外伤性嗅觉障碍大鼠嗅黏膜的组织学变化

目的 构建大鼠外伤性嗅觉障碍模型,观察不同时间点嗅黏膜组织学变化.方法 神经切断组(40只)和对照组(20只)大鼠均在显微镜下暴露左侧嗅球,沿筛板切断神经组切断大鼠左侧嗅神经.采用嗅觉诱发电位(olfactory evoked potentials,OEPs)和神经示踪验证造模效果.术后1天、5天、2周、3周、6周处理大鼠,处理前1天每组各取2只大鼠经鼻腔滴注辣根过氧化物酶(horseradish peroxidase,HRP).嗅黏膜及嗅球冰冻切片后观察嗅上皮的厚度、细胞数量的变化以及嗅神经的连续性,并且行免疫组化观察嗅上皮中的新生嗅感觉神经元(olfactory receptor neurons,ORNs).结果OEPs及神经示踪证实手术方法可以完全切断嗅神经.术后1天,切断侧黏膜中ORNs出现凋亡,两组大鼠双侧嗅上皮厚度和细胞数量比值无明显变化.5天时切断侧嗅上皮中细胞数量减少,上皮厚度变薄,嗅球中无HRP标记纤维.术后2、3周大鼠嗅球中出现蓝色标记,嗅上皮厚度和细胞数量逐渐增加,但仍然与对照组有差异,此时嗅上皮中出现大量的新生ORNs.经过6周的恢复,嗅上皮厚度及细胞数量基本恢复至对照组水平,嗅上皮中有较多的新生ORNs,其轴突与嗅球重新建立神经联系,但是上皮中仍然有一定数量的凋亡细胞.结论 嗅神经切断术可以作为制作外伤性嗅觉障碍的可靠方法;由于ORNs具有再生能力,大鼠嗅神经切断后嗅黏膜在一定时间内基本恢复至正常水平.     

鼻科学

20050442大鼠嗅球发育过程中的凋亡与增殖/陈福权…//第四军医大学学报.2004,25(20).1850～1852目的:探讨大鼠嗅球发育过程中细胞凋亡和增殖的变化规律。方法:胚胎14天,新生1天(postna talday,P1),P20及成年大鼠各10只,取嗅球以40g/L多聚甲醛固定,石蜡包埋,连续切片。采用TUNEL和免疫荧光染色方法检测大鼠嗅球中凋亡细胞和增殖细胞核抗原(PCNA)免疫反应阳性细胞的分布。结果:在胚胎14天和P1大鼠嗅球中,凋亡细胞广泛分布而PCNA免疫反应阳性细胞主要分布在室管膜周围区域,凋亡细胞和PCNA免疫反应阳性细胞在p20和成年大鼠嗅球中主要分…     

嗅觉神经元凋亡相关因子研究

成年哺乳动物的嗅觉受体神经元(olfactory receptor neurons,ORNs)可自我更新,成年人的嗅上皮也仍保持神经再生和分化能力,而嗅上皮的这种特性使嗅觉障碍的恢复和治疗成为可能.ORNs是脊椎动物中惟一一种胞体同外周环境直接接触的神经元.外界环境中细菌、病毒、吸烟以及物理性损伤都可以造成ORNs损伤和死亡.随着对嗅觉及嗅觉障碍等相关研究,在对嗅神经元的增殖与凋亡方面也有更加深入的进展.本文就嗅觉神经元的凋亡与其相关因子的关系进行了综述.     

缺血对大鼠嗅球影响的实验研究

目的：观察成年大鼠嗅球缺血性损伤后的病理改变,探讨缺血对嗅觉的影响。方法：选取40只成年SD大鼠（体重250-300g）,分成对照组、实验组（1周组、1月组、2月组）,每组10只。将实验组大鼠双侧颈总动脉结扎造成缺血,分别在1周、1个月、2个月时处死,光镜下观察嗅球病理改变,透射电镜下观察嗅球内细胞超微结构的变化。结果：光镜下可见实验组大鼠嗅球僧帽细胞胞核深染、颗粒细胞数目减少。透射电镜下1周组大鼠嗅球内僧帽细胞线粒体破坏,细胞变性、坏死;1月组僧帽细胞退行性变,神经纤维髓鞘断裂、板层脱落。结论：缺血可损伤神经细胞和神经纤维,可能造成或加重嗅觉障碍。     

嗅神经切断对小鼠嗅感觉神经元的影响

目的分析嗅神经切断对小鼠嗅感觉神经元（olfactory receptor neurons，ORN）凋亡情况的影响，并探讨这一造模方法的可靠件。方法取2月龄C57小鼠33只，随机分组后，实验组小鼠行嗅神经切断术，通过辣根过氧化物酶（horseradish peroxidase，HRP）顺行神经示踪验证造模成功与否。以脱氧核苷酸转移酶介导的牛物素dUTP缺口末端标记技术（TdT mediated deoxyuridine triphosphate—biotin nick end labelling，TUNEI。）观察术后8h、2d、3d和5d嗅上皮中ORN的凋亡情况，同时在蛋白和mRNA水平观察成熟ORN的特异性标记蛋白——嗅标记蛋白（olfactory marker protein，OMP）在嗅上皮巾的表达情况。结果嗅神经切断术后嗅球中无HRP标记。TUNEL和OMP阳性反应发生于ORN，嗅神经切断术后TUNEL阳性细胞数显著增多，并于术后第2天达到高峰，与此同时OMPmRNA的表达水平开始显著下降，并住术后第5天降至更低，嗅上皮的厚度也相应变薄。结论本实验所采取的造模方法可以较可靠地切断小鼠的嗅神经，并造成小鼠嗅上皮中ORN的同步凋亡。     

嗅感觉神经元凋亡的信号转导途径及基因调控

哺乳动物嗅上皮内衰老和受损伤的嗅感觉神经元(olfactory receptor neurons,ORNs)以凋亡的方式被机体清除,ORNs凋亡的发生、发展是在基因精确调控下通过两条信号转导途径来完成的.与凋亡相伴行的足ORNs的再生,二者的平衡确保了嗅上皮层ORNs的新旧更替和嗅觉功能的维持.本文通过复习ORNs凋亡和再生的相关文献,阐述ORNs的损伤因素及其凋亡的信号转导途径与基因调控.     

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。     

大鼠嗅感觉上皮发育及嗅细胞凋亡的研究

目的 探讨神经特异性烯醇酶（NSE）、嗅细胞标记蛋白（OMP）及细胞凋亡在不同胎龄大鼠嗅黏膜中的表达。方法 免疫组化方法检测NSE及OMP在不同孕期大鼠嗅上皮中的表达及其规律。TUNEL方法检测不同孕期大鼠嗅上皮中凋亡细胞并计算细胞凋亡指数（AI）。结果 E13d鼻腔黏膜中即有NSE阳性表达,细胞数量多。E13d嗅上皮中未见OMP阳性表达细胞。在E14d,嗅上皮中出现嗅OMP阳性细胞,数量少,随胎龄增加阳性细胞数量增多,至17d达高峰并逐渐趋于稳定。E13～E15d细胞凋亡数量较稳定,至E16d凋亡细胞数量明显增加,达到高峰,E18～E21d凋亡细胞数量逐渐减少渐趋于稳定。E16dAI与其他d数差异有统计学意义。结论 E14d嗅黏膜中已有发育成熟的嗅细胞,数量少,胚胎发育后期大鼠嗅化学感受器发育已趋于成熟。在嗅上皮发育过程中存在细胞凋亡,E16d嗅细胞凋亡出现一高峰,细胞凋亡在嗅觉发育过程中起重要作用。     

前庭学

20020261豚鼠发育过程前庭上皮细胞增殖和凋亡的关系/胡金玮…//中华耳鼻咽喉科杂志一2001,36(3)一183~186 目的:研究豚鼠胚胎正常发育过程中前庭上皮细胞增殖和细胞凋亡的变化,探讨两者之间的关系及在前庭终器发育中的作用。方法:选用不同发育期正常豚鼠20只,取前庭上皮组织切片,用免疫组化法检测增殖细胞核抗原(PCNA),以末端脱氧核昔酸转移酶介导的生物dUTP缺口末端标记技术(TUNEL)和透射电镜观察细胞凋亡。结果:PCNA阳性细胞为支持细胞,毛细胞无阳性反应,随年龄增加,阳性细胞数逐渐减少。TUNEL阳性反应可发生于毛细胞和支持细胞…     

慢性庆大霉素损伤后豚鼠前庭上皮细胞的凋亡和再生观察

目的：观察慢性庆大霉素中毒后豚鼠前庭上皮的损伤和修复过程，探讨前庭毛细胞凋亡和再生的关系。方法：采用末脱氧核苷酸转移酶介导的dUTP缺口末端标记（terninal deoxyuncleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling，TUNEL）法，观察前庭感觉上皮细胞的凋亡，采用5′-溴-2-脱氧尿苷（bromodeoxyuridine，Brdu）免疫细胞化学法观察前庭上皮的再生过程。结果：对照组没有观察到TUNEL和Brdu阳性细胞。停止庆大霉素注射1天至3周均可观察到TUNEL阳性细胞，但1天组阳性细胞数量最多，3周仍有阳性细胞存在，阳性细胞主要位于毛细胞层。停止庆大霉素注射后1天组支持细胞层和毛细胞层均有Brdu标记阳性细胞，3d组支持细胞层阳性细胞显著增多，1周组毛细胞层也出现较多阳性细胞，2周组Brdu标记阳性细胞主要位于毛细胞层，3周仍可观察到Brdu标记阳性细胞。结论：慢性庆大霉素耳中毒后豚鼠前庭上皮细胞凋亡增加，凋亡与增殖伴随存在，前庭毛细胞的损伤可能是其增殖的触发因素。增殖过程表现为支持细胞增生向毛细胞增生移行的趋势，支持细胞增殖并转化成毛细胞可能是前庭上皮细胞损伤修复机理之一。     

Apoptosis in the normal olfactory epithelium of the adult guinea pig

Among nerve cells of vertebrates, the olfactory epithelia are uncommon in their capacity for cell turnover. Apoptosis is well known to play a key role in maintaining homeostasis in continuously renewing tissues. We examined whether true apoptosis occurred in the normal olfactory epithelia of healthy adult guinea pigs using nucleic acid labeling. Subsequently, apoptosis was recognized in olfactory nerve cells, indicating that apoptosis might play a role in turnover of the olfactory epithelium.     

Pathology of the olfactory epithelium: smoking and ethanol exposure

OBJECTIVE: To investigate the effects of tobacco smoke on the olfactory epithelium. Cigarette smoking has been associated with hyposmia; however, the pathophysiology is poorly understood. The sense of smell is mediated by olfactory sensory neurons (OSNs) exposed to the nasal airway, rendering them vulnerable to environmental injury and death. As a consequence, a baseline level of apoptotic OSN death has been demonstrated even in the absence of obvious disease. Dead OSNs are replaced by the mitosis and maturation of progenitors to maintain sufficient numbers of neurons into adult life. Disruption of this balance has been suggested as a common cause for clinical smell loss. This current study will evaluate the effects of tobacco smoke on the olfactory mucosa, with emphasis on changes in the degree of OSN apoptosis. STUDY DESIGN: A rat model was used to assess the olfactory epithelium after exposure to tobacco smoke. METHODS: Rats were exposed to tobacco smoke alone (for 12 weeks), smoke plus dietary ethanol (for the final 5 weeks), or to neither (control). Immunohistochemical analysis of the olfactory epithelium was performed using an antibody to the active form of caspase-3. Positive staining for this form of the caspase-3 enzyme indicates a cell undergoing apoptotic proteolysis. RESULTS: Control rats demonstrated a low baseline level of caspase-3 activity in the olfactory epithelium. In contrast, tobacco smoke exposure triggered a dramatic increase in the degree of OSN apoptosis that affected all stages of the neuronal lineage. CONCLUSIONS: These results support the following hypothesis: smell loss in smokers is triggered by increased OSN death, which eventually overwhelms the regenerative capacity of the epithelium.     

Apoptosis in the Aging Olfactory Epithelium

Objectives Olfactory receptor neurons undergo apoptosis at a baseline rate, probably secondary to environmental damage even in the absence of gross disease. The study demonstrates age‐related changes in expression of genes known to regulate apoptosis in the rat olfactory mucosa. These results are compared with gene expression in young rats and rats that have undergone surgical deafferentation of the olfactory receptor neurons. Study Design The olfactory mucosae from three groups of rats were studied: young, normal rats (age, 12 wk); old, normal rats (age, 24 mo); and young rats 9 days after bilateral removal of the olfactory bulb. Bulbectomy is known to produce an initial wave of apoptotic cell death in the population of olfactory neurons. At 9 days after the injury, the olfactory mucosae consist of an enhanced population of regenerating neurons destined to also undergo apoptosis, since their synaptic target (bulb) has been removed. Methods Ribonuclease protection assays and histological analysis of the three groups were performed. Results Ribonuclease protection assay analysis indicates that age induces increases in the expression of pro‐apoptotic genes in the olfactory mucosae similar to the increase seen after deafferentation (bulbectomy). Specifically, the expression of procaspase‐3 and bax was increased in aged animals and bulbectomized animals when compared with young, normal animals. Conclusions Aging induces changes in gene expression in the olfactory mucosae that appear to favor apoptosis, probably associated with increased fragility of olfactory receptor neurons in older animals. These changes may, at least in part, explain the age‐related decline in olfactory sensation and loss of olfactory receptor neurons seen in elderly patients.     

Lipopolysaccharide-induced apoptosis of olfactory receptor neurons in rats

CONCLUSION: Daily intranasal perfusion of lipopolysaccharide (LPS) for 14 days in rats induced apoptosis of olfactory receptor neurons (ORNs) over >3 but <7 days. OBJECTIVES: Smoking is one of the factors causing olfactory dysfunction. LPS is a major glycolipid component of the gram-negative bacterial cell wall and an active component of cigarette smoke. We studied whether LPS is one of the causes of tobacco-induced olfactory dysfunction by examining apoptosis in the olfactory epithelium after local exposure to LPS. MATERIALS AND METHODS: Rats received intranasal instillation of LPS or saline. Histochemical changes in the olfactory epithelium were examined using antibodies against single-stranded DNA, Bcl-2, Bax, and Caspase-3. We used different concentrations of LPS to examine the dose dependency and observed changes in the olfactory epithelium for a week after exposure cessation to see the duration of the effect of smoking. RESULTS: We found that numbers of cells positive for ssDNA, Bcl-2, Bax, and Caspase-3 were increased on the exposed side. The number of ssDNA-positive cells reached a maximum on the first day and decreased to normal levels on the seventh day after cessation of exposure.     

Pathology of the olfactory mucosa: implications for the treatment of olfactory dysfunction

Objective: The pathology of the olfactory mucosa is poorly understood; however, most cases of hyposmia and anosmia appear to be associated with a decline in the number of functioning mature olfactory sensory neurons (OSNs). Under normal conditions, OSNs undergo apoptotic cell death at a baseline rate likely secondary to their exposed location in the nose. Regeneration of mature OSNs from precursors in the epithelium allows the animal to maintain an adequate number of neurons necessary for olfactory sensation. In many cases of olfactory dysfunction, this balance is apparently disturbed, with a net loss of OSNs. The current study will examine normal and diseased olfactory tissue for the presence of data demonstrating that the preferred mechanism of OSN cell death is apoptotic in both health and disease. The potential therapeutic implications will be discussed. Study Design: Histologic analysis of human and animal olfactory tissue. Methods: Normal and diseased human and animal olfactory mucosa were assessed for immunohistochemical evidence of apoptosis. Results: Increased activity of the apoptotic effector enzyme caspase‐3 was demonstrated in diseased olfactory mucosa in comparison with normal controls. Conclusion: These results indicate that a common pathway may mediate OSN cell death from a diverse set of pathologic insults including aging, trauma, and sinusitis. Interference with this pathway of cell death is currently the subject of intense pharmacotherapeutic research for the management of stroke and meningitis. These drugs may ultimately prove useful in the treatment of clinical olfactory dysfunction.     

Treatment of olfactory dysfunction, II: studies with minocycline

OBJECTIVES/HYPOTHESIS: The treatment of anosmia has changed minimally since the early 1970s, despite dramatic advances in the understanding of the molecular biology of olfaction. Recent studies from the authors' laboratory have suggested that most common causes of clinical olfactory dysfunction, including rhinosinusitis, appear to be associated with increased apoptotic death of olfactory sensory neurons. This appears to result in a decline in the number of functioning mature olfactory sensory neurons, despite the capacity of the olfactory epithelium for regeneration. The current study evaluated the ability of the antibiotic minocycline to inhibit olfactory sensory neuron apoptosis. This drug is known to inhibit apoptosis separate from its anti-infective properties. Olfactory sensory neuron apoptosis was triggered by surgical deafferentation ("bulbectomy"), the standard experimental model. Earlier studies have indicated that bulbectomy and sinusitis invoke similar proteolytic enzyme cascades in olfactory sensory neurons. STUDY DESIGN: Histological analysis of animal olfactory tissue. METHODS: Mice underwent unilateral olfactory bulbectomy to induce apoptotic olfactory sensory neuron death, with and without 45 mg/kg intraperitoneal minocycline given 12 hours before surgery and every 12 hours until death. Mice were killed at 2 and 4 days after bulbectomy and assessed for activation of capsase-3 and olfactory sensory neuron survival by immunohistochemical analysis. RESULTS: Minocycline resulted in partial suppression of cell death at 2 days after surgery when compared with untreated animals. CONCLUSION: Minocycline inhibits olfactory sensory neuron death in the face of a potent pro-apoptotic stimulus. This drug is well tolerated and is currently undergoing human trials for the management of a variety of neurological disorders associated with apoptosis. The current results suggest that minocycline may be efficacious in the management of peripheral olfactory loss as well.     

二甲胺四环素对嗅感觉神经元凋亡的保护作用

目的：观察二甲胺四环素对慢性鼻窦炎大鼠嗅感觉神经元（OSNs）凋亡的保护作用。方法：将大鼠随机分为对照组（A组）、鼻窭炎造模组（B组）和鼻窦炎加二甲胺四环素组（C组）,于不同时间取材,用苏木精-伊红染色观察嗅上皮厚度及上颌窦黏膜病理变化,用免疫组织化学染色检测Caspase-3表达阳性细胞。结果：苏木精-伊红染色镜检发现上颌窦黏膜病理学改变：A组明显轻于B组和C组,B、C两组嗅上皮厚度差异有统计学意义（P〈0．05）,免疫组织化学镜检发现B、C两组Caspase-3阳性细胞表达差异有统计学意义（P〈0．05）。结论：二甲胺四环素能抑制鼻窦炎大鼠模型Caspaser-3的表达,可能成为治疗嗅觉障碍的有效药物。     

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.