Ultrastructure of coronary microvessels during heart reperfusion after prolonged ischemia under conditions of various forms of artificial hypothermia

It has been found that, in children with Roger’s disease corrected under conditions of two fundamentally different procedures of anesthetic management, myocardial reperfusion after cardiac arrest under artificial hypothermic circulation is accompanied with obstruction of more than 70% of coronary bed microvessels, one-third of them being blocked with hydropic endothelial cells or their cystiform fragments. It hampers or even totally prevents their functioning in the postoperative period. The content of necrotic cells increases, while the three “working” cell types demonstrate a decrease in myocropinocytotic transport. Circulatory arrest during perfusionless hypothermia and immersion reperfusion do not result in a dramatic change of general morphology of microvessels as compared to the control group maintaining the endothelial cell population unaltered. Ultrastructural organization of endothelial microvessels displayed no features of intracellular regeneration. However, a heterogenic response of the structures responsible for transendothelial transfer of macromolecules provides the basis for the recovery of endothelium structure and function, as a patient’s temperature reaches the standard value.     

The role of specific endothelial granules in changes of myocardium microvessel reperfusion at open-heart operations under hypothermia

For patients with congenital heart defects, a comparative analysis was carried out of specific endothelial granules and morphological properties of perfusion capacity of microvessels at various stages of reperfusion after prolonged circulatory arrest. At all stages of reperfusion, the quantity of specific granules in microvessel endothelium dramatically dropped, as compared with the control group. At the early stages of reperfusion, the level of granule exocytosis does not lead to any statistically significant changes in perfusion characteristics in the right atrium microvessels, as compared with the control group. Conversely, at the late stages of reperfusion, degranulation of endothelial cells is enhanced, and physiological mechanisms intended to remove the microvessels from circulation are replaced with pathological ones. These results suggest the existence of a threshold in exocytosis of specific endothelial granules, whose exceeding may switch on some pathological mechanisms in blood flow regulation.     

定量研究脑缺血及再灌注下微血管的通透性

利用示踪剂ＦＬＮａ在脑缺血及再灌注的动物模型上,通活体观察和测定血液、脑等脏 荧光强度,以及对软脑膜微血管荧光光图象的平滑处理与定量分析,研究软脑膜微血管的通透性,探讨脑缺血及再灌注对微血管通透性的影响及内在规律。实验结果表明：缺血、缺血及再灌注会引起微血管内皮细胞的损伤,导致微血管通透性增大,这种损伤一般发生在缺血或再灌注早期,早然各脏器微血管都受到损伤,但其荧光值不同,说明各脏器抗缺血与缺氧的     

Morphological characteristics of the microvasculature in healing myocardial infarcts.

Myocardial infarction (MI) is associated with an angiogenic response, critical for healing and cardiac repair. Using a canine model of myocardial ischemia and reperfusion, we examined the structural characteristics of the evolving microvasculature in healing MI. After 7 days of reperfusion, the infarcted territory was rich in capillaries and contained enlarged, pericyte-poor "mother vessels" and endothelial bridges. During scar maturation arteriolar density in the infarct increased, and a higher percentage of microvessels acquired a pericyte coat (60.4 +/- 6.94% after 28 days of reperfusion vs 30.17 +/- 3.65% after 7 days of reperfusion; p<0.05). The microvascular endothelium in the early stages of healing showed intense CD31/PECAM-1 and CD146/Mel-CAM immunoreactivity but weak staining with the Griffonia simplicifolia lectin I (GS-I). In contrast, after 28 days of reperfusion, most infarct microvessels demonstrated significant lectin binding. Our findings suggest that the infarct microvasculature undergoes a transition from an early phase of intense angiogenic activity to a maturation stage associated with pericyte recruitment and formation of a muscular coat. In addition, in the endothelium of infarct microvessels CD31 and CD146 expression appears to precede that of the specific sugar groups that bind the GS-I lectin. Understanding of the mechanisms underlying the formation and remodeling of the microvasculature after MI may be important in designing therapeutic interventions to optimize cardiac repair.     

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.     

Energy metabolism and mechanical recovery after cardioplegia in moderately hypertrophiedrats

It is well established that severe hypertrophy induces metabolic and structural changes in the heart which result in enhanced susceptibility to ischemic damage during cardioplegic arrest while much less is known about the effect of cardioplegic arrest on moderately hypertrophied hearts. The aim of this study was to elucidate the differences in myocardial high energy phosphate metabolism and in functional recovery after cardioplegic arrest and ischemia in mildly hypertrophied hearts, before any metabolic alterations could be shown under baseline conditions.Cardiac hypertrophy was induced in rats by constriction of the abdominal aorta resulting in 20% increase in heart weight/body weight ratio (hypertrophy group) while sham operated animals served as control. In both groups, isolated hearts were perfused under normoxic conditions for 40 min followed by infusion of St.Thomas' Hospital No. 1 cardioplegia and 90 min ischemia at 25øC with infusions of cardioplegia every 30 min. The changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were followed by³¹ P nuclear magnetic resonance (NMR) spectroscopy. Systolic and diastolic function was assessed with an intraventricular balloon before and after ischemia.Baseline concentrations of PCr, ATP and Pi as well as coronary flow and cardiac function were not different between the two groups. However, after cardioplegic arrest PCr concentration increased to 61.8 ± 4.9 mol/g dry wt in the control group and to 46.3 ± 2.8 mol/g in hypertrophied hearts. Subsequently PCr, pH and ATP decreased gradually, concomitant with an accumulation of Pi in both groups. PCr was transiently restored during each infusion of cardioplegic solution while Pi decreased. PCr decreased faster after cardioplegic infusions in hypertrophied hearts. The most significant difference was observed during reperfusion: PCr recovered to its pre-ischemic levels within 2 min following restoration of coronary flow in the control group while similar recovery was observed after 4 min in the hypertrophied hearts. A greater deterioration of diastolic function was observed in hypertrophied hearts.Moderate hypertrophy, despite absence of metabolic changes under baseline conditions could lead to enhanced functional deterioration after cardioplegic arrest and ischemia. Impaired energy metabolism resulting in accelerated high energy phosphate depletion during ischemia and delayed recovery of energy equilibrium after cardioplegic arrest observed in hypertrophied hearts could be one of the underlying mechanisms.     

Fahraeus-effect-reversal (FER) in compaction stasis (CS): microrheological and haemodynamic consequences of intravascular sedimentation of red cell aggregates

Red cell aggregate sedimentation under gravitation produces pronounced and rapid "phase separation effects" culminating in "compaction stasis" (CS), i.e. almost complete stuffing of microvessels by RBC. This can be readily observed and monitored in microvessels of vertically placed mesentery preparations by a horizontally aimed intravital microscope as shown by (GOBEL et al. VIRCHOW's Arch., 1988,). "Layered flow", floatational plasma skimming and progressive increase in local tube hematocrit (HT) up to 100% ("compaction stasis") occurs during induced low flow states in vivo (here preferentially in postcapillary venules), as well as in vitro (non-permeable artificial micro tube networks). Quantitative densitometry and velocimetry in vertically placed microvessels demonstrates that the process of RCA sedimentation results in progressive vertical skewing of the velocity profile, culminating in standstill of the RBC-sediment in the dependent vessel half, with superfluent plasma and small aggregates in the upper vessel half. The theory of compaction stasis is developed: in striking contrast to the situation under high shear conditions, red cells travel on slower trajectories than plasma: due to "red cell undervelocity" the average residence times of RBC in a venule is much higher than that of plasma. Consequently, CS can be explained as the result of a FAHRAEUS effect reversal since the principle of mass conservation requires that HT much greater than HD. Network aspects and hemodynamic consequences are also incorporated into the theory.     

缺血再灌后血管内皮空泡的形成与内皮的损伤

目的：在活体上探讨缺血再灌后血灌内上细胞损伤及白细胞、血小板与内皮之间粘附的变化。方法：用失血及与再回输血液造成缺血再灌流模型,在高倍显微镜下观察肠系膜微血管内皮损伤及血细胞粘附的变化。结果：缺血再灌后1－3h细静脉、集合毛细血管内出现白细胞、血小板的粘附,血管内皮水肿、管壁增厚,有的血管内皮细胞的胞浆形成圆丘形的空泡,空泡从血管内皮突入管胺、空泡直径10－30μm多出现的细动脉内,在同一根血管内可同时出现几个空泡,大的空泡几科占据血管腔的2／3。结论：缺血再灌后血管内皮水肿及空泡形成,显示内皮细胞的严重损伤。     

Neovascularization in human atherosclerosis

In the absence of disease, microvessels provide vessel wall nutrients to the tunica media, while the intima is fed by oxygen diffusion from the lumen. As disease evolves and the tunica intima thickens, oxygen diffusion is impaired, and microvessels become the major source for nutrients to the vessel wall. Microvessels serve as a port of entry for inflammatory cells, from the systemic circulation to the nascent atherosclerotic lesion. As disease progress, microvessels also play a role in intraplaque hemorrhage, lipid core expansion, and plaque rupture. In addition, microvessels are also involved in stent restenosis, and plaque regression. Therefore, microvessels are a pivotal component of atherosclerosis, and proper patient risk-stratification in the near future may include the detection of increased neovascularization in atherosclerotic lesions. This review divided in two parts summarizes the current understanding of atherosclerosis neovascularization, starting with the normal anatomy and physiology and progressing to more advanced stages of the disease. We will review the structure and function of vasa vasorum in health and disease, the mechanisms responsible for the angiogenic process, the role of the immune system, including inflammation and Toll-like receptors, and the pathology of microvessels in early atherosclerotic plaques. Furthermore, the review addresses the advanced stages of atherosclerosis, summarizing the progressive role for microvessels during disease progression, red blood cell extravasation, lipid core expansion, plaque rupture, healing, repair, restenosis, and disease regression, offering the clinician a state-of-the-art, "bench to bedside" approach to neovascularization in human atherosclerosis.     

Fas-Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation-arrested testes

To elucidate the mechanism of maturation arrest, known as one of the male infertility, we addressed whether germ cell apoptosis occurs during maturation arrest, and if so, whether Fas and Fas ligand expressions are involved in the apoptosis. By electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), typical apoptotic features were frequently found around the spermatocytic stage in maturation arrest, compared to that in normal testes. When paraffin-embedded sections reacted with anti-Fas antiserum, staining for Fas was found in the plasma membranes of spermatocytes in the maturation-arrested testes, while no positive spermatogenic cells were seen in the normal testes. On the other hand, positive immunostaining for Fas ligand was restricted to Sertoli cells in the maturation-arrested testes as well as in the normal testes, although the intensity of staining for Fas ligand in normal testicular Sertoli cells was much weaker than that of maturation-arrested ones. Thus, these findings demonstrate that "maturation arrest" is characterized by frequent apoptosis of spermatocytes, and that Fas and Fas ligand staining are associated with a high frequency of apoptosis.     

In Vitro Recapitulation of Functional Microvessels for the Study of Endothelial Shear Response,Nitric Oxide and [Ca2+]i

Microfluidic technologies enable in vitro studies to closely simulate in vivo microvessel environment with complexity. Such method overcomes certain constrains of the statically cultured endothelial monolayers and enables the cells grow under physiological range of shear flow with geometry similar to microvessels in vivo. However, there are still existing knowledge gaps and lack of convincing evidence to demonstrate and quantify key biological features of the microfluidic microvessels. In this paper, using advanced micromanufacturing and microfluidic technologies, we presented an engineered microvessel model that mimicked the dimensions and network structures of in vivo microvessels with a long-term and continuous perfusion capability, as well as high-resolution and real-time imaging capability. Through direct comparisons with studies conducted in intact microvessels, our results demonstrated that the cultured microvessels formed under perfused conditions recapitulated certain key features of the microvessels in vivo. In particular, primary human umbilical vein endothelial cells were successfully cultured the entire inner surfaces of the microchannel network with well-developed junctions indicated by VE-cadherin staining. The morphological and proliferative responses of endothelial cells to shear stresses were quantified under different flow conditions which was simulated with three-dimensional shear dependent numerical flow model. Furthermore, we successfully measured agonist-induced changes in intracellular Ca²⁺ concentration and nitric oxide production at individual endothelial cell levels using fluorescence imaging. The results were comparable to those derived from individually perfused intact venules. With in vivo validation of its functionalities, our microfluidic model demonstrates a great potential for biological applications and bridges the gaps between in vitro and in vivo microvascular research.     

"Ischemia/reperfusion" for stem cells of two "critical" cell renewal systems of organism

In this article is presented the result of the experiments on mice-hybrids F1(CBA x C57B1/6), which indicates the presence of the reaction of "ischemia/reperfusion" for stem cells of two "critical" cell renewal systems of organism (bone marrow and intestinal epithelium) during the irradiation under the conditions of hypoxic radioprotector application. The additional injection of the source of nitric oxide radicals-sodum nitroprusside (SNT) to the mice right after the irradiation under the conditions of hypoxic protection by serotonin, resulted the substantial increase of the survival rate of hematopoietic stem cells (registered by the methods of endogenous and exogenous colony forming in spleen) and stem cells of intestinal epithelium (registered by the method of intestinal "microcolonies"). The similar radioprotective effect was also registered during the test of survival rate of mice under tests of "bone marrow" and "intestinal" forms of radiation lethality that is evidence of the importance of the realization of phenomenon "ischemia/reperfusion" in the reaction of whole organism on the acute radiation injury. As SNP weakens the manifestation apoptosis and necrosis through competition with active forms of oxygen (AFO) during the period of "reperfusion" on basis of the found out phenomenon experimental model for studying mechanisms of stem cells damage in vivo induced by AFO and for the search of the modifiers weakening or strengthening such damage can be developed.     

Origin of periendothelial cells in microvessels derived from human microvascular endothelial cells

In microvessels, periendothelial cells expressing alpha smooth muscle actin (alphaSMA) interact with the endothelial cells and are essential for vessel maturation and stabilization. In adult tissues, the cellular origin of the periendothelial cells is still not clear, in particular in humans. To determine the origin of human periendothelial cells, we used a recently developed 3D co-culture system that mimics human skin connective tissue. This system is composed of normal human dermal fibroblasts (NHDF), human dermal microvascular endothelial cells (HMEC-1), and a collagen matrix. In this system, "microvessels" composed of an endothelial lumen associated with periendothelial cells develop. Using this co-culture system, we (i) labelled fibroblasts with the vital dye CFDA-SE, cultured them with unlabelled endothelial cells, and observed that only endothelium-associated CFDA-SE-labelled cells express alphaSMA; (ii) infected endothelial cells with a retrovirus stably expressing eGFP, cultured them with unlabelled fibroblasts, and observed that cells expressing alphaSMA did not co-express eGFP, but were associated with the eGFP-expressing endothelial cells of the microvessels. Together, these results indicate that periendothelial cells arise by differentiation from fibroblasts and that they require interaction with endothelial cells to do so.     

Effect of mannitol on blood recirculation in the brain after interruption of the blood flow

Premedication with mannitol before circulatory arrest not only promotes the reflow in the brain microvessels but also increases their diameters, thereby making more favourable conditions for blood supply of the brain after circulatory arrest. The data were obtained with the aid of automatic morphometry on microscopic slices of the dog brain perfused with Indian ink.     

A single cell model of myocardial reperfusion injury: Changes in intracellular Na+ and Ca2+ concentrations in guinea pig ventricular myocytes

To investigate the contribution of the changes in intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i) to myocardial reperfusion injury, we made an ischemia/reperfusion model in intact guinea pig myocytes. Myocardial ischemia was simulated by the perfusion of metabolic inhibitors (3.3 mM amobarbital and 5 M carbonyl cyanide m-chlorophenylhydrazone) with pH 6.6 and reperfusion was achieved by the washout of them with pH 7.4. [Na+]i increased from 7.9 ± 2.0 to 14.0 ± 3.4 mM (means ± S.E., p < 0.01) during 7.5 min of simulated ischemia (SI) and increased further to 18.8 ± 3.0 mM at 7.5 min after reperfusion. [Ca2+]i, expressed as the ratio of fluo 3 fluorescence intensity, increased to 133 ± 8% (p < 0.01) during SI and gradually returned to the control level after reperfusion. Intracellular pH decreased from 7.53 ± 0.04 to 6.31 ± 0.04 (p < 0.01) and recovered quickly after reperfusion. Reperfusion with the acidic solution or the continuous perfusion of hexamethylene amiloride (2 M) prevented the reperfusion-induced increase in [Na+]i. When the duration of SI was prolonged to 15 min, the cell response after reperfusion varied, 16 of 37 cells kept quiescent, 21 cells showed spontaneous Ca2+ waves, and 4 cells out of these 21 cells became hypercontracted. In quiescent cells, both [Na+]i and [Ca2+]i decreased immediately after reperfusion. In cells with Ca2+ waves, [Na+]i transiently increased further at the early phase of reperfusion, while [Ca+]i declined. In hypercontracted cells, [Na+]i increased as much as in Ca2+ wave cells, but [Ca2+]i increased extensively and both ion concentrations continued to increase. Reperfusion with the Ca2+-free solution prevented both the [Ca2+]i increase and morphological change. In the presence of ryanodine (10 M), the increase in [Ca2+]i after reperfusion was augmented and some cells became hypercontracted. We concluded that (1) Na+/H+ exchange is active both during SI and reperfusion, resulting in the additional [Na+]i elevation on reperfusion, (2) the [Na+]i level after reperfusion and the following Ca2+ influx via Na+/Ca2+ exchange are crucial for reperfusion cell injury, and (3) the Ca2+ buffering capacity of sarcoplasmic reticulum would also contribute to the Ca2+ regulation and cell injury after reperfusion.     

Isolation of morphologically and functionally intact gastric mucosal microvessels rapid communication.

Gastric mucosal microvessels were isolated after arterial perfusion of the rat stomach with magnetized iron oxide suspension. After homogenization of scrapped gastric mucosa, microvessels were initially separated with a high power magnet and further separated and purified by using a nylon sieve. Aliquots of purified microvessels were assessed for viability, histologic appearance, ultrastructure and generation of prostacyclin. Microvessels were plated on Matrigel and cultured in DMEM with high glucose and 10% FBS for 1, 3 or 5 days. After 1, 3 and 5 days of culturing, endothelial viability was assessed with Fast green exclusion, and the basal and stimulated (with calcium ionophore) generation of prostacyclin was determined by assaying aliquots of the incubating medium for 6-keto PGF(1alpha). At 1 and 3 hrs after isolation, microvessels demonstrated intact morphologic structures as reflected by transmission EM and 92+/-4% of viable endothelial cells. The microvessels plated on Matrigel maintained good viability for at least 5 days and generated prostacyclin at the baseline and following ionophore stimulation. These data demonstrate that isolated microvessels cultured under optimal conditions are fully viable and functional.     

Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro

Skeletal myoblasts grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated "reserve cells" that share several characteristics with muscle satellite cells. The mechanism of determination of reserve cells is poorly understood. We find that the expression level of the metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in reserve cells than in myotubes. Inhibition of ADAM12 expression in differentiating C2C12 cultures by small interfering RNA is accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin alpha7A isoform). Overexpression of ADAM12 in C2C12 cells under conditions that promote cell cycle progression leads to upregulation of p130 and p27, cell cycle arrest, and downregulation of MyoD. Thus, enhanced expression of ADAM12 induces a quiescence-like phenotype and does not stimulate differentiation. We also show that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity as well as the cytoplasmic domain of ADAM12 are required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain is not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of reserve cells during myoblast differentiation.     

Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.

When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.     

Effects of different oxidizing agents on neutral amino acid transport systems in isolated bovine brain microvessels

Using isolated bovine brain microvessels as an in vitro model of the blood-brain barrier (BBB) we have evaluated the role of free radical generating solutions on some amino acid transport systems operating on the endothelial cell membrane. Fe(2+)/ascorbate, phenylhydrazine and CuSO(4) did not affect any of the transport system tested, while exposure of bovine brain microvessels to tert-butylhydroperoxide (t-BHP) caused a reduced capacity to take up small neutral amino acids via the Na(+)-dependent A-system. The presence of glucose during t-BHP treatment did not prevent this inhibition, which was partially counteracted when the isolated microvessels were incubated with 5mM inosine before the oxidative stress. Incubation of the isolated capillaries with 5mM dithiothreitol, after exposure to t-BHP, resulted in a 50% recovery of the alpha-methylaminoisobutyrate (MeAIB) uptake by the A-system. Treatment with t-BHP, which had no effect on the L-system of neutral amino acid transport, caused a significant decrease of the intracellular levels of ATP, of glutathione (GSH), and of gamma-glutamyltranspeptidase (GGT) activity, while no significant modification of hexokinase (HK) or of alkaline phosphatase (ALKP) activities were observed. Oxidative damage of the BBB appears therefore to impair essentially the metabolic pathways which ensure the energy requirement for the endothelial cells, thus inhibiting the energy-dependent amino acid transport system "A".