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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

Immunohistological identification of the activated lymphocytes in human palatine tonsil

Employing various monoclonal antibodies and immunoperoxidase technique, we studied the distribution of the activated lymphocytes in human tonsillar tissue. Both the activated T-cells positive for IL2-receptor (IL2-R) and the activated B-cells defined by L29 were seen in the interfollicular area. Vast majority of the lymphocytes in the germinal center was stained with the L29, the anti-transferrin-receptor antibody, and the Ki-67 antibody which reacts with cycling cells in G1, S, or G2 + M phase. On the other hand, scarcely any cells in the mantle zone were stained with those. The Ki-67 positive cells in the germinal center were identified as two types of staining pattern, i.e., with strong nucleolar staining found in the dark zone and with nuclear staining found in the light zone. Small number of IL2-R positive cells was found in the mantle zone. The cell number per unit area of the activated lymphocytes in the interfollicular area as well as in the germinal center was decreased as the increment of patients' age. From these results, the immunological activation system of T- and B-cells in the tonsils was discussed.     

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.     

Migration of cochlear lateral wall cells

The role of apoptosis and proliferation in maintenance of cochlear lateral wall cells was examined. The methods employed for detection of apoptosis were the Hoechst fluorescence stain and TUNEL (TdT-mediated dUTP-biotin nick-end-labeling) assay, and proliferations were 5-bromo-2'-deoxyuridine (BrdU) incorporation and presence of the proliferating cell nuclear antigen. The incidence of apoptosis in the strial marginal cell was 50% greater (32.9+/-3.7%) than strial intermediate and basal cells but similar to spiral ligament cells. Although division of marginal strial cells was rarely detected, a significant number of proliferating cells in the remaining stria vascularis and spiral ligament were observed. These data implied that replacement of marginal cells arose elsewhere and could be followed by a BrdU-deoxythymidine pulse-chase study. At 2 h post injection, nuclear BrdU in marginal cells was not detected; however, by 24 h post injection, 20-25% of marginal cell nuclei were BrdU-positive. These observations are consistent with the hypothesis that marginal cells were replaced by underlying cells. Cell migration appears to be an important mechanism for preserving the function and structure of the stria vascularis.     

神经特异性烯醇化酶及嗅标记蛋白在不同胎龄胎儿嗅黏膜中的表达

目的　探讨神经特异性烯醇化酶 (neuron specificenolase ,NSE)及嗅标记蛋白 (olfactorymarkerprotein ,OMP)在不同胎龄胎儿嗅黏膜中的表达。方法　以免疫组化方法检测NSE及OMP在1 2、1 6、2 0、2 4、2 8和 34周 6例不同胎龄胎儿嗅黏膜中的表达。结果　NSE免疫阳性反应在孕 1 2～ 34周的胎儿嗅黏膜中均有表达 ,各胎龄胎儿嗅黏膜切片中均可见大量阳性着色的双极嗅神经细胞。孕1 2周时 ,阳性细胞数量很多 ,排列紧密 ,胞体多位于嗅上皮中下部且阳性嗅神经上皮占据胎儿鼻腔上2 /3的黏膜 ,随着孕龄的增大 ,阳性细胞逐渐成多层次排列 ,所占鼻腔黏膜面积却逐渐减小 ,至孕 34周时 ,仅局限于鼻腔上 1 /3黏膜。OMP免疫反应在孕 1 2周胎儿鼻腔上 2 /3黏膜切片中仅发现少量阳性着色的双极神经细胞 ,明显少于同龄胎儿NSE阳性反应细胞。随着胎龄的增加 ,OMP阳性细胞逐渐增多 ,且胞体多位于嗅上皮中上部 ,但其数量仍相对少于同龄NSE阳性细胞。结论　人胚胎 1 2周时嗅黏膜中已有大量的嗅神经细胞 ,其中少数嗅神经细胞已开始发育成熟。随着胎龄的增大 ,嗅上皮面积逐渐缩小 ,但成熟的嗅神经细胞逐渐增多 ,胚胎发育后期的胎儿嗅化学感受器发育已趋于成熟     

Nucleoplasm staining patterns and cell cycle-associated expression of Ki-67 in middle ear cholesteatoma

OBJECTIVES: To compare Ki-67 expression patterns in middle ear cholesteatoma with the corresponding retroauricular and external auditory canal skins, and to determine the cell cycle-dependent localization of Ki-67. MATERIAL AND METHODS: MIB-1 monoclonal antibody was used for comparative assessment of proliferative activity of middle ear cholesteatoma, external auditory canal skin, and retroauricular skin samples on formalin-fixed paraffin-embedded tissue sections. Primary keratinocytes from cholesteatoma tissue were isolated and subjected to kinetic analysis of the cell cycle. RESULTS: Higher proliferative activity was established in cholesteatoma in comparison with retroauricular and external auditory canal skins. Three different staining patterns have been described. Kinetic analysis revealed continuous expression of Ki-67 during all active phases of the cell cycle and remained "silent" in resting cells. CONCLUSION: The established correlation between the staining patterns and cell cycle-associated expression of Ki-67 specifies Ki-67 as a reliable and stable marker of proliferation for middle ear cholesteatoma.     

High-dose glucocorticoids inhibit proliferation of rat olfactory epithelium

Glucocorticoids (GCs) are commonly prescribed for treatment of olfactory dysfunction. However, the effects of GCs on olfactory epithelium are not well known. We investigated the effects of high-dose GCs on proliferating cells of olfactory epithelium. Five adult male rats (300 g) received a single daily subcutaneous dose of vehicle containing 0.3 mg dexamethasone (DEX) for 9 days (DEX+ group), and a control group received vehicle alone (DEX- group). We compared sections from the two groups for numbers of Ki67-positive cells. The mean number of Ki67-positive cells per 500 olfactory epithelial cells was 9.6 for the DEX+ group and 58 for the DEX- group (significant difference). We conclude that high-dose GC suppressed proliferation of olfactory epithelium. We suggest that high-dose GC suppresses cytokines and growth factors, resulting in secondary suppression of proliferating ability.     

Expression of neuron-specific enolase and olfactory marker protein in the developing olfactory mucosa of human fetuses]

OBJECTIVE: To study the expression of neuron-specific enolase (NSE) and olfactory marker protein (OMP) in the developing olfactory mucosa of human fetuses. METHOD: The expression of NSE and OMP in the olfactory mucosa of 6 human fetuses (12, 16, 20, 24, 28 and 34 weeks) was studied using the technique of immunohistochemistry. RESULTS: NSE immunological positive reactions were seen in all 6 fetal mucosa from gestational 12 (G12) to G34, with plenty of positive-stained dual-pole neuron cells. At G12, the positive cells aligned tightly, the cell bodies were localized in the lower portion of olfactory epithelium and the positive-stained area occupied upper 2/3 of fetal nasal mucosa. With the development, the positive cells gradually became multilayer, but the density and the relative area of positive-cells reduced. At G34, the positive cells were located only in upper 1/3 of nasal mucosa. OMP-positive reactions were localized in a few dual-pole neurons at G12, the number was much less than NSE-positive cells in the same fetus. With the development, the OMP-positive cells gradually increased with most of the cell bodies located in the upper portion of epithelium, but number still relatively less than the NSE-positive cells at the same age. CONCLUSION: At G12, there were lots of olfactory neuron in the olfactory mucosa and only a few olfactory neurons had became mature. With the development, the olfactory epithelial area reduced but the number of mature olfactory neurons increased. At the last trimester, fetal olfactory sensor was almost matured.     

Fate of newly formed periglomerular cells in the olfactory bulb

It is well established that olfactory receptor cells are replaced during life. Periglomerular (PG) cells of the olfactory bulb have recently been demonstrated to be produced following proliferation and migration of periventricular neuronal precursor cells even in adulthood. The purpose of the present study was to examine the fate of newly formed PG cells in adult rodents. Using 5-bromodeoxyuridine (BrdU), we carried out a quantitative immunohistochemical analysis of BrdU-positive cells in the bulbar glomerular layer at different survival periods. Each number of BrdU-positive PG cells per 100 olfactory glomeruli was 34.1 +/- 3.3 (1 week), 57.2 +/- 2.7 (2 weeks), 28.0 +/- 4.7 (4 weeks) and 25.9 +/- 1.6 (8 weeks). These results indicate that bulbar PG cells, similar to olfactory receptor cells, are mostly replaced during life, and that the olfactory system is composed of disposable neuronal networks centrally as well as peripherally.     

Cyclin D(1) and D(3) expression in vestibular schwannomas

OBJECTIVES: The G1 regulators of the cell cycle, cyclin D(1) and D(3), have been implicated in the regulation of Schwann cell proliferation and differentiation. The purpose of this study is to evaluate cyclin D(1) and D(3) protein expression and the corresponding clinical characteristics of vestibular schwannomas. STUDY DESIGN AND METHODS: Tissue sections of 15 sporadic vestibular schwannomas were prepared. Immunohistochemical analysis of the vestibular schwannomas was performed with anticyclin D(1) and anticyclin D(3) antibodies. The immunoreactivity was evaluated in comparison with adjacent vestibular nerves. Tissue sections of breast carcinoma and prostate carcinoma were used as positive controls for cyclin D(1) and D(3) staining, respectively. Patient demographics, tumor characteristics, and cyclin D expression were reviewed, and statistical analysis was performed. RESULTS: While the breast carcinoma control expressed abundant cyclin D(1) protein, none of the 15 vestibular schwannomas showed detectable cyclin D(1) staining. In contrast, seven of 15 vestibular schwannomas stained positive for the cyclin D(3) protein. Cyclin D(3) staining was taken up in the nucleus of schwannoma tumor cells in greater proportion than Schwann cells of adjacent vestibular nerve. Although sample size was small, no significant difference in the average age of presentation, tumor size, and male to female ratios for the cyclin D(3)(+) or cyclin D(3)(-) groups was found. CONCLUSION: The Cyclin D(1) protein does not appear to play a prominent role in promoting cell cycle progression in vestibular schwannomas. In contrast, cyclin D(3) expression was seen in nearly half of the tumors examined, suggesting that it may have a growth-promoting role in some schwannomas. Further studies are needed to define its cellular mechanism.     

Expression of cell cycle regulatory proteins and analysis of apoptosis in normal nasal mucosa and in nasal polyps

BACKGROUND: The etiopathogenesis of nasal polyps still is to be clarified. Although hyperplasia is a typical feature of these pathological processes, little attention has been paid to specific aspects of cellular growth in polyps. We have evaluated the expression and localization of some of the regulatory proteins that direct the cell through the specific sequence of events culminating in mitosis or apoptosis in nasal polyps. METHODS: Twenty samples of nasal polyps and 20 samples of normal nasal mucosa have been analyzed for apoptotic index by detecting the DNA 3'OH ends deriving from DNA fragmentation. Moreover, they have been evaluated by immunohistochemical staining for expression of Ki-67, cyclins A and B1, p53, p21, p27, murine double minute clone 2, and Bcl-2. RESULTS: We have identified a greater proportion of proliferating cells in the lining epithelial cells of the polyps when compared with the normal mucosa as stained with anti-Ki-67 antibodies. An overexpression of p53, MDM2, and Bcl-2 and an increased apoptosis were observed in nasal polyps compared with the normal mucosa, whereas no variation of p27 expression was observed. The p21 and cyclins A and B1 were rarely expressed in both pathological and normal tissue. CONCLUSION: The p53-based control system of cell cycle progression appears to be altered in nasal polyps, potentially leading to an abrogation of the DNA damage checkpoint. Evaluation of the expression of the regulatory proteins that direct the cells throughout their cycle in nasal polyps may allow a better understanding of the biological behavior and clinical outcome of these benign pathological entities.     

DNA拓扑异构酶I抑制剂诱导鼻咽癌上皮细胞凋亡的实验研究

拟从细胞凋亡角度出发，探讨能够诱导鼻咽癌上皮细胞凋亡的因素，为有效地防治该恶性肿瘤提供新的思路。方法：光镜下观察细胞的形态变化；流式细胞仪检测细胞周期变化及亚二倍体比例；ＤＮＡ凝胶电泳及二苯胺法测定ＤＮＡ片段化程度。结果：一定浓度的拓扑异构酶Ｉ抑制剂喜树碱（ＣＰＴ）体外作用于人低分化鼻咽癌上皮细胞系ＣＮＥ－２Ｚ细胞一定时间后，镜下可见细胞体积缩小，染色质凝聚，胞膜突起及凋亡小体形成等形态变化。２￣     

Evaluation of proliferative activity in human head and neck tumors using the monoclonal antibody Ki-67

The monoclonal antibody Ki-67 is directed against a human nuclear antigen occurring in proliferating cells. As a rule it is absent in quiescent cells. Seven cases of squamous cell carcinomas with different pathohistological features are investigated by immunostaining. The antibody Ki-67 is suitable for use as a marker for proliferative compartments of head and neck tumors. Squamous cell carcinomas show a correlation between Ki-67 labeling index and histological differentiation. In highly differentiated parts of the carcinomas, an additional granular immunostaining in the entire cytoplasm of some squamous epithelial cells was observed.