Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.
Adult ; Caspases/metabolism* ; Cell Death ; Enzyme Activation ; Humans ; Male ; Mitochondria/pathology* ; Necrosis ; Nitrosative Stress/physiology* ; Permeability ; Peroxynitrous Acid/pharmacology* ; Phosphatidylserines/metabolism* ; Spermatozoa/ultrastructure*

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named "MitoQuant". This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.
Animals ; Axonal Transport ; physiology ; Cerebral Cortex ; cytology ; metabolism ; Drosophila melanogaster ; cytology ; metabolism ; Embryo, Mammalian ; Gene Expression ; Lab-On-A-Chip Devices ; Microscopy, Confocal ; Mitochondria ; metabolism ; ultrastructure ; Motor Neurons ; metabolism ; ultrastructure ; Movement ; Mutation ; Primary Cell Culture ; RNA-Binding Protein FUS ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Software

OBJECTIVETo preliminarily investigate the long-term structural and functional injuries of mitochondria in rat brain caused by sepsis.

METHODSWistar rats were randomly assigned into sepsis and control groups. A rat model of sepsis was prepared by an intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) of gram-negative bacteria, and the survival assay was performed. Eight rats in the sepsis group were sacrificed at 12, 24, 48, or 72 hours after LPS injection, while rats in the control group were sacrificed after an intraperitoneal injection of an equal volume of normal saline. Mitochondria were extracted from rat brain tissue. Mitochondrial membrane potential (MMP) and mitochondrial swelling level were determined by flow cytometry, and the activities of electron transport chain complexes (I-V) were measured using enzyme assay kits. Hematoxylin-eosin (HE) staining and electron microscopy were used to observe morphological changes in brain tissue and mitochondria.

RESULTSThe sepsis group had a significantly lower survival rate than the control group (P<0.01). The MMP and activities of electron transport chain complexes (I-V) in the sepsis group, which were significantly lower than those in the control group (P<0.05), were reduced to the lowest levels at 48 hours and partially recovered at 72 hours. The mitochondrial swelling level in the sepsis group, which was significantly higher than that in the control group (P<0.05), increased to the peak level at 48 hours and partially recovered at 72 hours. Hematoxylin and Eosin staining revealed substantial damages in the structure of brain tissue, and electron microscopy showed mitochondrial swelling, and vacuolization in a few mitochondria.

CONCLUSIONSIn the rat model of LPS-induced sepsis, both structural and functional injuries are found in cerebral mitochondria, and achieve the peak levels probably at around 48 hours.

Animals ; Brain ; pathology ; physiopathology ; ultrastructure ; Lipopolysaccharides ; toxicity ; Male ; Membrane Potential, Mitochondrial ; Mitochondria ; physiology ; ultrastructure ; Rats ; Rats, Wistar ; Sepsis ; chemically induced ; mortality ; physiopathology

BACKGROUNDLymphocyte function and homeostasis is associated with immune defence to infection. Apoptosis of lymphocytes might be a considerably important component which has an impact on immunity to infections in people with hyperglycemia. The aim of this study was to explore the mitochondrial apoptosis pathway of lymphocyte in diabetic patients.

METHODSSixty patients with type 2 diabetes mellitus and fifty healthy volunteers were included in this study. Annexin V and propidiumiodide (PI) were joined in the isolated lymphocytes and the rate of lymphocyte apoptosis was calculated with flow cytometry. Observation of the lymphocytes was done using transmission electron microscopy; mitochondria had been extracted and then mitochondrial membrane potential (MMP) was detected to assess mitochondrial function; the mRNA level of Bcl-2, cytochrome c (Cyt-C), caspase-9 and caspase-3 were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSApoptosis rate of lymphocyte was significantly higher in diabetic group than that in normal control group (P < 0.05). Transmission electron microscopy showed lymphocyte shrinkage and breakage, chromatin condensation and less mitochondria; a fall in MMP levels was also evident; Bcl-2 concentration was reduced and the expressions of caspase-9, caspase-3 and Cyt-C were elevated (P < 0.05) in diabetic patients.

CONCLUSIONSThe rate of lymphocyte apoptosis was significantly higher in type 2 diabetic patients than that in normal population. Mitochondrial apoptosis pathway may play a very important role in decreasing function of lymphocyte in diabetes.

Aged ; Aged, 80 and over ; Apoptosis ; physiology ; Caspase 3 ; genetics ; Caspase 9 ; genetics ; Diabetes Mellitus, Type 2 ; metabolism ; Female ; Humans ; Lymphocytes ; cytology ; metabolism ; ultrastructure ; Male ; Microscopy, Electron, Transmission ; Mitochondria ; metabolism ; Real-Time Polymerase Chain Reaction ; bcl-2-Associated X Protein ; genetics

OBJECTIVETo probe the changes of fast component of brainstem auditory evoked potentials (FC-BAEP), slow component of brainstem auditory evoked potentials (SC-BAEP) and the mitochondrial ultrastructures of the neurons in the brainstem in the rat pups with hyperbilirubinemia.

METHODS7 days old SD rat pups were randomly divided into control group (C, 17 rat pups) and two test groups (T1, 17 rat pups and T2, 17 rat pups). Bilirubin solutions (2 g/L) were injected into the abdominal cavity of the rat pups in the group T1 and T2 at the postnatal day 7 and 10. Six hours after the second injection, seven rat pups of each group were randomly selected to test serum bilirubin concentration via a micro-gauge. FC-BAEP and SC-BAEP were examined with an evoked potential recorder in the rest rat pups of each group at postnatal day 17 and 20. At the postnatal day 20, the endocardial perfusion was performed in these rat pups for the fixation of the brain, and then the brains were taken out. The cochlear nuclei were used for observation via electron microscope.

RESULTSSix hours after the injection of bilirubin solution at the postnatal day 10, the serum bilirubin concentrations of the rat pups in group T1 and T2 were increased significantly. Except for II-IV inter-peak latency(IPL), all the peak latency(PL) and IPL of FC-BAEP evoked via three sound stimulating rates (10/s, 40/s,80/s) at the postnatal day 17 prolonged significantly in the rat pups of group T1 and T2, and the PL in group T2 were much longer than that in group T1. Except for II-IV IPL of FC-BAEP evoked via sound stimulating rates of 10/s and 40/s, all the PL and IPL at the postnatal day 20 prolonged significantly in the rat pups of group T1 and T2. The PL of SC-BAEP evoked via sound stimulating rate of 10/s at the postnatal day 17 and 20 in the rat pups of group T1 and T2 prolonged significantly, and the PL at the postnatal day 17 in group T2 were much longer than that of group T1. The changes of mitochondria of the neurons in the cochlear nuclei at the postnatal day 20 in the rat pups of group T1 and T2 were characterized by swell, the slurred membranes, the broken crista and so on.

CONCLUSIONThere were the abnormal changes of FC-BAEP, SC-BAEP and the mitochondrial ultrastructures of the neurons in the brainstem in the rat pups with hyperbilirubinemia. The PL and IPL of FC-BAEP and SC-BAEP could be taken as the objective and sensitive indexes for early monitoring the bilirubin-induced hearing loss and brain injury.

Animals ; Animals, Newborn ; Brain Stem ; pathology ; Evoked Potentials, Auditory, Brain Stem ; physiology ; Hearing Loss ; etiology ; physiopathology ; Hyperbilirubinemia ; complications ; physiopathology ; Male ; Mitochondria ; ultrastructure ; Neurons ; ultrastructure ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of diazoxide (DE) on the myocardial ultrastructure and opening of maitochondrial permeability transition pore (MPTP) in donor rat heart suffered from long-term hypothermic preservation.

METHODSThe Langendorff model of isolated rat heart was used. The hearts were stored in 4 degrees C Celsior solution containing different concentration of DE (15, 30, or 45 micromol/L) for 9 h followed by 60 min of reperfusion. The recovery of rate-pressure product (RPP) was observed. The opening of MPTP and myocardial mitochondria ultrastructure were also evaluated.

RESULTS(1) As compared with the celsior solution preserved group, DE (30 micromol/L) increased recovery of RPP during reperfusion and inhibited the opening of MPTP. DE also alleviated the myocardial mitochondrial ultrastucture damage induced by long-term hypothermic preservation. (2) The above effects of DE were attenuated by a mitoK(ATP) channel inhibitor 5-hydroxydecanoate and a MPTP opener atractyloside.

CONCLUSIONIn the donor rat heart, DE protects myocardial mitochondria ultrastructure against long-term hypothermic preservation injury via inhibiting the opening of MPIP.

Animals ; Cryopreservation ; Diazoxide ; pharmacology ; Heart ; In Vitro Techniques ; Male ; Mitochondria, Heart ; physiology ; ultrastructure ; Mitochondrial Membrane Transport Proteins ; drug effects ; metabolism ; Organ Preservation Solutions ; pharmacology ; Potassium Channels ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Apoptosis/*drug effects/physiology ; BH3 Interacting Domain Death Agonist Protein/physiology ; Drug Design ; Genes, bcl-2 ; Humans ; Mitochondria/physiology/ultrastructure ; Mitochondrial Membranes/*metabolism/physiology ; Multigene Family ; Proto-Oncogene Proteins c-bcl-2/*antagonists & inhibitors ; Signal Transduction

A vast portion of human disease results when the process of apoptosis is defective. Disorders resulting from inappropriate cell death range from autoimmune and neurodegenerative conditions to heart disease. Conversely, prevention of apoptosis is the hallmark of cancer and confounds the efficacy of cancer therapeutics. In the search for optimal targets that would enable the control of apoptosis, members of the BCL-2 family of anti- and pro-apoptotic factors have figured prominently. Development of BCL-2 antisense approaches, small molecules, and BH3 peptidomimetics has met with both success and failure. Success-because BCL-2 proteins play essential roles in apoptosis. Failure-because single targets for drug development have limited scope. By examining the activity of the BCL-2 proteins in relation to the mitochondrial landscape and drawing attention to the significant mitochondrial membrane alterations that ensue during apoptosis, we demonstrate the need for a broader based multi-disciplinary approach for the design of novel apoptosis-modulating compounds in the treatment of human disease.
Apoptosis/*drug effects/physiology ; BH3 Interacting Domain Death Agonist Protein/physiology ; Drug Design ; Genes, bcl-2 ; Humans ; Mitochondria/physiology/ultrastructure ; Mitochondrial Membranes/*metabolism/physiology ; Multigene Family ; Proto-Oncogene Proteins c-bcl-2/*antagonists & inhibitors ; Signal Transduction

OBJECTIVETo study the influence of conditioned medium of rat brain microvascular endothelial cells on mitochondrial function of cortical neurons and the protective effect of Tongluo Jiunao Injection (TJI) on it.

METHODSFour kinds of conditioned endothelial cell (EC) cultured medium were prepared, i.e. the N-CM medium prepared by EC cultured in the normal conditioned medium without any treatment; the NT-CM prepared by EC cultured in N-CM and treated with TJI 1 microl/ml for 10 h; the I-CM prepared by EC cultured in the non-glucose kreb medium under hypoxia condition; and the IT-CM by EC pre-treatce with TJI 1 microl/ml for 4 h and cultured as that of I-CM. The levels of neuronic mitochondrial activity, membrane potential (MMP) and cytochrome C (Cyt C) were determined before and after the glucose-oxygen deprived model neurons of brain cortex being cultured with different kinds of conditioned EC cultured medium for assessing the effects of these media on mitochondria of injured neuron.

RESULTSAs compared with those of the normal neuron, the mitochondrial activity and MMP of all injured neurons decreased and Cyt C level increased significantly. But comparison of these indexes among neurons cultured with different conditioned EC culture media showed that the greatest extent abnormality revealed in the N-CM cultured neurons, which even greater than that in the model neuron; while that was less in the N-CM cutured neuron than in model neuron; as for those cultured in the NT-CM and IT-CM, i.e. the TJI treated cuture medium, the abnormal changes were reduced significantly when compared with those cultured in medium untreated with TJI (N-CM and I-CM), respectively (all P < 0.05).

CONCLUSIONThe paracrine secretion of the brain microvascular endothelial cells has evident regulatory effect on survival of the injured neurons, which might possibly be related to its protective effect on neuron mitochondrial function, and TJI could enhance the protective effect.

Animals ; Capillaries ; cytology ; Cells, Cultured ; Cerebral Cortex ; blood supply ; cytology ; Culture Media, Conditioned ; pharmacology ; Cytochromes c ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; drug effects ; ultrastructure ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; physiology ; Neurons ; cytology ; drug effects ; ultrastructure ; Rats

AIMTo ascertain the protective effect of oxyphenamone, a novel inodilator, against myocardial ischemia-reperfusion injury.

METHODSA model of global myocardial ischemia-reperfusion injury (I-R) was established by arresting the infusion to heart 40 min followed by reperfusion 30 min in the preparations of rat Langendorff' s hearts. The protective effects of oxyphenamone were evaluated by the cardiac function, the activity of creatine phosphokinase (CPK) in coronary efflux, the myocardial energy metabolism and antioxidation, the membrane fluidity, the activity of ATPase, the calcium content and ultrastructure of mitochondria.

RESULTSAdministration of oxyphenamone to the infused heart before cardiac arrest and during reperfusion ameliorated the decreases of myocardial contractile force and coronary flow and the increase of the activity of CPK in coronary efflux induced by cardiac arrest-reperfusion (A-R). At the concentration of 1-10 micromol x L(-1) oxyphenamone dose-dependently antagonized the decrease of myocardial adenosine triphosphate (ATP) and creatine phosphate (PCr) induced by A-R, increased the activities of myocardial superoxide dismutase (SOD) and glutathione peroxidase (GSH-px), decreased mitochondrial malondialdehyde (MDA) content and increased membrane fluidity, glutathione (GSH) content and the activities of GSH-px and ATPase of mitochondria. The parameters mentioned above even maintained at normal level when high concentration of oxyphenamone (10 micromol x L(-1)) was applied. Oxyphenamone also antagonized the mitochondrial calcium overload and the ultrastructure damage of mitochondria induced by A-R obviously. Addition of oxyphenamone (1-100 micromol x L(-1)) to the system of Fe2+-cysteine or Fe2+-H2O2, which could produce oxygen free radicals, decreased MDA content and increased GSH and membrane fluidity of mitochondria significantly.

CONCLUSIONWith the results of examinations of the cardiac physiological function, myocardial energy metabolism and antioxidation and the calcium content and ultrastructure of mitochondria, it is indicated that oxyphenamone could protect the isolated rat heart against cardiac arrest-reperfusion injury markedly and the mechanism of its action may be related to the antioxidative effect of this agent.

Animals ; Calcium ; metabolism ; Coronary Circulation ; drug effects ; Female ; In Vitro Techniques ; Male ; Membrane Fluidity ; drug effects ; Mitochondria, Heart ; metabolism ; physiology ; ultrastructure ; Myocardial Contraction ; drug effects ; Myocardial Reperfusion Injury ; metabolism ; physiopathology ; Myocardium ; metabolism ; Organic Chemicals ; pharmacology ; Protective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Wistar