甲酸脱钙液的浓度对骨组织脱钙后切片染色效果的研究

目的探讨骨组织经不同浓度的甲酸脱钙液24小时脱钙后切片染色效果,选择最佳甲酸脱钙工作液。方法350例骨组织标本每例取材3份,分别用40％、30％、20％的甲酸脱钙液常温下脱钙24小时,观察三种不同浓度的甲酸脱钙液脱钙后骨组织的切片和HE染色情况。结果骨组织经40％甲酸脱钙液脱钙后,切片完整,但HE染色核质偏浅,胞浆偏红;30％甲酸脱钙液脱钙后,切片完整,HE染色核质清晰,胞浆鲜艳;20％甲酸脱钙液脱钙后,切片不够完整,HE染色核质偏浅,胞浆偏浅。结论脱钙液脱钙后应保持组织切片完整,染色效果良好,且30％的甲酸脱钙液是较理想的工作液。     

甲酸脱钙液的浓度对骨组织脱钙后切片染色效果的研究

目的探讨骨组织经不同浓度的甲酸脱钙液24小时脱钙后切片染色效果,选择最佳甲酸脱钙工作液。方法350例骨组织标本每例取材3份,分别用40％、30％、20％的甲酸脱钙液常温下脱钙24小时,观察三种不同浓度的甲酸脱钙液脱钙后骨组织的切片和HE染色情况。结果骨组织经40％甲酸脱钙液脱钙后,切片完整,但HE染色核质偏浅,胞浆偏红;30％甲酸脱钙液脱钙后,切片完整,HE染色核质清晰,胞浆鲜艳;20％甲酸脱钙液脱钙后,切片不够完整,HE染色核质偏浅,胞浆偏浅。结论脱钙液脱钙后应保持组织切片完整,染色效果良好,且30％的甲酸脱钙液是较理想的工作液。     

骨髓穿刺组织石蜡制片方法改进的探讨

由于骨髓组织含有大量的钙盐而非常坚硬,不能直接进行石蜡制片,而经过脱钙处理后,才能进行切片、HE染色、特殊染色和免疫组化染色。脱钙过度或不当均可影响上述染色。因此,脱钙时间和脱钙液的选择十分重要。传统的方法用14％硝酸脱钙液脱钙常出现脱钙过度或不足,难以掌握,常造成组织切片不完整、组织结构破坏、细胞模糊不清,     

不同的脱钙液在骨组织制片中的比较应用

目的比较不同的脱钙液对骨组织的脱钙能力的差异,探讨其对HE染色的影响。方法选取50例手术切除的新鲜骨标本,运用4种常用的脱钙液(14%硝酸、10%盐酸、20%甲盐酸和EDTA脱钙液)进行脱钙,比较其脱钙效果和对HE染色的影响。结果不同的脱钙液对骨组织的脱钙能力、HE染色效果影响有显著区别。结论 14%硝酸脱钙能力最强,用时间最短,染色的效果最差;10%盐酸对骨组织的损伤小,HE染色的效果最好,但用时较长;20%甲盐酸脱钙能力和HE染色效果都介于10%盐酸和EDTA脱钙液之间;EDTA脱钙液脱钙用时最长,对骨组织的损伤最小。     

微波-EDTA脱钙技术在免疫组织化学染色中的应用研究

目的探讨微波(MW)—乙烯二胺四乙酸钠(EDTA)快速脱钙技术对骨及软组织抗原活性的影响。方法根据实验条件将待脱钙组织分为4组，即MW—EDTA(15℃)，Jenkins脱钙-固定液(20℃)，10％EDTA(20℃)和10％硝酸(20℃)，其中Mw—EDTA方法中EDTA的浓度分别是4％、7％、10％、13％和16％。应用免疫组织化学路AB染色，光镜观察免疫组织化学染色结果。结果MW—EDTA脱钙的免疫组织化学染色阳性细胞显著高于Jenkins脱钙—固定液(20℃)、10％ED—TA(20℃)及10％硝酸脱钙(20℃)3种脱钙方法，其时间明显缩短。其中用10％和13％浓度的EDTA脱钙的组织，其免疫组织化学染色阳性细胞又明显多于EDTA浓度为4％、7％和16％者。结论以Mw—10％EDTA技术进行组织脱钙，其脱钙时间短、免疫组织化学染色效果好。     

两种EDTA脱钙方法的比较

目的寻求切片制作更容易、染色效果更好的脱钙方法。方法对兔下颌骨分别使用15％EDTA微波脱钙法及15％EDTA低温脱钙法进行脱钙,从切片制作、免疫组化染色等方面进行比较。结果以15％EDTA低温脱钙法对兔下颌骨进行脱钙后,制作切片时脱片率明显降低,染色效果更好。结论以科学实验为目的对骨组织进行脱钙时,选用15％EDTA低温脱钙法能取得更准确的实验结果。     

微波-EDTA脱钙技术在免疫组织化学染色中的应用研究

目的探讨微波(MW)-乙烯二胺四乙酸钠(EDTA)快速脱钙技术对骨及软组织抗原活性的影响.方法根据实验条件将待脱钙组织分为4组,即MW-EDTA(15℃),Jenkins脱钙-固定液(20℃),10%EDTA(20℃)和10%硝酸(20℃),其中MW-EDTA方法中EDTA的浓度分别是4%、7%、10%、13%和16%.应用免疫组织化学LSAB染色,光镜观察免疫组织化学染色结果.结果MW-EDTA脱钙的免疫组织化学染色阳性细胞显著高于Jenkins脱钙-固定液(20℃)、10%ED-TA(20℃)及10%硝酸脱钙(20℃)3种脱钙方法,其时间明显缩短.其中用10%和13%浓度的EDTA脱钙的组织,其免疫组织化学染色阳性细胞又明显多于EDTA浓度为4%、7%和16%者.结论以MW-10%EDTA技术进行组织脱钙,其脱钙时间短、免疫组织化学染色效果好.     

家兔鼻腔病理标本制备及组织结构解析

目的 研究家兔鼻腔的解剖学和组织学结构,为吸入性毒性研究提供有效的病理学评价方法。方法 采集正常成年家兔的鼻腔,经10%中性福尔马林溶液固定,三氯甲烷脱脂,甲酸溶液脱钙后,从鼻腔顶端开始连续切片,HE染色,对其中具有标志性的解剖学和组织学结构部位的4个切面的主要上皮类型和组织结构进行评价。结果 切面Ⅰ在紧靠门齿后修取,主要排列有鳞状上皮带有少量密集的变移上皮,该部位仅适用于灌注给药试验的毒性病理学评价。第一个腭脊处修取切面II,主要排列有变移上皮和呼吸上皮。紧靠第一个上前臼齿前修取切面III,紧靠第一个上臼齿前修取切面IV,切面III和切面IV排列有呼吸上皮和嗅上皮,通常还分布有鼻相关性淋巴组织。与大鼠、小鼠、狗、猴相比,家兔鼻腔的相对体积与人类似。结论 通过家兔明显的颅骨解剖标志物确定鼻腔的4个组织切面,可完整涵盖所有鼻腔内的组织结构特征,这为临床前药物安全性评价中鼻腔的科学性评估提供支持和帮助。     

术中冰冻切片快速免疫组化HE染色技术诊断硬化性肺细胞瘤和肺腺癌的应用对比

目的 探讨术中冰冻切片快速免疫组化诊断硬化性肺细胞瘤（PSP）和肺腺癌的可行性及意义。方法 收集安徽医科大学第一附属医院2018年1月至2019年7月PSP术中标本12例设为PSP组以及肺腺癌术中标本26例设为肺腺癌组,使用术中冰冻切片HE染色和术中冰冻切片快速免疫组化检测甲状腺转录因子-1（TTF-1）、雌激素受体（ER）和细胞增殖核抗原（Ki-67）在两种疾病标本中的表达,以石蜡切片免疫组化结果为金标准,对比术中冰冻切片快速免疫组化和HE染色两种方法诊断PSP和肺腺癌的正确率。结果 术中冰冻切片快速免疫组化和术中冰冻切片HE染色诊断PSP正确率分别为100.00%,58.33%,差异有统计学意义（P=0.037）。术中冰冻切片快速免疫组化结果显示,PSP组TTF-1阳性、ER阳性和Ki-67阴性表达率分别为100.00%、91.67%和83.33%,均高于肺腺癌组,差异有统计学意义（P < 0.001）。结论 术中冰冻切片快速免疫组化诊断PSP和肺腺癌显著优于术中冰冻切片HE染色,TTF-1、ER和Ki-67联合可用于术中冰冻切片快速免疫组化指标鉴别诊断PSP和肺腺癌。     

微波低温改良脱钙在钙化软组织免疫组化染色中的应用

目的 探讨运用微波低温改良脱钙法对提高钙化软组织在免疫组织化学染色中的效果.方法 选取本院送检伴有钙化灶状的甲状腺组织标本62例,每例采用室温和微波低温进行脱钙,免疫组化染色光镜观察效果.结果 运用微波低温改良脱钙在免疫组化染色中明显比室温下的10％硝酸脱钙定位准、染色阳性细胞显示多、假阴性减少,背景清晰.结论 微波低温改良脱钙法能更好的运用于钙化软组织的免疫组化染色.     

51O例股骨头标本常规脱水与超声波快速脱水对切片效果的比较研究

目的通过510例股骨头标本HE制片结果的分析,比较常规脱水与超声波快速脱水的效果。方法股骨头标本用锯锯成2cm×2cm×0.3cm的薄片二份,用30％的甲酸脱钙液进行脱钙16～18小时,分别用常规脱水的方法及超声波快速脱水的方法脱水处理后,经石蜡包埋、切片、染色制成HE切片,观察染色后的组织结构的清晰度及完整性。结果股骨头标本经二种脱水方法处理后制成的HE切片,阅片比较发现超声波快速脱水法优良率略高于常规脱水法,两种方法比较,P〉0．05,差异无统计学意义。但超声波组织处理仪能明显缩短病理制片工作时间。结论骨组织脱钙后采用超声波快速脱水的方法可以代替常规脱水,HE切片效果良好,缩短了病理检验报告时间,更好地满足了临床工作的需求。     

Histochemical localisation of carboxylesterase activity in rat and mouse oral cavity mucosa

Vinyl acetate (VA) is widely used within the chemical industry, in the manufacture of polyvinyl alcohol, and as polyvinyl acetate emulsions in latex paints, adhesives, paper and paper board coatings. Chronic oral exposure of rodents to high concentrations of VA induces tumours within the oral cavity. Carboxylesterase-dependent hydrolysis of VA is thought to be critical in the development of nasal tumours following inhalation exposure of animals to VA. Therefore, carboxylesterase activity was determined histochemically in the oral cavities of male F344 rats and BDF mice in order to explore the potential role of carboxylesterase-dependent hydrolysis of VA in the development of oral tumours. Following fixation in 10% neutral buffered formalin heads were decalcified in neutral saturated EDTA, embedded in resin, sectioned at six levels (three each for the upper and lower jaws), and carboxylesterase activity revealed in the tissue using -naphthyl butyrate as substrate. The localisation of carboxylesterase activity in freshly dissected rat oral tissue was compared to that of the resin sections and found to be identical, thus validating the decalcification process. A similar pattern of carboxylesterase activity was observed for the two species. Staining was low in areas surrounding the teeth, and medium/high in the buccal mucosa, the central/posterior upper palate and those regions of the lower jaw not proximal to the teeth. In general the intensity of staining was greater in sections from the rat compared to those from the mouse. By comparison, carboxylesterase activity was considerably higher in mouse nasal olfactory epithelium than in any of the oral tissues. Thus the mucosa of the oral cavity has the potential to hydrolyse VA to its metabolites, acetic acid and acetaldehyde, and the presence of carboxylesterases at this site is consistent with, and may be an important determining factor in, the development of oral cavity tumours following exposure to VA.     

Nasal Molds as Predictors of Fine and Coarse Particle Deposition in Rat Nasal Airways

Abstract

Important data for human risk assessment of inhaled chlorine are provided by a recent rodent cancer bioassay (Wolf et al., 1995) and a chronic inhalation toxicity study in rhesus monkeys (Klonne et al., 1987). To improve interspecies comparisons based upon these data sets, the tissues from these studies were reexamined to (a) map the location of responses to assess the potential role of local chlorine dosimetry, (b) generate quantitative data on selected endpoints to compliment subjective scores, and (c) further characterize the responses in relation to interspecies differences and potential human health risks. Chlorine-induced lesions, which were confined to the respiratory tract, exhibited both similarities and differences among rodents and primates. At equivalent airborne concentrations (～2.5 ppm), chlorine-induced responses were less severe in rhesus monkeys, but extended more distally in the respiratory tract to involve the trachea, while treatment-induced lesions were confined to the nose in rats and mice. Quantitation of septal fenestration, intraepithelial mucus, intraepithelial eosinophilic material, eosinophil infiltration (detected during the present study), and olfactory sensory cell loss generally supported previously reported subjective pathology scores and clarified concentration-response relationships. In both rodents and rhesus monkeys, airflow-driven regional dosimetry patterns were considered to play a major role in lesion distribution. The present work highlights the need for understanding regional respiratory-tract dosimetry and mechanisms of tissue response for inhaled chlorine in laboratory animals and humans.     

Histochemical localization of formaldehyde dehydrogenase in the rat

Formaldehyde dehydrogenase (FDH) activity has been demonstrated biochemically in the olfactory and respiratory mucosae and in the liver of the rat, but the cellular localization of this enzyme has not been investigated. A histochemical procedure was developed to permit cellular localization of FDH. This allowed us to examine the relationship between distribution of FDH and formaldehyde-induced toxicity. Cold-processed glycol methacrylate embedded tissues were used to localize FDH activity in the rat respiratory tract, kidney, liver, and brain. Five- or ten-micrometer tissue sections were incubated in a reaction mixture containing formaldehyde (HCHO), glutathione (GSH), NAD+, nitroblue tetrazolium, pyrazole, and disulfiram. A blue formazan precipitate was formed at the site of FDH activity. Epithelial cell cytoplasm of both the respiratory and the olfactory mucosae of the nose stained for FDH, and olfactory sensory cell nuclei were also positive. Underlying Bowman's and seromucous glands were weakly positive. The lung had FDH activity located mainly in the Clara cells of the airways, with only diffuse weak activity in the lung parenchyma. Liver had activity in the cytoplasm of the hepatocytes, while in the kidney FDH was most prominent in the brush border of the P2 segment of the proximal tubules. Brain white matter stained strongly for FDH, while in gray matter only the neuropil exhibited weak activity. Corresponding tissue sections were stained for sulfhydryls; these sections indicated that GSH is likely to be present in all cells with FDH activity. For the respiratory tract these results demonstrate distinct differences between the location of FDH activity and previously reported nonspecific aldehyde dehydrogenase activity in the nose (M. S. Bogdanffy, H. W. Randall, and K. T. Morgan, 1986, Toxicol. Appl. Pharmacol. 82, 560-567). While high aldehyde dehydrogenase activities were found in tissues with low toxicities due to acetaldehyde exposure and vice versa, FDH activity was observed in tissues whether or not they exhibited a toxic response to inhaled HCHO. While not able to account for the localized toxicity of HCHO, the presence of FDH and glutathione in the epithelial layer of the nasal cavity presents a barrier to inhaled formaldehyde at low concentrations and may partially explain the observed nonlinearity of HCHO toxicity.     

A Distributed-Parameter Model for Formaldehyde Uptake and Disposition in the Rat Nasal Lining

DNA–protein cross-links (DPX) serve as a dosimeter for inhaled formaldehyde and are associated with tumor induction in rat nasal passages after chronic exposure to 6 ppm and above. To determine the role of epithelium-specific morphometry in formaldehyde-induced patterns of injury, we developed a mathematical model that links airflow-driven formaldehyde uptake with DPX formation in regions of the rat nose with high and low tumor incidence. A three-dimensional, anatomically accurate computational fluid dynamics model of rat nasal airflow and inhaled gas uptake was integrated with a physiologically based mathematical model incorporating tissue thickness, formaldehyde diffusion, its removal by enzymatic and nonenzymatic processes, and DNA distribution in the nasal mucosa to predict DPX formation. The model implicitly incorporates the reversible conversion of formaldehyde to methylene glycol. Where possible, parameter values were taken from the literature or estimated using published correlations. The Michaelis–Menten kinetic constants V_max and K_m, as well as a first-order constant for formaldehyde removal, were left as fitted parameters. The resultant model fit to the experimentally measured DPX in the high- and low-tumor-incidence regions of the rat nasal passages was very good. Sensitivity analysis indicates that among the fitted parameters, model fits are most sensitive to V_max and that predictions were sensitive to changes in tissue thickness when all other parameters are held constant. The model structure facilitates extrapolation to primates and humans and application to other soluble, reactive gases.     

实验动物冰冻组织切片制备关键要点的探讨

目的 探讨实验动物组织冰冻切片制备的关键要点和注意事项,以提高切片质量。方法 采用美国赛默飞世尔恒温式冷冻切片机,对30例SD大鼠和30例食蟹猴的皮肤、肌肉、乳腺、卵巢、子宫、胃、大肠、甲状腺、肝脏、肾脏、脾脏、淋巴结、脑、脂肪等新鲜组织进行常规冰冻切片取材、包埋、切片、染色后封片。结果 镜下检查组织切面平整,细胞形态完整、清晰,染色良好。结论 不同组织需要区别对待,制作优良的冰冻切片就需要对取材、组织速冻、温度选择、组织包埋、切片、固定等多个环节进行严格谨慎地操作。     

Physiologically based clearance/extraction models for compounds metabolized in the nose: an example with methyl methacrylate

Airstream clearance (with units of volume/time) is the volumetric flow from which chemical would have to be completely removed to account for the net loss in the nose. Extraction is the proportion of airflow from which the chemical is completely removed. Over the past several years we have developed physiologically based clearance-extraction (PBCE) models for the nose to assess the physiological, biochemical, and anatomical factors that control airstream clearance. A generic clearance equation was derived for single airway/tissue compartments that had a separate air region and either one, two, or three underlying tissue regions. For all of these structures, airstream clearance (Cl(sys)) has a common form-Equation (1)-related to tissue clearance (Cltot), gas-phase diffusional clearance (PAgas), airflow (Q), and the mucus air partition coefficient (Hmuc:a). Clsys = CltotHm:aPAgasQ/CltotHm:a(Q + PAgas) + PAgasQ. A physiologically based clearance-extraction (PBCE) model for the whole nose combined three separate nasal tissue regions, each with a four-compartment tissue stack (air, mucus, epithelial tissue, and submucosal region). A steady-state solution of the PBCE model successfully described literature results on the steady-state extraction of methyl methacrylate (MMA) and several other metabolized vapors. Model-derived tissue dosimetry estimates, that is, the amount of MMA metabolized in the target epithelial compartment of the olfactory region, for rats and humans provide dosimetric adjustment factors (DAFs) required in calculating a human reference concentration (RfC) from rodent studies. Depending on the assignment of esterase activities to sustentacular and submucosal regions, the DAFs from the PBCE model varied between 1.6 and 8.0, compared to the default value of 0.145. From the experience with MMA, a minimal data set could be defined for building the PBCE model. It consists of mucus:air and blood:air partition coefficients, metabolic constants for enzymatic hydrolysis in nasal tissues from rat and human tissues, immunohistochemistry of the distribution of these activities in rats and human olfactory tissues, and extraction studies in anesthetized rats to assess the total nasal metabolism of the test compound.