Neutrophil myeloperoxidase deficiency associated with canine hepatozoonosis.

Hepatozoonosis is a very important disease in dogs in Nigeria. Hepatozoonosis was reported in Nigeria in 18 dogs. The clinical signs included fever, anorexia, loss of weight, lameness, oculonasal discharge and conjunctivitis. Hematologic findings included leukocytosis due to neutrophilia and eosinophilia. Parasitemia varied from 1 to 9% of the circulating neutrophils in the peripheral blood smears of the dogs examined. Hepatozoon canis gametocytes were identified in circulating neutrophils of dogs. Peripheral blood smears from dogs confirmed to have natural H. canis infection were cytochemically stained for myeloperoxidase. Parasitized neutrophils were myeloperoxidase deficient while non-parasitized neutrophils were myeloperoxidase positive. This is considered important, because deficiency of the enzyme may be responsible for poor response of H. canis to chemotherapeutic agents.

Beneficial effects of low doses of ethanol on reoxygenation injury following anoxia in rat hearts.

We investigated the effect of ethanol on adverse effects of anoxia and reoxygenation in isolated rat hearts. Perfusion of the anoxic Krebs-Henseleit medium for 40 min followed by 30 min of perfusion with aerobic medium produced considerable myocardial cell injury. Incorporation of ethanol (21.7 mM), in both anoxic and aerobic perfusion media resulted in a significant reduction of cell injury and inhibition of creatine phosphokinase release. The contraction bands were reduced to 0.24 as compared to 1.14 per field in the non-treated hearts. The tissue Ca++ was decreased to 8.72 mumol/gm/dry dry wt as compared to 20.17 mumol/gm/dry wt), as compared to the nontreated anoxic tissue (4.41 mumol/gm/dry wt). However, the inclusion of only ethanol in the anoxic medium did not decrease the damage, suggesting that maximal injury occurred during reoxygenation. Ethanol appears to inhibit myofibril contractures and preserve the structural integrity of plasma membrane during anoxia and reoxygenation. This study suggests a beneficial effect of ethanol in low doses on the post anoxic reperfusion injury in the myocardium.

Morphological and biochemical evidence of protective effect by ONO-3144, a free radical scavenger, on the reoxygenation injury in the anoxic myocardium.

We have investigated the effect of ONO-3144 (2:aminomethyl-4-tert-butyl-propionylphenol), which accelerates the conversion of prostaglandin G2 to H2 and acts as a scavenger for free radicals, on the reoxygenation injury in the anoxic heart. Rat hearts were perfused retrogradely with Krebs-Henseleit (KH) medium for 30 minutes in Group I. In Group II, the hearts which were perfused with anoxic KH medium for 40 minutes were reoxygenated for 30 minutes. Group III was similar to Group II except that 4 mg ONO-3144/liter was added in anoxic medium. Group IV was similar to group III except ONO-3144 was present both during anoxia and reoxygenation. Coronary effluent was collected for the measurement of creatine phosphokinase (CPK). Tissue from each group was processed for electron microscopy, adenosine triphosphate (ATP), and tissue calcium. A six-fold increase in CPK leakage that was observed after reoxygenation of anoxic heart was prevented by ONO-3144. Tissue ATP was reduced from 21.65 +/- 1.1 mumol/gm dry weight (Group I) to 4.83 +/- 0.8 mumol/gm dry weight (Group II). A significant amount of ATP (9.05 +/- 1.22 mumol/gm dry weight) was preserved in the treated Group IV. The number of normal cells obtained by morphometrical analysis increased significantly from 24.2 +/- 8.4% (Group II) to 70.00 +/- 4.0% (Group IV), and severely injured cells were also reduced to 19.8 +/- 2.8% in the same group as compared to 54.8% in the untreated Group II. At the electron microscopic level, the cellular membranes, mitochondria, vascular endothelium, and glycogen deposits were well preserved in Group IV. The treatment during anoxia only did not minimize the reoxygenation damage (Group III). Thus, treatment with ONO-3144 provides a great protection against reoxygenation injury to the myocyte and vascular endothelium of the anoxic myocardium, perhaps by scavenging active oxygen species.

Moderating effect of low doses of ethanol on reoxygenation injury in the anoxic myocardium.

We have investigated the action of ethanol on the reoxygenation injury in isolated rat hearts. Perfusion of the anoxic Krebs-Henseleit medium for 40 minutes followed by 30 minutes of perfusion with aerobic medium produced considerable myocardial cell injury. Incorporation of ethanol (21.7 mMol), in both anoxic and aerobic perfusion media resulted in a marked reduction of cell injury and inhibition of creatine kinase. Contraction band necrosis was reduced to 0.25 as compared to 1.14 per field in the nontreated hearts. The tissue Ca++ was decreased to 8.2 as compared to 12.12 mumol/g/dry weight in the non-treated hearts and tissue ATP was increased by 50% in the treated tissue (9.33 mumol/g/dry wt) as compared to the non-treated anoxic tissue (5.5 mumol). Thus, ethanol appears to lessen myofibrilar contraction bands and preserve plasma membrane integrity during anoxia and reoxygenation. This suggests a scavenging role of free radicals which include hydroxy radicals or closely related species, in the pathogenesis of anoxic cell injury and beneficial effect of ethanol in low doses on the post anoxic reoxygenation injury.

Prevention of lactate production and myocyte injury in isolated rat hearts perfused with perfluorochemical emulsion.

The ability of an oxygenated perfluorocarbon (PFC) emulsion to prevent early signs of myocardial cell stress associated with Krebs-Henseleit (KH) perfusion was evaluated in isolated rat hearts supported in a Langendorff apparatus. Throughout the two-hour perfusion, hearts in both perfusion groups had similar left ventricular pressure and rates of left ventricular pressure development (dP/dt). After 2 h, coronary perfusate flow was 6.6 +/- 1.0 ml/min in the KH group and 2.2 +/- 0.1 ml/min in the PFC group. Oxygen content was 1.5 +/- 0.1 ml/100 ml and 3.4 +/- 0.1 ml/100 ml, in KH and PFC groups, respectively. Perfusate lactate levels rose from zero to 24.0 +/- 8.0 microgram/ml in the KH group and to 10.0 +/- 3.0 micrograms/ml in the PFC group. Reduction in myocardial cell injury and in total calcium content was also observed in hearts perfused with PFC emulsion. At the electron microscopic level, mitochondrial structure was preserved in both normal and injured myocytes of hearts perfused with PFC. We conclude that early signs of anaerobic metabolism are retarded by perfusion with PFC emulsions and that ventricular contractile performance is maintained at approximately one-half coronary perfusate flow in the PFC hearts. The mechanism whereby coronary flow is regulated downward in the PFC perfused hearts remains unanswered.

Cardiac injury in short duration anoxia and modification by diltiazem, a calcium channel blocking agent.

The protective effects of diltiazem were studied in isolated perfused rat hearts after 10, 20 and 30 minutes of anoxia and 10 minutes of reoxygenation. The hearts were treated with diltiazem, 4 mg/liter, during anoxia and reoxygenation. The myocardial tissue was processed for electron microscopy and tissue calcium was measured. Four types of cell injury ranging from normal to severe were observed. The prominent morphologic changes in the nondiltiazem-treated tissue were contraction bands, distortion and calcification of mitochondria and loss of glycogen. In the treated group, a partial reduction of cell injury was noted. The mitochondria were usually well preserved, but contraction bands were present. The tissue calcium decreased after treatment with diltiazem. The observations suggest that diltiazem decreases tissue calcium and protects mitochondria more than other cellular components against calcium overload, and this protection may be responsible for the beneficial action of this drug.

Heterogeneity of cell response to early anoxia and reoxygenation in rat heart.

Heterogeneous cell injury and increased tissue calcium levels were observed in the isolated rat hearts perfused with anoxic medium for 5, 10, 15, 20, 25 and 30 min. These changes were intensified after reoxygenation of the anoxic hearts. The cell injury was quantitated using a score of 0, 1, 2 or 3 (0 being normal and 3 being severe injury). All four classes of cells were present even after 5 min of anoxia. The injury and calcium content increased with increase in duration of anoxia. Reduction in the percentage of injured cells was observed on reoxygenation of hearts perfused with anoxic medium for 5 to 20 min. However, reoxygenation of hearts initially perfused with anoxic medium for 25 to 30 min resulted in a higher percentage of injured myocytes. Beneficial effects of reoxygenation were observed in the anoxic hearts when the percentage of cells with an injury score of 1 and glycogen deposit were high, coupled with low calcium levels.

Effects of diltiazem on anoxic injury in the isolated rat heart.

The effect of diltiazem, a calcium channel blocking agent, on anoxic injury was studied in isolated perfused rat hearts. Anoxia for 60 minutes caused a considerable release of creatine kinase and significant cell injury. Reoxygenation of these anoxic hearts for 20 minutes accelerated the creatine kinase release and caused severe cell injury. Reoxygenation of anoxic hearts with diltiazem at a rate of either 2 or 4.5 mg/liter did not reduce creatine kinase release significantly (probability [p] greater than 0.05). However, the higher dosage of diltiazem (4.5 mg/liter during both anoxic and reoxygenation phases resulted in significant (p less than or equal to 0.05) preservation of healthy tissue. The data suggest that diltiazem in the higher concentration prevents cell injury and reduces mitochondrial damage in anoxic injury.

Ultrastructural alterations of the mitochondrial ATPase in the calcium paradox as revealed by negative staining.

We have used a simple negative staining technique to study the structural alterations of mitochondria from biopsies of hearts subjected to the calcium paradox and treatment with diltiazem, a calcium channel blocker. A significant (P less than 0.05) decrease in the number of spheres on the mitochondrial membranes occurs during the calcium paradox (58.0 +/0 4.1/micrometer vs. control 80.5 +/- 6.5). Treatment with diltiazem prevented the loss of spheres from mitochondrial membranes during the calcium paradox (75.5 +20 5.0 micrometer). We found that this negative staining technique can be used for quick assessment of the condition of mitochondria in biopsies from normal and pathological organs.