脂质过氧化在肾缺血和缺血再灌注损伤中的变化

本实验观察大鼠肾缺血75min及缺血60min后再灌注15min时肾组织脂质过氧化(LPO)和有关酶类变化。结果显示,肾脏缺血和缺血/再灌注后肾皮质和髓质中脂质过氧化产物丙二醛(MDA)含量均显著增高。超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性无明显变化。缺血/再灌注后黄嘌呤氧化酶(XO)活性显著升高。上述结果提示,肾缺血和缺血/再灌注时的LPO增强与氧自由基(OFR)产生增多有关。但上述二种情况时OFR产生机制不尽相同。     

Ultrastructural correlates of ischaemic contracture during global subtotal ischaemia in the rat heart.

The development of left ventricular ischaemic contracture and its correlation with ultrastructural and sarcolemmal permeability defects were studied in isolated rat hearts during global subtotal ischaemia. With acetate as substrate the hearts exhibited a rise in diastolic tension after 8-10 min at which time small foci of contracted myocytes were scattered throughout the myocardium. In hearts with 5% of the maximum diastolic tension (termed 5% contracture), the foci were situated predominantly in the subendocardium and papillary muscle. Contracted myocytes in these foci were capable of excluding ionic lanthanum thus demonstrating retention of normal sarcolemmal permeability properties. With 30% contracture ultrastructural damage had spread to the subepicardium and with further contracture there was an associated increase in the number and size of foci in all regions. In these foci, swelling of the tubular sarcolemmal system and occasionally of the sarcoplasmic reticulum appeared to precede myofibrillar contraction. At 50% contracture lanthanum influx into contracted cells became more frequent. Hearts developed full contracture by 15-18 min at which time most myocytes were contracted and retained lanthanum intracellularly. The heterogeneity of the response at a cellular level may offer a possible explanation for the lack of correlation between contracture and tissue ATP. A possible sequence of structural injury leading to impaired calcium homeostasis is also suggested.     

Transmural changes in mast cell density in rat heart after infarct induction in vivo

The cardiac distribution of mast cells was investigated after the induction of acute myocardial infarction in the rat. The left anterior descending coronary artery (LAD) was occluded by ligation in the infarct group, whereas in sham rats only a superficial ligature was placed beside the LAD. Rats of both groups were killed at 4, 7, 14, 21, 35, and 85 days following surgery. Hearts were excised and formalin-fixed. Mast cell densities were monitored in subepicardial and subendocardial layers of the left ventricle (LV) in 6 μm thick toluidine blue-stained cross-sections. In control (non-operated) animals, mast cell densities were comparable in the LV subepicardial and subendocardial layers (1·5–2·0 cells per mm²). Following infarction, the mast cell density at the subepicardial site of the infarction gradually increased, reaching a maximum of 25 cells per mm² on day 21, while a non-significant increase was observed at the subendocardial site. In the non-infarcted regions, the mast cell density increased transiently to reach a maximum of 7 cells per mm² on day 35 in the subepicardial layer. Again, changes in mast cell density in the subendocardial layer were non-significant. In the sham group, a gradual increase to 9 cells per mm² on day 21 and a subsequent decrease to 5 cells per mm² on day 85 were observed in the subepicardial layers. These findings indicate a massive accumulation of mast cells in the subepicardial layers of the infarcted region and a small but significant effect of the surgical procedure on cardiac mast cell deposition, especially in the outer layers of the left ventricle.     

Diminished toxicity of ouabain in the hypertrophied rat heart

The responsiveness to ouabain of hypertrophied rat hearts has been investigated either in vivo using an isolated Langendorff rat heart perfused at various external calcium concentrations, or in vitro on purified sarcolemma vesicles. (i) The physiological study shows that at 0.25 mM CaCl₂, the positive inotropic effect of 10^–5 M ouabain was diminished in hypertrophied hearts (p<0.02). At 0.5 mM CaCl₂, the drug has no effect in controls, but it has a slight positive inotropic effect in hypertrophied hearts. At 2.50 mM CaCl₂, ouabain has a negative inotropic effect accompanied by extrasystoles in controls, but in hypertrophied hearts it still has a positive inotropic effect and is not arrhythmogenic. (ii) After the pretreatment of the hearts with 2.5 mM CaCl₂, the responsiveness of the (Na⁺, K⁺)-ATPase activity to ouabain was studied: the sarcolemma from hypertrophied heart contains half as many low affinity forms of (Na⁺, K⁺)-ATPase for ouabain (35%±6) than in controls (80%±2). Assuming that the low affinity forms are responsible for the toxic effect, these data correlate well with some of the physiological findings and suggest that the diminished toxicity for ouabain in hypertrophied hearts rather reflects a modification of the properties of the (Na⁺, K⁺)-ATPases than a change in the myocardial calcium metabolism.Supported by a grant from Caisse Nationale d'Assurances Maladie des Travailleurs Salariés (1986)     

Inotropic effect of ouabain in hypertrophied rat heart

The contribution of the heterogeneous digitalis receptors to the inotropic effect of ouabain was studied in hypertrophied rat hearts (aortic stenosis) by using isolated Langendorff heart preparations. Development and washout of the biological effects as well as the dose/ response curves revealed two inotropic components of high and low drug sensitivity. Maximal inotropy was observed with 100 M ouabain in both control and hypertrophied rat hearts. The high-sensitivity component accounted for only one-third of the response in control hearts but for two-thirds in hypertrophied hearts. The respective apparent affinities (10–20 nM and 10–20 M) of the two inotropic components found in isolated hearts were similar to those of the high- and low-affinity Na⁺,K⁺-ATPase activities detected in isolated cardiac sarcolemma. Onset and reversion of the pharmacological effects of ouabain occurred at respective rates that were similar to those of the association and dissociation of ouabain to the Na⁺,K⁺-ATPase level. In hypertrophied heart, the high- and low-sensitivity components (or receptors) reacted seven- and threefold, respectively, more slowly than the corresponding sites in normal hearts. These alterations in inotropic responsiveness and propertries of the digitalis receptors in cardiac hypertrophy suggest that new regulations should be taken into account in the adaptation to pressure overload.     

Transmural gradient of glycogen metabolism in the normal rat left ventricle

The changes of glycogen metabolism with the location of tissue within the ventricle wall have been explored in the rat myocardium. The hearts were cut in 100 microns thick serial sections and all sections were analyzed for their content in glycogen, glucose-6-phosphate, UDPG and glycogen enzymes and for glucose incorporation into glycogen and for the 2-deoxyglucose uptake after the intravenous injection of the 14C-labelled sugars. The rate of glycogen turnover was significantly higher in the subendocardial myocardium (P less than 0.01) and the levels of glucose-6-phosphate and the total (i.e. a + b) activity of glycogen phosphorylase were significantly higher in the subepicardial tissue (P less than 0.01 in both instances). No significant transmural gradient of UDPG was found and transmural changes of total (i.e. I + D) synthase activity were barely significant. These changes in glycogen metabolism may be related to regional differences in the cardiac work load and to a differentiation of the subendocardial and subepicardial heart fibers.     

Myocardial contracture and accumulation of mitochondrial calcium in ischemic rabbit heart

The relationship between myocardial contracture and cell calcium was studied in electrically paced, isolated perfused rabbit hearts. Isovolumic left ventricular dP/dt and end-diastolic pressure were utilized as indexes of contractility and ventricular stiffness. After 60 min of low flow (ischemia) without or with reperfusion at high flow for 10 min, calcium was measured in the mitochondrial fraction and used as an indicator of intracellular calcium. Low flow led to ventricular standstill and contracture, and reperfusion produced partial mechanical recovery with end-diastolic pressure remaining markedly elevated. Nifedipine (10(-7) M), an antagonist of myocardial calcium uptake, prevented contracture and permitted nearly complete mechanical recovery without elevation in diastolic pressure. Increases in mitochondrial calcium paralleled the severity of contracture and the lack of diastolic relaxation after reperfusion. Mitochondrial calcium did not increase in hearts protected by nifedipine. Results demonstrate a close relationship between mechanical changes induced by ischemia and accumulation of intracellular calcium.     

Passive electrical properties of normal and hypertrophied rat myocardium

We determined the electrical constants of epicardial and endocardial preparations from both normal and hypertrophied rat hearts. This was done by comparative analysis of the spatial decay of steady-state electronic voltage deflection produced by injection of a hyperpolarizing constant-current pulse. We used a two-dimensional finite disk model to obtain the apparent membrane resistance, (Rm)app, and internal longitudinal resistivity (Ri), (Rm)app was significantly larger in epicardial (565 +/- 222 omega . cm2) than endocardial (375 +/- 137) preparations from normal hearts. This regional difference disappeared in hypertrophied hearts (epicardium 421 +/- 138, endocardium 383 +/- 121 omega . cm2). Ri was similar for normal endocardial (272 +/- 169 omega . cm) and epicardial (326 +/- 152) preparations, as well as for hypertrophied endocardial (251 +/- 108) and epicardial (312 +/- 59) preparations. We determined the effective membrane capacity (Ceff) by measuring the ratio of applied charge to the displacement of membrane potential. Ceff was larger for normal hearts (epicardium 9.7 +/- 2.5 micro F/cm2, endocardium 7.5 +/- 3.0) than for hypertrophied hearts (epicardium 4.1 +/- 1.4, endocardium 4.7 +/- 1.2). From the values for Ceff we calculated the effective membrane resistance, (Rm)eff. (Rm)eff was larger for normal (epicardium 5,392 +/- 2,613 omega . cm2, endocardium 3,013 +/- 2,096) than for hypertrophied (epicardium 1,552 +/- 633, endocardium 1,838 +/- 826) preparations. Our results show that the amount of electrically effective membrane area is decreased in hypertrophied myocardium, despite the increased total area per hypertrophied cell. One functional implication of this finding is that activation of contraction by spread of surface electrical depolarization into the T-tubules may be impaired in hypertrophied cardiac muscle.     

Release of endothelin during reperfusion of ischemic myocardium. ET-1 release from reperfused heart.

Endothelin, the most potent endothelium-derived vasoconstrictor, was found to be released from the isolated ischemic rat heart after a prolonged period of reperfusion. No endothelin release was observed up to 1 hour of reperfusion following 30 minutes of global ischemic arrest. Although most of the endothelin release occurred as a burst after 1 hour of reperfusion, the endothelin release increased progressively at a slower rate up to 2 hours of reperfusion. The calcium slow channel blocker, nifedipine, and the phospholipase inhibitor, mepacrine, but not the free radical scavengers, superoxide dismutase (SOD) and catalase, prevented the endothelin release from the heart. Unlike endothelin release that did not occur until after 1 hour of reperfusion, lipid peroxidation assessed by thiobarbituric acid reactive product formation, and cellular injury as monitored by creatine kinase (CK) release, occurred upon reperfusion, and increased progressively with the duration of reperfusion. All the interventions including nifedipine, mepacrine, and SOD plus catalase, prevented the lipid peroxidation and CK release significantly. Coronary flow was reduced significantly after 1 hour of reperfusion, when endothelin release occurred, an event that also coincided with myocardial endothelial injury as assessed by transmission electron microscopy. These results indicate that endothelin is released only after a prolonged period of reperfusion, and the release process seems to be controlled by calcium and phospholipase, but not by free radicals.     

Metabolic and physical changes during calcium paradox induced in the rat heart.

Early changes in metabolic and physical properties were determined in rat hearts during calcium paradox. Calcium paradox was induced under constant perfusion pressure (60 mmHg) or constant coronary flow rate (9.8 ml/min). Within 30 s after calcium repletion, in either case, NADH increased, despite a decrease in ATP and increases in ADP and AMP. Surface spectrophotometry showed a deoxygenation of the myoglobin, thereby indicating myocardial oxygen depletion. These changes were predominant under conditions of constant pressure perfusion. In association with a rapid development of contracture, there were also a reduction in coronary flow (18%) in constant pressure perfusion, and an increase in perfusion pressure (208%) under constant flow perfusion. Thus, tissue deoxygenation has to be given due attention in the early development of calcium paradox, particularly in case of a constant pressure perfusion. Under constant flow perfusion, the physical stress due to high pressure perfusion against contracture may play an important role in the development of calcium paradox. This may be the first reported evidence for tissue anoxia in calcium paradox.     

Role of platelet-activating factor in the reperfusion injury of rabbit ischemic heart.

This study shows that the administration of the PAF receptor antagonist SDZ 63.675 (5 mg/kg body weight) before reperfusion significantly reduced the hematologic and hemodynamic alterations, as well as the size of necrotic area in rabbits subjected to 40 minutes of coronary occlusion and reperfusion. Pretreatment with SDZ 63.675 prevented the reduction of platelet counts in the blood obtained from the right ventricle (86.6 +/- 2.8% of the control preischemia value) and the transient bradycardia (85.0 +/- 2.8%), the systemic hypotension (58.0 +/- 2.8%), and the increase in right ventricular pressure (125.0 +/- 3.6%) that were evident in the first minutes of reperfusion in untreated control rabbits. Two as well as 24 hours after reperfusion, the infarct size, judged by staining with tetrazolium, was significantly reduced in rabbits treated with SDZ 63.675 (infarct size in control animals, 66.0 +/- 2.9% and 63.46 +/- 2.09% of the risk region at 2 or 24 hours, respectively, compared with 38.9 +/- 5.2% and 37.11 +/- 2.44% of the risk region at 2 and 24 hours in rabbits treated with SDZ 63.675). This result was confirmed by histologic examination of cardiac tissue 24 hours after reperfusion. In addition, SDZ 63.675 markedly reduced the accumulation of 111In-oxine-labeled platelets that occurs 15 minutes after reperfusion in the central ischemic area of the heart and in the lungs. These results suggest that PAF plays a role in the evolution of myocardial injury observed during reperfusion.     

Proliferative and morphologic changes in rat colon following bypass surgery.

In this study the proliferative and morphologic changes that occur in the colon of normal and dimethylhydrazine-treated rats following surgical bypass of the middle third of the colon are reported. Proliferative changes were measured by estimating accumulated mitotic indexes following vinblastine treatment and morphologic changes were observed with the use of light microscopy and scanning electron microscopy. Data were collected on Days 0, 7, 14, 30, and 72 after surgery. The results show that surgical bypass produces contrasting effects in the segments proximal to and distal to the suture line. In the proximal segment there was morphologic evidence of hyperplasia, although proliferative activity was unchanged except for an increase at 7 days in normal rats. In the distal segment there was a long-lived increase in the mitotic index, although morphologic changes were not seen. The results for DMH-treated rats were similar to those in normal rats. Groups of isolated dysplastic epithelial cells were often seen in the submucosa adjacent to sutures up to 72 days after surgery. Increased lymphoid infiltration was seen in segments proximal to but not distal to the suture line. It is hypothesized that the different responses of the proximal and distal segments may be related to the different embryologic origins of those segments. It is also hypothesized that the seeding of the submucosa with epithelial cells during suturing may be a factor in tumor recurrence.     

Ischaemic contracture and myocardial perfusion in isolated rat heart

Summary The development of left ventricular contracture and myocardial perfusion defect was studied in isolated rat hearts during global ischaemia of 90 min duration. The left ventricular pressure was measured by a balloon catheter inserted into the ventricle and filled with water. The pressure reached the maximum at 16 min of ischaemia. The left ventricular volume and compliance (passive distensibility) were measured by the same balloon, the former by connecting the balloon to an open catheter and the latter by applying a constant additional volume (0.020 ml) into the balloon. The left ventricular volume and compliance both decreased progressively for 20 min of ischaemia after which they remained low for the rest of the observation period (90 min). The myocardial perfusability was tested by infusing 0.1 per cent sodium fluorescein in isotonic saline into the cannulated aortic root of the isolated heart preparation. The percentage perfused with the fluorescent tracer in horizontal frozen myocardial sections was estimated by point counting from colour photographs taken under ultraviolet light. The proportion of the perfused area decreased gradually from 100% at 0 min of ischaemia to 93, 67, 43 and 37% at 15, 30, 60 and 90 min of ischaemia, respectively. It was concluded that ischaemic contracture of the left ventricle is followed by the development of a myocardial perfusion defect in isolated ischaemic rat heart.     

Ischaemia-reperfusion induced alterations of mitochondrial function in hypertrophied rat heart

The impact of in vivo ischaemia and ischaemia-reperfusion (I-R) on mitochondrial respiratory function was investigated in hypertrophied (HP) hearts with aortic constriction compared with control hearts using an open-chest rat surgical model. Moreover, mitochondrial susceptibility to superoxide radicals (O₂P^-) in vitro was examined in HP and control hearts with or without I-R. With the site I substrates pyruvate-malate, mitochondrial state 4 (basal) respiration and the respiratory control index (RCI) were not affected by either ischaemia alone or I-R in both HP and control hearts. State 3 (ADP-stimulated) respiration was increased with I-R in control hearts, but showed a reduction after I-R in the HP hearts. Exposure of mitochondria to O₂P^- (20 nm hypoxanthine in the presence of 0.13 unit mL^-1 xanthine oxidase) significantly increased state 4 respiration, whereas state 3 respiration and RCI were decreased in all treatment groups. I-R hearts in both HP and control showed greater increases in state 4 respiration with O₂P^- than either sham or ischaemic hearts. HP hearts exhibited a significantly lesser extent of inhibition in state 3 respiration and RCI by O₂P^- compared with control hearts. These changes in mitochondrial respiratory properties were not observed with the site II substrate succinate. Myocardial reduced vs. oxidized glutathione ratio was significantly decreased after I-R in both control and HP hearts. Malondialdehyde content showed an increase with I-R, but the increase was significant only in control hearts. These data indicate that short-term in vivo I-R does not impair heart mitochondrial respiratory function, but renders the organelles more vulnerable to imposed oxidative stress. Mitochondria from the HP hearts are more resistant to free radical damage under normal and ischaemic conditions; however, this advantage is severely compromised after reperfusion.     

缺血心肌梗死模型大鼠中等强度运动后梗死面积的变化

背景：目前对心肌梗死的急性期是否能进行适量的运动,且适量的运动是否对梗死心肌修复与再生有积极的意义,尚存在很大的争议。 目的：观察中等强度运动对心肌梗死大鼠梗死面积的影响。 方法：将48只SD大鼠随机分为雌、雄训练组和雌、雄对照组,每组12只。结扎大鼠左冠状动脉前降支建立左心室前壁梗死模型。造模24 h,训练组大鼠进行30 min/d的跑台训练,强度为20 m/min,0% grade,共持续12 d;对照组造模后正常饲养。2周后,对大鼠心肌行常规Masson’s Trichrome染色,观察梗死面积和左心室几何参数的变化。 结果与讨论：造模2周,训练组和对照组大鼠的死亡率十分接近(22% vs. 20%)。训练组(雌、雄)的心肌梗死面积明显小于对照组(P < 0.05),梗死边缘区室壁厚度大于对照组(P < 0.05),梗死区室壁最小厚度也大于对照组,但差异无显著性意义 (P > 0.05)。说明心肌梗死急性期即进行中等强度的跑台训练不但没有明显降低大鼠的存活率,还能有效减小缺血心肌的梗死面积、改善左心室重构,且上述作用没有性别差异。     

Ultrastructural changes in the rat endometrium during the normal estrous cycle

Dynamic changes in the endometrial stroma during the following 5 stages of the estrous cycle in normal rats were examined by transmission electron microscopy. Diestrus: Macrophages migrated into the endometrial stroma from blood vessels. Proestrus: Eosinophils migrated into the endometrial stroma from blood vessels. They possessed specific crystalloid granules and small granules. Estrus: The endometrial stroma was swollen and stromal cells degenerated. Eosinophils contained a few or no crystalloid granules, while the number of small granules increased. Metestrus-1: Epithelial projections protruded through the basal lamina and established focal adhesions to stromal cells. Stromal cells also adhered to one another. Metestrus-2: Most eosinophils were engulfed by macrophages. In this report, we discuss the interaction of epithelial cells with endometrial stromal cells during the normal estrous cycle.     

Performance of the hypertrophied left ventricle in spontaneously hypertensive rat. Effects of changes in preload and afterload.

Isolated hearts from adult spontaneously hypertensive rats (SHR; Okamoto 1969), with established hypertension, were investigated in an antegrade perfusion apparatus where preload and afterload could be varied independently. Frank-Starling curves were constructed at constant afterloads ranging from 50 mmHg to 150 mmHg. As earlier reported, the SHR hearts exhibited a rightward shift of their Frank-Startling relationships compared to those from the normotensive control hearts, though visible only at afterloads up to about 100 mmHg. At higher afterloads the SHR hearts performed significantly better then the NCR ones as their maximal stroke volume was significantly greater compared to that of controls. Thus, left ventricular hypertrophy obviously increases the work capacity of the heart, though at the cost of an altered Frank-Startling relation dependent on the reduced diastolic compliance. For such reasons the myocardial hypertrophy in established SHR hypertension must be considered a physiologic adaptation and not a degenerative phenomenon, though naturally degenerative processes may later become superimposed.     

Endothelial superoxide production in the isolated rat heart during early reperfusion after ischemia. A histochemical study.

This paper describes a histochemical study of superoxide generation in buffer-perfused, isolated rat hearts during the first 2 minutes of reperfusion after 60 minutes of warm ischemia. Superoxide radical production was demonstrated by a modification of Karnovsky's manganese/diaminobenzidine technique, in which superoxide oxidizes Mn++ to Mn ions, which in turn oxidize diaminobenzidine to form amber, osmiophilic polymers, observable by light or electron microscopy. Isolated hearts were rendered ischemic, reperfused with oxygen equilibrated buffer containing Mn++ and diaminobenzidine, fixed by perfusion with Trump's solution, and processed for light and electron microscopy. The method consistently demonstrated evidence of superoxide generation near the luminal surfaces of arterial, capillary, and venular endothelial cells during the first 2 minutes of reoxygenation after ischemia. The histochemical reaction was absent or markedly reduced in non-manganese-treated or nonischemic hearts, as well as in hearts perfused with calcium-free or oxygen-free buffers. The histochemical differences were statistically significant on quantitative morphometric analysis. These results provide direct, visual evidence of the existence and endothelial localization of a burst of superoxide radicals in intact, postischemic myocardium and suggest the pathophysiologic importance of calcium-dependent endothelial cell activation in the initiation of reperfusion injury.