Enduring changes in Purkinje cell electrophysiology following transient exposure to AMPA: correlates to dark cell degeneration

Purkinje cells (PCs) are selectively vulnerable to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-mediated delayed toxicity that is manifested as dark cell degeneration (DCD) rather than necrosis. The purpose of the present study was to utilize electrophysiologic changes induced by AMPA to gain mechanistic insights into its cytotoxic actions. The whole-cell configuration of the patch clamp technique was used to record spontaneous electrical activity and ionic currents of Purkinje neurons from cerebellar slices using an experimental paradigm known to produce DCD in response to AMPA. Initial electrophysiologic responses to AMPA consisted of a large transient depolarization and inward current that declined by 75% 20 min into the 30-min exposure to 30 microM AMPA. Cellular responses temporarily continued towards basal levels following removal of AMPA. A sustained membrane depolarization (and underlying persistent inward current), an abundance of apparent excitatory synaptic events, and loss of electro- and chemoresponsiveness were observed 60-75 min into the expression phase (following AMPA removal). These events correspond temporally to the development of DCD in Purkinje cells and may represent an electrophysiological signature of AMPA receptor-mediated delayed neurotoxic events. Antagonists of the AMPA receptor present concomitantly with AMPA are known not to affect DCD and failed to alter the electrophysiologic changes. The secondary depolarization and loss of electroresponsiveness were prevented by antagonists present after removal of AMPA, at a time when DCD also is prevented. Electrical clamping of the PC membrane to equivalent depolarized membrane potentials (V(m)s) obtained with AMPA failed to elicit any long lasting alterations in PC physiology. Collectively, morphological and electrophysiological data indicate that induction of DCD is not strongly dependent on ionotropic mechanisms elicited by AMPA receptors, but that expression of DCD does possess an ionotropic element.     

Primary phagocytosis of viable neurons by microglia activated with LPS or Abeta is dependent on calreticulin/LRP phagocytic signalling

ABSTRACT: BACKGROUND: Microglia are resident brain macrophages that can phagocytose dead, dying or viable neurons, which may be beneficial or detrimental in inflammatory, ischaemic and neurodegenerative brain pathologies. Cell death caused by phagocytosis of an otherwise viable cell is called 'primary phagocytosis' or 'phagoptosis'. Calreticulin (CRT) exposure on the surface of cancer cells can promote their phagocytosis via LRP (low-density lipoprotein receptor-related protein) on macrophages, but it is not known whether this occurs with neurons and microglia. METHODS: We used primary cultures of cerebellar neurons, astrocytes and microglia to investigate the potential role of CRT/LRP phagocytic signalling in the phagocytosis of viable neurons by microglia stimulated with LPS (lipopolysaccharide) or nanomolar concentrations of amyloid-beta peptide1-42 (Abeta). Exposure of CRT on the neuronal surface was investigated using surface biotinylation and western blotting. A phagocytosis assay was also developed using BV2 and PC12 cell lines to investigate CRT/LRP signalling in microglial phagocytosis of apoptotic cells. RESULTS: We found that BV2 microglia readily phagocytosed apoptotic PC12 cells, but this was inhibited by a CRT blocking antibody or LRP-blocking protein (Receptor-Associated Protein: RAP). Activation of primary rat microglia with LPS or Abeta resulted in loss of co-cultured cerebellar granule neurons, and this was blocked by RAP or antibodies against CRT or against LRP, preventing all neuronal loss and death. CRT was present on the surface of viable neurons, and this exposure did not change in inflammatory conditions. CRT antibodies prevented microglia-induced neuronal loss when added to neurons, while LRP antibodies prevented neuronal loss when added to the microglia. Pre-binding of CRT to neurons promoted neuronal loss if activated microglia were added, but pre-binding of CRT to microglia or both cell types prevented microglia-induced neuronal loss. CONCLUSIONS: CRT exposure on the surface of viable or apoptotic neurons appears to be required for their phagocytosis via LRP receptors on activated microglia, but free CRT can block microglial phagocytosis of neurons by acting on microglia. Phagocytosis of CRT-exposing neurons by microglia can be a direct cause of neuronal death during inflammation, and might therefore contribute to neurodegeneration and be prevented by blocking the CRT/LRP pathway.     

Apoptotic bodies in the cerebellum of Japanese patients with Creutzfeldt-Jakob disease

The aim of this study was to provide morphological evidence for neuronal apoptosis in Creutzfeldt-Jakob disease (CJD) using hematoxylin-eosin (HE)-stained specimens. A microscopic examination showed typical apoptotic bodies were found in the granular layer of the cerebellum in 13 of 14 Japanese patients with CJD, while no apoptotic bodies were observed in any other areas of the examined CJD brains. Most of the fragmented nuclei of the apoptotic cells were labeled by in situ end-labeling of fragmented DNA, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling. To the authors' knowledge, this is the first report demonstrating neuronal apoptotic bodies in the human neurodegenerative disorders on HE-stained sections. The present findings indicate that apoptosis plays a major role in the neuronal loss of the cerebellar granule cells in CJD.     

Neuroprotection of microglia conditioned media from apoptotic death induced by staurosporine and glutamate in cultures of rat cerebellar granule cells

Microglia, the immune cells of the mammalian CNS, have often been indicated as dangerous effector cells for their activation in response to traumatic CNS injuries or immunological stimuli and for their involvement in many chronic neurodegenerative diseases. Recently, several in vitro and in vivo studies have emphasized that microglial activity is essential in promoting neuronal survival. We have tested the efficacy of media directly conditioned by microglia or conditioned by microglia after having been exposed to apoptotic neurons, towards neuroprotection of rat cerebellar granule cells (CGCs) challenged with staurosporine or glutamate. Apoptotic death of CGC caused by staurosporine, as well as by a mild excitotoxic stimulus delivered through sub-chronic glutamate treatment, was significantly counteracted by microglia conditioned media. On the other hand, an acute excitotoxic insult delivered through a short pulse of glutamate exposure in the absence of magnesium and resulting in a mix of apoptotic and necrotic death was only marginally counteracted by microglia conditioned media. The present results extend the available information regarding the neuroprotective role of microglia and support the usefulness of employing the culture approach for perspective identification of neuroprotective factors released by these cells. Furthermore, the use of media previously exposed to apoptotic neurons to elicit the neuroprotective response of microglia, indicate the feasibility to re-create also in the isolated culture conditions, at least some of the elements at the basis of neuron/microglia cross-talk.     

Morphological evidence of neutrophil-tumor cell phagocytosis (cannibalism) in human gastric adenocarcinomas

The phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis has been documented by light microscopy in various human carcinomas, but little is known about the cellular pathological processes and the morphological changes involved. In an attempt to clarify the nature of this phenomenon, the authors' ultrastructural studies on the relationships among neutrophils and tumor cells in human gastric carcinomas are reviewed and analyzed. At the electron microscopy level, apoptotic neutrophils were found within vacuoles of adenocarcinoma cells in 2 cases. They showed either early apoptotic morphology with perinuclear chromatin aggregation but cytoplasm integrity or late apoptotic morphology with uniform, collapsed nucleus and tightly packed cytoplasmic granules. A light microscopy review of 200 cases of resected gastric carcinomas identified 22 cases (11%) that were characterized by neutrophil-tumor cell phagocytosis (cannibalism). TUNEL staining confirmed the presence of apoptotic neutrophils within the cytoplasm of the tumor cells. This study provides light and electron microscopic evidence of apoptotic neutrophils phagocytosed by gastric adenocarcinoma cells. The morphological features of neutrophil-tumor cell phagocytosis (cannibalism) would suggest a particular mechanism of tumor-immune escape in human gastric carcinoma.     

Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate

In cerebellar granule cells a rapid necrotic cell death has been observed during and immediately after glutamate exposure, followed by a delayed apoptotic type of neuronal death in a subpopulation of the surviving neurons. In some experimental models the DNA fragmentation characteristic of apoptosis is readily detected. In other systems apoptosis may occur only in a limited number of cells, rendering DNA fragmentation undetectable using conventional DNA-staining techniques (e.g., ethidium bromide). We have used a sensitive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. This method is based on the introduction of thymine dimers into DNA after separation by pulse field gel electrophoresis, followed by detection with thymine dimer specific antibodies. Applying this method to cerebellar granule cells in culture, we detected an increase in the amount of HMW DNA fragments characteristic of apoptosis as early as 4 h after glutamate exposure. The N-methyl-D-aspartic acid (NMDA)-receptor antagonist MK801 protected against the fragmentation, whereas no protection was observed using the non-NMDA-receptor antagonist CNQX.     

Transmission electron microscopic analysis of malathion-induced cytotoxicity in granulosa cells of caprine antral follicles

Malathion, one of the most abundantly used organophosphate pesticides, has immoderate potency as a cytotoxic and genotoxic compound that induces toxicity in granulosa cells, resulting in its apoptosis. Thus, the present study aims to employ ultrastructural analysis for assessing the cytotoxicity of malathion at nanomolar concentrations (1 nM and 10 nM) in granulosa cells of caprine antral follicles at different exposure durations. Transmission electron microscopy revealed diminished cell–cell contact and cellular integrity, presence of crescent-shaped nucleus, chromatin condensation, and pyknosis with nuclear membrane folding, accumulation of lipid droplets with occurrence of cytoplasmic protrusions in granulosa cells treated with 1 nM malathion, whereas at 10 nM concentration, along with apoptotic attributes, prominent association of nucleus, endoplasmic reticulum, mitochondria and lipid droplets, nucleus invagination into lipid droplets, apical localization of lipid bodies, and occurrence of autophagic body were observed as compared to healthy granulosa cells in control with normal intact cellular integrity, well-developed cellular association, and doubled membrane nuclear lamina with homogenously dispersed chromatin surrounded by intact mitochondria with well-developed cristae. Thus, the results of ultrastructural analysis clearly suggest that nanomolar concentration of malathion induces apoptotic hallmarks within the granulosa cells of antral follicles that play a consequential role in increasing the incidence of follicular atresia, thereby affecting the overall fertility.     

NMDA-evoked excitotoxicity increases tissue transglutaminase in cerebellar granule cells

In neuronal cells, excessive activation of glutamate receptors causes excitotoxic damage culminating in apoptotic and necrotic cell death. The molecular mechanism of excitotoxicity has been associated with excessive Ca(2+) influx and overload, triggering biochemical events that lead to cell death and tissue degeneration. Following mild insults via NMDA-receptor activation, central neurons undergo several biochemical modifications recognizable as early events in apoptotic machinery.Tissue transglutaminase, the most ubiquitous among cell transglutaminases, catalyzes the Ca(2+)-dependent protein cross-linking probably associated with morphological changes in several neurodegenerative disorders. The possible involvement of this enzyme in excitotoxicity-mediated events was investigated in primary cultures of cerebellar granule cells exposed for 30 min to NMDA (100 microM) in Locke's buffer. Under these conditions time-dependent increases in transglutaminase activity were observed. Tissue transglutaminase expression reached the highest levels within 3-4 h of NMDA exposure. Similarly, high levels of incorporation of fluorescent substrates were observed in living cells. Confocal laser microscopy analysis showed that fluorescein-labelled structures were distributed within the cytoplasm and close to the membranes of NMDA-exposed cells.These effects were dependent on the Ca(2+) influx triggered by the excitotoxic stimulus. Morphological changes in NMDA-treated cells gave evidence of significant cell damage which appeared within 5-6 h of NMDA exposure.These results suggest that increases in tissue transglutaminase may be associated to the effects of NMDA-induced excitotoxicity. Therefore, it is reasonable to hypothesize that if tissue transglutaminase levels and activity are up-regulated under such conditions, the protein cross-linking could be likely involved in excitotoxic response.     

Morphological Evidence of Neutrophil-Tumor Cell Phagocytosis (Cannibalism) in Human Gastric Adenocarcinomas

The phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis has been documented by light microscopy in various human carcinomas, but little is known about the cellular pathological processes and the morphological changes involved. In an attempt to clarify the nature of this phenomenon, the authors' ultrastructural studies on the relationships among neutrophils and tumor cells in human gastric carcinomas are reviewed and analyzed. At the electron microscopy level, apoptotic neutrophils were found within vacuoles of adenocarcinoma cells in 2 cases. They showed either early apoptotic morphology with perinuclear chromatin aggregation but cytoplasm integrity or late apoptotic morphology with uniform, collapsed nucleus and tightly packed cytoplasmic granules. A light microscopy review of 200 cases of resected gastric carcinomas identified 22 cases (11%) that were characterized by neutrophil-tumor cell phagocytosis (cannibalism). TUNEL staining confirmed the presence of apoptotic neutrophils within the cytoplasm of the tumor cells. This study provides light and electron microscopic evidence of apoptotic neutrophils phagocytosed by gastric adenocarcinoma cells. The morphological features of neutrophil-tumor cell phagocytosis (cannibalism) would suggest a particular mechanism of tumor-immune escape in human gastric carcinoma.     

Granule neuron DNA damage following deafferentation in adult rats cerebellar cortex: a lesion model

Neuronal programmed cell death is regulated by a neurotrophic supply from targets and afferent inputs. The relative contribution of each component varies according to neuronal type and age. We have previously reported that primary cultures of cerebellar granule cells undergo apoptosis when deprived of depolarising KCl concentrations, suggesting a significant role of afferent inputs in the control of cerebellar granule cells survival. This issue was investigated by setting up various in vivo lesional paradigms in order to obtain partial or total deafferentation of the cerebellar granule layer in adult rats. At different times after surgery, cerebellar sections were subjected to TUNEL staining in order to detect possible DNA damage. One week after unilateral pedunculotomy, few scattered groups of apoptotic granule neurons were observed in the homolateral hemisphere. On the contrary, total deafferentation obtained by a new experimental paradigm based on an "L-cut" lesion induced massive and widespread apoptotic death in the granule layer of the deafferentated area. The time window of DNA fragmentation in granule layer was one to seven days after the "L-cut". Selective Purkinje cell deafferentation obtained by 3-acetylpyridine injection did not result in TUNEL staining in the cerebellar cortex. The current finding that mossy fiber axotomy induces granule cell apoptotic death points out for the first time the crucial role of afferent inputs in mature granule cell survival. Moreover, the in vivo lesional model described here may prove to be an useful tool for investigating cellular and molecular mechanisms of neuronal death triggered by deafferentation.     

Freeze-fracture organization of chromatin and cytoplasm in neurons and astroglia of rat cerebellar cortex

Summary The cytology of the cell nucleus and cytoplasm of neurons and astroglia of the rat cerebellar cortex has been investigated by freeze-fracture electron microscopy. The main differential characteristics in the cytoplasm of the several cell types of the cerebellar cortex were: (1) the organization of endoplasmic reticulum elements, including special configurations of lamellar bodies and hypolemmal complexes, (2) the polarity, extension and arrangement of Golgi cisterns and associated tubulovesicular elements; (3) the connection pattern among different membrane-bounded cellular compartments; and (4) the architecture of endomembranes (i.e. presence of pits and fenestrations). In the nucleus, the main differential features were the the three-dimensional view of the nuclear envelope, the distribution of nuclear pores and the aggregation pattern of chromatin, visualized as clusters of nuclear particles in cross-fractures. The quantitative analysis of chromatin revealed four peaks of nuclear particle sizes (8, 12, 17 and 21 nm) that may correspond to variable degrees of coiling of the polynucleosomal chain in the chromatin fibre. Significant differences were observed in the proportion, numerical density and size distribution of aggregated nuclear particles in heterochromatin domains among the different cell types of the cerebellar cortex. The percentage of nuclear particles in aggregates varied from 10% in Purkinje cells to 64% in granule cells. Astrocytes and Bergmann glia showed intermediate values (about 40%). The percentage of nuclear particles in aggregates snowed a significant (P < 0.05) negative linear correlation with the nuclear volume, the number of pores per unit nuclear volume and the total number of pores per nucleus. In granule cells and astroglia, heterochromatin domains had a greater percentage of large nuclear particles (>10 nm) than did euchromatin domains, whereas in interneurons, Purkinje and Golgi cells heterochromatin and euchromatin showed a similar proportion of large particles. Nuclear particles in euchromatin exhibited a similar pattern of distribution in all cerebellar cells.     

Apoptotic DNA binds to HLA class II molecules inhibiting antigen presentation and participating in the development of anti-inflammatory functional behavior of phagocytic macrophages

Resident macrophages are mainly responsible for the clearance of apoptotic cells from tissue by phagocytosis. Phagocytosis of apoptotic cells is not accompanied by activation of inflammatory mechanisms, unlike what happens when necrotic phenomena occur. We analyzed the effect of phagocytosis of apoptotic bodies on macrophage cell functions. After phagocytosis of apoptotic cells macrophages were unable to present an exogenous antigen to autologous antigen-specific T-cell lines. The inhibition was mediated by different mechanisms including binding of apoptotic DNA to human leukocyte antigen (HLA) class II molecules of macrophages, decreased expression of co-stimulatory molecules and increased secretion of tumor growth factor beta (TGFbeta). When dendritic cells were cultured with macrophages phagocytosing apoptotic cells, or with their supernatant, impaired dendritic cell antigen presenting activity and reduced tumor necrosis factor alpha (TNFalpha) secretion were found. Our results suggest that: (1) the phagocytosis of apoptotic bodies inhibits macrophage antigen presentation; (2) such inhibition is mediated by the binding of apoptotic DNA to macrophage HLA class II molecules as well as by the activation of biological mechanisms that induce an anti-inflammatory functional behavior in macrophages; and (3) macrophages phagocytosing apoptotic cells inhibit antigen presentation of neighboring dendritic cells via TGFbeta secretion. These events are likely related to the preservation of healthy tissues from the onset of inflammation.     

Acute effect of ethanol on firing patterns of Purkinje cells in the rat cerebellar slice preparation

This study examined the acute effects of ethanol (EtOH) on the firing patterns of Purkinje cells (PCs) using an intracellular recording in slice preparation of rat cerebellum. The experiments were performed in sagittal cerebellar slices (400 micrometer) of adult Sprague-Dawley rats (80-100g). Ethanol was applied by a bath superfusion with a known concentration expressed as the percentage of solution by volume (v/v) at 0.1, 0.5, 1, 2, and 4%. The result of the Chi-square test illustrated that the firing patterns were altered significantly after EtOH (p=0.007). However, the firing patterns that were altered by EtOH application were not affected by EtOH concentration (p= 0.1296). Among the 54 PCs tested, 30 PCs did not display any spontaneous firing activity and 24 PCs displayed spontaneous spike activity, either spiking in the simple manner (n=14) or cyclicly oscillating (n=10). In the presence of EtOH, 31 PCs were quiet, 22 PCs exhibited simple spiking activity and 1 PC continued to oscillate. Most PCs that displayed spontaneous activity before EtOH application progressively slowed their spike activity after EtOH superfusion. Especially, it was evident that 9 out of 10 oscillating PCs stopped their regular cyclic activity. In addition, 9 out of 14 PCs that displayed simple spike activity ceased to fire after EtOH application. Eleven out of 30 quiet PCs began to fire irregularly after EtOH application and this phenomenon usually occurred with membrane depolarization. EtOH induced spontaneous activity in 36.7% (11/30) of the quiescent PCs. In conclusion, there was differential EtOH sensitivity in the vitro slice preparation. EtOH depressed the endogenously generated spontaneous activity, especially the oscillatory firing activity. In contrast, the silent PCs were excited after EtOH application. Since this differential sensitivity persists in the presence of tetrodotoxin (TTX), it is suggested that this differential sensitivity is peculiar to the PCs.     

Granule neuron DNA damage following deafferentation in adult rats cerebellar cortex: a lesion model

Neuronal programmed cell death is regulated by a neurotrophic supply from targets and afferent inputs. The relative contribution of each component varies according to neuronal type and age. We have previously reported that primary cultures of cerebellar granule cells undergo apoptosis when deprived of depolarising KCl concentrations, suggesting a significant role of afferent inputs in the control of cerebellar granule cells survival. This issue was investigated by setting up various in vivo lesional paradigms in order to obtain partial or total deafferentation of the cerebellar granule layer in adult rats. At different times after surgery, cerebellar sections were subjected to TUNEL staining in order to detect possible DNA damage. One week after unilateral pedunculotomy, few scattered groups of apoptotic granule neurons were observed in the homolateral hemisphere. On the contrary, total deafferentation obtained by a new experimental paradigm based on an “L-cut” lesion induced massive and widespread apoptotic death in the granule layer of the deafferentated area. The time window of DNA fragmentation in granule layer was one to seven days after the “L-cut”. Selective Purkinje cell deafferentation obtained by 3-acetylpyridine injection did not result in TUNEL staining in the cerebellar cortex.The current finding that mossy fiber axotomy induces granule cell apoptotic death points out for the first time the crucial role of afferent inputs in mature granule cell survival. Moreover, the in vivo lesional model described here may prove to be an useful tool for investigating cellular and molecular mechanisms of neuronal death triggered by deafferentation.     

'LE cells' result from phagocytosis of apoptotic bodies induced by antinuclear antibodies

Lupus erythematosus (LE) cells are believed to represent phagocytosis by granulocytes of cell nuclei whose DNA has been 'depolymerized' and opsonized by serum factors, most likely antinuclear antibodies and C3b. Since it is known that certain antinuclear antibodies are capable of inducing apoptosis after intracellular penetration; and that the resulting apoptotic bodies can be ingested by non-professional phagocytes, we decided to investigate the possibility that LE cells could result from the phagocytosis of apoptotic bodies induced by antinuclear antibodies. We demonstrate herein, through different methodological approaches, that the ingested material within LE cells corresponds to apoptotic bodies, and that the LE cell phenomenon can be reproduced, in the absence of other serum factors, by penetrating murine monoclonal anti DNA antibodies.     

外源性谷氨酸致新生鼠脑兴奋毒性神经变性的形态学研究

目的 :探讨外源性谷氨酸对新生鼠脑细胞的兴奋毒性作用。方法 :新生 7天SD大鼠 ,背部皮下注射单钠谷氨酸 (MSG) 2mg/ g ,在 2h、6～ 8h、2 4～ 2 8h分别光镜和电镜观察脑细胞的形态改变。结果 :光镜下 ,2h未见明显异常 ,此后可见多处脑细胞肿胀 ,进而细胞固缩。电镜着重观察海马神经元 ,变化一为神经元胞体和树突巨大膨胀 ,伴随着内质网膜的空泡化及线粒体由最早期的浓缩到巨大膨胀 ,轴突基本正常 ,核染色质由簇状集聚于核膜下呈钟面排列到向中央积聚成轮廓不规则的团块 ;变化二为胞浆和胞核均浓缩 ,胞奖中有大小不等的空泡。结论 :外源性谷氨酸能引起未成熟脑细胞的死亡 ,为某些食品添加剂对儿童脑细胞的影响的相关研究提供一定的形态学基础。     

Effect of histone deacetylase inhibitors on the cell nucleus and nucleolus of leukemic myeloblasts in vitro - a cytochemical study

The present study was designed to provide complementary information on the effects of histone deacetylase inhibitors (HDACi's) such as trichostatin A (TSA) and sodium valproate (VAP) on nuclei and nucleoli of leukemic myeloblasts represented by cultured Kasumi-1 cells. The number of apoptotic cells and bodies with characteristic chromatin condensation and fragmentation was greater after TSA treatment. However, in contrast to TSA, myeloblasts treated with VPA recovered and started to proliferate again. TSA-treated myeloblasts with a fine chromatin structure exhibited an intense phagocytosis of cell fragments. The decreased number and translocation of silver-stained proteins of nucleolus organiser regions (AgNORs) in large nucleoli of myeloblasts treated with HDACi's indicated that these cells entered apoptosis and/or ageing without preceding terminal maturation. The nucleolar asynchrony observed in an increased number of treated cells with both HDACi's studied here possibly represented myeloblasts resistant to such treatment. In conclusion, this study demonstrates that the chromatin structure and nucleoli visualised by simple cytochemical procedures provides useful information on the effects of HDACi's on myeloblasts and facilitated detection of these effects at the single cell level.     

葡萄糖及游离脂肪酸对人血管内皮细胞凋亡的影响

为了观察葡萄糖、游离脂肪酸（FFAs）对人血管内皮细胞凋亡的影响及葡萄糖与FFAs是否具有协同作用，将培养细胞随机分为五组进行干预：对照组；葡萄糖处理组；FFAs处理组；葡萄糖与FFAs联合作用组；渗透压对照组。通过电镜、DNA片段琼脂糖凝胶电泳、流式细胞仪检测细胞凋亡和细胞周期。结果：高糖、高棕榈酸组见到凋亡小体等典型凋亡改变；高糖和高FFAs使细胞阻滞在G0／G1期，凋亡峰和凋亡率明显增高（P〈0．05），并呈剂量一时间依赖性；两者联用凋亡率明显高于两者单独使用（P〈0．05）；低浓度FFAs组细胞凋亡率与对照组无显著差异（P〉0．05）。高糖和高FFAs诱导内皮细胞凋亡具有时间一效应、浓度一效应关系且具有协同作用，可能参与了糖尿病血管并发症的发病过程。     

Cell death in retinoblastoma: electron microscopic, immunohistochemical, and DNA fragmentation studies

Apparent cell loss by apoptosis occurs in carcinomatous tissue. To investigate cell death in retinoblastoma (Rb), ultrastructural examination, ApopTag staining, electrophoresis to detect apoptotic DNA fragmentation, and flow cytometric studies were performed. Immunostaining for the oncogenic products bcl-2 and p53 was also carried out. Relationships between the proliferation fraction (PF), apoptotic index (AI), and the distribution of bcl-2 and p53 were investigated according to the degree of histologic differentiation of Rb. Ultrastructurally, two patterns of cell death were seen. Necrotic cells exhibited vacuolation of cytoplasmic organelles with a marked lytic change in the karyoplasm and cytoplasm. In contrast, apoptotic cells were characterized by crescentic margination of chromatin, condensation of karyoplasm and cytoplasm, and fragmentation of the nucleus. Differentiated Rb had a low AI value (< 1%), whereas undifferentiated Rb had a high AI value (> 8%). The PF of undifferentiated RB (31%) was significantly higher than that of differentiated RB (14%). Analysis of DNA fragmentation using 3'-end labeling with terminal transferase indicated that undifferentiated Rb has increased DNA cleavage. The distribution of apoptotic bodies within Rb was inversely correlated with the expression of bcl-2. A majority of tumor cells of differentiated Rb were negative for p53, whereas 20-40% of tumor cells of undifferentiated Rb showed a positive reaction for p53. These findings suggest that the degree of susceptibility to apoptosis is closely related to PF, is inversely related to the degree of differentiation of Rb, and is protected by oncogene bcl-2.