E. Yu. Plotnikov

The mitochondrion as Janus Bifrons

The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of heme, steroids, iron-sulfur clusters, and reactive oxygen and nitrogen species. Mitochondria also play an important role in the Ca2+-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.

Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 μM) and NBQX (10 μM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase—oligomycin, an uncoupler of oxidative phosphorylation—CCCP, a chelator of intracellular Ca2+-BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport—ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+]i. Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.

Functional activity of mitochondria in cultured neural precursor cells.

We studied mitochondrial transmembrane potential of neural precursor cells forming neurospheres in culture. Uneven energization of mitochondria in neurosphere cells was detected. Heterogeneity of cells by the mitochondrial potential increased with neurosphere enlargement during culturing. Decrease in the mitochondrial potential in the central cells in large spheres, presumably caused by insufficient diffusion of oxygen and nutrients, can provoke their damage and death. Population of cells with high mitochondrial potential responded to addition of the nuclear dye by a decrease in mitochondrial potential, which can indicate functioning of ABCG2 complex in these cells, characteristic of undifferentiated stem cells. These data will help to create optimum conditions for culturing of neural stem cells for the maintenance of their maximum functional and proliferative activity.

Effects of Ischemic and Hypoxic Preconditioning on the State of Mitochondria and Function of Ischemic Kidneys

Laser confocal microscopy showed that fluorescence of tetramethylrhodamine ethyl ether probe specifically accumulating in energized mitochondria significantly decreased in renal tubular epithelium after 40-min thermal ischemia, while fluorescence of dichlorodihydrofluorescein and diaminofluorescein probes in the same structures increased under these conditions, which attests to increased generation of ROS and NO, respectively. These forms were generated predominantly in mitochondria of tubular epitheliocytes. Hypoxic preconditioning (a series of sessions of breathing hypoxic mixture) preserved functional activity of mitochondria and prevented activation of ROS and NO generation. Ischemic preconditioning of the kidney consisting of three preliminary episodes of vascular clamping (5 min with 5 min reperfusion periods) also increased the percentage of functionally active mitochondria and prevented activation of NO synthesis without appreciably modifying ROS production. Both protective methods significantly reduced the severity of postischemic dysfunction of the kidney.

Morphological Changes in the Kidneys of Rats with Postischemic Acute Renal Failure after Intrarenal Administration of Fetal Mesenchymal Stem Cells from Human Bone Marrow

Chronic experiments on outbred albino rats were performed to compare the dynamics of histological signs for postischemic renal injury (90-min thermal ischemia) after intraparenchymal injection of cultured fetal MSC from human bone marrow. Functional indexes of the ischemic kidney were predetermined. In the early period after ischemia (day 4), administration of human bone marrow MSC was followed by the increase in blood flow in the microcirculatory bed and decrease in the degree of alteration in renal tubules. An increase in the area of zones with histological signs for normal function of tubules was accompanied by the improvement of biochemical indexes for renal function. In the delayed period, a protective effect of cell therapy was manifested in the prevention of death of renal tubules. Mild calcification of the necrotic tubular epithelium served as a marker of this process. Human bone marrow MSC were labeled with the fluorescent probe Calcein. These cells migrated from the site of injection, spread in the interstitium, and retained viability for 7 days. During this period, some cells were incorporated into the lumen of renal tubules.

Effect of anesthetics on efficiency of remote ischemic preconditioning

Remote ischemic preconditioning of hind limbs (RIPC) is an effective method for preventing brain injury resulting from ischemia. However, in numerous studies RIPC has been used on the background of administered anesthetics, which also could exhibit neuroprotective properties. Therefore, investigation of the signaling pathways triggered by RIPC and the effect of anesthetics is important. In this study, we explored the effect of anesthetics (chloral hydrate and Zoletil) on the ability of RIPC to protect the brain from injury caused by ischemia and reperfusion. We found that RIPC without anesthesia resulted in statistically significant decrease in neurological deficit 24 h after ischemia, but did not affect the volume of brain injury. Administration of chloral hydrate or Zoletil one day prior to brain ischemia produced a preconditioning effect by their own, decreasing the degree of neurological deficit and lowering the volume of infarct with the use of Zoletil. The protective effects observed after RIPC with chloral hydrate or Zoletil were similar to those observed when only the respective anesthetic was used. RIPC was accompanied by significant increase in the level of brain proteins associated with the induction of ischemic tolerance such as pGSK-3β, BDNF, and HSP70. However, Zoletil did not affect the level of these proteins 24 h after injection, and chloral hydrate caused increase of only pGSK-3β. We conclude that RIPC, chloral hydrate, and Zoletil produce a significant neuroprotective effect, but the simultaneous use of anesthetics with RIPC does not enhance the degree of neuroprotection.

Mitochondrial regulation of production of reactive oxygen species and nitrogen in rat cells of kidney during ischemia/reperfusion

Injuries caused by limited blood supply may occur in all organs during different surgical operations. In particular, induced ischemia of kidney is a risk factor, especially during organ transplantation and operations on organs of abdominal cavity, as well as during strong arterial hypotension. Often under natural (in thrombosis) and always under artificial conditions (during surgical operation) ischemia is followed by reperfusion and reoxygenation of tissues, which occurs during blood supply recovery. Such alternation of hypoxia and reoxygenation leads to the state of oxidative stress, which has been well described for brain tissues [1], myocardium [2], and other organs. Oxidative stress is an acute and substantially irreversible destructive process that severely limits the strategy of intervention in the vital functions of organs. Postischemic injuries of kidneys result in destruction of tubules, reversed flow of filtrate from the tubule lumen [3], and perturbation of filtration in the glomerules [4].

Methods of Detection of Mesenchymal Stem Cells in the Kidneys during Therapy of Experimental Renal Pathologies

We studied the possibility of using different methods for identifi cation of mesenchymal multipotent stromal cells in the renal parenchyma under conditions of experimental thermal ischemia of the kidneys and acute pyelonephritis. In vivo and in vitro methods of identifi cation of mesenchymal multipotent stromal cells by magnetic resonance imaging and immunological and immunohistochemical methods were compared. Labeling of stem cells with iron-containing particles followed by their histological identifi cation and immunohistochemical staining with species-specifi c antibodies were the most informative methods. Active migration of the cells to the renal tissue was detected by these methods in experimental acute pyelonephritis with infl ammation foci.

Quantification of Mitochondrial Morphology in situ

The structural organization of mitochondria reflects their functional status and largely is an index of cell viability. The indirect parameter to assess the functional state of mitochondria in cells is the degree of their fragmentation, i.e., the ratio of long or branched mitochondrial structures to round mitochondria. Such evaluations requires an approach that allows to create an integral pattern of the three-dimensional organization of mitochondrial reticulum using confocal images of mitochondria stained with a fluorescent probe. In the present study, we tested three approaches to analyzing the structural architecture of mitochondria under normal conditions and fission induced by oxidative stress. We revealed that, while the most informative is a three-dimensional reconstruction based on series of confocal images taken along the Z-dimension, with some restrictions it is plausible to use more simple algorithms of analysis, including one that uses unitary twodimensional images. Further improvement of these methods of image analysis will allow more comprehensive study of mitochondrial architecture under normal conditions and different pathological states. It may also provide quantification of a number of mitochondrial parameters determining the morphofunctional state of mitochondria—primarily, their absolute and relative volumes—and give additional information on threedimensional organization of the mitochondrion.

The Influence of Proinflammatory Factors on the Neuroprotective Efficiency of Multipotent Mesenchymal Stromal Cells in Traumatic Brain Injury

We studied the neuroprotective potential of multipotent mesenchymal stromal cells in traumatic brain injury and the effect of inflammatory preconditioning on neuroprotective properties of stem cells under in vitro conditions. To this end, the effects of cell incubation with LPS or their co-culturing with leukocytes on production of cytokines IL-1α, IL-6, TNFα, and MMP-2 and MMP-9 by these cells were evaluated. Culturing under conditions simulating inflammation increased the production of all these factors by multipotent mesenchymal stromal cells. However, acquisition of the inflammatory phenotype by stromal cells did not reduce their therapeutic effectiveness in traumatic brain injury. Moreover, in some variants of inflammatory preconditioning, multipotent mesenchymal stromal cells exhibited more pronounced neuroprotective properties reducing the volume of brain lesion and promoting recovery of neurological functions after traumatic brain injury.