Wolfgang J. Mergner

Studies on the subcellular pathophysiology of acute lethal cell injury.

Summary In this paper we have summarized the effects of acute lethal injury on the cell. Such injuries are defined as injuries that result in cell death within a relatively short period of time usually minutes or hours. Following death; the cell undergoes necrosis. Ultrastructural and biochemical methods are needed to study pathophysiology. The cell passes through a series of stages numbered 1 through 7. Stages 1 through 4 are reversible while 5 through 7 are irreversible. Injuries resulting in acute cell death and necrosis include direct damage to the cell membrane, for example by antibody and complement or non-penetrating mercurials or interference with mitochondrial energy supply as in ischemia. More complex injuries such as chemical toxicity in vivo probably act through these means. A hypothesis of progression through the stages and the reversibility is discussed.

The use of bilateral adult renal allografts - a method to optimize function from donor kidneys with suboptimal nephron mass.

Alternatives to traditional organ donor usage has allowed expansion of the organ donor pool to help compensate for the increasing disparity between recipients and donors. The use of bilateral adult renal transplants is a novel idea to salvage older donor kidneys with suboptimal nephron mass that would otherwise be destined for discard. Ten renal transplants were performed utilizing both kidneys from adult cadaver donors with diminished nephron mass determined by calculated glomerular filtration rate or biopsy evidence of significant glomerulosclerosis (>10%). Nine of ten (90%) recipients have satisfactory renal function at a mean follow-up of 7 months. The single case of graft failure was due to documented medical non-compliance. Mean serum creatinine at 6 months was 1.5 mg/dl. Mean measured creatinine clearance was 43.2+/-3.4. These preliminary findings suggest that the use of bilateral renal transplants provide satisfactory early function and allows salvage of older donor kidneys with suboptimal nephron mass.

Cellular change in human disease: A new method of pathological analysis*

Abstract The concept of evaluation of cellular alterations as a method for studying the pathogenesis and the manifestations of human disease is presented. Such study is based on evaluation by chemical and morphologic methods performed with human tissues obtained at immediate autopsy and is discussed in the context of cellular reaction to injury. The techniques discussed are readily available and comparable in resources required and difficulty of execution to many other specialized laboratory procedures presently in use. Now is an appropriate time to apply established cell biological principles to the study of human disease.

Aging changes in the human aortic valve in relation to dystrophic calcification

To elucidate the pathogenesis of aging changes and their relation to age associated calcification, a morphological study of 27 human aortic valves was carried out. Nine valves were obtained from immediate autopsies and 18 valves from routine autopsies done within four hours after death. Calcium deposition was present deep in the zona fibrosa along a zone of lipid accumulation. Fibrocytes in the zona fibrosa showed predominant age associated changes, i.e., a massive accumulation of residual bodies in the cytoplasm probably derived from autophagic vacuoles. Light microscopic lipid accumulation corresponded with both intracellular accumulation of electron dense spherules and membranous vesicles derived from degenerate fibrocytes. Calcium deposition in various stages, including needle shaped hydroxyapatite crystals, was seen in close association with these cellular degradation products rather than collagen or elastic fibers. Dystrophic calcification in the aortic valve appears to result from cellular aging and death followed by petrification of cellular degradation products, which may progress to calcific aortic stenosis.

Increased Prevalence of Aortic Fatty Streaks in Cholesterol-Fed Rabbits Administered Intravenous Cocaine: The Role of Vascular Endothelium*1

Several recent postmortem studies suggest an increased prevalence of atherosclerosis in young habitual cocaine abusers. However, little is known about the effects of cocaine abuse on the vascular endothelium and its relationship to atherosclerosis. Therefore, the consequence of chronic administration of intravenous cocaine on the induction of aortic sudanophilia was examined. Male New Zealand White rabbits were fed a 0.5% cholesterol diet for 10 wk. During this period, animals were randomized to receive either cocaine-hydrochloride (0.25 mg/kg) intravenously (n = 17) twice daily; or an equivalent volume of 0.9% physiologic saline, control group (n = 16). Mean values for total circulating leukocytes and platelets and total plasma cholesterol and triglycerides were similar in both groups throughout the protocol. At the completion of the study, aortic sudanophilia was measured and expressed as a percentage of regional involvement (R1 = proximal 4 cm, R2 = middle 6 cm, and R3 = distal 10 cm). Statistical significance among groups was achieved in the proximal thoracic aorta (p = 0.057). No significant differences in sudanophilia were noted in the middle and distal segments. When animals were placed in subgroups according to percent total plaque involvement, there was a significant increased distribution of rabbits with a greater extent of sudanophilia in the cocaine-treated group as compared with control (p = 0.01, chi-square analysis). Immunocytochemical studies using the macrophage-specific and muscle actin-specific monoclonal antibodies demonstrated that sudanophilic areas in both groups were predominantly composed of macrophage-derived foam cells. Evaluation of plaque morphology showed an increase in intimal plaque thickness and in the number of macrophages and smooth muscle cells in cocaine-treated animals; however, group differences were not statistically significant. Because no significant differences were found in the cellular composition of atherosclerotic plaques between groups, further studies were performed to assess the effects of cocaine on the permeability function of cultured endothelial cell monolayers as a possible mechanism of increased sudanophilia. Cocaine (100 μM)-treated endothelial cell monolayers demonstrated an increased permeability to horseradish peroxidase during all time intervals studied (0-6 hr). Permeability differences were statistically significant at 30 min and 1 hr (p = 0.003 and 0.02, respectively). Collectively, these observations suggest that administration of cocaine to cholesterol-fed rabbits increases the prevalence of aortic sudanophilia via at least one possible mechanism involving enhanced vascular permeability.

The application of electron microscopy and cellular biochemistry to the autopsy: Observations on cellular changes in human shock*

A method based on the utilization of electron microscopy, morphometric analysis, tissue culture, and biochemical analysis in the study of human autopsies is described. In this method rapid sampling immediately following somatic death is conducted in order to make meaningful the application of such techniques. In addition to describing the procedure, we present some new findings relating to cellular changes associated with shock. As in cellular pathobiology, it is of utmost importance that ultrastructural changes be correlated with alterations in chemistry and function.

Cocaine: an independent risk factor for aortic sudanophilia. A preliminary report

Several recent autopsy reports indicate an increased prevalence of coronary atherosclerosis in ischemic heart disease temporally associated with cocaine abuse. The objective of this study was to conduct a retrospective analysis of sudanophilic lesions in young asymptomatic individuals who abused cocaine. Twenty-six cases (15-34-year-old black males) were examined from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. Sixteen subjects (mean age 25 +/- 1 years) had a positive toxicologic screen for cocaine and/or its major metabolites at autopsy and were confirmed habitual cocaine abusers. The remaining 10 cases (mean age 24 +/- 2 years) were subjects with a negative toxicologic screen at autopsy and no history of illicit drug abuse. Post-mortem blood was collected for lipoprotein analysis and determination of smoking status. The aorta and right coronary arteries were stained with Sudan IV and the degree and extent of sudanophilia was quantitated by image analysis. Multiple linear regression analysis of cocaine, age, smoking status, VLDL+LDL-C/HDL-C ratio and HDL-C as predictor variables of percentage intimal surface involvement, revealed an association between cocaine abuse and the extent of sudanophilia in both the thoracic and abdominal aorta (P = 0.002 and 0.049, respectively). Analysis of risk factors or of cocaine abuse as predictors of sudanophilia did not achieve statistical significance in the right coronary artery. These preliminary results suggest that habitual use of cocaine, through unknown mechanism(s), increases aortic sudanophilia independent of traditional risk factors.

Long-term culture of human aortas. Development of atherosclerotic-like plaques in serum-supplemented medium.

SummarySegments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures.

Myocardial protection by perfluorochemical infusion during transient ischemia produced by balloon coronary occlusion.

To assess the efficacy of the perfluorochemical Fluosol-DA 20% for myocardial protection during repeated periods of balloon occlusion of the left circumflex coronary artery, 25 anesthetized dogs were randomized to receive either oxygenated perfluorochemical, (Fluosol-DA [F]; n = 10) or oxygenated Ringers lactate (R; n = 6), at the rate of 30 ml/min, during inflation of the balloon. A control group (C; n = 9) received no infusion. A total of eight inflations were performed, each lasting 90 seconds, followed by an equivalent deflation time. Hemodynamics, ECGs, regional myocardial function, and biochemical parameters were studied. Significant differences were noted in ST segment elevation at 90 seconds of inflation in the F (1.5 mm +/- 0.6), C (3.7 mm +/- 0.75), and R (2.9 mm +/- 0.75) groups (F vs C or R p less than 0.05). This was associated with significant improvement in radial shortening in the jeopardized zone at 45 seconds into occlusion in the F group compared to the C and R groups (F = 21.1% +/- 5.1 vs C = 3.5% +/- 4.5 or R = 1.1% +/- 3.2; p less than 0.05). Results of electron microscopy showed reversible changes of ischemia within the mitochondria, and these were most marked in the C and R groups compared to the F group. Endothelial swelling was mild and was present only focally in the R and C groups. Thus perfluorochemicals may enhance the safety and efficacy of balloon angioplasty.

Studies on the pathogenesis of ischemic cell injury: IV. Alteration of ionic permeability of mitochondria from ischemic rat kidney

Abstract This study reports reversible and irreversible changes in ionic permeability of mitochondria isolated at serial time intervals from ischemic rat kidney. It was found that mitochondrial permeability changes occur shortly after the onset of ischemia and accelerate until 30–60 minutes. At later intervals no further acceleration occurs. Contraction of swollen mitochondria correlates well with the swelling rate until 60 minutes; after this ischemic interval the rate of contraction declines drastically. There was a good correlation between the rate of contraction and the total amount of swelling or the initial rate of swelling before 1–2 hours of ischemic time. Changes in ΔOD of mitochondrial responses were correlated with electron micrographs of samples before swelling, after swelling and after addition of contraction medium. On the basis of the data presented it is proposed that permeability changes of the mitochondrial inner membrane represent important indicators of alterations occurring in the fine structure of the inner membrane. It is furthermore proposed that the initial alteration is reversible.