KISEP Original Articles J Rhinol 4(2), 1997

Regional Differences in Proliferative Activity of Nasal Epithelium in Rat

Sang Hag Lee, M.D., Jae Yong Lee, M.D. and Heung Man Lee, M.D.

ABSTRACT 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.

KEY WORDS：Proliferating cell nuclear antigen·Respiratory epithelium·Olfactory epithelium.

acteristics in the nervous system of vertebrates in that there is INTRODUCTION continuous turnover and replacement of the sensory neurons in the olfactory system. Mature olfactory neurons die and are Nasal mucosa is the initial site of contact with inhaled irri- replaced from undifferentiated neuroblasts over the entire life tants such as altered airflow, atmospheric pollutants, allergens span of the individuals. Furthermore, a number of experim- and virus infections, and functions to protect itself and lower ental techniques have been applied to induce degeneration of respiratory tract from these environmental toxicants. It is ge- the olfactory neurons in order to study subsequent neuroge- 4-6) nerally believed that the nasal epithelium plays an important nesis and replacement of this cell population. These studies role in these protective functions by absorbing water-soluble clearly demonstrated replacement of receptor cells, indicating gases, trapping and clearing inhaled particles. that the olfactory epithelium is significantly recovered follo- A number of histopathological studies revealed that in lab- wing injury. However, the pattern, density or location of olfa- oratory rodents, inhalation of harmful toxicants results in sig- ctory cell proliferation in normal nasal mucosa has not been nificant morphologic alterations to surface epithelia lining the determined precisely. nasal respiratory tract, increasing the rate of surface epithelial The present study was undertaken to determine the distr- cell proliferation.1-3) This proliferative response is believed ibution of epithelial cells undergoing DNA replication in the necessary to replace the damaged epithelium and to protect the various regions of the rat nasal cavity. DNA replication was nasal mucosa from further insult by repairing and adapting the detected with immunohistochemical method using antibody epithelial barrier. However, little attention has been paid to the against proliferating cell nuclear antigen (PCNA). PCNA is nasal epithelium in terms of its proliferative response in the auxiliary protein for DNA polymerase delta, which is expre- steady state without inhalation of toxicants. ssed in late G1, during S and early G2 phases of the prolife- 7)8) The olfactory epithelium presents unique cytological char- rating cell. Department of Otorhinolaryngology-Head and Neck Surgery, College of Medicine, Korea University, Seoul, Korea MATERIALS AND METHODS Address correspondence and reprint requests to Sang Hag Lee, M.D., Department of Otorhinolaryngology-Head and Neck Surgery, College of Medicine, Korea University, 126-1, 5 Ka, Tissue preparations Anam-Dong, Sungbuk-Ku, Seoul 136-705, Korea Tel：82-2-920-5486, 5761, Fax：82-2-925-5233 Ten healthy adult Sprague-Dawley rats (200-300 g) were Accepted for publication on September 14, 1997 used in this experiment. They were anesthetized with intrap-

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Lee et al：Proliferative Activity of Nasal Epithelium / 121 eritoneal injection of sodium pentobarbital (30 mg/Kg) and continuously along the basement membrane of epithelium perfused through the aorta with normal saline, followed by (Fig. 1). These sqaumous and non-ciliated cuboidal/transiti- 150 ml of 4% paraformaldehyde in 0.1 M phosphate buffer onal type of epithelial cells were transformed to pseudostratified (pH 7.4). After perfusion fixation, the nasal mucosa was tot- ciliated respiratory epithelium in the posterior direction where ally separated from nasal septum and lateral wall of nasal ca- positive reaction became scanty and was limited to the basal vity, and immersed in 20% sucrose solution at 4℃ for 6 hours. cells. In this anterior portion of ciliated respiratory epithelium, They were embedded in OCT compounds (Tissue Tek® Miles PCNA-positive cells were scattered with some distance along Scientific, Naper-ville, USA) and quickly frozen in liquid ni- the basement membrane, and multiple layers of positive cells trogen. Whole tissue samples were serially sectioned from up- were rarely seen (Fig. 2). PCNA-positive reaction became per to lower portion in nasal septum and from frontal to rostral more sparse in the posterior portion of ciliated respiratory ep- portion in lateral wall of nasal cavity including turbinates. ithelium, and the positive nuclei existed with wider distance. After being sectioned at 15 μm in a cryostat (Leica CM 1800, This distribution pattern of cell nuclei positive for PCNA were Germany) and selected one every 5-section intervals, they were observed in all sections from upper to lower portion of nasal mounted on poly-L-lysine-coated glass slides and processed septum. In olfactory epithelium, the nuclei immunoreactive for for PCNA immunolabeling. PCNA were observed in the basal cell layers, but not in the intermediate or upper olfactory neuronal cells. The labeling Immunohistochemistry for PCNA was distinct in some area, but absent in adjacent other portions (Fig. 3). However, compared with the respiratory mucosa, the Cryostat sections were air-dried and immersed in methanol olfactory epithelium did not show the region-specific immu- mixed with 0.6% H2O2 for 30 minutes for blocking endogen- ous peroxidase. After washing in phosphate-buffered saline (PBS, pH 7.4) for 5 min, the sections were boiled in citrate- buffer (pH 6.0, 3 microwave exposure cycles at 650 W, 5 mi- nutes) and incubated in 0.05% saponin (Sigma Chemical Co- mpany, USA) for 30 minutes followed by rinsing in PBS again. They were incubated in normal horse serum for 30 minutes, and after washing in PBS, incubated with a 1：100 dilution of anti-PCNA monoclonal mouse antibody (DAKO Co., USA) in moist chamber at 4 ℃ overnight. The sections were was- hed in PBS and incubated with biotinylated anti-mouse IgG secondary antibody for 1 hour, followed by streptavidin-con- jugated peroxidase for 1 hour (Vectastain® ABC Kit PK 4002, Vector Lab., Burlingame, CA, USA). The immunoreactivity Fig. 2. The posterior portion of nasal septal mucosa (anterior po- rtion of pseudostratified ciliated respiratory epithelium). In this was visualized by diaminobenzidine (D-AB)-H2O2 solution portion, PCNA-positive reaction is not prominent and sparse (5 mg DAB/10 ml PBS＋5 μl 30% H2O2). The immunost- even if it exists (×100). ained sections were dehydrated in a graded ethanol series and mounted with Permount® (Fisher Scientific, New Jersey, USA). They were observed under light microscopy and the epithelial cell nuclei with intensely brown red, granular or diffuse stai- ning pattern corresponding to S phase PCNA were regarded as PCNA-positive.

RESULTS

Nasal septum

In respiratory portion, distinctly positive reaction was obs- erved in the basal and suprabasal layers of squamous and non- ciliated cuboidal/transitional epithelium, which were located Fig. 1. The anterior portion of nasal septal mucosa (squamous and non-ciliated cuboidal/transitional epithelium). These epit- in anterior portion of nasal cavity. In these epithelia immuno- helia show distinctly PCNA-positive reaction in the basal layers labeled cells were observed in the basal cell layers, and existed (×100).

122 / J Rhinol 4(2), 1997 nolabeling, that is, anterior-posterior gradient of PCNA-positive cuboidal/transitional epithelium which were located anteriorly nuclei was not observed in olfactory epithelium of nasal septal in nasal cavity (Figs. 5 and 6). These positive cells existed in mucosa. Schematic illustration of the distribution pattern of the basal cell layers. In the anterior end of nasoturbinate and PCNA-positive cells in the nasal septal mucosa is shown in maxilloturbinate, there was some portion covered with squ- Fig. 4. amous epithelium in which abundant positive reaction was observed. These dense PCNA-positive cells in the squamous Lateral wall of nasal cavity including turbinates and non-ciliated cuboidal/transitional epithelium became sca- nty and scattered as directed posteriorly where the epithelium In nasal mucosa of turbinates and lateral wall of nasal was substituted for pseudostratified ciliated respiratory epith- cavity, the distribution pattern of epithelial cell nuclei positive elium. In this anterior portion of ciliated respiratory epithelium, for PCNA was similar compared with septal mucosa. Positive PCNA immunolabeling was limited to the basal cells and in- reaction for PCNA was observed in both respiratory and olf- continuously distributed (Fig. 7). The posterior portion of actory mucosa. In respiratory portion, densely clustered positive pseudostratified ciliated respiratory epithelium, PCNA-positive nuclei were noted mainly in the squamous and non-ciliated cells became more sparse, and existed with wider distance. As observed in the septal mucosa, respiratory portion of turbina- tes and lateral wall of nasal cavity showed anterior-posterior gradient of PCNA positivity. In olfactory epithelium, as in nasal septal mucosa, the nuclei immunoreactive for PCNA were observed in the basal cell layers, and the labeling was

Fig. 3. The olfactory portion of nasal septal mucosa. This olfac- tory epithelium shows PCNA-positive cells in basal cell layers and this positive reaction is distinct in some area while adjacent other portions are absent of PCNA immunolabeling (×100).

Fig. 5. The coronal section of the turbinates at the level of squ- amous and non-ciliated cuboidal/transitional epithelium. These epithelia shows distinctly PCNA-positive reaction in the basal layers (×100).

Fig. 4. Schematic illustration of distribution pattern of PCNA- positive epithelial cells in normal nasal septal mucosa. Black dots represent PCNA-positive epithelial cells. In respiratory por- tion, positive reaction is mainly seen in the squamous and non- ciliated cuboidal/transitional epithelium which are located in the anterior portion of nasal cavity. These types of epithelial cells are transformed to pseudostratified ciliated epithelium in the posterior direction where positive reaction become scanty. In the olfactory epithelium, the nuclei immunoreactive for PCNA are distinct in some area, but absent in other adjacent portions： I-squamous epithelium, II-non-ciliated cuboidal/transitional ep- Fig. 6. The coronal section of the lateral wall of transitional epi- ithelium, III-pseudostratified ciliated respiratory epithelium, IV- thelium. PCNA-positive reaction is prominently seen in the basal olfactory epithelium. layers (×100).

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distinct and clustered in some portion, while adjacent other portions were absent of positive reaction (Fig. 8). The olfac- tory epithelium did not show the region-specific distribution of PCNA-positive nuclei that was observed in the respiratory mucosa. Schematic illustration of the distribution pattern of PCNA-positive cells in the turbinates and lateral wall of the nasal cavity is shown in Fig. 9.

DISCUSSION

This is the first study in which immunohistochemistry with antibodies to PCNA has been used to reveal proliferative act- Fig. 7. The coronal section of lateral wall of nasal cavity at the ivity of epithelial cells lining the nasal mucosa of normal rat. level of anterior portion of pseudostratified ciliated respiratory This immunohistochemical method is relatively simple com- epithelium. This portion shows scanty and scattered PCNA-po- sitive nuclei limited to basal cell layers (×100). pared to the use of tritiated thymidine or bromdeoxyuridine and demonstrates the usefulness of this technique in localizing the epithelium with proliferative ability in nasal tissue.2)9) This study also shows that cell turnover occurs without overt signs of cell damage or death under normal conditions. Cell prolif- eration in the non-olfactory epithelium was most prominent in the anterior region of the nose, showing region-specific dist- ribution while in the olfactory region, the epithelial cells with actively proliferative ability were more widely distributed. The non-olfactory epithelium of the rat nasal cavity is co- mposed of three morphologically distinct epithelial populati- ons. These populations have specific locations within the nasal cavities and are composed of different epithelial cell types. These regions include：stratified squamous epithelium in the Fig. 8. The coronal section of nasal mucosa of the turbinates and nasal vestibule；ciliated pseudostratified respiratory epithel- lateral wall of nasal cavity at the level of olfactory epithelium. This epithelium shows PCNA-positive cells in basal cell layers and ium in the main cavity of the nasal airway；a narrow region this positive reaction is distinct in some area while adjacent of non-ciliated, cuboidal, transitional epithelium lying between other portions are absent of PCNA immunolabeling (×40). the squamous and respiratory epithelium in the anterior aspect of the main nasal cavity. These specifically defined epithelial regions are similarly observed in all commonly used labora- tory animals.1-3)10) It seems that the distribution pattern of these epithelial population is a normal findings and not a patholo- gical change in nasal respiratory epithelium. Air stream alone is thought to be the physiological irritant which includes various kinds of toxicants；virus, bacteria, ch- emical pollutants, physical factors and other substances. In the present study, increased numbers of PCNA stained nuclei was predominantly observed in the squamous and transitional epithelium. These findings indicate that exposure of normal nasal surface epithelium to inhaled air is accompanied by ep- Fig. 9. Schematic illustration of distribution pattern of PCNA-po- ithelial cell proliferation in even normal nasal breathing. Pa- sitive epithelial cells in normal nasal mucosa of the lateral wall rticularly, squamous and transitional epithelia of the nasal of nasal cavity including turbinates. Black dots represent PCNA- positive epithelial cells. In both respiratory and olfactory mucosa, cavity are assumed to be more intensely stimulated by the distribution pattern of the positive reaction for PCNA is identical continuous physiological air stream and may be particularly to that of the septal mucosa：I-squamous epithelium, II-non- ciliated cuboidal/transitional epithelium, III-pseudostratified ci- vulnerable to inhaled air because of their proximal location in liated respiratory epithelium, IV-olfactory epithelium. the nasal airway. These assumptions are supported by previous

124 / J Rhinol 4(2), 1997 studies showing that surgical closure of the nostril induces the isting results, this study has systemically analyzed the distri- regeneration of atrophic or metaplastic respiratory epithelium bution pattern of actively proliferating zone in the olfactory in human and dog.11)12) mucosa and produced two major findings. First, basal cells only The previous studies reported the effects of inhaled toxic- demonstrated PCNA immunopositivity in the olfactory epi- ants on surface epithelia lining the nasal cavity. These studies thelium of rat, indicating the origin of new cells. Second, the suggested that the distribution of toxicant-induced lesions in distribution of basal cells with proliferation activity did not the nasal cavity is thought to arise from a combination of the show the region-specific pattern in the whole nasal cavity. That effects of regional uptake patterns, local clearance processes, is, the neurogenesis of olfactory nerve cells is most likely to and differences in the metabolic capacity or inherent sensiti- occur in some area, but not in adjacent or other areas. These vity of various cell types to a given toxicants.13)14) There may findings were constantly noted in septal and ethmoturbinates also be differences in the susceptibility of transitional and sq- mucosa which was covered by the olfactory mucosa. Taken to- uamous epithelium in anterior nasal cavity to inhaled toxicants. gether, it seems that the neurogenesis of olfactory mucosa, at Both epithelia lining the anterior nasal cavity normally contain least, is independent of air stream inhaled into the nasal cavity. few mucous cells, while numerous mucous cells are present in In summary, the results of present study suggest that mitotic the ciliated respiratory epithelium. 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