Micheline Federman

Triggering of Plaque Disruption and Arterial Thrombosis in an Atherosclerotic Rabbit Model

BACKGROUND It is now recognized that plaque disruption and thrombosis, a process often triggered by activities of the patient, is generally the cause of the onset of acute coronary syndromes. Understanding of disease onset could be greatly enhanced by the availability of a suitable animal model of plaque disruption and thrombosis. The aim of this study was to replicate and further characterize an atherosclerotic rabbit model of triggering of arterial thrombosis that was introduced by Constantinides and Chakravarti more than 30 years ago but not subsequently used. Aortic plaques were induced by a high-cholesterol diet, by mechanical balloon injury of the artery, or by a combination of the two. Triggering was attempted by injection of Russells viper venom (RVV), which is a proteolytic procoagulant, and histamine. METHODS AND RESULTS A total of 53 New Zealand White rabbits were exposed to one of four preparatory regimens: rabbits in group I (n = 9) were fed a regular diet for 8 months; rabbits in group II (n = 13) were fed a diet of 1% cholesterol for 2 months alternated with 2 months of a regular diet for a total of 8 months; rabbits in group III (n = 5) underwent balloon-induced arterial wall injury, then were given a regular diet for 8 months; and rabbits in group IV (n = 14) underwent balloon-induced arterial wall injury, then were given a diet of 1% cholesterol for 2 months followed by a regular diet for 2 months for a total of 4 months. After completion of the preparatory regimen, triggering of plaque disruption and thrombosis was attempted by injection of RVV (0.15 mg/kg IP) and histamine (0.02 mg/kg IV). In group I, normal control rabbits without atherosclerosis, only one small thrombus was noted in 1 of 9 rabbits. In group II, cholesterol-fed rabbits, thrombosis occurred in 3 of 13 rabbits. Thrombus occurred in all rabbits in group III (5 of 5) and in 10 of 14 rabbits in group IV. Although the frequency of thrombosis was not significantly different between groups I and II, possibly due to a small sample size, it was significantly different among all four groups (P < .001). Also, the frequency and amount of thrombus formation were significantly different among all four groups (P < .001; P < .0001) but not between groups I and II. Rabbits with atherosclerosis (those in groups II and IV) demonstrated plaque disruption and overlying platelet-rich thrombus formation similar to that observed in patients with acute coronary syndromes. The surface area covered by thrombus was 2 mm2 in group I, 15.3 +/- 19.2 mm2 in group II, 223 +/- 119 mm2 in group III, and 263 +/- 222 mm2 in group IV. Rabbits in groups III and IV had the greatest amount of thrombus, and this amount was significantly greater than in rabbits in groups I and II (P < .001 and P < .03, respectively). CONCLUSIONS A suitable animal model is available for the study of plaque disruption and arterial thrombosis. Hypercholesterolemia and mechanical arterial wall injury seemed to produce plaques vulnerable to triggering of disruption and thrombosis, whereas normal arteries were relatively resistant to triggering. This model provides a method to evaluate agents that might decrease the occurrence of vulnerable plaques or the amount of thrombus formed after triggering. Most important, the model can be used to identify the features of vulnerable plaques and the pharmacological stressors that trigger plaque disruption and thrombus formation.

Developmental differences in cytosolic calcium accumulation associated with surgically induced global ischemia: Optimization of cardioplegic protection and mechanism of action ☆ ☆☆ ★ ★★ ♢

OBJECTIVE The effect of cardioplegic solutions with high concentrations of potassium or magnesium (or both) on cytosolic calcium accumulation was investigated with fura-2 in isolated perfused mature (n = 24) and aged (n = 24) rabbit hearts. METHODS We compared cytosolic calcium accumulation before ischemia (control), during 30 minutes of ischemia and 30 minutes of reperfusion under global ischemia, or after treatment with potassium (20 mmol/L), magnesium (20 mmol/L), or both. RESULTS Cytosolic calcium accumulation was increased during global ischemia in the mature heart (from 178.7 +/- 24.2 in the control group to 393.6 +/- 25.5 nmol/L; p < 0.005) and in the aged heart (from 187.4 +/- 18.7 in the control group to 501.0 +/- 46.1 nmol/L; p < 0.005). Potassium reduced cytosolic calcium accumulation during ischemia in both the mature and aged hearts (300.9 +/- 23.2 and 365.2 +/- 27.7 nmol/L, respectively; p < 0.05 vs global ischemia). Magnesium and potassium/magnesium completely controlled cytosolic calcium accumulation in the mature heart (198.7 +/- 27.5 nmol/L; p < 0.01 vs global ischemia and p < 0.05 vs potassium: 182.3 +/- 22.7 nmol/L; p < 0.05 vs global ischemia and potassium, respectively). Magnesium and potassium/magnesium attenuated cytosolic calcium accumulation in the aged heart (261.3 +/- 26.7, 262.3 +/- 25.2 nmol/L, respectively; p < 0.01 vs global ischemia). These changes in cytosolic calcium accumulation correlated with improved post-ischemic ventricular function. To investigate the mechanism(s) of magnesium-supplemented cardioplegic inhibition of cytosolic calcium accumulation, we performed parallel studies (n = 43) using nifedipine, ryanodine, and dimethylthiourea. Nifedipine with or without ryanodine reduced cytosolic calcium accumulation. Dimethylthiourea did not alter cytosolic calcium accumulation during global ischemia. Our results suggest that cytosolic calcium accumulation during global ischemia was mainly increased via the sarcolemmal 1-type calcium channel and the sarcoplasmic reticulum calcium-release channel. The modulating action of potassium/magnesium cardioplegia on cytosolic calcium accumulation during ischemia would appear to act through the inhibition of the myocardial 1-type calcium channel and the sarcoplasmic reticulum calcium-release channel. CONCLUSION Senescent cardiac dysfunction correlates with increased ischemia-induced cytosolic calcium accumulation. Magnesium-supplemented potassium cardioplegia ameliorates this age-related phenomenon at normothermia and may have important implications in myocardial protection in the elderly population.

Malabsorption and wasting in AIDS patients with microsporidia and pathogen-negative diarrhea.

ObjectiveTo define the clinical syndrome, nutritional status and malabsorptive status in patients with HIV and chronic diarrhea and either microsporidia or no identified pathogen. PatientsHIV-positive patients from an urban, hospital-based infectious disease clinic with chronic diarrhea who had undergone exhaustive gastrointestinal and stool studies for enteric pathogens and were found to have either microsporidia or no pathogen. MethodsPatients were evaluated for clinical history, physical, body composition, nutritional and malabsorptive studies including D-xylose, Schilling test, determinations of 24 h stool fat, weight and nitrogen, and 24 h urea nitrogen. ResultsTen patients with microsporidia were studied, four of whom were infected with Septata intestinalis, six with Enterocytozoon bieneusi; nine patients had no identified pathogen. Patients in both groups were comparable in stage of HIV disease, and demonstrated abnormal nutritional status and malabsorptive parameters. Patients with no pathogen had significantly longer duration of symptoms prior to presentation; however, patients with microsporidia had significantly greater malabsorption of fat, D-xylose, vitamin B12, and significantly lower serum levels of zinc. Nutritional status and malabsorption were similarly depressed in patients infected with either species of microsporidia. ConclusionHIV-infected patients with chronic diarrhea associated with either microsporidial infection or with no identified pathogen had abnormal parameters of absorption and malnutrition, and those infected with microsporidia demonstrated more severe malabsorption.

Magnesium cardioplegia reduces cytosolic and nuclear calcium and DNA fragmentation in the senescent myocardium.

Previous reports have indicated that the senescent myocardium is less tolerant to surgically induced ischemia and that diminished functional recovery is associated with alterations in cytosolic calcium ([Ca2+]i) accumulation. Recently, increased [Ca2+]i has been suggested to alter nuclear calcium ([Ca2+]n) accumulation. To investigate the relation between [Ca2+]i and [Ca2+]n, we subjected mature and aged rabbit hearts to normothermic global ischemia, either without treatment or after treatment with potassium cardioplegia, magnesium cardioplegia, or a combination of potassium and magnesium cardioplegia. The relation between altered [Ca2+]n and DNA fragmentation was also investigated. Our results indicate that [Ca2+]i was increased during 30 minutes of normothermic global ischemia without treatment in both the mature and aged hearts (p < 0.05). Accumulation of [Ca2+]i during global ischemia was reduced with the use of potassium, magnesium, and a combination of potassium and magnesium cardioplegia (p < 0.05 versus untreated ischemia) in both the mature and aged hearts. Levels of [Ca2+]n were unaffected by global ischemia or cardioplegia in the mature myocardium; however, in the aged myocardium, [Ca2+]n was increased during global ischemia and with potassium cardioplegia and was associated with increased nuclear DNA fragmentation (p < 0.05). The use of magnesium and a combination of potassium and magnesium cardioplegia attenuated [Ca2+]n accumulation and nuclear DNA fragmentation (p < 0.05). Control of [Ca2+]i and [Ca2+]n was associated with enhanced functional recovery during reperfusion. These results indicate that during normothermic ischemia, there is increased [Ca2+]i and [Ca2+]n in the aged myocardium, and increased [Ca2+]n is associated with increased nuclear DNA fragmentation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone cell cilia: Vestigial or functional organelles?

Cilia have been observed protruding from osteocytes in fetal rat calvaria by electron microscopy, raising the possibility that they might be concerned with the movement of fluids through the canalicular system of dense bone and, therefore, with the transport of calcium and other ions into and out of the skeleton.

Adenosine-enhanced ischemic preconditioning modulates necrosis and apoptosis: effects of stunning and ischemia–reperfusion

BACKGROUND Adenosine-enhanced ischemic preconditioning extends the protection of ischemic preconditioning by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. METHODS The effects of adenosine-enhanced ischemic preconditioning on necrosis and apoptosis were investigated in the sheep heart using models of stunning (15 minutes regional ischemia, 120 minutes reperfusion) and ischemia-reperfusion (30 and 60 minutes regional ischemia, 120 minutes reperfusion). Ischemic preconditioned hearts received 5 minutes regional ischemia, 5 minutes reperfusion before ischemia. Adenosine-enhanced ischemic preconditioned hearts received a 10 mmol/L adenosine bolus (10 mL) through the left atrium coincident with ischemic preconditioning. Adenosine hearts received a 10 mmol/L bolus (10 mL) of adenosine. Regional ischemic hearts received no pretreatment. RESULTS Minimal apoptosis (< 45 per 3,000 myocytes) was observed in the stunning models but was significantly increased with ischemia-reperfusion in regional ischemic hearts after 30 minutes (p < 0.05 versus ischemic preconditioning, adenosine, or adenosine-enhanced ischemic preconditioning) and in adenosine and ischemic preconditioned hearts after 60 minutes ischemia (p < 0.05 versus adenosine-enhanced ischemic preconditioning). DNA laddering was apparent after 60 minutes ischemia in regional ischemia, adenosine, and ischemic preconditioning but not in adenosine-enhanced ischemic preconditioned hearts. CONCLUSIONS Adenosine-enhanced ischemic preconditioning significantly ameliorates necrosis and apoptosis in the regional ischemic blood-perfused heart.

Diazoxide amelioration of myocardial injury and mitochondrial damage during cardiac surgery

BACKGROUND Recently, we have shown that the selective opening of mitochondrial ATP-sensitive potassium channels with diazoxide significantly decreases myocardial injury. The purpose of this study was to determine the effects of diazoxide on apoptosis and the mechanisms modulating apoptosis and myocardial injury in a blood-perfused model of acute myocardial infarction. METHODS Pigs (32 to 42 kg) undergoing total cardiopulmonary bypass underwent left anterior descending coronary artery occlusion for 30 minutes. The aorta was cross-clamped and magnesium-supplemented potassium cold-blood cardioplegia (DSA; n = 6) or magnesium-supplemented potassium cardioplegia containing 50 micromol/L diazoxide (DZX; n = 6) was administered, followed by 30 minutes of global ischemia and 120 minutes of reperfusion. Left ventricular tissue samples from DSA and DZX hearts were obtained after reperfusion. Apoptosis was determined by TUNEL, caspase-3 and PARP cleavage, and caspase-3 activity. Bax and bcl-2 levels were determined and tissue morphology was examined by light and transmission electron microscopy. RESULTS Apoptosis, as estimated by TUNEL-positive nuclei/3,000 myocardial cells, was 120.3 +/- 48.8 in DSA hearts and was significantly decreased to 21.4 +/- 5.3 in DZX hearts (p < 0.05 vs control). Caspase-3 and poly-ADP-ribose polymerase cleavage and pro-apoptotic bax protein levels were significantly decreased with diazoxide (p < 0.05 vs DSA). Light and transmission electron microscopy indicated severe disruption of tissue with capillary dilatation, mitochondrial cristae damage, and evidence of increased presence of mitochondrial granules in DSA as compared with DZX hearts. CONCLUSIONS The addition of diazoxide (50 micromol/L) to cardioplegia significantly decreases regional myocardial apoptosis and mitochondrial damage, and provides an additional modality for achieving myocardial protection.

Transmission electron microscopy of intracellular particles of polyethylene from joint replacement prostheses: size distribution and cellular response.

The objectives of this transmission electron microscopy study of peri-implant tissues retrieved at revision arthroplasty were to (1) determine the size distribution of intracellular polyethylene particles, and (2) assess the cellular response to phagocytosed polyethylene particles as revealed by the condition of the cellular organelles. The frequency distributions of intracellular polyethylene particle sizes for 15 cases of total hip replacement showed that more than 75% of the particles had lengths of less than 0.5 microm. More than 90% of the particles were less than 1.0 microm in size. In comparison, the frequency distribution for the particles in cellscomprising tissue retrieved from three total knee replacement prostheses showed that only 43% of the particles were less than 0.5 microm in length and 72% were less than 1 microm in size. There was no statistically significant difference in the mean particle length between the specimens from the hip and knee patients. The majority of the cells containing polyethylene were without signs of degeneration. The cytoplasmic and nuclear membranes were intact. Several electron lucent voids which once contained polyethylene particles were seen surrounded by several healthy appearing mitochondria, which displayed sharp membranes and intact cristae. There were no signs of a cytotoxic response to polyethylene at the ultrastructural level.

Magnesium Cardioplegia Enhances mRNA Levels and the Maximal Velocity of Cytochrome Oxidase I in the Senescent Myocardium During Global Ischemia

BACKGROUND The aged myocardium accumulates significantly more cytosolic calcium [Ca2+]i during ischemia, and functional recovery is more severely compromised as compared with the mature heart. Cardioplegia ameliorates these phenomena. The mechanism by which increased calcium accumulation reduces functional recovery in the senescent myocardium is unknown, but it has been suggested that futile calcium cycling in the mitochondria leading to depletion of ATP stores during normothermic global ischemia may be involved. METHODS AND RESULTS To investigate the effect of cardioplegia on mitochondrial calcium ([Ca2+]mt) accumulation and the expression of cytochrome oxidase I (COX I) during global ischemia, mitochondria were isolated from mature (age, 15 to 20 weeks) and aged (age > 130 weeks) rabbit hearts after Langendorff perfusion. Five perfused heart groups were investigated: 30 minutes of global ischemia without treatment (control), with potassium (K, 20 mmol/L), magnesium (Mg, 20 mmol/L), or potassium and magnesium (K/Mg) cardioplegia. No significant difference in [Ca2+]mt was evident in mature hearts with any protocol. In aged hearts, [Ca2+]mt was increased in global ischemia but was ameliorated with Mg and K/Mg cardioplegia. COX I mRNA levels in aged hearts were lower in both control and global ischemia but were increased with cardioplegia. Maximal velocities for COX I were significantly increased with Mg cardioplegia both in the mature and the aged myocardium. CONCLUSIONS K and/or Mg cardioplegia ameliorates [Ca2+]mt accumulation in aged hearts during normothermic global ischemia and increases COX I mRNA levels to a level not significantly different from that found in mature hearts.

Cutaneous metastasis of neuroendocrine carcinoma of uterine origin

We report a case of a 33‐year‐old woman who presented with the rapid development of multiple dermal and subcutaneous nodules on the chest, back, abdomen, axilla, neck, and scalp. These nodules were firm, tender, and non‐ulcerated and measured from 0.2 to 1.2 cm in diameter. A punch biopsy of one of the subcutaneous nodules in the scalp showed a poorly differentiated carcinoma in the reticular dermis with light microscopic, immunohistochemical, and ultrastructural features supporting neuroendocrine differentiation. The past medical history was significant for a recent total abdominal hysterectomy for carcinoma of the cervix at another hospital, originally characterized as a poorly differentiated adenocarcinoma of the cervix and lower uterine segment. Review of the microscopic slides of the uterine neoplasm revealed features suggesting a more appropriate classification as a primary neuroendocrine carcinoma; this was subsequently confirmed by immunophenotyping and ultrastructural evaluation. This is the first reported case of a uterine neuroendocrine carcinoma metastasizing exclusively to the skin. It further illustrates the important contribution of dermatopathology to the understanding of a systemic pathologic process.