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.     

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.     

Globosal basal cells are identified as proliferating cells in mouse olfactory epithelium

Although anti-bromodeoxyuridine (BrdU) antibodies are used for detecting proliferating cells, they may stain proliferating cells in phases following the S-phase, except for G1. Anti-Ki67 antibodies are widely used for detecting proliferating cells in all phases of the cell cycle (G1-, S-, G2-, and M-phase), but not in resting cells (G0-phase). Anti-cyclin D1 antibodies are used to detect proliferating cells in the G1-phase. The present study investigated the olfactory epithelium of mice by double immunostaining using antibodies to BrdU, Ki67. cyclin D1, and cytokeratin 14 (CK14). The cells positive to the anti-BrdU antibody, the anti-Ki67 antibody, and the anti-cyclin D1 antibody differed from the cells positive to the anti-CK14 antibody. Thus, we confirmed that the proliferating cells in all the phases of the cell cycle, including the G1-phase, were globosal basal cells, which were the precursors of the olfactory cells.     

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.     

Age-related changes in dividing cells of the olfactory epithelium of the maturing guinea pig

Changes in dividing cells of the olfactory epithelium from guinea pigs of different ages were examined by immunohistochemical staining using anti-proliferating cell nuclear antigen antibody. Numerous dividing cells were scattered diffusely in the basal layer of the olfactory epithelium at 1 and 2 months following birth and then gradually decreased with maturation until 4 months. Findings then remained constant between 4 and 24 months. Subsequently, cell numbers were found to decrease as animals became older. The number of olfactory receptor cells did not vary significantly between 1 and 30 months. Although no correlation could be found between the numbers of dividing cells and olfactory receptor cells, it is still possible that the longevity of the olfactory receptor cells changes to maintain the overall size of the neuronal population. Received: 18 February 1998 / Accepted: 9 April 1998     

Immunohistochemical localization of proliferating cells and epidermal growth factor receptors in mouse olfactory epithelium

We investigated age-related changes in proliferating cells and epidermal growth factor receptors (EGFRs) in mouse olfactory epithelium using an immunohistochemical method with the antiproliferating cell nuclear antigen (PCNA) antibody and the antihuman EGFRs antibody. Many PCNA-positive cells occurred in the surface and basal layers of the olfactory epithelium in the embryonal and neonatal mice. However, only some PCNA-positive cells occurred in the basal layer of adult mice, and only a few presented in the basal layer of aged mice. EGFRs were observed in all layers of the olfactory epithelium at the embryonal and neonatal stages, but were not identified in the olfactory epithelium at the adult or aged stages. We believe that a decrease in EGFRs in the olfactory epithelium induces the inhibition of cell proliferation, with the resultant atrophy of the olfactory epithelium.     

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.     

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.     

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.     

Upregulation of neural growth-associated protein and neural cell adhesion molecule in mouse olfactory epithelium and axons after unilateral removal of the olfactory bulb

The expression of synaptosomal-associated protein (SNAP-25), neural growth-associated protein (GAP-43) and neural cell adhesion molecule (NCAM) were studied in mouse olfactory cells and axons for 2 weeks following unilateral bulbectomy. The olfactory cells and axons in the control olfactory epithelium were positive for SNAP-25 but levels decreased in the atrophic olfactory epithelium 3 days after bulbectomy. There was no expression of SNAP-25 in the olfactory epithelium on the bulbectomy side 7 days after bulbectomy, indicating that this protein may be a good marker for the degeneration of olfactory cells. The expression of NCAM was still found in the atrophic olfactory epithelium at 7 days after bulbectomy, while the expression of NCAM in the olfactory epithelium of the bulbectomy side was stronger than that on the control side at 14 days after bulbectomy. The expression of GAP-43 in the olfactory axonal bundles of the bulbectomy side at 3 and 4 days after bulbectomy was stronger than that on the control side. These results suggest that upregulation of NCAM may be related to the regeneration of the olfactory cells, with upregulation of GAP-43 probably playing a role in axonal regeneration after bulbectomy. Received: 6 January 1998 / Accepted: 22 April 1998     

Changes in epidermal growth factor receptors in olfactory epithelium associated with aging

To clarify the mechanisms of age-related changes in the olfactory epithelium, we investigated age-related changes in epidermal growth factor receptors (EGFRs) in the epithelium. We examined 3 mice at each of the following stages: embryonal (embryonic days 14 and 16), neonatal (postnatal days 1 and 7), adult (postnatal weeks 5 and 12), and aged (postnatal years 1.5 to 2). The olfactory epithelium of each mouse was stained with sheep anti-human EGFR polyclonal IgG by an immunohistochemical method. The EGFRs were observed in all layers of the olfactory epithelium at the embryonal and neonatal stages. They were identified only in the basal layer of the olfactory epithelium at adult and aged stages, and the number of regions in which EGFRs were identified in the basal layer of the olfactory epithelium decreased in aged mice compared to adult mice. We believe that a decrease in EGFRs in the olfactory epithelium induces the inhibition of cell proliferation, with resultant atrophy of the olfactory epithelium.     

Developmental changes in expression of a calcium-binding protein (spot 35-calbindin) in the Nervus terminalis and the vomeronasal and olfactory receptor cells.

The detailed localization of spot 35-calbindin and its ontogenic change were studied in Nervus terminalis, the vomeronasal organ and the olfactory epithelium of the rat by immunohistochemistry. At the embryonic days 12 and 13 (E 12-13), calbindin-immunoreactive cells were found in the outermost layer of the presumptive olfactory bulb and within the olfactory placode. At E 14 to the postnatal day 1 (P 1), intense calbindin-immunoreactivity was localized in ganglionated fiber bundles of Nervus terminalis coursing through the mesenchymal spaces on both sides of the nasal septum. Nervus terminalis decreased the immunoreactivity abruptly after P 1 and it showed no distinct immunoreactivity for calbindin at P 7 and thereafter. On the other hand, numerous receptor cells in the olfactory epithelium and the thicker vomeronasal epithelium exhibited weak to moderate immunoreactivity for calbindin at E 18-P 1. Their immunoreactivity decreased in intensity progressively after P 7 and no distinct immunoreactivity for calbindin was detected in most of the receptor cells, whereas moderate immunoreactivity was detected in most of the vomeronasal and olfactory nerves at P 28 and P 63.     

Immunohistochemical localization of epidermal growth factors in mouse olfactory epithelium.

Our object was to clarify changes in epidermal growth factor (EGF) occurring in the olfactory epithelium with aging. We investigated the changes in EGF in the murine olfactory epithelium with aging by an immunohistochemical method. We examined six mice at each of the following stages: embryonal (embryonic days 14 and 16), neonatal (postnatal days 1 and 7), adult (postnatal weeks 5 and 12), and aged (postnatal years 1.5-2). The olfactory epithelium of each mouse was stained with anti-EGF polyclonal IgG using an immunohistochemical method. EGF were observed in the whole layer of the olfactory epithelium at all stages. The mesenchyme under the epithelium was stained in the embryonal stage, and the circumference of the olfactory gland cells was also stained, although weakly, in the adult stage. We believe that EGF contribute to cell proliferation, growth and turnover of the olfactory epithelium. However, the decrease in EGF was less than expected in the aged mice.     

Assessment of bromodeoxyuridine-labeled S-phase cells in experimentally induced precancerous lesions in the rat's tongue

Precancerous lesions and early invasive carcinomas were produced in the tongues of rats by oral administration of 0.001% 4-nitroquinoline 1-oxide in drinking water. The distribution and densities of S-phase cells were then studied in an attempt to clarify the mechanism of carcinogenesis from the viewpoint of cell cycle. Bromodeoxyuridine (BrdU)-labeled S-phase cells were demonstrated by an indirect peroxidase method, using anti-BrdU monoclonal antibody, and their percentage was determined as the labeling index (LI). The average BrdU LI was significantly higher in the precancerous lesions than in the normal epithelium. There was a wide range of LI found in cells showing such changes as hyperplasia, hyperparakeratosis, dysplasia and papilloma, but the differences among them were not significant. These findings also showed that there could be considerable differences in proliferative activity among lesions of the same grade, while the difference in histology did not mean a difference in proliferative activity. Additionally, many BrdU-labeled cells were seen in a few layers over the basement membranes of noncancerous lesions immediately adjacent to early invasive carcinoma, suggesting that these layers had a higher possibility for advancing to early invasive carcinoma.     

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。     

Cell kinetic study on experimental tongue carcinoma in rats

Tongue carcinomas were produced in rats by oral administration of 0.001% 4-nitroquinoline 1-oxide (4NQO) in drinking water, and the biological characteristics and tumor kinetics were correlated. Bromodeoxyuridine (BrdU) was used to investigate cell kinetics of rat tongue tumors in vivo. BrdU-labeled S-phase cells were demonstrated by an indirect peroxidase method using anti-BrdU monoclonal antibody, and their percentage, the labeling index (L.I.), was determined. The average BrdU L.I. in normal tongue epithelium, well differentiated and moderately differentiated squamous cell carcinoma was 5.7 +/- 1.1, 8.8 +/- 3.7 and 14.9 +/- 5.4, respectively. The differences among the three groups are significant. These results indicate that the BrdU L.I. correlates well with the degree of differentiation of tongue epithelium. Furthermore, the distribution patterns of BrdU-labeled cells in the tissue sections of each group were different. If these cell kinetics studies using BrdU are applied to human tongue carcinomas, the data may provide information on the biological characteristics and help in establishing the diagnosis and in choosing treatment regimens.     

Cell renewal in the eustachian tube epithelium of guinea pigs.

The turnover time of the epithelial cells of a normal eustachian tube was estimated in guinea pigs by means of lightmicroscopic autoradiography with thymidine-H3. The cell migration from the basal layer to the tubal lumen was chased by sacrificing the animals at different intervals after tnymidine-H administration. This study revealed that the generative cells were the basal cells in the tubal epithelium and that the different types of maturative cells in the epithelium have a different life span. The turnover time of non-ciliated secretory cells was about 145 hrs (about 6 days), while that of ciliated cells and goblet cells more than 6 days.     

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.     

Degeneration and regeneration of olfactory epithelial neurons after olfactory nerve sectioning]

The morphological changes of olfactory mucous membrane have been investigated with light microscope and T.E.M. within a period of 3 days to 4 weeks after unilateral olfactory nerve sectioning. No obvious morphological changes of supporting cells and Bowman's gland cells were seen in any group postoperatively. A degeneration in some neurons began to appear on the third day following the operation and reached its peak about one week after operation. No changes of the basal cells were seen on the third day. After one week, however, they changed into globose type and some of which showed mitotic activity. Within 2-4 weeks, the globose cells differentiated gradually into mature neurons. Four weeks after the operation, the regenerated olfactory epithelium appeared similar to that of the control group. The main feature of neuron degeneration was the increase in plasma electron density. In serious cases, the organelle systems disappeared and the nuclei were pyknotic. The results obtained from this experiment shows that after the sectioning of olfactory nerve, degeneration and regeneration processes of olfactory neuron will occur; and the regenerated neuron may possibly be developed from basal cells.