多视场多参数形态定量分析模块在神经研究领域中的应用

缺血性神经元损伤的性质是神经研究关注的热点。但目前用于区分凋亡与坏死细胞的ＴＵＮＥＬ技术,较易受主观因素的影响。本实验和图像分析技术对缺血动物脑的ＴＵＮＥＬ染色切片进行多场多参数形态定量分析,即细胞形态这亘参数（核面积、周长、直径、形状因子）和含 密度（平均光密度、积分光密度）。本实验结果显示图像分析能提高ＴＵＮＥＬ鉴别凋亡与坏死的灵敏度和精确性,并提供了一种简便快捷的形态定量方法,是在神经研究中     

低氧与缺血诱导培养海马和皮质神经元钙应答反应的比较

目的：钙作为重要信使参与多种生理和病理代谢过程,而且在缺血性神经元损伤机制中起着重要的作用。本实验模拟脑缺血的病理生化改变,用激光扫描共聚焦显微镜（LSCM）和Fluo-3荧光探针标记技术,观察低氧缺血状态下体外培养的海马,皮质神经元内钙浓度的变化。方法：用100μmol/L氰化钠造成细胞低氧;100μmol/L氰化钠和3.5mmol/L碘醋酸盐模拟在体完全性脑缺血;1mmol/LL-谷氨酸模拟在体脑缺血时兴奋性氨基酸大量释放;无葡萄糖介质剥夺细胞能量代谢的底物。结果：低氧使海马神经元[Ca^2 ]i显著升高,但有两种不同的钙振荡现象,谷氨酸引起海马神经元[Ca^2 ]i持续升高,但峰值低于低氧组,缺血组未见[Ca^2 ]i大幅升高,葡萄糖缺如不引起[Ca^2 ]i升高,结论：低氧和谷氨酸引起的神经元损害是能量依赖性的,轻度酸中毒可阻止胞内Ca^ 升高,糖对神经元具有保护作用,但单纯无糖引起的神经元损害与Ca^2 超载机制无关。     

大鼠视网膜缺血后Pavalbumin免疫反应神经元的变化

本文观察了大鼠视网膜缺血后Ｐａｒｖａｌｂｕｍｉｎ（ＰＶ）免疫反应神经元的变化。动物分为缺血１０ｍｉｎ组，１５ｍｉｎ组，３０ｍｉｎ组及６０ｍｉｎ组等４组。     

汉防己碱对缺血神经元钾通道的作用

目的和方法：研究汉防己碱（Ｔｅｔ）对缺血状态下大鼠皮层神经元膜电压依赖性钾通道变化的影响。方法：分离大鼠皮层神经元和建立细胞贴附膜片钳技术。结果：在缺血状态下，钾通道开放时间常数τ１和τ２分别由对照组的０４２９ｍｓ和８２０９ｍｓ增加至１８５５ｍｓ和１７４６４ｍｓ（Ｐ＜００１），开放概率由０１１４增加到０１５１（Ｐ＜００１）。Ｔｅｔ（７５，１５和３０μｍｏｌ／Ｌ）浓度依赖性抑制由缺血所诱导的钾通道开放。Ｔｅｔ７５和１５μｍｏｌ／Ｌ改变了通道的关闭模式，３０μｍｏｌ／Ｌ改变了通道的开放模式。结论：缺血可导致神经元电压依赖性钾通道开放显著增加，对胞外钾离子聚积起重要作用，从而参与神经元的损伤，Ｔｅｔ可抑制上述改变而对缺血脑细胞产生保护作用     

大鼠坐骨神经局部缺血后其神经元胞体超微结构的病理变化

采用结扎大鼠一侧髂总动脉致该侧坐骨神经局部缺血的方法，研究了其相关神经元胞体超微结构的病理变化。结扎髂总动脉的实验动物分别存活４，８，１０，１４天后，经升主动脉灌流杀死，取出腰髓第５节段和第５腰脊神经节，透射电镜下观察了腰髓前角细胞和仍神经节细胞超微结构的变化。     

图像分析系统对缺血心肌琥珀酸脱氢酶活性的定量检测

目的；研究早期心肌缺血的病理变化，探讨发病机理，方法；对实验性心肌缺血的心肌切片进行琥珀酸脱氢酶染色，并用图像分析仪检测酶活性的变化。结果：正常心肌和阴性对照组中琥珀酸氢酶的活性是均衡的，缺血组中缺血６０ｍｉｎ后，琥珀酸脱氢酶活性开始出现明显的变化，缺血区域随时间延长而加重，图像分析蓝色色值与正常对照组相比，差异有显著性，结论：图像分析系统对缺血心肌组化染色结果的一检测，具有高度的精确性，蓝色色值     

TUNEL法原位检测肿瘤组织细胞凋亡的改进

原位末端标记技术是当今较为流行的一类定量凋亡细胞评判方法,以脱氧核苷酸末端转移酶(TdT) 介导的核苷酸(dUTP) 缺口末端标记法(TUNEL )应用最为广泛.在对各种组织进行凋亡研究时,多数采用从各种试剂公司购买的试剂盒.虽然试剂盒使用方便,但是有时结果并不理想,如非特异性染色过多,背景染色深.对动物组织的研究更是如此.我们在TUNEL染色过程中如何更好地利用试剂盒,在避免假阳性、消除非特异性反应、降低背景染色等方面进行了探索和改进,获得了满意的实验结果,现介绍如下.     

坐骨神经局部缺血后相关神经元胞体乙酰胆碱酯酶变化的定量分析

目的:探讨周围神经局部缺血后相关神经元胞体乙酰胆碱酯酶(acetylcholine esterase,AchE)含量的变化。方法:采用结扎髂总动脉的方法,造成一侧坐骨神经局部缺血,术后大鼠分别存活8d和14d,取腰髓第5节段和双侧第5腰背根节。Leica振荡切片机连续切片,Karnovsky-Root直接显示AchE的方法使细胞呈色,采用MiVnt图像分析系统对AchE阳性细胞数和平均灰度值进行定量分析。结果:缺血8d组实验侧背根节和脊髓前角AchE阳性细胞数显著低于对照侧、平均灰度值显著高于对照侧;缺血14d组此变化较缺血8d组更为明显。结论:周围神经局部缺血可导致相关神经元胞体AchE含量明显降低。     

淫羊藿苷对缺血/再灌致神经元损伤的保护作用研究

目的 :观察淫羊藿苷对体外模拟缺血 /再灌致神经元损伤的保护作用 ,初步探讨其可能的作用机制。方法 :原代培养大鼠乳鼠大脑皮层神经元 ,分别在缺氧和缺糖 (6h) /再复氧和复糖培养后不同时间点 (0、3、1 2、2 4h)检测细胞活度、LDH活性、细胞凋亡率和细胞内游离钙离子浓度。结果 :淫羊藿苷 0 .2 5、0 .5和1mg·L 1明显减轻缺氧和缺糖 /复氧和复糖致神经细胞损伤 ,提高神经元MTT值 ,减少乳酸脱氢酶释放 ,减少凋亡和抑制细胞内游离钙离子浓度的升高。结论 :淫羊藿苷具有保护神经元抵抗模拟缺血性损伤的作用 ;其阻止细胞内钙离子超载可能是…     

CREB与脑缺血神经元损伤

cAMP反应元件结合蛋白(CREB)是位于细胞核内的转录因子,在脑缺血缺氧保护机制中起到了十分重要的作用。在脑缺血缺氧后,CREB通过多种途径被激活,再通过激活下游抗凋亡因子(如BDNF、Bcl-2+2等)及参与阻止Ca内流等多种途径起到保护神经元作用。     

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa

Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).     

Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labelling of DNA fragmentation

The process of palate fusion was examined in 13- and 14-day-old mouse fetuses by using in situ staining for nuclear DNA fragmentation (TUNEL method) and immunofluorescent staining for keratin, with special reference to the disruption of the midline epithelial seam. TUNEL-positive cells were found in the disappearing midline seam and the oral and nasal epithelial triangles at some late stages of palate fusion, but not in the palatal shelves prior to contact or in the intact midline epithelial seam. It seems that DNA fragmentation or apoptosis is required for the midline epithelial seam to disrupt, but may not be necessary for initial contact of palatal shelves or for the epithelial fusion of opposing palatal shelves. A similar sign of apoptotic cell death was observed in the disappearing epithelial seam between the fusing nasal septum and dorsal palate. We have demonstrated that apoptotic programmed cell death does occur at some stages of palate fusion, although the present results do not exclude the possibility of epithelial-mesenchymal transformation and the oral and nasal migration of midline epithelial cells.     

Apocrine change in fine-needle aspiration biopsy: nuclear morphometry and DNA image cytometry

The aim of this study was to investigate the potential of computerized nuclear morphometry and DNA image cytometry in characterizing the apocrine change of mammary epithelium in fine-needle aspiration biopsy (FNAB). The effect of two different sample processing techniques on the results was also studied. Mean nuclear areas in air-dried smears ranged from 59.0 microm2 to 151.0 microm2 and in ethanol-fixed samples from 32.3 microm2 to 63.4 microm2. The DNA histograms of apocrine cells usually showed a dominant peak in the diploid region. In some cases the mode of the peak was slightly shifted to the right or left in respect to the control peak. One case had a tetraploid cell population, suggesting atypical apocrine change. After histological investigation this case was diagnosed as infiltrating carcinoma. The patient had earlier been treated with x-ray irradiation for a mediastinal lymphoma. Findings of nuclear morphometry and DNA cytometry in apocrine metaplasia are here described in a systematic study for the first time. The data suggest that these methods may help in distinguishing premalignant and malignant apocrine lesions from typical apocrine metaplasia of mammary epithelial cells.     

Active caspase 3 and DNA fragmentation as markers for apoptotic cell death in primary and metastatic liver tumours

AIMS: The induction of tumour cell death by apoptosis is a major goal of cancer therapy and the in situ detection of apoptosis in tumour tissue has become an important diagnostic parameter. Different apoptosis detection methods assess distinct biochemical processes in the dying cell. Thus, their direct comparison is mandatory to evaluate their diagnostic value. The aim of this study was to compare the immunohistochemical detection of active caspase 3 and single-stranded DNA in primary and metastatic liver tumours as markers of apoptotic cell death. METHODS: We studied detection of active caspase 3 and single-stranded DNA in 20 primary hepatocellular carcinomas (HCC) and 20 liver metastases from colorectal carcinomas (CRC) using immunohistochemistry on paraffin sections. RESULTS: Our results reveal that both methods are suitable and sensitive techniques for the in situ detection of apoptosis, however, they also demonstrate that immunohistochemistry for active caspase 3 and single-stranded DNA have differential sensitivities in HCC and CRC. CONCLUSION: The sensitivity of apoptosis detection using immunohistochemistry for active caspase 3 and single-stranded DNA may be tumour cell type dependent.     

Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways

It was shown recently that exposure of the developing rat brain during the peak of synaptogenesis to commonly used general anesthetics can trigger widespread apoptotic neurodegeneration in many regions of the developing rat brain and persistent learning/memory deficits later on in life. To understand the mechanism by which general anesthetics induce apoptotic neuronal death we studied two common apoptotic pathways--the intrinsic and the extrinsic pathway--at different time points during synaptogenesis. We found that the intrinsic pathway is activated early on during anesthesia exposure (within two hours), as measured by the down-regulation of bcl-x(L), up-regulation of cytochrome c and the activation of caspase-9 in 7-day-old rats (the peak of synaptogenesis), but remains inactivated in 14-day-old rats (the end of synaptogenesis). The extrinsic pathway is activated later on (within six hours of anesthesia exposure), as measured by the up-regulation of Fas protein and the activation of caspase-8 in 7-day-old rats, but remains inactivated in 14-day-old rats. Anesthesia-induced apoptotic neurodegeneration is age dependent with vulnerability closely correlating with the timing of synaptogenesis, i.e. the developing brain is most sensitive at the peak of synaptogenesis (7 days old) and least sensitive at the end of synaptogenesis (14 days old).     

Comparison of ploidy analysis by flow cytometry and image analysis in hydatidiform mole and non-molar abortion

Determination of DNA ploidy is useful in the diagnosis and classification of hydatidiform mole. Most reports of ploidy analysis in molar tissue have used DNA flow cytometry. Although image analysis cytometry offers theoretical advantages over flow cytometry, there have been few reports of ploidy analysis by image analysis in hydatidiform mole. We selected 47 cases and measured DNA ploidy by flow cytometry and image analysis cytometry in complete hydatidiform mole, partial hydatidiform mole and non-molar abortion. The two cytometry modalities were compared using kappa statistics. There was reasonable overall agreement between the two modalities (κ= 0.69) and when ploidy was stratified into diploid/polyploid and triploid categories there was near perfect agreement (κ= 0.93). Aneuploid cell populations, which were not evident on flow cytometry, were identified by image analysis in a significant proportion of complete and partial hydatidiform moles and in a small number of non-molar abortions. Flow cytometry and image analysis cytometry yield comparable ploidy information, useful in the diagnosis and classification of hydatidiform mole. Image analysis cytometry offers greater sensitivity in the detection of small non-diploid cell populations but the significance of this latter finding is uncertain.     

Proliferative activity determined by DNA flow cytometry and proliferating cell nuclear antigen (PCNA) immunohistochemistry as a prognostic factor in prostatic carcinoma.

Proliferative activity was measured in 165 paraffin-embedded prostatic carcinomas using DNA flow cytometric analysis of the S-phase (SPF) and G2/M-phase fractions and CAS 200 image analysis of the proliferating cell nuclear antigen (PCNA) expression defined immunohistochemically by PC10 and 19A2 monoclonal antibodies. No significant associations were found between the flow cytometric and the two immunohistochemical measures of cell proliferation. Of the four indices, only SPF, S + G2/M, and immunostaining with 19A2 antibody were associated with the poor histological grade of the tumour. High SPF and S + G2/M were significantly associated with poor 10-year overall survival (P < 0.001) and prostatic carcinoma-specific survival (P < 0.01). Multivariate analyses of prostatic carcinoma-specific survival in patients with non-metastatic disease (M0-stage) indicated that only S + G2/M, T-stage, and histological grade (only if re-evaluated by a single pathologist) had independent prognostic significance. High-level PCNA staining (> 16 per cent of cells stained) with 19A2 antibody was associated with poor prognosis only in univariate analysis, and PC10 immunostaining had no prognostic value. In conclusion, a high proliferative activity as defined by flow cytometric S+G2/M is an independent predictor of poor survival in patients with non-metastatic prostatic carcinoma. PCNA immunostaining from formalin-fixed, paraffin-embedded prostatic carcinomas has little, if any, prognostic value.     

Diagnostic and prognostic value of DNA image cytometry in myelodysplasia.

The DNA content of erythropoietic cells from 10 patients with refractory anaemia (RA) with megaloblastic changes, who subsequently developed acute non-lymphoblastic leukaemia (ANL), and from seven patients with megaloblastic marrow aspirates due to pernicious anaemia were compared by DNA image cytometry. The DNA distribution, the rate of aneuploid cells exceeding 5c (5cER), and the square deviation index of DNA values from the normal 2c-peak (2cDI) were recorded. Both variables were of diagnostic and prognostic importance for epithelial tumours, malignant lymphomas, and dysplastic lesions. A rate of 5cER greater than 0 was found in eight of 10 myelodysplastic, but in none of seven control cases. Hypodiploidy was equally pronounced in both groups of patients. The 5cE had the highest discriminative value of all variables calculated. The 2cDI was not significantly different in either group. In pernicious anaemia the 2cDI depended mainly on the percentage of S cells, reflecting the defect of DNA synthesis. In RA with megaloblastosis the 2cDI correlated with the percentage of G2 cells, reflecting G2 arrest. In the myelodysplastic group the 2cDI correlated positively with the length of time until ANL developed, indicating the prognostic relevance of 2cDI. Our findings show that in megaloblastic anaemia DNA image cytometry can distinguish myelodysplasia from pernicious anaemia and that it also provides prognostic information.     

Comparison of DNA histograms by standard flow cytometry and image cytometry on sections in Barrett's adenocarcinoma

Background  

The purpose of this study was to compare DNA histograms obtained by standard flow cytometry (FC) and high fidelity image cytometry on sections (ICS) in normal gastrointestinal mucosa and Barrett's adenocarcinoma (BAC).