposure, regenerating epithelial cells were observed, indicating that these cells represented the first step of the repair process after exposure. Nonetheless, epithelial degeneration in the nasal mucosa without erosion was observed for 4–7 days after exposure. Such epithelial lesions were more severe in the olfactory mucosa and appeared earlier than in other sites in the respiratory mucosa. The present study revealed that perchloroethylene gas exerted a more potent harmful action on the olfactory mucosa than on the general respiratory mucosa.     

Fenestrated endothelia in vessels of the nasal mucosa

Summary The ultrastructure of the vessels in the normal respiratory and olfactory mucosa of the nasal septum was studied in 15 adult rabbits. Capillaries with continuous and fenestrated endothelia could be observed in both tissues. In the respiratory part, many of the fenestrated capillaries were located subepithelially with their fenestrations facing the adjacent epithelium. Fenestrated capillaries of the olfactory mucosa were found mainly in the deeper parts of the tunica propria and usually revealed only a small number of fenestrations in their endothelial lining. It was also demonstrated that muscularized veins sometimes displayed fenestrated areas in their attenuated endothelia. These results are discussed and compared with the appropriate literature. Our results confirm that there is a link between the morphological peculiarities of the vascular wall and the functional behaviour of the nasal mucosa. These findings also emphasize that endothelial fenestrations seem to be characteristic for a certain segment of the microcirculatory system rather than for a distinct type of capillary vessel.     

Fenestrated endothelia in vessels of the nasal mucosa. An electron-microscopic study in the rabbit

The ultrastructure of the vessels in the normal respiratory and olfactory mucosa of the nasal septum was studied in 15 adult rabbits. Capillaries with continuous and fenestrated endothelia could be observed in both tissues. In the respiratory part, many of the fenestrated capillaries were located subepithelially with their fenestrations facing the adjacent epithelium. Fenestrated capillaries of the olfactory mucosa were found mainly in the deeper parts of the tunica propria and usually revealed only a small number of fenestrations in their endothelial lining. It was also demonstrated that muscularized veins sometimes displayed fenestrated areas in their attenuated endothelia. These results are discussed and compared with the appropriate literature. Our results confirm that there is a link between the morphological peculiarities of the vascular wall and the functional behaviour of the nasal mucosa. These findings also emphasize that endothelial fenestrations seem to be characteristic for a certain segment of the microcirculatory system rather than for a distinct type of capillary vessel.     

Localization of antileukoprotease in middle ear mucosa

The localization of antileukoprotease was studied immunohistologically in normal middle ear mucosa specimens obtained at autopsy and in chronically inflamed middle ear mucosa specimens obtained at middle ear surgery for chronic otitis media. In the sections of normal as well as in the sections of chronically inflamed middle ear mucosa, antileukoprotease localization was confined to PAS-positive goblet cells of surface epithelium and to PAS-positive goblet-like cells of submucosal glands and crypts, whereas ciliated mucosal cells and stratified squamous epithelial cells were devoid of anti-leukoprotease. In comparison with normal middle ear mucosa, an increased number of goblet cells--and thus an increased number of cells containing antileukoprotease--was present in the chronically inflamed middle ear mucosa. Since antileukoprotease is a potent inhibitor of granulocyte elastase and Cathepsin G, it was concluded that this proliferation of the respiratory epithelium during inflammatory processes in the middle ear indicates an increased activity of the biologic defence system against the action of granulocyte proteases.     

Immunohistochemical observations of dividing cells in olfactory epithelium using anti-BrdU antibody

Summary To examine the dividing cells in the olfactory epithelium, an experiment using a novel immunohistochemical technique with bromodeoxyuridine (BrdU) was performed. Tissue specimens were obtained from the olfactory epithelium of guinea pigs at days 7 and 21 after olfactory nerve axotomy. BrdU uptake was detected in the epithelial cell layer directly above the basal cell layer rather than in the basal cells per se. The BrdU-immunoreactive cells were found more numerously at 7 days than at 21 days after axotomy. The basal cells showed no immunoreaction to the anti-BrdU antibody on either day. The cells reacting with the anti-BrdU antibody also showed no reaction to the anti-cytokeratin antibody used to identify the basal cells. These findings indicate that the cells showing mitotic activity have characteristics different from those of basal cells, which has been considered previously to be the precursors of regenerating olfactory receptor cells.     

上呼吸道感染后嗅觉障碍患者嗅上皮超微结构观察

目的通过对上呼吸道感染后嗅觉障碍的患者鼻腔大体及嗅上皮超微结构的研究，从形态学上观察嗅觉减退或丧失的超微结构改变。方法选择上呼吸道感染后嗅觉减退或丧失患者10例，用T＆T嗅觉测试法测试患者的嗅觉功能。常规前鼻镜、鼻内镜下对鼻腔大体结构进行观察，鼻内镜下钳取嗅区黏膜行透射电镜超微结构观察。结果上呼吸道感染后嗅觉障碍患者嗅黏膜超微结构有以下变化：①嗅上皮结构层次仍能保持，但细胞间隙增宽；②上皮表面嗅泡明显减少，即使嗅泡存在，其末端的纤毛也明显减少，部分嗅泡呈空泡状改变；③微绒毛细胞和支持细胞表面的微绒毛减少或缺失；④支持细胞的细胞核变形或固缩，嗅细胞的树突水肿变形，细胞器减少。结论上呼吸道感染后嗅觉功能障碍与嗅黏膜上皮超微结构的改变密切相关。患者嗅泡及嗅泡内纤毛缺失，微绒毛细胞及支持细胞的微绒毛减少是引起嗅觉减退的主要原因，支持细胞胞核的变形及嗅细胞树突的形态学改变与嗅觉改变相关。     

Immunohistochemical method for the diagnosis of olfactory disturbance

To confirm the diagnosis and determine the cause of the olfactory disturbance, we used an immunohistochemical method to examine biopsy specimens of the olfactory mucosa from a patient who complained of anosmia after head injury. Neuron-specific enolase immunoreactivity was found in the olfactory vesicles and dendrites of the receptor cells in the olfactory epithelium. S-100 protein immunoreactivity was found in the ductal cells of Bowman's gland in the olfactory epithelium and in the acinar cells of Bowman's gland in the lamina propria. This suggests that the olfactory receptor cells and Bowman's gland were normal. The olfactory disturbance in this patient was not caused by nerve transection due to the head injury, but by already existing chronic sinusitis. Immunohistochemical methods are useful for diagnosing olfactory disturbance when used in combination with biopsy of olfactory mucosa.     

Optical recording of the intrinsic signal from the human olfactory cleft

OBJECTIVES: Endoscopy of the human olfactory cleft is important for both research in human olfaction and clinical examination with regard to olfactory disorders. However, endoscopy only provides information on the morphology and functional status of the epithelium, and it does not allow discrimination between respiratory and olfactory mucosa. To obtain information on the functional status of the olfactory mucosa, I used endoscopy to investigate the optical intrinsic signal recording from the human olfactory cleft. METHODS: A light-emitting diode (617 nm) light source and a cooled charge-coupled device camera were prepared for endoscopy of the olfactory cleft. Subjects were exposed to various odors presented in front of their nostrils. In addition, blanks were used for control. RESULTS: When normosmic subjects sniffed the odors, the intensity of the signal from the olfactory mucosa changed, which was not the case when blank stimuli were presented. Different odors activated different response patterns. A decrease of the oxyhemoglobin level in the activated olfactory epithelium is suspected to be responsible for this observation. CONCLUSIONS: The optical intrinsic signals were recorded from the human olfactory cleft with an endoscope. This technique may be applicable to basic research in olfaction and to a clinical test for the assessment of olfactory disorders.     

Effects of bacterial toxins on air-exposed cultured human respiratory sinus epithelium

The present study was designed to investigate the effects of the bacterial toxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on air-exposed cultured human respiratory sinus epithelium. The morphological changes, proliferation, and differentiation of sphenoid sinus mucosa were examined after incubation with different LPS or LTA concentrations. Air-exposed cultured sinus mucosa differentiated from pseudostratified respiratory epithelium to squamous ciliated epithelium with few goblet cells. High concentrations of bacterial toxins induced a significant increase in mucus production and a decrease in ciliated cells. Ki67 immunostaining showed an increased cell proliferation after incubation with moderate levels of LPS or LTA. High concentrations of bacterial toxins, on the other hand, induced a decreased proliferation. Involucrin expression was clearly altered by incubation with high levels of bacterial toxins, indicating an increased degree of terminal differentiation. These results indicate that the bacterial toxins LPS and LTA both induce comparable dose-dependent morphological changes in sinus epithelium.     

放射线损伤对小鼠嗅上皮细胞凋亡及再生的影响

目的研究放射线损伤后小鼠嗅上皮细胞凋亡和再生情况,探讨放射治疗后嗅觉障碍的致病机制。方法以4Gy的X射线照射小鼠鼻部,建立放射线损伤小鼠嗅黏膜的动物模型分别于照射前及照射后第1,3,6,12,60天分批处死动物取材;TUNEL法检测嗅上皮中细胞凋亡,BrdU掺入免疫组化检测基底细胞再生。结果放射线损伤后小鼠嗅上皮凋亡细胞显著增多（P〈0.01）,基底细胞再生也相应增多（P〈0.01）,但再生细胞少于凋亡细胞（P〈0.01）。结论放射线损伤可促进嗅上皮细胞凋亡及基底细胞再生,但再生细胞少于凋亡细胞,二者失衡可能是放射治疗后嗅觉障碍的致病机制之一。     

Proliferation markers, proliferating cell nuclear antigen, Ki67, 5-bromo-2'-deoxyuridine, and cyclin D1 in mouse olfactory epithelium

We immunohistochemically identified proliferating cells in the olfactory epithelium of mice, using an anti-proliferating cell nuclear antigen (PCNA) antibody, an anti-Ki67 antibody, an anti-5-bromo-2'-deoxyuridine (BrdU) antibody, and an anti-cyclin D antibody. Positive cells stained by the 4 antibodies were identified mainly in the basal layer. The mean numbers of positive cells stained by the 4 antibodies in 500 olfactory epithelium cells from each animal were as follows: PCNA-positive cells 42, Ki67-positive cells 23, BrdU-positive cells 13.7, and cyclin D1-positive cells 9.2. PCNA may be detected in both proliferating and resting cells. Ki67 is an intranuclear antigen expressed in proliferating, but not resting, cells. Anti-BrdU antibodies might stain proliferating cells only following the S-phase, but not the G1-phase. Cyclin D1 is a protein that works during the G1-phase of the cell cycle. When we stain proliferating cells using proliferating cell markers, it is important to consider the cell cycle phases during which each marker stains.