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- － 120 － 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