Studies on the pathogenesis of ischemic cell injury. VI. Mitochondrial flocculent densities in autolysis

Flocculent densities in the matrix of mitochondria have become quite important in cell pathology since, when prominent, they indicate irreversible cell injury. The morphology and chemical nature of these flocculent densities have been studied in Kidney after various periods of autolysis in vitro in whole tissue samples and in isolated mitochondria. After 30 to 60 min of ischemia, flocculent densities were seen only occasionally and they were most prominent in samples subjected to mechanical damage during isolation. However, in 2- and 4-h samples numerous densities were seen. The size of the densities increased with time, being about 1,400 A in diameter at 4 h. Densities were also seen in mitochondria isolated in medium containing EDTA. They were seen only in the mitochondrial matrix, and could occasionally be found in condensed mitochondria. Small densities were generally round but larger one varied in shape and often appeared as aggregates of smaller densities. Digestion of the densities from water-soluble glycol methacrylate embedded samples was successful with pronase, but neither acid nor lipid solvents were effective. calcium or inorganic phosphate content of isolated mitochondria did not show an increase parallel to the occurrence of flocculent densities. The results suggest that the densities consist predominantly of protein and are probably formed through denaturation of proteins of the mitochondrial matrix and/or the inner membrane.     

Studies on the pathogenesis of ischemic cell injury. XI. P/O ratio and acceptor control.

Acceptor control index, P/O ratio and inner membrane permeability were examined in isolated mitochondria following periods of renal ischemia for 15, 30, 60, 120, and 240 min. It was noted that the P/O ratio remained unchanged until 1-2 h after the onset of ischemia. A similar change was noted in the contraction rate of isolated ischemic mitochondria after swelling in KCl and addition of ATP+Mg2+. Both changes are probably indications of a basic membrane alteration which correlates with the occurrence of irreversibility of cell injury. In contrast, the swelling rate in KCl and the acceptor control index are altered almost simultaneously with the onset of ischemia. Therefore, acceptor control index and the rate of swelling are affected prior to the point of irreversible cell injury. They are not, therefore, good as indicators of irreversible changes in the inner membrane of mitochondria leading to the "point-of-no-return."     

Altered calcium homeostasis in the pathogenesis of myocardial ischemic and hypoxic injury

Pathological calcification, observed in infarcted myocardium under certain conditions, is the most severe manifestation of abnormal calcium (Ca2+) homeostasis induced by ischemia and related forms of myocardial injury. Specialized techniques for measurement of intracellular electrolytes, i.e., electron probe X-ray microanalysis, and intracellular free Ca2+, i.e. carboxylate indicators including fura-2, are providing new insights into regulation of intracellular Ca2+ and the role of altered Ca2+ homeostasis in the pathogenesis of myocardial cell injury. Several lines of investigation indicate that increased intracellular Ca2+ develops in association with other electrolyte alterations, altered cell volume regulation, and altered membrane phospholipid composition during the progression of myocardial cell injury.     

Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury

Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H₂O₂-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H₂O₂-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H₂O₂-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases.     

Protection from DNA damage during an ischemic cell injury

During an ischemic cell injury, cellular NAD levels are lowered and DNA is damaged in a manner similar to other types of injury. Nicotinic acid given to an animal prior to the ischemic insult, caused very little change in the NAD levels but minimized the DNA damage of the injury. This protection is eliminated by 3-aminobenzamide which indicates a possible involvement of poly (ADP-ribose) synthetase in the process.     

Structural alterations of the inner mitochondrial membrane in ischemic liver cell injury

Mitoplasts were prepared from 3-h ischemic livers in an attempt to define the structural alterations in the inner membrane that may account for the functional deficiencies of ischemic mitochondria. Mitoplasts from both control and ischemic livers had similar specific activities of cytochrome oxidase and succinate-cytochrome c reductase. With both preparations, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was 10-fold lower than in the mitochondria from which they were prepared. Ischemic mitoplasts had no respiratory control with ADP, and had a slightly reduced phospholipid to protein ratio and an increased cholesterol to protein ratio. As a result, the cholesterol to phospholipid molar ratio was increased from the control of 0.04 to 0.08. There were also differences in the content of individual phospholipid species. Phosphatidylcholine increased by 15%, while cardiolipin decreased by 60%. There were increases in sphingomyelin and in the lysophospholipids of phosphatidylcholine, ethanolamine, and cardiolipin. Pretreatment with chlorpromazine did not prevent these changes. Linoleic acid was decreased by 35% in ischemic phospholipids, and the content of free fatty acids was increased 4-fold. Electron spin resonance spectroscopy of mitoplasts spin labeled with either 5- or 12-doxyl stearic acid revealed an increased molecular order (decreased fluidity) of ischemic inner mitochondrial membranes consistent with the increased cholesterol to phospholipid ratio. The data indicate activation of a phospholipase A in ischemic mitochondria with the resulting accumulation of products of lipid hydrolysis. This conclusion further emphasizes the close similarity between the structural and functional consequences of ischemia in the intact animal and the effect on isolated mitochondria of the activation of the endogenous phospholipase A. In both cases the major functional alterations are attributable to changes in the permeability of the inner mitochondrial membrane induced by the accumulation of lysophospholipids.     

Programmed cell death in spinal cord injury pathogenesis and therapy

Spinal cord injury (SCI) always leads to functional deterioration due to a series of processes including cell death. In recent years, programmed cell death (PCD) is considered to be a critical process after SCI, and various forms of PCD were discovered in recent years, including apoptosis, necroptosis, autophagy, ferroptosis, pyroptosis and paraptosis. Unlike necrosis, PCD is known as an active cell death mediated by a cascade of gene expression events, and it is crucial for elimination unnecessary and damaged cells, as well as a defence mechanism. Therefore, it would be meaningful to characterize the roles of PCD to not only enhance our understanding of the pathophysiological processes, but also improve functional recovery after SCI. This review will summarize and explore the most recent advances on how apoptosis, necroptosis, autophagy, ferroptosis, pyroptosis and paraptosis are involved in SCI. This review can help us to understand the various functions of PCD in the pathological processes of SCI, and contribute to our novel understanding of SCI of unknown aetiology in the near future.