Enzyme and immunohistochemical assessment of myocardial damage after ischaemia and reperfusion in a closed-chest pig model

The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated. Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation. Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains. Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin. In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically. Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion. The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity. We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally. Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S. Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells. Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium.     

Comparison of triphenyltetrazolium chloride (TTC) staining versus detection of fibronectin in experimental myocardial infarction

Staining with triphenyltetrazolium chloride (TTC), although controversial, has frequently been used for the delineation of myocardial infarction. This study was performed further to explore the reliability of the TTC method. In 24-h experiments pigs were subjected to closed-chest occlusion of the left anterior descending coronary artery for 30, 60 or 90 min followed by reperfusion with or without superoxide dismutase (SOD) as an adjunct. One TTC-stained slice from each heart was stabilized by microwave irradiation, gelatin-embedded, frozen in hexane chilled with dry ice and cryosectioned. Serial sections were stained with antibodies against fibronectin in order to identify irreversibly injured myocytes and with van Gieson histologically to confirm the necrotic tissue. A close correspondence of the infarct size was found between TTC stained slices and anti-fibronectin stained sections. The infarct size in the van Gieson stained sections also showed good correspondence but the area of infarction tended to be larger. In the experimental group subjected to 30 min ischaemia and with SOD as an adjunct, the estimated infarcted area in the TTC stained slices was significantly smaller than the area estimated from the anti-fibronectin stained sections. In sections viewed in the light microscope an inverse pattern of TTC and anti-fibronectin staining was observed. It was confirmed at the light microscopic level that myocytes containing an abundance of TTC deposits lacked fibronectin whereas myocytes stained with antifibronectin in general lacked TTC staining except for a zone approximately 0.5 mm wide which was located at the intersection between damaged and surviving myocytes where small TTC deposits were present. The width of the stained zone did not differ among the experimental groups. Thus, differences in estimated infarct size by the three methods used reflect problems in correctly delineating the border between living and dead myocardium rather than an interference by SOD on TTC staining.     

Evidence for transcytosis of exogenous superoxide dismutase and catalase from coronary capillaries into dog myocytes

Infusion of superoxide dismutase (SOD) and catalase (CAT) into the coronary circulation protects myocardial tissue from free radical injury and improves recovery of myocardial function after a short episode of ischaemia. To investigate the ultrastructure of myocardium treated with SOD and CAT, these enzymes were injected into the left atrium of dogs prior to and during 15 min of regional myocardial ischaemia, allowing 30 min of reperfusion, and then fixing the tissue for electron microscopy. The exogenous SOD + CAT was found to promote recovery of both function and structure in these hearts. In addition, electron dense material was unexpectedly found in vesicles of capillary endothelia, between capillaries and myocyte, and in vesicles within myocytes. This occurred only in hearts treated with SOD and/or CAT, suggesting SOD and CAT was concentrated and transported across the capillary endothelium and into myocytes. The rate of transcytosis, as measured by the number of intra-endothelial vesicles, was increased in tissue subjected to ischaemia and reperfusion in the presence of SOD and CAT. These observations suggest transcytosis of SOD and CAT is an important part of the process by which these enzymes provide protection to myocardium during reperfusion after ischaemia.     

Occurrence of oxidative stress during myocardial reperfusion

Reperfusion, without doubt, is the most effective way to treat the ischaemic myocardium. Late reperfusion may however cause further damage. Myocardial production of oxygen free radicals above the neutralizing capacity of the myocytes is an important cause of this reperfusion damage. There is evidence that prolonged ischaemia reduces the naturally occurring defence mechanisms of the heart against oxygen free radicals, particularly mitochondrial manganese superoxide dismutase, and intracellular pool of reduced glutathione. Consequently, reperfusion results in a severe oxidative damage, as evidenced by tissue accumulation and release of oxidized glutathione.An oxygen free radical-mediated impairment of mechanical function also occurs during reperfusion of human heart. In fact we observed during surgical reperfusion of coronary artery disease (CAD) patients, a prolonged and sustained release of oxidized glutathione;the degree of oxidative stress was inversely correlated with recovery of mechanical and haemodynamic function. These findings represent the rationale for therapeutic interventions which increase the cellular antioxidant capacities and improve the efficacy of myocardial reperfusion.     

Curcumin modulates free radical quenching in myocardial ischaemia in rats

This study was designed to investigate the protective effect of curcumin (CUR) against isoprenaline induced myocardial ischaemia in rat myocardium. The effect of single oral dose of curcumin (15 mg kg(-1)), administered 30 min before and/or after the onset of ischaemia, was investigated by assessing oxidative stress related biochemical parameters in rat myocardium. Curcumin pre and post-treatment (PPT) was shown to decrease the levels of xanthine oxidase, superoxide anion, lipid peroxides (LPs) and myeloperoxidase while the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities were significantly increased after curcumin PPT. Histopathological and transmission electron microscopical studies also confirmed the severe myocardial damage occurring as a consequence of isoprenaline induced ischaemia and they also showed the significant improvement effected by curcumin PPT. These findings provided evidence that curcumin was found to protect rat myocardium against ischaemic insult and the protective effect could be attributed to its antioxidant properties as well as its inhibitory effects on xanthine dehydrogenase/xanthine oxidase (XD/XO) conversion and resultant superoxide anion production.     

Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts.

Cardiovascular ageing is associated with an increase in cardiac susceptibility to ischaemia and reperfusion and production of reactive oxygen species has been suspected to be responsible for this age-associated particular vulnerability. To determine whether administration of antioxidant treatment could afford some protection against ischaemia and reperfusion during aging, isolated perfused hearts from adult and senescent rats were submitted to normoxia (180 min), prolonged low-flow ischaemia (15% of initial coronary flow;180 min) or low-flow ischaemia/reperfusion (45 min/30 min), without or with antioxidant enzymes (superoxide dismutase+catalase; 50IU/ml). Contractile function and coronary perfusion were measured and protein oxidation was quantitated in left ventricle after normoxia, ischaemia and ischaemia/reperfusion. Protein oxidation was higher in senescent than in adult hearts after ischaemia-reperfusion, in contrast to prolonged ischaemia. During prolonged ischaemia, antioxidant treatment prevented coronary vasoconstriction at both ages and delayed contractile dysfunction in senescent hearts but did not limit protein oxidation. During reperfusion, antioxidant treatment prevented coronary vasoconstriction and protein oxidation at both ages and considerably improved recovery of contractile function in senescent hearts. In conclusion, antioxidant treatment fully protects the senescent heart against ischaemia/reperfusion but not against prolonged ischaemia injury, indicating that oxidative stress plays a central role in the age-associated vulnerability to ischaemia-reperfusion.     

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.     

Problems related to infarct size measurements in the rat heart.

The feasibility of measuring the extent of hypoperfused myocardium and the infarct size was examined in rat hearts after occlusion of the left coronary artery. The extent of hypoperfused myocardium was examined by autoradiography and after perfusion with fluorescent microspheres. Both methods appeared unreliable in this model. Triphenyltetrazolium chloride (TTC) staining, however, provided a distinct demarcation line between viable myocardium, which was stained red, and the necrotic myocardium, consistent with the ultrastructural border between normal and severely damaged myocytes 5 h after coronary occlusion. TTC staining gives the best demarcation of ischemic tissues. In verapamil-treated rats, there was an apparent reduction in infarct size as compared with untreated rats; 20% reduction in infarct size 5 h after coronary occlusion and 12% reduction after 24 h. There was, however, a large postoperative mortality among the verapamil-treated rats. These problems, and the nonuniform infarct size in rats, may in part explain why infarct size limitation by verapamil has been reported from rat experiments, but not from clinical trials.     

The influence of lactate,pyruvate and glucose as exogenous substrates on free radical defense mechanisms in isolated rat hearts during ischaemia and reperfusion

Previous studies have shown that exogenous lactate impairs mechanical function of reperfused ischaemic hearts, while pyruvate improves post-ischaemic recovery. The aim of this study was to investigate whether the diverging influence of exogenous lactate and pyruvate on functional recovery can be explained by an effect of the exogenous substrates on endogenous protecting mechanisms against oxygen-derived free radicals. Isolated working rat hearts were perfused by a Krebs-Henseleit bicarbonate buffer containing glucose (5 mM) as basal substrate and either lactate (5 mM) or pyruvate (5 mM) as cosubstrate. In hearts perfused with glucose as sole substrate the activity of glutathione reductase was decreased by 32% during 30 min of ischaemia (p<0.10 versus control value), while the activity of superoxide dismutase and catalase was reduced by 27 and 35%, respectively, during 5 min of reperfusion (p<0.10 versus control value). The GSH level in the glucose group was reduced by 29% following 30 min of ischaemia and 35 min of reperfusion (p<0.10). In lactate- and pyruvateperfused hearts there were no significant decreases of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activity during 30 min of ischaemia, 5 min of reperfusion or 35 min of reperfusion. In pyruvate-perfused hearts the glutathione peroxidase activity was even increased by 43% during 30 min of ischaemia (p<0.05). Glutathione levels (reduced and oxidized) did not markedly change in the lactate and pyruvate groups. Thus, the endogenous defense mechanism against oxygen-derived free radicals is compromised at the onset of reperfusion when glucose as sole substrate is present, while addition of lactate or pyruvate prevents reduction of the endogenous capacity to scavenge oxygen-derived free radicals. The equivocal relationship between endogenous scavenging enzyme activity and haemodynamic recovery indicates that involvement of the endogenous antioxidants, if any, in functional recovery of the post-ischaemic heart is complex. Pyruvate may exert protective effects on mechanical function after mild ischaemia by functioning as exogenous scavenger in itself, as pyruvate is able to react with hydrogen peroxide.     

Selective staining methods for cartilage of rat fetal specimens previously treated with alizarin red S.

A convenient method for staining cartilage with several basic stains after alizarin red S staining of bone was investigated in rat fetuses. It was found that bromophenol blue was useful and effective for staining of the margin and center areas of cartilage, even in specimens stored in glycerin for over 10 years. The specimens were washed in running tap water for 1 hr, and subsequently were immersed in water or in 70% ethanol at pH 4 for 1 hr or longer. The specimens were then stained with 0.005% bromophenol blue in 40% ethanol adjusted to pH 4 for 2 hr, or with 0.001% bromophenol blue in 40% ethanol adjusted to pH 4 for 24 hr. Furthermore, the bromophenol blue stain color actually faded when the specimens were immersed in water or in 70% ethanol at pH 8. Descending order of the stain-effective action on fetal rat cartilage for the basic stains tested was bromophenol blue, aniline blue, Evans blue, methyl violet, trypan blue, and water blue.     

Luxol Fast Blue Combined With the Periodic Acid-Schiff Procedure for Cytological Staining of Kidney

Rat kidneys fixed in Regaud's fluid were stained by luxol fast blue (LFB), by the periodic acid-Schiff (PAS) method, and by LFB combined with PAS. When used separately the PAS stains the brush border, hyaline droplets and basement membranes reddish, the LFB stains the mitochondria, hyaline bodies and basement membranes greenish-blue. The combined LFB-PAS method stains the brush border reddish and the mitochondria dark blue, while the hyaline bodies and basement membranes are purplish colored. The LFB-PAS method provides color contrasts which show cytological features that are particularly significant in the kidney.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

Demonstrating the Osseous Skeleton of Human Embryos and Fetuses

Transparent human embryos and fetuses whose osseous skeletons are stained in toto by alizarin red S are successfully prepared when the KOH clearing of the soft tissues and the alizarin staining of the bones are performed simultaneously instead of independently. This modification minimizes the possibility of macerating and staining the soft tissues. Fetuses over 50 mm. CR length are skinned, eviscerated, decerebrated, defatted by dissection, fixed in 95% alcohol, bleached in H₂O₂, cleared and stained simultaneously in an aqueous solution of KOH (from 2% to 10% depending upon the size of the specimen) and .0001 to .00005% alizarin red S (solution has a pale lavender color). This solution is changed periodically to maintain the concentration of the KOH until the clearing of the tissues is complete and of the alizarin until the bones are properly stained. Tissues are dehydrated in increasing concentrations of glycerin and stored in white glycerin plus thymol.     

The use of alizarin red S to detect and localize calcium in gametophyte cells of ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca2+ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KCl to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca2+. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca2+ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

A Technique for Demonstrating Glands in Miracidia of Some Fasciolidae (Trematoda)

A technique (periodic acid-Schiff—Victoria blue) is described which selectively stains glands in miracidia of some fasciolide trematodes. In this method, sections of miracidia are first stained with periodic acid-Schiff and then with an iron-rescorcin lake of Victoria blue. Following this treatment, the semetory granules of the apical gland are blue and the anterior glands are red.     

Permanent Preservation of Whole Alizarin Red S Skeletons by Clearing and Embedding in Polyester Resins

A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method.     

Permanent preservation of whole alizarin red S skeletons by clearing and embedding in polyester resins

A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method.     

The Use of Alizarin Red S To Detect and Localize Calcium in Gametophyte Cells of Ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca²⁺ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KC1 to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca²⁺. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca²⁺ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.     

Aspiration biopsy cytology and special stains

Histochemical methods play a vital role in the histology laboratory. The application of histochemistry to aspiration biopsy cytology (ABC) has shown equal promise in the demonstration of distinctive intracytoplasmic and extracytoplasmic substances. In our examination of more than 8,000 fine needle aspirates, we have found that certain histochemical studies may be of special diagnostic importance. These include mucicarmine, periodic acid-Schiff, oil red O, Fontana-Masson argentaffin and Gomori's iron stains. With the exception of the oil red O stain for neutral lipids (alcohol soluble), each may be applied to the destained smear initially prepared according to the method of Papanicolaou. Modifications of these histologic stains have been made to suit the "rapid service" motto of our busy cytology laboratory.