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Biomedical subjects

I S Harper

Publications and source records attributed to I S Harper.

At least 19 recordsLinked to original sources
PubMed2001

Confocal imaging, visualization and 3-D surface measurement of small mammalian teeth.

The difficulties traditionally faced by functional morphologists in representing and interpreting three-dimensional objects can now be mostly overcome using available laser and computer imaging technologies. A practical method for three-dimensional imaging of small mammalian teeth using confocal microscopy is reported. Moulding and casting of the teeth were first performed, followed by confocal fluorescence imaging. Accuracy and precision of the scanned structures were tested in morphometric studies by using a new technique to measure the noise in the scan of a three-dimensional surface, and linear and angular dimensions of the scans were compared with measurements made using traditional morphological tools. It is shown that measurements can be taken with less than 4% difference from the original object. Teeth of the microchiropteran bat Chalinolobus gouldii were scanned and measured to show the potential of the techniques. Methods for visualizing the small teeth in three-dimensional space, and animating the teeth in occlusion, show the power of this approach in aiding a three-dimensional understanding of the structure and function of teeth and other three-dimensional structures.

Animals↗
PubMed2000

A revised model of platelet aggregation.

In this study we have examined the mechanism of platelet aggregation under physiological flow conditions using an in vitro flow-based platelet aggregation assay and an in vivo rat thrombosis model. Our studies demonstrate an unexpected complexity to the platelet aggregation process in which platelets in flowing blood continuously tether, translocate, and/or detach from the luminal surface of a growing platelet thrombus at both arterial and venous shear rates. Studies of platelets congenitally deficient in von Willebrand factor (vWf) or integrin alpha(IIb)beta(3) demonstrated a key role for platelet vWf in mediating platelet tethering and translocation, whereas integrin alpha(IIb)beta(3) mediated cell arrest. Platelet aggregation under flow appears to be a multistep process involving: (a) exposure of vWf on the surface of immobilized platelets; (b) a reversible phase of platelet aggregation mediated by the binding of GPIbalpha on the surface of free-flowing platelets to vWf on the surface of immobilized platelets; and (c) an irreversible phase of aggregation dependent on integrin alpha(IIb)beta(3). Studies of platelet thrombus formation in vivo demonstrate that this multistep adhesion mechanism is indispensable for platelet aggregation in arterioles and also appears to promote platelet aggregate formation in venules. Together, our studies demonstrate an important role for platelet vWf in initiating the platelet aggregation process under flow and challenge the currently accepted view that the vWf-GPIbalpha interaction is exclusively involved in initiating platelet aggregation at elevated shear rates.

Animals↗
PubMed1998

Mitochondrial Ca2+ transients in cardiac myocytes during the excitation-contraction cycle: effects of pacing and hormonal stimulation.

Using laser scanning confocal microscopy, our objective was to measure mitochondrial, nuclear, and cytosolic free ionized Ca2+ in adult rabbit cardiac myocytes loaded with Ca2+-indicating fluorophores. When myocytes were loaded with Fluo 3 at 37 degrees C, the fluorophore was loaded extensively into the cytosol and nucleus, but poorly into mitochondria, and Fluo 3 fluorescence transients after field stimulation were confined to the cytosol and nucleus. In contrast, after loading at 4 degrees C, Fluo 3 also entered mitochondria, and large transients of mitochondrial Fluo 3 fluorescence then occurred after stimulation. Isoproterenol (1 microM) increased the magnitude of Ca2+ transients and their subsequent rate of decay, an effect more marked in the cytosol and nucleus than in mitochondria. As pacing frequency was increased from 0.5 to 2 Hz, diastolic mitochondrial Ca2+ rose markedly in the absence but not in the presence of isoproterenol. Resting Ca2+ estimated by Indo 1 ratio imaging using UV/visible laser scanning confocal microscopy was about 200 nM in all compartments. During field stimulation, Ca2+ transiently increased to 671, 522, and 487 nM in cytosol, interfibrillar mitochondria, and perinuclear mitochondria, respectively. Isoproterenol increased these respective peak values to 1280, 750, and 573 nM. These results were consistent with those obtained in Fluo 3 experiments. We conclude that rapid mitochondrial Ca2+ transients occur during excitation-contraction coupling in adult rabbit cardiac myocytes, which may be important in matching mitochondrial metabolism to myocardial ATP demand during changes in cardiac output.

Aniline Compounds↗
PubMed1996

Mitochondrial free calcium transients during excitation-contraction coupling in rabbit cardiac myocytes.

Mitochondrial free Ca2+ may regulate mitochondrial ATP production during cardiac exercise. Here, using laser scanning confocal microscopy of adult rabbit cardiac myocytes co-loaded with Fluo-3 to measure free Ca2+ and tetramethylrhodamine methylester to identify mitochondria, we measured cytosolic and mitochondrial Ca2+ transients during the contractile cycle. In resting cells, cytosolic and mitochondrial Fluo-3 signals were similar. During electrical pacing, transients of Fluo-3 fluorescence occurred in both the cytosolic and mitochondrial compartments. Both the mitochondrial and the cytosolic transients were potentiated by isoproterenol. These experiments show directly that mitochondrial free Ca2+ rises and falls during excitation-contraction coupling in cardiac myocytes and that changes of mitochondrial Ca2+ are kinetically competent to regulate mitochondrial metabolism on a beat-to-beat basis.

Aniline Compounds↗
PubMed1996

The pH paradox in ischemia-reperfusion injury to cardiac myocytes.

During myocardial ischemia, a large reduction of tissue pH develops, and tissue pH returns to normal after reperfusion. In recent studies, we evaluated the role of pH in ischemia/reperfusion injury to cultured cardiac myocytes and perfused papillary muscles. Acidosis (pH < or = 7.0) protected profoundly against cell death during ischemia. However, the return from acidotic to normal pH after reperfusion caused myocytes to lose viability. This worsening of injury is a 'pH paradox' and was mediated by changes of intracellular pH (pH(i)), since manipulations that caused pH(i), to increase more rapidly after reperfusion accelerated cell killing, whereas manipulations that delayed the increase of pH(i) prevented loss of myocyte viability. Specifically, inhibition of the Na+/H+ exchanger with dimethylamiloride or HOE694 delayed the return of physiologic pH(i) after reperfusion and prevented reperfusion-induced cell killing to both cultured myocytes and perfused papillary muscle. Dimethylamiloride and HOE694 did not reduce intracellular free Ca2+ during reperfusion. By contrast, reperfusion with dichlorobenzamil, an inhibitor of Na+/Ca2+ exchange, decreased free Ca2+ but did not reduce cell killing. Thus, the pH paradox is not Ca(2+)-dependent. Our working hypothesis is that ischemia activates hydrolytic enzymes, such as phospholipases and proteases, whose activity is inhibited at acidotic pH. Upon reperfusion, the return to normal pH releases this inhibition and hydrolytic injury ensues. Increasing pH(i) may also induce a pH-dependent mitochondrial permeability transition and activate the myofibrillar ATPase, effects that increase ATP demand and compromise ATP supply. In conclusion, acidotic pH is generally protective in ischemia, whereas a return to physiologic pH precipitates lethal reperfusion injury to myocytes.

Animals↗
PubMed1995

Preconditioning with a single short episode of global ischemia in the isolated working rat heart: effect on structure, mechanical function, and energy metabolism for various durations of sustained global ischemia.

PURPOSE: Preconditioning in the setting of global ischemia, using functional recovery during reperfusion as the endpoint, has recently been demonstrated in the isolated perfused rat heart. It has been suggested that its beneficial actions have a metabolic basis. The isolated rat heart has not been fully characterized with respect to the metabolic, functional, and structural changes associated with this phenomenon in the setting of global ischemia. The purpose of this study was to determine (1) the time course of protection conferred by a single episode (5 minutes) of preconditioning; (2) changes in tissue high energy phosphates, lactate, and glycogen levels at different time intervals; and (3) morphological appearance of the heart at the end of ischemia as well as after reperfusion. METHODS: Isolated perfused working rat hearts were used. Preconditioning consisted of a single episode of 5 minutes of global ischemia and 15 minutes of reperfusion. Preconditioned and non-preconditioned hearts were subjected to global ischemia of 20-35 minutes duration. Functional recovery, energy metabolism (high energy phosphates, lactate, and glycogen), and structural appearance were studied at different stages. RESULTS: The functional recovery of the preconditioned hearts was significantly higher than in the corresponding nonpreconditioned group during reperfusion for all durations of ischemia longer than 25 minutes. The degree of protection observed was less than reported previously. A minor degree of energy sparing was reflected by differences in the rate of depletion of glycogen and accumulation of tissue lactate during the sustained episode of ischemia. Semiquantitative light microscopy evaluation revealed that ischemia-induced structural damage was less in the preconditioned hearts, both at the end of the sustained ischemic episode as well as after reperfusion. CONCLUSIONS: A single episode of global ischemia successfully preconditions the isolated working rat heart. The protection elicited was demonstrated on a functional and structural level, and was accompanied by a small energy-sparing effect.

Animals↗
PubMed1994

Halothane protects the isolated rat myocardium against excessive total intracellular calcium and structural damage during ischemia and reperfusion.

A recent study from our laboratory demonstrated halothane to be a powerful protectant of the isolated rat heart during reperfusion after normothermic cardioplegic arrest. It was speculated that this protective effect might be due to prevention of excessive intracellular calcium. The aim of the present study was to evaluate the effect of halothane on the total intracellular calcium (Ca2+) content and on myocardial structure both at the end of normothermic cardioplegic arrest and at the end of reperfusion. Isolated perfused rat hearts were perfused for a control period of 30 min, followed by 40 min of normothermic cardioplegic arrest with or without reperfusion for 30 min. Halothane (1.5%) was administered continuously before and after arrest. Halothane caused a significant decrease of intracellular Ca2+ at the end of normothermic cardioplegic arrest and after reperfusion. Myocardial morphology was assessed by extensive light microscopy and ultrastructure was evaluated by electron microscopy. Grading of ischemic damage showed that exposure to normothermic cardioplegia resulted in marked ischemic injury, regardless of whether the hearts were treated with halothane. Reperfusion in the presence of halothane caused a significant reversal of ischemic damage and almost complete ultrastructural repair, whereas untreated hearts still exhibited severe edema, contracture, and contracture bands. Our results indicate that the beneficial effects of halothane on myocardial structural recovery during reperfusion is associated with a reduction in excessive intracellular Ca2+. The exact mechanism of this protective action is under investigation.

Animals↗
PubMed1993

Effect of captopril on changes in rats' hearts induced by long-term irradiation.

The aim of this study was to test the efficacy of captopril, an angiotensin-converting enzyme inhibitor and a known suppressor of fibrosis, in preventing late radiation-induced cardiac pathology. Myocardial functional, histochemical and ultrastructural-morphometric studies were done on perfused hearts of rats isolated 3 and 6 months after 60Co gamma irradiation with 20 Gy and age-matched controls. At each time the animals were divided into the following groups: nonirradiated controls; irradiated once with 20 Gy; irradiated as above and given daily doses of captopril; daily doses of captopril without irradiation. The results showed that captopril, while ameliorating the decrease in the indices of capillary function, increase in mast cells, fibrosis, number of atrial granules, and changes in nerve terminals, failed to prevent the progressive functional deterioration of the hearts after irradiation. These findings suggest that an intramyofiber derangement may be involved in the long-term myocardial complications of irradiation.

Animals↗
PubMed1991

Myocardial membrane cholesterol: effects of ischaemia.

Evidence has recently been presented that myocardial ischaemia is associated with a significant increased mitochondrial cholesterol content, suggesting a redistribution of cholesterol within the ischaemic cell (Rouslin et al. 1980, 1982). The aim of this study was therefore to determine the effects of different periods of ischaemia and reperfusion on the cholesterol content of myocardial mitochondria, sarcoplasmic reticulum and sarcolemma. Using the isolated perfused rat heart as experimental model, it was demonstrated that increasing periods of ischaemia (15-60 min) caused a progressive loss of cholesterol from the tissue as well as from the sarcolemma and sarcoplasmic reticulum, concomitant with a significant increase in mitochondrial cholesterol content. These compositional changes were associated with a marked increase in sarcolemmal and mitochondrial microviscosity, while that of the sarcoplasmic reticulum was reduced. To gain more insight into the mechanisms controlling intracellular cholesterol distribution, control and ischaemic hearts were perfused with either exogenous cholesterol or its precursor [U-14C]acetate as an indicator of endogenous cholesterol synthesis. Perfusion with exogenous cholesterol resulted in significant increases in the membrane cholesterol content of control hearts. However, hypoxic, low flow perfusion prevented cholesterol enrichment of the sarcolemmal and sarcoplasmic reticulum membranes, while the cholesterol content of the mitochondria was increased from 99.48 +/- 12.75 to 127.61 +/- 1.84 nmols/mg protein, indicating specific incorporation into this membrane system. Incorporation of [U-14C]acetate into cholesterol in the sarcoplasmic reticulum was increased by 120% in ischaemic conditions. However, a marked redistribution of newly synthesized cholesterol was observed within the ischaemic cell: under control conditions most of the labelled cholesterol was transferred to the sarcolemma and least to the mitochondria, while this distribution pattern was reversed in ischaemia. In view of the fact that exchange of cholesterol between membranes is affected by both phospholipid polar head-group composition and acyl chain length and saturation, it is suggested that prior ischaemia-induced membrane compositional changes might lead to intracellular cholesterol redistribution. Finally, to determine whether cholesterol loss affects sarcolemmal permeability, hearts enriched in sarcolemmal cholesterol were subjected to 15 or 30 min global ischaemia followed by reperfusion and the rate of enzyme release determined. However, enzyme release was similar in treated and untreated hearts, indicating that sarcolemmal cholesterol loss probably does not affect its permeability.

Animals↗
PubMed1990

Lanthanum probing of cell membrane permeability in the rat heart: pathological versus artefactual alterations.

The influence of fixation on membrane permeability has been examined in the isolated rat heart using lanthanum as a permeability probe. Normal and ischaemic hearts were probed at various stages during a conventional fixation programme with either ionic or colloidal lanthanum and compared with lanthanum saline administered prior to fixation. Fixation of the myocardium coincident with or followed by lanthanum probing resulted in an influx of the probe into most myocytes in normal tissue. Alterations in permeability after ischaemic episodes could not be distinguished from the artefact. However, lanthanum saline prior to fixation showed exclusion of the probe from normal tissue, while the increased permeability demonstrated after ischaemia was associated with declining myocardial performance during subsequent reperfusion. These results illustrate the need for caution in the application and evaluation of methods determining permeability in fixed tissue. Probes of differing size and charge permeated fixed tissue to varying degrees thereby implicating the formation of specific lesions during chemical fixation.

Animals↗
PubMed1990

Electron dense artefactual deposits in tissue sections: the role of ethanol, uranyl acetate and phosphate buffer.

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.

Animals↗
PubMed1990

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.

Animals↗
PubMed1989

Radiation-induced changes in the ultrastructure and mechanical function of the rat heart.

A time sequence study was performed to study the early effects of radiation on the ultrastructure of the rat heart. Wistar rats were exposed to 20 Gy electron irradiation to a field including the heart and a third of the lung. The hearts were excised at varying time intervals (1 h-180 days), and the ultrastructure of perfusion-fixed subepicardium and subendocardium studied. Changes were observed in both myocytes and interstitium at all time intervals. The most pronounced change observed in the myocyte was that of intercalated disc damage which reached a peak at 30 days post-irradiation. Mitochondrial damage, characterized by swelling and fenestration in areas of myofibrillar contracture, was focal and relatively scarce. Swelling of the capillary endothelial cells and collapse of the capillaries were marked up to 60 days. Of significance was the observation that the damage to both myocytes and interstitium receded after 60 days and the hearts exhibited an almost normal ultrastructure from 100 to 180 days post-irradiation. Mechanical function of these hearts followed a similar pattern: maximal depression was observed 60 days after irradiation. Thereafter the work performance of these hearts improved significantly, almost reaching control levels after 180 days.

Animals↗
PubMed1987

Ultrastructural observations on the effects of different substrates on ischaemic contracture in global subtotal ischaemia in the rat heart.

Ultrastructural morphology was examined in myocardia perfused with different substrates, following the hypothesis that ultrastructural changes or differences would indicate the mechanism of early ischaemic contracture. Substrates used were glucose, acetate and no substrate (substrate-free). Isolated rat hearts were subjected to global low flow ischaemia followed by perfusion fixation at either no contracture, early contracture (5%) or full contracture (100%). Total tissue adenosine triphosphate (ATP) levels were analysed from myocardia of each group. Glucose perfused myocardia did not develop contracture for at least 30 minutes and maintained ultrastructural and ATP normality. Substrate-free and acetate perfused myocardia developed contracture after 10-12 minutes of ischaemia at which time myofibrillar morphology was altered within small sparse foci of developing mild oedema with contracture and hyperextension of myofibrils. ATP levels were normal. At full ischaemic contracture, both substrate-free and acetate perfused myocardia showed severe mitochondrial damage, oedema, myofibrillar contracture and hyperextension with accompanying distortion and swelling of the sarcoplasmic reticulum.

Animals↗
PubMedDate not supplied

Substrate effects on sarcolemmal permeability in the normoxic and hypoxic perfused rat heart.

OBJECTIVES: Based on the hypothesis that provision of glucose is good and fatty acids are bad for the ischaemic myocardium, the aims of this study were to determine i) the effects of different substrates on sarcolemmal permeability during normoxia, low-flow hypoxia (HLF) and reperfusion, ii) whether increased membrane permeability is associated with ultrastructural damage and increased influx of Ca2+ into cells and iii) whether changes in membrane permeability correlate with myocardial function and high energy phosphate metabolism. METHODS: The isolated rat heart subjected to HLF was used as model of global ischaemia, and sarcolemmal permeability assessed by release of LDH from and influx of lanthanum and Ca2+ into myocardial tissue. Myocyte structural injury was also evaluated quantitatively, and mechanical activity was monitored throughout the experimental protocol. RESULTS: Regardless of the substrate used, HLF caused a 80-90% and 20-40% reduction in myocardial oxygen uptake and coronary flow rate, respectively. Palmitate (0.5 mM conjugated to 0.1 mM albumin) or substrate-free perfusion caused ultrastructural damage and loss of normal sarcolemmal integrity during both normoxia and HLF. Although reperfusion reversed injury in some cells, in general, myocytes exhibited myofibrillar contracture, while membrane integrity recovered to some extent, as indicated by reduced lanthanum influx. Intracellular Ca2+ increased significantly upon reperfusion. Mechanical function as well as tissue high energy phosphates were significantly depressed during both HLF and reperfusion. Glucose, on the other hand, protected against ischaemia-induced structural damage and loss of sarcolemmal integrity. Reperfusion in these experiments resulted in almost complete recovery of normal morphology, ultrastructure and sarcolemmal integrity, while intracellular Ca2+ remained unchanged. Mechanical function and tissue high energy phosphates were significantly higher in glucose-perfused hearts than in palmitate-perfused or substrate-free hearts. Glucose was also able to attenuate the harmful effects of palmitate on myocardial ultrastructure, membrane integrity, mechanical function, energy metabolism and prevented Ca2+ overloading during reperfusion. CONCLUSION: The results provide new evidence for the protective role of glucose during myocardial ischaemia and reperfusion. Although the exact mechanism of the beneficial actions of glucose remains to be established, the results suggest that glycolytic flux and thus glycolytically derived ATP protect against ischaemic damage via preservation of membrane integrity.

Adenine Nucleotides↗