Degree Of Mitochondrial Swelling Research Articles

Activation of Pink1/Parkin-mediated mitochondrial autophagy alleviates exertional heat stroke-induced acute lung injury in rats.

To investigate the role of Pink1/Parkin-mediated mitochondrial autophagy in exertional heat stroke-induced acute lung injury in rats. Sixty SD rats were divided into four groups: normal group (CON group), normal Parkin overexpression group (CON + Parkin group), exertional heat stroke group (EHS group), and exertional heat stroke Parkin overexpression group (EHS + Parkin group). Adeno-associated virus carrying the Parkin gene was intravenously injected into the rats to overexpress Parkin in the lung tissue. An exertional heat stroke rat model was established, and survival curves were plotted. Lung micro-CT was performed, and lung coefficient and pulmonary microvascular permeability were measured. Compared with the EHS group, the survival rate of rats in the EHS + Parkin overexpression group was significantly increased, lung coefficient and pulmonary microvascular permeability were reduced, and pathological changes such as exudation and consolidation were significantly reduced. The levels of inflammatory factors IL-6, IL-1β, TNF- α, and ROS were significantly decreased; the degree of mitochondrial swelling in type II alveolar epithelial cells was reduced, and no vacuolization was observed. Lung tissue apoptosis was reduced, and the colocalization fluorescence of Pink1 and Parkin, as well as LC3 and Tom20, were increased. The expression of Parkin and LC3-II/LC3-I ratio in lung tissue were both increased, while the expression of P62, Pink1, MFN2, and PTEN-L was decreased. Impairment of Pink1/Parkin-mediated mitochondrial autophagy function is one of the mechanisms of exertional heat stroke-induced acute lung injury in rats. Activation of the Pink1/Parkin pathway can alleviate acute lung injury caused by exertional heat stroke.

Cyclophilin D Regulates Oxidative Stress and Apoptosis via Mitochondrial Permeability Transition Pore in Acute Acalculous Cholecystitis.

Acute acalculous cholecystitis (AAC) is characterized by acute onset, rapid progression, high mortality, and various complications. Cyclophilin D (CypD) regulates the mitochondrial permeability transition pore (MPTP) and is involved in the occurrence of ischemia-reperfusion injury and inflammation; however, the role of CypD in AAC remains unclear. Guinea pigs of 300-350 g were randomly divided into three groups, namely the sham group, the common bile duct ligation-24h group (CBDL-24h group), and the CBDL-48h group. Western blot and qRT-PCR were applied to analyze the differential expression of CypD in each group, and transmission electron microscopy was employed to detect changes in mitochondrial structure. Inhibiting the activity of CypD by Cyclosporine A (CsA), we evaluated the difference of mitochondrial utilizing mitochondrial swelling, reactive oxygen species (ROS) detection and mitochondrial membrane potential. Compared with the sham group, the prolongation of obstruction aggravated gallbladder inflammation and upregulated CypD expression in the CBDL-24h and CBDL-48h groups. The degree of mitochondrial swelling was increased, and the opening of MPTP was prolonged in the CBDL-24h and 48h groups. Decreasing the expression of CypD could repress the opening of MPTP, prevent manipulation of the mitochondrial membrane potential, and ultimately diminish the levels of intracellular ROS and apoptosis. CypD plays a proinflammatory role in the development of AAC by regulating the opening of MPTP. Inhibiting the activity of CypD could reduce the levels of ROS and apoptosis, rescue the function of mitochondria and finally alleviate AAC. Therefore, CypD might serve as a potential therapeutic target for ACC.

Effects of mitophagy on aerobic exercise intervention for depression in rats

Objective: This experiment was designed to observe the effects of aerobic exercise on depressive behavior in rats induced by chronic unpredictable mild stress (CUMS), and to explore the possible mechanism by detecting the proteins related to mitochondrial autophagy. Methods: SD rats were randomly divided into three groups: blank control group (C, n=12), depression model group (D, n=12) and post-depression exercise group (D+E, n=12). Group D and D+E were modeled with CUMS for 28 days, and group D+E underwent aerobic exercise intervention for 4 weeks after model establishment. Then the behavior of rats was evaluated. The concentrations of whole brain dopamine and norepinephrine were determined by ELISA kits. The morphology and structure of mitochondria in the frontal lobe were observed with the transmission electron microscope (TEM). Mitochondrial autophagy lysosomes were localized by immunofluorescence colocalization. The expressions of LC3 and P62 proteins in the frontal lobe were measured with Western Blotting. The relative content of mitochondrial DNA was detected using Real-time PCR. Results: ①Compared with group C, the sucrose preference ratio in group D was decreased significantly(P＜0.01); Compared with group D, the sucrose preference ratio in group D+E was increased significantly (P＜0.01). ②In the open field experiment, compared with group C, group D had a significant decrease in activity, average speed and total distance (P＜0.05); Compared with group D, the average rate of activity in group D+E was significantly higher (P＜0.05). ③ELISA results showed that the levels of whole brain dopamine and norepinephrine were significantly lower in group D rats than those in group C (P＜0.05). ④Under transmission electron microscopy, compared with group C, group D had different degrees of mitochondrial swelling, decreased crest density, and intermembrane space dilation.; Compared with group D, a significant increase in mitochondrial autophagosomes and autophagic lysosomes was observed in neurons in group D+E. Increased co-localization of mitochondria with lysosomes in the D+E group could be observed under fluorescence microscopy. ⑤Compared with group C, the expression of P62 was increased significantly(P＜0.05), and LC3II/LC3I ratio was decreased significantly (P＜0.05) in group D; Compared with group D, LC3II/LC3I ratio was significantly higher in group D+E than that in group D (P＜0.05). ⑥Compared with group C, the relative number of mitochondrial DNA in the frontal lobe of group D was increased significantly (P＜0.05). Conclusion: Aerobic exercise has a significant improvement effect on depression induced by CUMS in rats, and its mechanism may be related to the upregulation of the level of linear autophagy.

Effect of methylene blue preconditioning on ischemia-reperfusion injury in isolated rat lungs

Objective To evaluate the effect of methylene blue(MB)preconditioning on ischemia-reperfusion(I/R)injury in isolated rat lungs. Methods Eighteen pathogen-free healthy male Sprague-Dawley rats, aged 3 months, weighing 240-320 g, were divided into 3 groups(n=6 each)using a random number table: sham operation group(group Sham), lung I/R group(group I/R)and methylene blue preconditioning group(group MB). A model of isolated lung I/R injury was established in pentobarbital sodium-anesthetized rats.MB 2 mg/kg was intraperitoneally injected at 2 h before stopping perfusion in group MB.Isolated lungs were perfused for 20 min, followed by 45-min ischemia, and then reperfused for 60 min in I/R and MB groups.At 60 min of reperfusion, the activity of lactic dehydrogenase(LDH)in the perfusate was detected, wet weight(W)and dry weight(D)was determined, W/D ratio was calculated, and the levels of malondialdehyde(MDA), ATP, reactive oxygen species(ROS)and superoxide dismutase(SOD)were measured in lung tissues.Mitochondria and cytoplasm were isolated from lung tissues for determination of mitochondrial membrane potential(MMP), degree of mitochondrial swelling and content of cytochrome C(Cyt c)in cytoplasm.Apoptotic cells in lung tissues were detected using TUNEL, and apoptotic index was calculated. Results Compared with group Sham, the activity of LDH in perfusate, W/D ratio, levels of ROS, MDA and Cyt c in cytoplasm and apoptosis index were significantly increased, the degree of mitochondrial swelling was aggravated, and the content of ATP and MMP were decreased in I/R and MB groups, and the SOD activity was significantly decreased in group I/R(P<0.05). Compared with group I/R, the activity of LDH in perfusate, W/D ratio, levels of ROS, MDA and Cyt c in cytoplasm and apoptosis index were significantly decreased, the degree of mitochondrial swelling was attenuated, and the activity of SOD, content of ATP and MMP were increased in group MB(P<0.05). Conclusion Methylene blue preconditioning can reduce I/R injury in isolated rat lungs, and the mechanism may be related to improving mitochondrial function and inhibiting cell apoptosis. Key words: Methylene blue; Ischemic preconditioning; Reperfusion injury; Lung

Correlation between uncoupling protein 2 expression and myocardial mitochondrial injury in rats with sepsis induced by lipopolysaccharide

To investigate the correlation between uncoupling protein 2 (UCP2) expression and myocardial mitochondria injury in rats with sepsis induced by lipopolysaccharide (LPS). The rat model of sepsis was established through an intraperitoneal injection of LPS. Forty male Sprague-Dawley rats were randomly and equally divided into control group (an intraperitoneal injection of normal saline), sepsis 6 h group (LPS-6 h group), sepsis 12 h group (LPS-12 h group), sepsis 24 h group (LPS-24 h group), and sepsis 48 h group (LPS-48 h group). The serum and heart tissues were harvested at corresponding time points and myocardial mitochondria was extracted. The microplate reader was applied to measure creatine kinase (CK), creatine kinase-MB (CK-MB), and reactive oxygen species (ROS). Flow cytometry was applied to measure the degree of mitochondrial swelling and mitochondrial membrane potential (MMP). Western blot was used to measure the expression level of UCP2. Electron microscopy was applied to observe the morphological changes in heart tissues and myocardial mitochondria. Compared with the control group, the LPS groups had significantly increased serum levels of CK, CK-MB, and myocardial ROS, as well as a significantly increased degree of mitochondrial swelling (P<0.05), and these values reached their peaks at 24 hours after LPS injection. The LPS groups had a significant decrease in MMP (P<0.05), which reached the lowest level at 24 hours after LPS injection. Western blot showed that the LPS groups had a significant increase in the expression level of myocardial UCP2 compared with the control group (P<0.05), which reached its peak at 24 hours after LPS injection. The results of electron microscopy showed mitochondrial swelling, partial rupture of the mitochondrial membrane, and cavity formation in rats in the LPS groups. The most severe lesions occurred in the LPS-24 h group. In rats with LPS, the ROS level in the myocardial mitochondria and the degree of mitochondrial swelling were positively correlated with the expression level of UCP2 (r=0.796 and 0.893, respectively; P<0.05), while MMP was negatively correlated with the expression level of UCP2 (r=-0.903, P<0.05). In the rat model of sepsis, the myocardium and myocardial mitochondria have obvious injuries, and the expression level of UCP2 is closely correlated with mitochondrial injury. Therefore, UCP2 might play an important role in myocardial mitochondrial injury in sepsis.

Protective effect of continuous intravenous infusion of Isoproterenol on myocardial mitochondria of early septic rats and the corresponding mechanism

Objective To investigate the protective effect of continuous intravenous infusion of Isoproterenol (ISO)on myocardial mitochondria of early septic rats and the corresponding mechanism. Methods Thirty Sprague Dawley (SD)rats were randomly divided into 5 groups (6 cases per group): control group, endotoxin group, ISO small-dose group, ISO medium-dose group and ISO large-dose group.Endotoxin group and ISO intervene group received same management apart from drug intervention: receiving intravenous injection of lipopolysaccharide (LPS)10 mg/kg followed by an continuous intravenous infusion of 9 g/L saline 1 mL/h or ISO 0.06 μg/(kg·min), 0.30 μg/(kg·min)and 0.60 μg/(kg·min). Control group received intraperitoneal injection and continuous intravenous infusion with the same amount of 9 g/L saline.The primary endpoint of the study was 24 hours after injection of 9 g/L saline or LPS.Serum creatine kinase (CK)and creatine kinase isoenzyme (CK-MB), oxidative and nitrosative stress levels and swelling of isolated heart mitochondrion were detected.The pathological changes of the myocardium and morphologic changes of the heart mitochondria were observed through light microscope and scanning electron microscope, respectively. Results The levels of CK, CK-MB, nitric oxide (NO)content, inducible nitric oxide synthase (iNOS)activity and malondialdehyde (MDA)content in endotoxin group were increased compared with control group (all P<0.05), while the superoxide dismutase (SOD) activity decreased [(11.543±1.080) U/mg prot vs (9.892±0.815) U/mg prot, P<0.05]. The morphology of the heart mitochondria significantly changed (such as swelling, disordered arrangement, crest fracture, fusion and cavitations, and so on). ISO intervention significantly decreased the levels of CK, CK-MB and mitochondrial swelling (all P<0.05)and increased the SOD activity (all P<0.05). The levels of NO content, iNOS activity and MDA content were significantly decreased in small-dose group[(10.823±2.240) μmol/g prot vs (7.917±2.203) μmol/g prot, (0.045±0.008) U/mg prot vs (0.033±0.003) U/mg prot, (1.663±0.618) mmol/mg prot vs (0.768±0.312) mmol/mg prot, all P<0.05], while the levels of iNOS activity and MDA content were significantly increased in medium- and large-dose group (all P<0.05); compared with medium-dose group, the degree of mitochondrial swelling in large-dose group increased (1.160±0.186 vs 1.393±0.128, P<0.05). The pathological changes of the myocardium mitochondria significantly improved. Conclusions The myocardium and myocardial mitochondria of early septic rats were damaged, continuous intravenous infusion of low-dose ISO revealed protective effect on these damages, and the corresponding mechanism may relate to the decrease of the oxidative and nitrosative stress. Key words: Isoproterenol; Lipopolysaccharide; Sepsis; Oxidative stress; Mitochondrion

Effects of grape seed proanthocyanidin extract on pentylenetetrazole-induced kindling and associated cognitive impairment in rats.

Numerous studies have demonstrated the antioxidant effects of grape seed proanthocyanidin extract (GSPE). The generation of free radicals and the ensuing apoptosis may contribute to the pathogenesis of epilepsy; therefore, in the present study, we examined the effects of GSPE on cognitive impairment and neuronal damage induced by chronic seizures in rats. Seizures were induced by a daily intraperitoneal (i.p.) injection of pentylenetetrazole (PTZ; 35 mg/kg/day, 36 days). Two other groups were treated with GSPE (100 or 200 mg/kg/day, orally) for 24 days and then for 36 days prior to each PTZ injection. After the final PTZ injection, hippocampus-dependent spatial learning was assessed using the Morris water maze (MWM). The rats were then sacrificed for the measurement of hippocampal malondialdehyde (MDA, a measure of lipid peroxidation) and glutathione (GSH, a measure of endogenous antioxidant capacity) levels, and for the expression of pro-apoptotic factors [cytochrome c (Cyt c), caspase-9 and caspase-3]. The mitochondrial generation of reactive oxygen species (ROS), degree of mitochondrial swelling, neuronal damage and mitochondrial ultrastructure were also examined. Performance in the MWM was markedly impaired by PTZ-induced seizures, as evidenced by longer escape latencies during training and fewer platform crossings during the probe trial. This cognitive decline was accompanied by oxidative stress (MDA accumulation, ROS generation, reduced GSH activity), an increased expression of pro-apoptotic proteins, as well as damage to CA1 pyramidal neurons and the mitochondria. Pre-treatment with GSPE dose-dependently reversed PTZ-induced impaired performance in the MWM, oxidative stress, mitochondrial ROS generation, the expression of pro-apoptotic proteins and neuronal and mitochondrial damage. Thus, GSPE may reverse the hippocampal dysfunction induced by chronic seizures, by reducing oxidative stress and preserving mitochondrial function.

Hydrogen-rich saline attenuates neuronal ischemia–reperfusion injury by protecting mitochondrial function in rats

BackgroundHydrogen, a popular antioxidant gas, can selectively reduce cytotoxic oxygen radicals and has been found to protect against ischemia–reperfusion (I/R) injury of multiple organs. Acute neuronal death during I/R has been attributed to loss of mitochondrial permeability transition coupled with mitochondrial dysfunction. This study was designed to investigate the potential therapeutic effect of hydrogen-rich saline on neuronal mitochondrial injury from global cerebral I/R in rats. Materials and methodsWe used a four-vessel occlusion model of global cerebral ischemia and reperfusion, with Sprague–Dawley rats. The rats were divided randomly into six groups (n = 90): sham (group S), I/R (group I/R), normal saline (group NS), atractyloside (group A), hydrogen-rich saline (group H), and hydrogen-rich saline + atractyloside (group HA). In groups H and HA, intraperitoneal hydrogen-rich saline (5 mL/kg) was injected immediately after reperfusion, whereas the equal volume of NS was injected in the other four groups. In groups A and HA, atractyloside (15 μL) was intracerebroventricularly injected 10 min before reperfusion, whereas groups NS and H received equal NS. The mitochondrial permeability transition pore opening and mitochondrial membrane potential were measured by spectrophotometry. Cytochrome c protein expression in the mitochondria and cytoplasm was detected by western blot. The hippocampus mitochondria ultrastructure was examined with transmission electron microscope. The histologic damage in hippocampus was assessed by hematoxylin and eosin staining. ResultsHydrogen-rich saline treatment significantly improved the amount of surviving cells (P < 0.05). Furthermore, hydrogen-rich saline not only reduced tissue damage, the degree of mitochondrial swelling, and the loss of mitochondrial membrane potential but also preserved the mitochondrial cytochrome c content (P < 0.05). ConclusionsOur study showed that hydrogen-rich saline was able to attenuate neuronal I/R injury, probably by protecting mitochondrial function in rats.

By improving regional cortical blood flow, attenuating mitochondrial dysfunction and sequential apoptosis galangin acts as a potential neuroprotective agent after acute ischemic stroke.

Ischemic stroke is a devastating disease with a complex pathophysiology. Galangin is a natural flavonoid isolated from the rhizome of Alpina officinarum Hance, which has been widely used as an antioxidant agent. However, its effects against ischemic stroke have not been reported and its related neuroprotective mechanism has not really been explored. In this study, neurological behavior, cerebral infarct volumes and the improvement of the regional cortical blood flow (rCBF) were used to evaluate the therapeutic effect of galangin in rats impaired by middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia. Furthermore, the determination of mitochondrial function and Western blot of apoptosis-related proteins were performed to interpret the neuroprotective mechanism of galangin. The results showed that galangin alleviated the neurologic impairments, reduced cerebral infarct at 24 h after MCAO and exerted a protective effect on the mitochondria with decreased production of mitochondrial reactive oxygen species (ROS). These effects were consistent with improvements in the membrane potential level (Δψm), membrane fluidity, and degree of mitochondrial swelling in a dose-dependent manner. Moreover, galangin significantly improved the reduced rCBF after MCAO. Western blot analysis revealed that galangin also inhibited apoptosis in a dose-dependent manner concomitant with the up-regulation of Bcl-2 expression, down-regulation of Bax expression and the Bax/Bcl-2 ratio, a reduction in cytochrome c release from the mitochondria to the cytosol, the reduced expression of activated caspase-3 and the cleavage of poly(ADP-ribose) polymerase (PARP). All these data in this study demonstrated that galangin might have therapeutic potential for ischemic stroke and play its protective role through the improvement in rCBF, mitochondrial protection and inhibiting caspase-dependent mitochondrial cell death pathway for the first time.

Effects of sevoflurane pretreatment on myocardial injury in patients undergoing cardiac valve replacement with cardiopulmonary bypass

Objective To investigate the effects of sevoflurane pretreatment on the myocardial injury in patients undergoing cardiac valve replacement with cardiopulmonary bypass (CPB).Methods Twenty NYHA class Ⅱ or Ⅲ patients,aged ＜ 60 yr,undergoing cardiac valve replacement with CPB,were randomly divided into 2 groups (n =10 each):sevoflurane group (group S) and control group (group C).The patients were premeditated with intramuscular morphine and scopolamine.Anesthesia was induced with iv injection of midazolam 0.05-0.08 mg/kg,fentanyl 10-15 μg/kg and pipecuronium 0.08-0.10 mg/kg.Anesthesia was maintained with intermittent iv boluses of midazolam,fentanyl and pipecuronium and in addition sevoflurane was inhaled before aortic clamping and the end-tidal concentration was rapidly increased to 1.0％ and maintained at the level for 5 min in group S.Blood samples were taken from the central vein before skin incision (T1),immediately after aortic clamping (T2 ),at 0 and 30 min after aortic unclamping (T3-4),and at 2,6,12 and 24 h after operation (T5-8) for determination of the concentration of serum cardiac troponin Ⅰ (cTnI) and activities of creatine kinase (CK) and creatine kinase isoenzyme-MB (CK-MB).Myocardial specimens were taken from right auricle before aortic clamping and at 10 min after aortic unclamping for electron microscopic examination.Results The concentration of serum cTnI and activities of CK and CK-MB were significantly increased at T4-8 in both groups ( P ＜ 0.05).The serum cTnI concentration at T4-8 and the activities of CK and CK-MB at T8 were significantly lower in group S than in group C ( P ＜0.05).Different degrees of mitochondrial swelling were observed after aortic unclamping in both groups,but the changes were milder in group S than in group C.Conclusion Sevoflurane pretreatment can attenuate the myocardial injury in patients undergoing cardiac valve replacement with CPB. Key words: Anesthetics, inhalation; Cardiopulmonary bypsss; Myocardial reperfusion injury; Heart valve prosthesis implantation

Acetaminophen reduces mitochondrial dysfunction during early cerebral postischemic reperfusion in rats

Acetaminophen, a popular analgesic and antipyretic, has been found to be effective against neuronal cell death in in vivo and in vitro models of neurological disorders. Acute neuronal death has been attributed to loss of mitochondrial permeability transition coupled with mitochondrial dysfunction. The potential impact of acetaminophen on acute injury from cerebral ischemia–reperfusion has not been studied. We investigated the effects of acetaminophen on cerebral ischemia–reperfusion-induced injury using a transient global forebrain ischemia model. Male Sprague–Dawley rats received 15 mg/kg of acetaminophen intravenously during ischemia induced by hypovolemic hypotension and bilateral common carotid arterial occlusion, which was followed by reperfusion. Acetaminophen reduced tissue damage, degree of mitochondrial swelling, and loss of mitochondrial membrane potential. Acetaminophen maintained mitochondrial cytochrome c content and reduced activation of caspase-9 and incidence of apoptosis. Our data show that acetaminophen reduces apoptosis via a mitochondrial-mediated mechanism in an in vivo model of cerebral ischemia–reperfusion. These findings suggest a novel role for acetaminophen as a potential stroke therapeutic.

Ultrastructural pathology of human hydrocephalic cerebral cortex

The ultrastructural pathology of cerebral cortex in human hydrocephalus is described. Cortical biopsies of 17 patients of ages ranging from 10 days to 21 years old examined in our laboratory are described. Nerve cells show moderate and severe swelling characterized by dilation of endoplasmic reticulum canaliculi and perinuclear cistern, edema and degenerative changes of Golgi apparatus, variable degrees of mitochondrial swelling, and fragmentation of plasma membrane. The neighboring neuropil exhibits notable enlargement of extracellular space and signs of synaptic plasticity and degeneration. The astrocytes display edematous changes and phagocytic activity. Glycogen rich- and glycogen-depleted astrocytes are observed. Oligodendroglial cells appear normal in certain cases and in others show moderate hydropic changes. Numerous myelin figures are observed in some unidentified nerve cell processes. The capillary wall shows evident signs of blood-brain barrier dysfunction featured by increased endothelial vesicular and vacuolar transport, closed and open interendothelial junctions, and in severely edematous areas thinning and breakdown of capillary basement membrane. The role of ischemia, oxidative stress, increased calcium concentration, activation of N-methyl-d-aspartate (NMDA) receptors, and disturbance of ion homeostasis are discussed in relationship with the fine structural alterations of hydrocephalic brain parenchyma.

Effect of drug–light interval on the mode of action of Photofrin photodynamic therapy in a mouse tumor model

Our objective was to examine the effect of time intervals between Photofrin injection and laser irradiation [i.e., drug-light interval (DLI)] on the mode of action of Photofrin photodynamic therapy (PDT). Kunming mice transplanted with sarcoma-180 cells were used as an animal model. The tumor-bearing mice in the control group were given neither photosensitizer nor laser irradiation. PDT groups were given intravenous (i.v.) injection of Photofrin (7.5 mg/kg) prior to being irradiated with a 630 nm laser at 120 J/cm(2) at different DLIs (1 min-48 h). Tumors and overlying skin were visually examined daily. Histopathological and electron microscopic examinations were carried out 48 h after PDT. Survival rates were recorded. The mice in the groups that had experienced short DLIs (<60 min) showed stronger skin reactions than the groups subjected to long DLIs (>6 h). Histological examination showed that antitumor effects were achieved mainly by the destruction of tumor blood vessels and the formation of thrombosis at short DLIs, whereas, at long DLIs, the tumor cells were killed directly by PDT-mediated cytotoxicity. Electron microscopy revealed various degrees of mitochondrial swelling. The survival rate of the mice subjected to long DLIs was slightly higher than that of the mice subjected to short DLIs. Both vascular (e.g., tumor vessel destruction) and cellular (e.g., cytotoxicity) effects contributed to Photofrin PDT-induced tumor ablation.

Oxidative damage to mitochondria is mediated by the Ca(2+)-dependent inner-membrane permeability transition.

The ability of O2 metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. Under 2,4-dinitrophenol-uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in O2 consumption supported by NAD(+)-linked substrates, but showed almost no change in O2 consumption in the presence of succinate and ascorbate. Oxidative stress caused the loss of intramitochondrial nicotinamide nucleotides, and addition of NAD+ fully prevented any fall in O2 consumption with NAD(+)-linked substrates. The activity of electron-transfer complex I (NADH oxidase and NADH-cytochrome c oxidoreductase) and the energy-dependent reduction of NAD+ by succinate were unaltered by oxidative stress. Exposure to free radicals also had an uncoupling effect at all three coupling sites. The degree of mitochondrial swelling was closely correlated with the inhibition of State-3 oxidation of site-I substrates and with the increase in State-4 oxidation of succinate. The immunosuppressive agent cyclosporin A completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, Ca2+ release, sucrose trapping, uncoupling and selective inhibition of the mitochondrial respiration of site-I substrates). The same protective effect was found when Ca2+ cycling was prevented, either by chelating Ca2+ with EGTA or by inhibiting Ca2+ reuptake with Ruthenium Red. These findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cyclosporin A-sensitive and Ca(2+)-dependent membrane transition, and not by direct impairment of the mitochondrial inner-membrane enzymes.

Close Correlations between Mitochondrial Swelling and ATP-Content in the Ischemic Canine Myocardium: A Combined Morphometric and Biochemical Study

This study investigates how far mitochondrial swelling in the ischemic heart is influenced by factors pertaining to anaerobic energy turnover. Canine hearts were arrested by aortic cross clamping or cardioplegically with St. Thomas or HTK solution and incubated at 25 degrees C in the solution used for cardiac arrest. Samples of the left ventricle were taken at the end of cardiac arrest and during ischemia for structural evaluation and biochemical analysis. The extracellular pH in the interventricular septum was measured. Mitochondrial swelling was determined with the surface to volume ratio, a parameter independent of the reference space. Values obtained for different swelling were related to defined metabolite concentrations and pHe values to establish possible correlations between structural and biochemical parameters in the ischemic heart. At the onset of ischemia and during the breakdown of creatine phosphate (CP) to 3 mumol/g wet weight mitochondrial volume depends on the method of cardiac arrest and does not increase significantly in any of the three groups. The degree of mitochondrial swelling after depletion of CP correlates with the decline in ATP independent of the form of cardiac arrest. Characteristic values of the surface to volume ratio ascertained at different times of ischemia for all groups correspond to determined ATP concentrations. Acid pHe values seem to intensify mitochondrial swelling. With increased lactate concentrations mitochondria swell, but first initially the degree of swelling differs significantly in the forms of cardiac arrest investigated. Thus, the surface to volume ratio is a powerful and valid ultrastructural parameter, which makes correlations between mitochondrial structure and metabolism possible and furthermore indicates a strong correlation between mitochondrial swelling and ATP-concentration in the ischemic heart.

Cataract in retinitis pigmentosa. An analysis of cataract surgery results and pathological lens changes.

The result of cataract surgery and lens pathoanatomical examination were presented in patients with retinitis pigmentosa (RP). A retrospective study of 24 cataract extractions in 15 RP patients was carried out, with special regard to visual outcome and surgical complications. The results were compared to an age matched randomly selected control group. In the retinitis pigmentosa group the post-operative visual acuity was generally good, and the surgical complications except for one case of central venous thrombosis were not more frequent than in the control group. Seven lenses from 5 patients with RP were analysed using transmission electron microscopy and quantitative microradiography. In the lenses with a progressive subcapsular edema, extensive extracellular vacuolization was found in the lens epithelium. Focal degeneration of epithelial cells was seen both in the more stationary posterior subcapsular cataracts and the progressive subcapsular cataracts. Different degrees of mitochondrial swelling was found in the epithelial cells of the stationary cataracts. The subcapsular lens fiber changes consisted of an extensive swelling of hydrated cells together with a significant reduction in their dry mass content. Migrating elongated nuclei-containing cells were found on the posterior capsule in both stationary and progressive cataracts.

Histological, ultrastructural and mitochondrial oxidative phosphorylation studies in liver of rats chronically treated with oral valproic acid

The effects of oral administration of valproic acid (VPA) have been studied on the liver of female adult Wistar rats, at doses of 60 and 120 mg/kg/day during 4 and 18 weeks (periods I and II). Other groups received the same doses of VPA with phenobarbital (PB). The animals developed periportal steatosis, more intense at the end of period II and in groups non-induced by PB, and also some degree of mitochondrial swelling. An uncoupling of the oxidative phosphorylation was also evident with both doses in the non-induced groups, and only with the high dose of VPA in the induced group. An in vitro study on isolated liver mitochondria showed uncoupling only with very high concentration of VPA (10 mM). We comment on these findings and propose a chronic model of experimental hepatotoxicity of oral VPA in the rat.

Electrolyte versus Blood Cardioplegia: Randomized Clinical and Myocardial Ultrastructural Study

In 40 consecutive patients undergoing coronary artery bypass, one of two solutions for cardioplegia, each containing 30 mEq/L of K + was used randomly. The groups were comparable except for intramyocardial temperature. With electrolyte solution (Group A), it was 16.5° ± 0.34°C, while with blood from the pump-oxygenator (Group B) it was 20.3° ± 0.41°C ( p < 0.001). After bypass left atrial pressure (LAP) was 11.9 ± 0.67 torr in Group A and 8.1 ± 0.49 torr in Group B ( p < 0.001). CPK-MB was elevated in 45% of Group A patients versus 15% in Group B ( p < 0.05). No patient died. Two myocardial infarctions occurred in Group A and one in Group B. Stereological morphometric electron microscopy was performed on biopsy specimens taken from the left ventricle (1) before perfusion, (2) after cardioplegia, and (3) 30 minutes after reperfusion. Group A showed marked intracellular edema, mitochondrial swelling, pronounced depletion of glycogen stores, and focal myofibrillary disorganization. Group B showed near normal myocardial ultrastructure with increased glycogen stores and minimal mitochondrial swelling. Morphometric analysis revealed a statistically significant increase in the degree of mitochondrial swelling (51%) in Group A compared with Group B after reperfusion ( p < 0.001). Thus, blood K + cardioplegia resulted in better preservation of myocardial ultrastructure, lower ventricular filling pressure, and lesser CPK-MB release compared with this particular electrolyte cardioplegia.

Pea mitochondrial lipids and their oxidation during mitochondria swelling.

AbstractThe composition and oxidation of lipids in pea mitochondria induced by swelling in presence of some transitional metal ions and ascorbic acid has been studied.Among the major mitochondrial lipids found were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and diphosphatidylglycerol. The major fatty acids present in these lipids were linoleic, oleic and palmitic acids. The average unsaturation for mitochondria total fatty acids, expressed as Double Bond Index was 1.44, while the unsaturation for fatty acids of individual lipids varied between 1.02 and 1.73 being highest for an unidentified phospholipid and 1.44 for phosphatidylcholine present in mitochondria as the major lipid.Mitochondria isolated from pea seeds germinated for 4 days were found to be tightly coupled and to have an ADP/O ratio of 1.3. The swelling assay in 0.1 M‐KCl for 1‐day‐old mitochondria has shown a swelling ability matching those of 4 and up to 8 days of age. Isolated mitochondria aged for 24 h at 2–4º C showed a swelling comparable to that of the fresh preparation. Swelling in the presence of ascorbic acid did not induce mitochondrial lipid oxidation. However, this was brought about in the presence of Cu2+ ions. The presence of Fe2+ ions induced the highest degree of oxidation, and instead of a concurrent swelling, it induced contraction. At the same level of ions, Fe2+ was more effective than Fe3+. Contrary to iron used at 4% of its physiological level, Mn2+ and Co2+ used at 12 to 24% of their levels found in pea seeds were ineffective in inducing oxidation. Nevertheless, the degree of mitochondrial swelling was highest in the medium containing KCl, lower in the presence of Co2+ and ascorbic acid and lowest in the presence of Fe3+. These results suggested that mitochondrial lipid oxidation does not depend on the degree of swelling.The lipid peroxides formed within mitochondria, up to 8 mg of mitochondrial protein, could not be detected by dropping mercury electrode polarography but were readily determined colorimetrically using thiobarbituric acid reagent.