Time course of leukocyte response and free radical release in an early reperfusion injury of the superior mesenteric artery

The sequence of changes in circulating immune cells and in free radical production was studied during the small intestine reperfusion. Rat small intestine ischemia/reperfusion was induced by a 45 min superior mesenteric artery occlusion followed by a 4-hour reperfusion. Samples of peripheral blood were collected every 20 min during reperfusion. While the number of polymorphonuclear leukocytes increased significantly both in the sham-operated controls and the experimental group (about 400 per cent at the end of reperfusion), a decrease in lymphocyte counts to 60 per cent was observed in the experimental group only. Although there were no changes in the counts of total T lymphocytes, a significant reduction in B cell counts was observed. Flow-cytometrical measurements showed no changes in the Tc subpopulation, while the Th subpopulation increased in the experimental group only. Free radical generation in blood (luminometric measurements) increased gradually and reached an eight-fold level by the end of reperfusion in both groups. Thus, it has been shown that the increase in free radical production is mainly due to the increased number of polymorphonuclear leukocytes mobilized already at the initial stages of reperfusion. The reduction in B lymphocyte population is probably due to homing mechanisms     

Diminution of "reperfusion injury" in reperfused ischemic myocardium by phenothiazines. A quantitative morphological study

The effect of pretreatment by phenothiazines--Chlorpromazine (CPR) /Spofa/ and Trifluoperazine (TFP) /Smith Kline and French/ on reperfusion injury of ischemic myocardium were studied. Reperfusion of ischemic myocardium following an ischemic period exceeding 40 min resulted in morphological, physiological and biochemical changes identical with those induced by enhanced cytosolic Ca2+ concentration. Left descending coronary ligation was performed on 70 dogs divided into four group. Group I: permanent occlusion (5 dogs--60 min, 5 dogs--120 min, 5 dogs--180 min); group II: 15 dogs (60 min occlusion + 120 min reperfusion); group III: 20 dogs (60 min occlusion, 15 mg CPR, reperfusion 120 min); group IV: 20 dogs (60 min occlusion, 2 mg TFP + 120 min reperfusion). CPR or TFP were administered 30 min after the ligation. The effect of drugs was quantified on tetrazolium stained gross sections and studied from physiological, biochemical and ultrastructural points of view. Treatment of animals with phenothiazines, known as calmodulin inhibitors, considerably improved the ultrastructure of myocytes in area at risk, and allowed for the recovery of at least 60 per cent of injured myocytes after reflow restoration. Ultrastructural findings tightly correlate with physiological and biochemical results.     

Necrosis and apoptosis: sequence of liver damage following reperfusion after 60 min ischemia in rats

This study evaluated the time-dependent modes of cell death that occur during the course of reperfusion after 60 min ischemia. The serum ALT level increased immediately after reperfusion, peaked at 6 h and then declined gradually thereafter. This was supported by the H&E staining of the liver tissues taken at 2 h reperfusion, which revealed massive peri-portal necrosis. The succinate driven mitochondrial-swelling rate, release of cytochrome c into the cytoplasm, increase in caspase-3 activity and TUNEL stained tissue were measured to determine the changes in the biochemical markers of apoptosis. The biochemical markers of apoptosis increased by 2 h of reperfusion, peaked at 6 h and remained elevated throughout the 24 h reperfusion period. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), inhibited MPT opening, the release of cytochrome c and caspase-3 activation. This indicates that necrotic death occurs particularly in the peri-portal region in the initial period of reperfusion, and delayed apoptotic death occurs primarily in the peri-central region in the liver tissues undergoing I/R.     

Leukocyte Mobilization, Chemiluminescence Response, and Antioxidative Capacity of the Blood in Intestinal Ischemia and Reperfusion

Intestinal ischemia and reperfusion elicits changes in leukocyte counts and increased production of reactive oxygen species (ROS). The purpose of this study was to investigate whether these changes were followed by and/or connected with changes in the extracellular antioxidative capacity in a rat superior mesenteric artery (SMA) occlusion/reperfusion model. The SMA was occluded for 45 min and then allowed to be reper-fused. Changes of leukocyte, polymorphonuclear (PMN), and lymphocyte counts, chemiluminescence (CL) of whole blood samples as a marker of ROS production, and the total antioxidative capacity of the serum were quantified at the end of ischemia and in 1 h intervals during the postischemic period up to 4 h. The myeloperoxidase (MPO) activity in the serum and intestinal tissue samples was also determined. The MPO activity in the intestinal tissue samples was significantly elevated at the end of ischemia, and this elevation lasted for the whole postischemic period. The oxidative challenge to the body induced a fast mobilization of extracellular antioxidative mechanisms already at the end of ischemia, which was followed by a significant increase in PMN counts and whole blood CL starting at the 2nd hour after reperfusion. The increased CL activity of whole blood was attributed to the increase of the circulating PMNs. No significant changes were observed in leukocyte and lymphocyte counts. It is concluded that compensatory mechanisms of the oxidative-antioxidative balance of the body react very quickly if challenged.     

Temperature-Dependent Transformation of Cells Infected with a Mutant of Bryan Rous Sarcoma Virus

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.     

Oxygen free radical-induced damage during colonic ischemia/reperfusion in rats

Reperfusion injury following ischemia is thought to be the consequence of reactive oxygen species. Role of these free radicals on the damaging effects of ischemia in colon has been investigated. A rat experimental model was used in which colon was subjected to ischemia and reperfusion and mucosal damage was assessed by biochemical and histological studies. Activity of myeloperoxidase, a neutrophil marker, was increased after ischemia (I) and ischemia/Reperfusion (I/R). Lipid peroxidation products such as malonaldehyde and conjugated diene did not show any change in the experimental colonic mucosa as compared to control. Mucosal level of low molecular weight thiols were found to be altered after I/R. A decrease in -tocopherol level was noticed after ischemia and the decrease was prominent after reperfusion. Histology indicated morphological changes in colon due to ischemia and reperfusion and the damage was more severe after reperfusion. These results suggest that colonic mucosal damage occurs during I/R and free radicals generated by the infiltrated neutrophils may play a role in this damaging process.     

核基质与细胞凋亡

阐述了凋亡过程中,核基质所发生的形态、生化变化及相关凋亡基因的表达,尤其是凋亡早期便出现核基质蛋白的降解.核基质是细胞核最基本的组分,对维持细胞核形态结构和功能非常重要,其主要由核纤层,核内骨架及核孔复合体构成,在DNA复制、转录、RNA加工转运等事件中起支持作用.多少年来,关于凋亡时细胞核形态及生化改变的分子机理一直未阐明,最近对核基质与细胞凋亡的研究取得了重大进展.     

Myocardial ischemia and reperfusion injury in rats: lysosomal hydrolases and matrix metalloproteinases mediated cellular damage

The aim of this study was to evaluate the time course events of cellular damage during myocardial ischemia and reperfusion injury in rats and to find out a correlation between the structural alterations with respect to the biochemical changes. Cardiac biomarkers and lysosomal enzymes viz. cathepsin D, acid phosphatase and β-glucuronidase and matrix metalloproteinases (MMPs) were evaluated at different time points, in response to ischemia-reperfusion induced oxidative stress in an isolated rat heart model perfused in Langendorff mode. Microscopically, changes in myocardial architecture, myofibrillar degradation, and collagen (COL) integrity were studied using hematoxylin-eosin, Masson’s trichrome and toluidine blue staining techniques. A three-fold increase in the level of myoglobin was observed after 30 min of ischemia followed by 120 min of reperfusion as compared to 15 min ischemia, 120 min reperfusion. Similarly, a significant increase (P < 0.05) in the levels of lipid peroxides and superoxide anion coupled with a decrease in enzymatic and nonenzymatic antioxidant levels were observed. A concomitant increase in the activity of cathepsin D (24.07 ± 0.95) and a higher expression of MMPs after 120 min of reperfusion following 30 min ischemia were shown to correlate with the myocardial damage as shown by histopathology, suggesting that free radical induced activation of cathepsin D and MMPs could mediate early damage during myocardial ischemia and reperfusion.     

Cell-based high-content screening of small-molecule libraries

Advanced microscopy and the corresponding image analysis have been developed in recent years into a powerful tool for studying molecular and morphological events in cells and tissues. Cell-based high-content screening (HCS) is an upcoming methodology for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. Multiparametric characterization of responses to such changes can be analyzed using intact live cells as reporter. These disturbances are screened for effects on a variety of molecular and cellular targets, including subcellular localization and redistribution of proteins. In contrast to biochemical screening, they detect the responses within the context of the intercellular structural and functional networks of normal and diseased cells, respectively. As cell-based HCS of small-molecule libraries is applied to identify and characterize new therapeutic lead compounds, large pharmaceutical companies are major drivers of the technology and have already shown image-based screens using more than 100,000 compounds.     

Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.     

Process of apoptosis induced by TNF-α in murine fibroblast Ltk-cells: Continuous observation with video enhanced contrast microscopy

Apoptosis is originally defined by unique morphological changes of dying cells, and the biochemical hallmark associated with apoptosis is internucleosomal DNA fragmentation. However, few report has shown the precise time course of the apoptotic events. The present study was designed to try to clarify apoptotic processes using a video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy. The morphological changes of murine fibroblast Ltk-cells treated with TNF- were divided into four stages: (i) pre-apoptotic, (ii) cytoplasmic shrinkage, (iii) membrane blebbing, and (iv) ballooning. Almost of the cells underwent cytoplasmic shrinkage and membrane blebbing within 6 hours after TNF- exposure, and at about 9 hours, they were in the ballooning stage. Based on these data, we investigated the relationship between morphological changes and other biochemical features. The earliest event was exposure of phosphatidyl-serine at the cytoplasmic membrane, which was already observed in the pre-apoptotic stage. Loss of mitochondrial membrane potential was observed in the cytoplasmic shrinkage stage. Caspase-8/-3 activities already started increasing in the pre-apoptotic stage, and reached their peak at 6 hours after TNF- exposure. DNA fragmentation occurred in the late phase of the membrane blebbing.     

SECRETION OF α-AMYLASE BY THE EMBRYONIC CHICK PANCREAS IN VITRO

Pancreases taken from chick embryos secrete amylase in vitro when stimulated by cholinergic drugs. Rates of secretion increase with developmental age. The pancreas isolated together with the duodenal loop from the 8 day embryo is already capable of secretion in vitro. It is therefore concluded that the pancreas acquires the ability to secrete digestive enzymes more than 10 days before the beginning of the prominent biochemical and morphological changes associated with the maturation of the gland.     

Morphological alterations induced by doxorubicin on H9c2 myoblasts: nuclear,mitochondrial, and cytoskeletal targets

Doxorubicin (Dox) is a very potent antineoplastic agent used against several types of cancer, despite a cumulative cardiomyopathy that reduces the therapeutic index for treatment. H9c2 myoblast cells have been used as an in vitro model to study biochemical alterations induced by Dox treatment on cardiomyocyte cells. Despite the extensive work already published, few data are available regarding morphological alterations of H9c2 cells during Dox treatment. The purpose of the present work was to evaluate Dox-induced morphological alterations in H9c2 myoblasts, focusing especially on the nuclei, mitochondria, and structural fibrous proteins. Treatment of H9c2 cell with low concentrations of Dox causes alterations in fibrous structural proteins including the nuclear lamina and sarcomeric cardiac myosin, as well as mitochondrial depolarization and fragmentation, membrane blebbing with cell shape changes, and phosphatidylserine externalization. For higher Dox concentrations, more profound alterations are evident, including nuclear swelling with disruption of nuclear membrane structure, mitochondrial swelling, and extensive cytoplasm vacuolization. The results obtained indicate that Dox causes morphological alterations in mitochondrial, nuclear, and fibrous protein structures in H9c2 cells, which are dependent on the drug concentration. Data obtained with the present study allow for a better characterization of the effects of Dox on H9c2 myoblasts, used as a model to study Dox-induced cardiotoxicity. The results obtained also provide new and previously unknown targets that can contribute to understand the mechanisms involved in the cardiotoxicity of Dox.     

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.     

Modification of response of mouse neuroblastoma cells in culture by serum type.

The effect of various types of serum on morphological and biochemical changes in mouse neuroblastoma cells (clone NBP2) in culture was studied. The extent of spontaneous morphological differentiation varied markedly depending upon the type of serum and was maximal in agammaglobulin calf serum (CS). The extent of morphological differentiation after treatment of cells with cyclic AMP-stimulating agents was also dependent upon serum type and was least pronounced in fetal calf serum. The doubling time and extent of clumping varied with the type of serum. The activity of tyrosine hydroxylase (TH) in NB cells was dependent upon serum type and it was highest in newborn CS and agammaglobulin CS. Although elevation of intracellular levels of cyclic AMP in NBP2 clone invariably stimulates neurite formation and TH activity, these functions were increased in certain sera without a significant increase in the cellular cyclic AMP levels. The present study shows that neurite formation, growth rate and TH activity are regulated by more than one mode, one of which is mediated by cyclic AMP. The above changes are independently regulated in the sense that the expression of one can be increased in the absence of others.     

牛磺酸对大鼠肢体缺血/再灌注后肺组织损伤的保护作用

目的:观察大鼠肢体缺血再灌注(LIR)后肺组织形态学的变化及牛磺酸对其影响.方法:Wistar大鼠随机分为3组,对照组(control)、缺血/再灌注组(LIR)、牛磺酸 缺血/再灌注组(Tau LIR),各组动物通过大体、光镜和透射电镜观察肺组织形态学变化,并测定肺系数和肺通透指数及肺组织活性氧和MDA含量.结果:大鼠LIR后肺组织出现以肺泡毛细血管膜通透性增加为特征的组织细胞损伤,光镜下显示毛细血管扩张充血、血管周围间隙增大、肺泡腔中有大量蛋白渗出物,电镜下可见肺泡上皮细胞之间、毛细血管内皮之间的紧密连接松解;肺系数和肺通透指数升高;肺组织活性氧及MDA含量增加.提前给予外源性牛磺酸可使肺组织损伤变化减轻.结论:牛磺酸对大鼠LIR后肺损伤有保护作用,其保护机理之一与其抗氧化,保护细胞之间的紧密连接有关.     

Chromosome structure dynamics during the cell cycle: a structure to fit every phase

Chromosomes undergo dramatic morphological changes as cells advance through the cell cycle. Using powerful molecular and computational methods, several recent studies revealed an outstanding complexity of continuous structural changes accompanying cell cycle progression. In agreement with cell division being a fundamental cellular process, characteristic features of cell cycle stage‐specific genome structure are conserved from yeast to mouse. These studies further shine light on the critical roles that SMC complexes, already well known as fundamental regulators of chromosome topology, have in orchestrating structural dynamics throughout the cell cycle.     

Directional sensitivity of an identified wind-sensitive interneuron during the postembryonic development of the locust

Simultaneous extracellular recordings from both locust abdominal connectives show a differential activation of both bilateral homologues of an identified long projection interneuron (A4I1) in response to wind stimuli from different directions. Despite the previously shown extensive structural dynamics of sensory afferents and synaptic rearrangement of the direct afferent-to-interneuron connections during postembryonic development, a directional sensitivity is already present in first instar nymphs. Only quantitative changes in the strength of the directional response can be detected. Intracellular stainings of the A4I1 interneuron in first instar nymphs and adults show that general morphological features do not change during postembryonic development, in contrast to the presynaptic sensory afferents. This also holds for general morphological features of pleuroaxillary flight motoneurons. The output connections of A4I1 to these motoneurons and an unidentified intersegmental interneuron are already present in flightless nymphs.     

Bronchial circulation in pulmonary artery occlusion and reperfusion

Obstruction of pulmonary arterial blood flow results in minimal biochemical and/or morphological changes in the involved lung. If the lung is reperfused, a syndrome of leukopenia and lung edema occurs. We used the radiolabeled microsphere technique to measure the response of the bronchial circulation in rabbits to acute pulmonary artery occlusion (PAO) and to pulmonary artery reperfusion. We found that the bronchial blood flow (Qbr) decreased from a base line of 0.37 +/- 0.10 to 0.09 +/- 0.04 (SE) ml.min-1.g dry lung-1 (P less than or equal to 0.05) after 4 h of PAO. In a separate group of animals, Qbr 24 h after PAO remained low (0.20 +/- 0.07 ml.min-1.g dry lung-1, P = 0.06). Qbr during PAO was inversely correlated with the wet-to-dry ratio after reperfusion (r = -0.68, P = 0.06). Qbr did not change during 4 h of reperfusion. We speculate that a critical level of Qbr may be necessary during PAO to prevent ischemia/reperfusion injury from occurring.     

大蒜素对全脑缺血再灌注大鼠脑保护机制的研究

目的：通过大蒜素预处理,观察全脑缺血再灌注大鼠海马区ICAM-1 的表达,从而探讨大蒜素的脑保护机制。方法：雄性 Wistar 大鼠30 只,随机分为5 组：假手术组、缺血再灌注组、缺血再灌注+ 大蒜素10、20、30 mg/kg 组。采用四血管闭塞法制备大 鼠全脑缺血再灌注模型,于再灌注24 h 取出海马,硫堇染色观察海马组织的形态学改变,免疫组织化学染色测定海马CA1 区 ICAM-1 免疫反应阳性细胞面积和积分光密度值。结果：通过给予大鼠全脑缺血8 min 再灌注24 h处理,海马CA1 区组织形态学 改变显著,神经元密度明显降低;ICAM-1的表达显著增加。静脉给予大蒜素可使缺血再灌注海马组织形态学改变明显改善,存活 神经元数目增加,ICAM-1 表达显著较少。结论：大蒜素可以通过减少ICAM-1 的表达抑制全脑缺血再灌注后的炎症损失从而发 挥脑保护作用。