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Peripheral nerve injury remains a serious problem for medicine, with no effective method of treatment at the moment. The most prominent example of this problem is neonatal brachial plexus palsy, which results from the stretching of the brachial plexus nerves in the birth or perinatal period. Multipotent mesenchymal cells (MSCs) and the extracellular vesicles (EVs) they produce are known to have a marked neuroprotective effect in central nervous system injuries. We suggested that the use of MSCs-derived EVs may be an effective approach to the regeneration of peripheral nerves after injury. Sciatic nerve injury was modeled in rats via crushing, and then a gel containing MSCs–EVs was applied to the injured area. After 15 and 30 days, a histological, physiological, and functional assessment of nerve, dorsal root ganglia (DRG), and innervated muscles’ recovery was performed. Transplantation of EVs to the area of sciatic nerve injury significantly reduced muscle atrophy as compared to the control group. Functional recovery of the innervated muscles, as measured by the extensor postural thrust test, was revealed 30 days after the surgery. We associate the obtained results with EVs-induced neuroprotective mechanisms, which were expressed in a decrease in apoptotic neuronal death and an increase in regeneration-associated proteins NF-200 and GAP-43, as well as in DRG and damaged nerve. We suggest that the therapeutic scheme we used is efficient for the treatment of acute peripheral nervous system injuries and can be transferred to the clinics. However, additional studies are required for a more detailed analysis of neuroprotection mechanisms.

We studied the development of acute kidney injury and animal death in the model of combined injury caused by kidney ischemia/reperfusion with simultaneous systemic administration of mitochondria. It was found that intraperitoneal injection of isolated mitochondria led to the appearance of mitochondrial DNA in the peripheral blood that could activate innate immunity. After administration of mitochondria, as well as after renal ischemia/reperfusion, proinflammatory changes were observed, primarily leukocytosis and granulocytosis. The combination of ischemia/reperfusion with injection of mitochondria caused a sharp increase in animal death, which may indicate a direct link between activation of TLR-signaling and high mortality of patients with combined injuries and multiple-organ failure in intensive care units. Treatment with mitochondria-targeted antioxidant increased animal survival, which indicated the participation of mitochondrial ROS in the development of systemic inflammatory response and death caused by acute renal failure.

Appending lipophilic cations to small molecules has been widely used to produce mitochondria-targeted compounds with specific activities. In this work, we obtained a series of derivatives of the well-known fluorescent dye 7-nitrobenzo-2-oxa-1,3-diazole (NBD). According to the previous data [Denisov et al. (2014) Bioelectrochemistry, 98, 30-38], alkyl derivatives of NBD can uncouple isolated mitochondria at concentration of tens of micromoles despite a high pK(a) value (similar to 11) of the dissociating group. Here, a number of triphenylphosphonium (TPP) derivatives linked to NBD via hydrocarbon spacers of varying length (C5, C8, C10, and C12) were synthesized (mitoNBD analogues), which accumulated in the mitochondria in an energy-dependent manner. NBD-C10-TPP (C10-mitoNBD) acted as a protonophore in artificial lipid membranes (liposomes) and uncoupled isolated mitochondria at micromolar concentrations, while the derivative with a shorter linker (NBD-C5-TPP, or C5-mitoNBD) exhibited no such activities. In accordance with this data, C10-mitoNBD was significantly more efficient than C5-mitoNBD in suppressing the growth of Bacillus subtilis. C10-mitoNBD and C12-mitoNBD demonstrated the highest antibacterial activity among the investigated analogues. C10-mitoNBD also exhibited the neuroprotective effect in the rat model of traumatic brain injury.

Oxidative kidney injury was compared in newborn and adult rats under conditions of ischemia/reperfusion and in experimental model of systemic inflammation induced by endotoxin (LPS of bacterial cell wall) administration. Oxidative stress in the kidney accompanied both experimental models, but despite similar oxidative tissue damage, kidney dysfunction in neonates was less pronounced than in adult animals. It was found that neonatal kidney has a more potent regenerative potential with higher level of cell proliferation than adult kidney, where the level proliferating cell antigen (PCNA) increased only on day 2 after ischemia/reperfusion. The pathological process in the neonatal kidney developed against the background of active cell proliferation, and, as a result, proliferating cells could almost immediately replace the damaged structures. In the adult kidney, regeneration of the renal tissue was activated only after significant loss of functional nephrons and impairment of renal function.

The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe.

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.

Autophagy plays an important role in the pathogenesis of acute kidney injury (AKI). Although autophagy activation was shown to be associated with an increased lifespan and beneficial effects in various pathologies, the impact of autophagy activators, particularly, rapamycin and its analogues on AKI remains obscure. In our study, we explored the effects of rapamycin treatment in in vivo and in vitro models of ischemic and cisplatin-induced AKI. The impact of rapamycin on the kidney function after renal ischemia/reperfusion (I/R) or exposure to the nephrotoxic agent cisplatin was assessed by quantifying blood urea nitrogen and serum creatinine and evaluating the content of neutrophil gelatinase-associated lipocalin, a novel biomarker of AKI. In vitro experiments were performed on the primary culture of renal tubular cells (RTCs) that were subjected to oxygen-glucose deprivation (OGD) or incubated with cisplatin under various rapamycin treatment protocols. Cell viability and proliferation were estimated by the MTT assay and real-time cell analysis using an RTCA iCELLigence system. Although rapamycin inhibited mTOR (mammalian target of rapamycin) signaling, it failed to enhance the autophagy and to ameliorate the severity of AKI caused by ischemia or cisplatin-induced nephrotoxicity. Experiments with RTCs demonstrated that rapamycin exhibited the anti-proliferative effect in primary RTCs cultures but did not protect renal cells exposed to OGD or cisplatin. Our study revealed for the first time that the mTOR inhibitor rapamycin did not prevent AKI caused by renal I/R or cisplatin-induced nephrotoxicity and, therefore, cannot be considered as an ideal mimetic of the autophagy-associated nephroprotective mechanisms (e.g., those induced by caloric restriction), as it had been suggested earlier. The protective action of such approaches like caloric restriction might not be limited to mTOR inhibition and can proceed through more complex mechanisms involving alternative autophagy-related targets. Thus, the use of rapamycin and its analogues for the treatment of various AKI forms requires further studies in order to understand potential protective or adverse effects of these compounds in different contexts.

The use of stem cells is part of a strategy for the treatment of a large number of diseases. However, the source of the original stem cells for use is extremely important and determines their therapeutic potential. Mesenchymal stromal cells (MSC) have proven their therapeutic effectiveness when used in a number of pathological models. However, it remains an open question whether the chronological age of the donor organism affects the effectiveness of the use of MSC. The asymmetric division of stem cells, the result of which is some residential stem cells acquiring a non-senile phenotype, means that stem cells possess an intrinsic ability to preserve juvenile characteristics, implying an absence or at least remarkable retardation of senescence in stem cells. To test whether residential MSC senesce, we evaluated the physiological changes in the MSC from old rats, with a further comparison of the neuroprotective properties of MSC from young and old animals in a model of traumatic brain injury. We found that, while the effect of administration of MSC on lesion volume was minimal, functional recovery was remarkable, with the highest effect assigned to fetal cells, the lowest effect was recorded for cells isolated from adult rats and postnatal cells, having intermediate potency. MSC from the young rats were characterized by a faster growth than adult MSC, correlating with levels of proliferating cell nuclear antigen (PCNA). However, there were no differences in respiratory activity of MSC from young and old rats, but young cells showed much higher glucose utilization than old ones. Autophagy flux was almost the same in both types of cells, but there were remarkable ultrastructural differences in old and young cells.

One of the most important pathological consequences of renal ischemia/reperfusion (I/R) is kidney malfunctioning. I/R leads to oxidative stress, which affects not only nephron cells but also cells of the vascular wall, especially endothelium, resulting in its damage. Assessment of endothelial damage, its role in pathological changes in organ functioning, and approaches to normalization of endothelial and renal functions are vital problems that need to be resolved. The goal of this study was to examine functional and morphological impairments occurring in the endothelium of renal vessels after I/R and to explore the possibility of alleviation of the severity of these changes using mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decylrhodamine 19 (SkQR1). Here we demonstrate that 40-min ischemia with 10-min reperfusion results in a profound change in the structure of endothelial cells mitochondria, accompanied by vasoconstriction of renal blood vessels, reduced renal blood flow, and increased number of endothelial cells circulating in the blood. Permeability of the kidney vascular wall increased 48 h after I/R. Injection of SkQR1 improves recovery of renal blood flow and reduces vascular resistance of the kidney in the first minutes of reperfusion; it also reduces the severity of renal insufficiency and normalizes permeability of renal endothelium 48 h after I/R. In in vitro experiments, SkQR1 provided protection of endothelial cells from death provoked by oxygen-glucose deprivation. On the other hand, an inhibitor of NO-synthases, L-nitroarginine, abolished the positive effects of SkQR1 on hemodynamics and protection from renal failure. Thus, dysfunction and death of endothelial cells play an important role in the development of reperfusion injury of renal tissues. Our results indicate that the major pathogenic factors in the endothelial damage are oxidative stress and mitochondrial damage within endothelial cells, while mitochondria-targeted antioxidants could be an effective tool for the protection of tissue from negative effects of ischemia.

Previously, we have assembled a cellular model of pyelonephritis which contains a primary culture of renal tubular epithelial cells, mononuclear leukocytes, and bacterial lysate or lipopolysaccharide. After cocultivation of renal cells with leukocytes and bacterial lysate, proinflammatory changes were observed in the renal cells, followed by nitrosative and oxidative stress and cell death. The interaction of bacterial antigens not only with leukocytes, but also with epithelial cells of the renal tubules, was partially mediated by signaling pathways involving Toll-like receptors (TLR2 and TLR4). Activation of these receptors led to increased levels of oxidative stress and synthesis of proinflammatory cytokines (TNF, IL-6, IL-1α) in the renal epithelium, while TLR4 blockade decreased the severity of these processes. Apart from the fact that activation of inflammatory signaling in response to bacterial antigens is observed directly in the renal cells, the presence of leukocytes significantly amplifies the inflammatory response as measured by the level of cytokines generated in the ensemble. In the presence of activated leukocytes, higher expression of TLR2 on the surface of renal cells was observed in response to exposure to bacterial components, which might explain the increased inflammatory response in the presence of leukocytes. The synthesis of IL-1α in the epithelial cells of the renal tubules in this inflammatory model leads to its accumulation in the nuclei, which has been reduced by the TLR4 antagonist polymyxin. TLR2 agonists also led to increased levels of IL-1α. The elevation in the content of IL-1α in nuclei was accompanied by increased acetylation of nuclear proteins, which has been reduced to control values after exposure to protective agents (Trolox, mitochondria-targeted antioxidant SkQR1 or LiCl). The high level of acetylation of histones is probably regulated by proinflammatory cytokines, and to some extent it is a marker of inflammation, which can indirectly be reduced by protective agents.

We studied the possibility of in vivo tracing of multipotent mesenchymal stromal cells labeled with a radiophermaceutic preparation based on metastable isotope Technetium-99m and injected to rats with modeled traumatic brain injury. Accumulation of labeled cells occurred primarily in the liver and lungs. The cells distribution in internal organs greatly varied depending on the administration route. Cell injection into the carotid artery led to their significant accumulation in the damaged brain hemisphere, while intravenous injection was followed by diffuse cell distribution in all brain structures. Scintigraphy data were confirmed by magnetic resonance imaging and histological staining of cells. Visualization of stem cells labeled with Technetium-99m-based preparation by scintigraphy is an objective and highly informative method allowing real-time in vivo cell tracing in the body.

Traumatic brain injury is one of the leading causes of disability among the working-age population worldwide. Despite attempts to develop neuroprotective therapeutic approaches, including pharmacological or cellular technologies, significant advances in brain regeneration have not yet been achieved. Development of silk fibroin-based biomaterials represents a new frontier in neuroregenerative therapies after brain injury. In this study, we estimated the short and long-term effects of silk fibroin scaffold transplantation on traumatic brain injury and biocompatibility of this biomaterial within rat neuro-vascular cells. Silk fibroin microparticles were injected into a brain damage area 1 day after the injury. Silk fibroin affords neuroprotection as judged by diminished brain damage and recovery of long-term neurological functions. We did not detect considerable toxicity to neuro-vascular cells cultured on fibroin/fibroin-gelatin microparticles in vitro. Cultivation of primary cell cultures of neurons and astrocytes on silk fibroin matrices demonstrated their higher viability under oxygen-glucose deprivation compared to 2D conditions on plastic plates. Thus, we conclude that scaffolds based on silk fibroin can become the basis for the creation of constructs aimed to treat brain regeneration after injury.

A complex analysis of acute kidney injury (AKI) in pregnant women shows that it is caused by the interaction of gestation-associated pathologies and beneficial signaling pathways activated by pregnancy. Studies report an increase in the regeneration of some organs during pregnancy. However, the kidney response to the injury during pregnancy has not been addressed. We investigated the mechanisms of the pregnancy influence on AKI. During pregnancy, the kidneys were shown to be more tolerant to AKI. Pregnant animals showed remarkable preservation of kidney functions after ischemia/reperfusion (I/R) indicated by the decrease of serum creatinine levels. The pregnant rats also demonstrated a significant decrease in kidney injury markers and an increase in protective markers. Two months after the I/R, group of pregnant animals had a decreased level of fibrosis in the kidney tissue. These effects are likely linked to increased cell proliferation after injury: using real-time cell proliferation monitoring we demonstrated that after ischemic injury, cells isolated from pregnant animal kidneys had higher proliferation potential vs. control animals; it was also supported by an increase of proliferation marker PCNA levels in kidneys of pregnant animals. We suggest that these effects are associated with hormonal changes in the maternal organism, since hormonal pseudopregnancy simulated effects of pregnancy.

Neonatal hypoxia&ndash, ischemia is one of the main causes of mortality and disability of newborns. To study the mechanisms of neonatal brain cell damage, we used a model of neonatal hypoxia&ndash, ischemia in seven-day-old rats, by annealing of the common carotid artery with subsequent hypoxia of 8% oxygen. We demonstrate that neonatal hypoxia&ndash, ischemia causes mitochondrial dysfunction associated with high production of reactive oxygen species, which leads to oxidative stress. Targeted delivery of antioxidants to the mitochondria can be an effective therapeutic approach to treat the deleterious effects of brain hypoxia&ndash, ischemia. We explored the neuroprotective properties of the mitochondria-targeted antioxidant SkQR1, which is the conjugate of a plant plastoquinone and a penetrating cation, rhodamine 19. Being introduced before or immediately after hypoxia&ndash, ischemia, SkQR1 affords neuroprotection as judged by the diminished brain damage and recovery of long-term neurological functions. Using vital sections of the brain, SkQR1 has been shown to reduce the development of oxidative stress. Thus, the mitochondrial-targeted antioxidant derived from plant plastoquinone can effectively protect the brain of newborns both in pre-ischemic and post-stroke conditions, making it a promising candidate for further clinical studies.

To identify the technical aspects of the potential use of clinically approved perfluorodecalin (PFD, CThe selective excitation of the magnetically equivalentThe study showed the feasibility of the successful application of PFD for

Recent evidence suggests that mitochondria are one of the main factors in the pathogenesis in different organs including brain. The pathogenesis after brain damage is caused not only by the change in bioenergetics, but also involves impairment of alternative functions of mitochondria, particularly those related to the control of cell death. In this study we evaluated partial metabolic pathways under the simulation of a stroke by using the occlusion of the middle cerebral artery in rats. The analysis shows that the induced switch to a non-oxidative energy metabolism (glycolysis) due to the block of tissue oxygen supply does not ensure the adequate supply of the tissue with ATP. Moreover, the well-known acidification of the ischemic tissue is not associated with the so-called traditionally and incorrectly considered “lactic acidosis” (the generation of lactate from glucose by itself does not lead to excessive generation of protons), but occurs because of the consumption of tissue ATP under its reduced resynthesis. Incubation of mitochondria isolated from normal rat brain at neutral and slightly acidic pH, mimicking the intracellular pH of normal and ischemic tissues correspondingly, revealed serious changes in mitochondrial bioenergetics, partially reflected in the magnitude of respiratory control and the basal and maximally stimulated respiration rates. Measurement of available metabolites by 1H MR spectra of normal and ischemia-damaged brains showed a significant increase in lactate and myo-inositol and a moderate decrease in N-acetylaspartate 24 h after reperfusion. Remarkably, the administration of lithium chloride in the reperfusion phase normalized the levels of metabolites. Moreover, the introduction of lithium salts (chloride or succinate) in the bloodstream, restored after ischemia, reduced both the size of the ischemia-induced brain damage and the degree of brain swelling. Besides, post-ischemic introduction of lithium salts largely restored the neurological status of the animal.

Here, in addition to the previously coined term "mitobiota", we introduce the term "mitodiversity" for various phenotypic and genetic heterogeneities of mitochondria within the same cell or organ. Based on data on the mitochondrial transmembrane potential determined both in situ and in vitro under normal conditions and after organ ischemia/reperfusion, such heterogeneity is most evident under pathologic conditions. Herein, a part of the mitochondrial population with transmembrane potential typical of the normal state is sustained even under a pathological condition that, perhaps, underlies the development of ways of reversing pathology back to the normal state. The membrane potentials of isolated mitochondria were shown to directly correlate with the magnitude of side-scattered light depicting internal structure of mitochondria. We analyzed possible interpretations of data on mitochondrial membrane potential obtained using fluorescent probes. We suggest a possible mechanism underlying retention of fluorescent probes inside the cells and mitochondria.

Mitochondria-targeted antioxidant SkQ1 did not increase the content of cytochromes P450 in livers of rats that were given SkQ1 in drinking water for 5 days in a dose (2.5 µmol per kg body weight) that exceeded 10 times the SkQ1 therapeutic dose. SkQ1 did not affect the levels of cytochrome P450 forms CYP1A2, CYP2B6, and CYP3A4 in monolayer cultures of freshly isolated human hepatocytes, while specific inducers of these forms (omeprazole, phenobarbital, and rifampicin, respectively) significantly increased expression of the cytochromes P450 under the same conditions. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in liver, and the absence of the inducing effect cannot be explained by poor availability of hepatocytes to SkQ1 in vivo.

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.

Background aims Acute pyelonephritis is one of the most frequent infectious diseases of the urinary tract and a leading cause of kidney failure worldwide. One strategy for modulating excessive inflammatory responses in pyelonephritis is administration of mesenchymal multipotent stromal cells (MMSCs). Methods The putative protective effect of injection of MMSCs against experimental acute pyelonephritis was examined. We used in vivo experimental model of APN where bacteria are introduced in the bladder of rat. Three days after, intravenous injection of MMSCs was done. On the 7th day blood samples and kidneys were taken for further analysis. Results We found obvious signs of oxidative stress and inflammation in the kidney in acute pyelonephritis in rats. Particularly, pro-inflammatory cytokine tumor necrosis factor-α levels, malondialdehyde, nitrite and myeloperoxidase activity were significantly increased. Histologic evaluation revealed numerous attributes of inflammation and tissue damage in the kidney. Treatment with MMSCs caused a remarkable decrease of all of these pathologic signs in renal tissue. Also, activated leukocytes induced pre-conditioning-like signaling in MMSCs. We showed alterations of expression or activity of inducible nitric oxide synthase, transforming growth factor-β, matrix metalloproteinase-2 and glycogen synthase kinase-3β, which could mediate immunomodulation and protective effects of MMSCs. This signaling could be characterized as inflammatory pre-conditioning. Conclusions The beneficial capacity of MMSCs to alleviate renal inflammation was more pronounced when pre-conditioned MMSCs were used. This approach could be used to prime MMSCs with different inflammatory modulators to enhance their engraftment and function in an immunoprotected fashion.

In young rats, ischemic preconditioning (IPC), which consists of 4 cycles of ischemia and reperfusion alleviated kidney injury caused by 40-min ischemia. However,old rats lost their ability to protect the ischemic kidney by IPC. A similar aged phenotype was demonstrated in 6-month-old OXYS rats having signs of premature aging. In the kidney of old and OXYS rats, the levels of acetylated nuclear proteins were higher than in young rats, however, unlike in young rats, acetylation levels in old and OXYS rats were further increased after IPC. In contrast to Wistar rats, age-matched OXYS demonstrated no increase in lysosome abundance and LC3 content in the kidney after ischemia/reperfusion. The kidney LC3 levels were also lower in OXYS, even under basal conditions, and mitochondrial PINK1 and ubiquitin levels were higher, suggesting impaired mitophagy. The kidney mitochondria from old rats contained a population with diminished membrane potential and this fraction was expanded by IPC. Apparently, oxidative changes with aging result in the appearance of malfunctioning renal mitochondria due to a low efficiency of autophagy. Elevated protein acetylation might be a hallmark of aging which is associated with a decreased autophagy, accumulation of dysfunctional mitochondria, and loss of protection against ischemia by IPC.

Background Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C 10 ) spacer yields a fluorescent uncoupler, coined mitoFluo, that selectively accumulates in energized mitochondria (Denisov et al., Chem.Commun. 2014). Methods Proton-transport activity of mitoFluo was tested in liposomes reconstituted with bacteriorhodopsin. To examine the uncoupling action on mitochondria, we monitored mitochondrial membrane potential in parallel with oxygen consumption. Neuro- and nephroprotecting activity was detected by a limb-placing test and a kidney ischemia/reperfusion protocol, respectively. Results We compared mitoFluo properties with those of its newly synthesized analog having a short (butyl) spacer (C 4 -mitoFluo). MitoFluo, but not C 4 -mitoFluo, caused collapse of mitochondrial membrane potential resulting in stimulation of mitochondrial respiration. The dramatic difference in the uncoupling activity of mitoFluo and C 4 -mitoFluo was in line with the difference in their protonophoric activity on a lipid membrane. The accumulation of mitoFluo in mitochondria was more pronounced than that of C 4 -mitoFluo . MitoFluo decreased the rate of ROS production in mitochondria. MitoFluo was effective in preventing consequences of brain trauma in rats: it suppressed trauma-induced brain swelling and reduced a neurological deficit. Besides, mitoFluo attenuated acute kidney injury after ischemia/reperfusion in rats. Conclusions A long alkyl linker was proved mandatory for mitoFluo to be a mitochondria- targeted uncoupler. MitoFluo showed high protective efficacy in certain models of oxidative stress-related diseases. General significance MitoFluo is a candidate for developing therapeutic and fluorescence imaging agents to treat brain and kidney pathologies.

The fate of myoglobin in renal cells was explored in an animal model of rhabdomyolysis known as the pathology highly related to oxidative stress resulting in impairment of renal functioning. The working hypothesis was that the proper degradation of myoglobin in rhabdomyolytic kidney can activate the reparative processes in the tissue. We found that incubation of myoglobin with kidney cells causes its accumulation in the cytoplasm. In rhabdomyolytic rats, the level of heme and free iron in cytoplasm and mitochondria of kidney cells is remarkably increased while inhibition of proteolysis results in further elevation of myoglobin content in the renal tissue. Heme oxygenase and ferritin levels were found to be increased in the kidney tissue at rhabdomyolysis and simulating conditions performed by i/v injection of myoglobin. In addition, the level of peroxidized lipids was high in rhabdomyolytic kidney and became even higher after inhibition of proteolysis by aprotinin. Elevated levels of carbonylated proteins were also observed after rhabdomyolysis, however, if prior to induction of rhabdomyolysis the injection of myoglobin was done, the level of carbonylated proteins dropped versus unprimed kidney tissue thus affording protection to the kidney against oxidative stress. Injection of myoglobin to the rat results in impairment of renal functioning and inhibition of myoglobin degradation in the rhabdomyolytic animal aggravates acute renal failure, demonstrating that degradation of myoglobin is somehow beneficial although it may result in undesired release of free iron which can participate in toxic redox cycling.

A protective effect of a mitochondria-targeted antioxidant, a cationic rhodamine derivative linked to a plastoquinone molecule (10-(6'-plastoquinonyl)decylrhodamine-19, SkQR1) was studied in the model of open focal trauma of rat brain sensorimotor cortex. It was found that daily intraperitoneal injections of SkQR1 (100 nmol/kg) for 4 days after the trauma improved performance in a test characterizing neurological deficit and decreased the volume of the damaged cortical area. Our results suggest that SkQR1 exhibits profound neuroprotective effect, which may be explained by its antioxidative activity.

It is generally accepted that mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Due to this, high values of the membrane potential are highly dangerous, specifically under pathological conditions associated with oxidative stress. Mild uncoupling of oxidative phosphorylation is an approach to preventing hyperpolarization of the mitochondrial membrane. We confirmed data obtained earlier in our group that dodecylrhodamine 19 (C(12)R1) (a penetrating cation from SkQ family not possessing a plastoquinone group) has uncoupling properties, this fact making it highly potent for use in prevention of pathologies associated with oxidative stress induced by mitochondrial hyperpolarization. Further experiments showed that C(12)R1 provided nephroprotection under ischemia/reperfusion of the kidney as well as under rhabdomyolysis through diminishing of renal dysfunction manifested by elevated level of blood creatinine and urea. Similar nephroprotective properties were observed for low doses (275 nmol/kg) of the conventional uncoupler 2,4-dinitrophenol. Another penetrating cation that did not demonstrate protonophorous activity (SkQR4) had no effect on renal dysfunction. In experiments with induced ischemic stroke, C(12)R1 did not have any effect on the area of ischemic damage, but it significantly lowered neurological deficit. We conclude that beneficial effects of penetrating cation derivatives of rhodamine 19 in renal pathologies and brain ischemia may be at least partially explained by uncoupling of oxidation and phosphorylation.

Magnetic resonance tomography, staining with triphenyltetrazolium chloride, and tests for evaluation of functional disturbances “cylinder” and “limb stimulation” showed that daily intraperitoneal injection of dipeptide mimetic of nerve growth factor GK-2 (1 mg/kg) for 6 days to rats with experimental focal ischemia provoked by unilateral intravascular occlusion of a branch of the middle cerebral artery significantly improved neurological defi cit and decreased the infarction area.

A recently discovered key role of reactive oxygen species (ROS) in mitochondrial traffic has opened a wide alley for studying the interactions between cells, including stem cells. Since its discovery in 2006, intercellular mitochondria transport has been intensively studied in different cellular models as a basis for cell therapy, since the potential of replacing malfunctioning organelles appears to be very promising. In this study, we explored the transfer of mitochondria from multipotent mesenchymal stem cells (MMSC) to neural cells and analyzed its efficacy under normal conditions and upon induction of mitochondrial damage. We found that mitochondria were transferred from the MMSC to astrocytes in a more efficient manner when the astrocytes were exposed to ischemic damage associated with elevated ROS levels. Such transport of mitochondria restored the bioenergetics of the recipient cells and stimulated their proliferation. The introduction of MMSC with overexpressed Miro1 in animals that had undergone an experimental stroke led to significantly improved recovery of neurological functions. Our data suggest that mitochondrial impairment in differentiated cells can be compensated by receiving healthy mitochondria from MMSC. We demonstrate a key role of Miro1, which promotes the mitochondrial transfer from MMSC and suggest that the genetic modification of stem cells can improve the therapies for the injured brain.

Photochemically induced thrombosis of blood vessels in the prefrontal cortex in rats was shown to lead to ischemic infarcts in the lesion zone. Bilateral ischemic lesioning of the prefrontal cortex degraded measures of spatial memory when animals were tested in a Morris water maze with an invisible platform 20-24 days after surgery. Chronic intranasal administration of the peptide Met-Glu-His-Phe-Pro-Gly-Pro (Semax), a synthetic analog of ACTH(4-7), at a dose of 250 microg/kg/day during the first six days after photothrombosis, led to recovery of the animals' learning ability. The long-term antiamnestic action of the peptide observed here may result from its neuroprotective activity and its ability to stimulate the synthesis of neurotrophic factors.

We explored the neuroprotective properties of natural plant-derived antioxidants plastoquinone and thymoquinone (2-demethylplastoquinone derivative) modified to be specifically accumulated in mitochondria. The modification was performed through chemical conjugation of the quinones with penetrating cations: Rhodamine 19 or tetraphenylphosphonium. Neuroprotective properties were evaluated in a model of middle cerebral artery occlusion. We demonstrate that the mitochondria-targeted compounds, introduced immediately after reperfusion, possess various neuroprotective potencies as judged by the lower brain damage and higher neurological status. Plastoquinone derivatives conjugated with rhodamine were the most efficient, and the least efficiency was shown by antioxidants conjugated with tetraphenylphosphonium. Antioxidants were administered intraperitoneally or intranasally with the latter demonstrating a high level of penetration into the brain tissue. The therapeutic effects of both ways of administration were similar. Long-term administration of antioxidants in low doses reduced the neurological deficit, but had no effect on the volume of brain damage. At present, cationic decylrhodamine derivatives of plastoquinone appear to be the most promising anti-ischemic mitochondria-targeted drugs of the quinone family. We suggest these antioxidants could be potentially used for a stroke treatment.

The goal of the present study was to maximally alleviate the negative impact of stroke by increasing the therapeutic potency of injected mesenchymal multipotent stromal cells (MMSCs). To pursue this goal, the intercellular communications of MMSCs and neuronal cells were studied in vitro. As a result of cocultivation of MMSCs and rat cortical neurons, we proved the existence of intercellular contacts providing transfer of cellular contents from one cell to another. We present evidence of intercellular exchange with fluorescent probes specifically occupied by cytosol with preferential transfer from neurons toward MMSCs. In contrast, we observed a reversed transfer of mitochondria (from MMSCs to neural cells). Intravenous injection of MMSCs in a postischemic period alleviated the pathological indexes of a stroke, expressed as a lower infarct volume in the brain and partial restoration of neurological status. Also, MMSCs after cocultivation with neurons demonstrated more profound neuroprotective effects than did unprimed MMSCs. The production of the brain-derived neurotrophic factor was slightly increased in MMSCs, and the factor itself was redistributed in these cells after cocultivation. The level of Miro1 responsible for intercellular traffic of mitochondria was increased in MMSCs after cocultivation. We conclude that the exchange by cellular compartments between neural and stem cells improves MMSCs' protective abilities for better rehabilitation after stroke. This could be used as an approach to enhance the therapeutic benefits of stem cell therapy to the damaged brain. Significance The idea of priming stem cells before practical use for clinical purposes was applied. Thus, cells were preconditioned by coculturing them with the targeted cells (i.e., neurons for the treatment of brain pathological features) before the transfusion of stem cells to the organism. Such priming improved the capacity of stem cells to treat stroke. Some additional minimal study will be required to develop a detailed protocol for coculturing followed by cell separation.

Semax had a pronounced neuroprotective and antiamnesic effect during focal photoinduced ischemia of the prefrontal cortex. Intranasal administration of Semax for 6 days decreased the volume of cortical infarction and improved retention and performance of conditioned passive avoidance response.

Here we studied the cytoprotective effect of lithium chloride and sodium valproate in the in vivo model of neonatal cerebral ischemia/hypoxia and analyzed the influence of these substances on the death of the major neurovascular unit components in experimental ischemia in vitro. Lithium chloride and sodium valproate effectively prevented death of neurons, astrocytes, and endothelial cells in the oxygen-glucose deprivation. This treatment protected the brain of newborn rats from ischemia/hypoxia injury. The results suggest that lithium and sodium valproate can be used for the treatment of neurodegenerative pathologies associated with hypoxia and ischemia in newborns.

We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.

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.

We studied the effect of systemic administration of multipotent stem cells on impaired neurological status in rats with brain injury. It was found that transplantation of multipotent mesenchymal stromal cells of the bone marrow or human neural stem and progenitor cells to rats with local brain injury promoted recovery of the brain control over locomotor function and proprioceptive sensitivity of forelegs. The dynamics of neurological recovery was similar after transplantation of fetal neural stem and progenitor cells and multipotent mesenchymal stromal cells. Transplantation of cell cultures improved survival of experimental animals. It should be noted that administration of neural stem and progenitor cells prevented animal death not only in the acute traumatic period, but also in delayed periods.

Zinc chloride (0.02 mM, 3h) did not have any influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution (BSS). However, in the absence of glucose ZnCl(2) caused severe neuronal damage, decreasing cell survival to 12±2%. Either the blockade of ionotropic glutamate NMDA-receptors with MK-801 or APV or supplementation the medium with ruthenium red (mitochondrial Ca(2+) uniporter blocker) almost entirely protected CGNs from the toxic effect of ZnCl(2) during glucose deprivation (GD). However, NBQX (AMPA/kainate glutamate receptor blocker) did not show protective effect. Measurements of intracellular calcium ions concentration using fluorescent probe (Fluo-4 AM) and zinc ions (FluoZin-3AM) demonstrated that 1.5h-exposure to GD induced intensive increase of Fluo-4 fluorescence and small increase of FluoZin-3 fluorescence in neurons. The supplementation of medium with ZnCl(2) caused equal increase of FluoZin-3 fluorescence at both GD and normoglycemia, whereas the potentiation of Fluo-4 fluorescence by zinc was observed only under GD and could be prevented by MK-801. However, neither MK-801 nor NBQX could influence [Zn(2+)](i) increase caused by zinc addition under GD, while ruthenium red did cause significant increase of [Zn(2+)](i). This data implies that zinc ions during GD induce an additional overload of CGNs with calcium ions that get transported through activated NMDA-channel. Zinc and calcium ions accumulate in mitochondria and amplify individual destructive action on these organelles leading to neuronal death.

Oxidative stress-related renal pathologies apparently include rhabdomyolysis and ischemia/reperfusion phenomenon. These two pathologies were chosen for study in order to develop a proper strategy for protection of the kidney. Mitochondria were found to be a key player in these pathologies, being both the source and the target for excessive production of reactive oxygen species (ROS). A mitochondria-targeted compound which is a conjugate of a positively charged rhodamine molecule with plastoquinone (SkQR1) was found to rescue the kidney from the deleterious effect of both pathologies. Intraperitoneal injection of SkQR1 before the onset of pathology not only normalized the level of ROS and lipid peroxidized products in kidney mitochondria but also decreased the level of cytochrome c in the blood, restored normal renal excretory function and significantly lowered mortality among animals having a single kidney exposed to ischemia/reperfusion. The SkQR1-derivative missing plastoquinone (C12R1) possessed some, although limited nephroprotective properties and enhanced animal survival after ischemia/reperfusion. SkQR1 was found to induce some elements of nephroprotective pathways providing ischemic tolerance such as an increase in erythropoietin levels and phosphorylation of glycogen synthase kinase 3β in the kidney. SkQR1 also normalized renal erythropoietin level lowered after kidney ischemia/reperfusion and injection of a well-known nephrotoxic agent gentamicin.

A mitochondria-targeted chimeric compound consisting of a rhodamine derivative linked to a plastoquinone molecule (10-(6'-plastoquinonyl)decylrhodamine, SkQR1) was studied under conditions of acute brain or kidney damage. A protective effect of this compound was demonstrated in a model of focal brain ischemia, rat kidney ischemia/reperfusion, myoglobinuria (rhabdomyolysis, or crush syndrome), and pyelonephritis. We found that a single intraperitoneal injection of SkQR1 diminishes the size of the ischemic zone in the brain and improves performance of a test characterizing neurological deficit in ischemic animals. Control substance not containing plastoquinone appeared to be not neuroprotective. The data show that SkQR1 is a nephroprotectant and neuroprotectant, which can be due to the antioxidative action of this Skulachev cation.

The volumes of foci of injuries, evaluated by T2-suspended MRT images and analysis of histological sections stained by triphenyltetrazoleum chloride, were compared on a model of unilateral intravascular blocking of the middle cerebral artery branch. The two methods for evaluation of foci of lesions gave close results, correlating with the severity of neurological deficiency in animals subjected to ischemia, manifesting in behavioral tests.

BACKGROUND: Many ischemia-induced neurological pathologies including stroke are associated with high oxidative stress. Mitochondria-targeted antioxidants could rescue the ischemic organ by providing specific delivery of antioxidant molecules to the mitochondrion, which potentially suffers from oxidative stress more than non-mitochondrial cellular compartments. Besides direct antioxidative activity, these compounds are believed to activate numerous protective pathways. Endogenous anti-ischemic defense may involve the very powerful neuroprotective agent erythropoietin, which is mainly produced by the kidney in a redox-dependent manner, indicating an important role of the kidney in regulation of brain ischemic damage. The goal of this study is to track the relations between the kidney and the brain in terms of the amplification of defense mechanisms during SkQR1 treatment and remote renal preconditioning and provide evidence that the kidney can generate signals inducing a tolerance to oxidative stress-associated brain pathologies. METHODOLOGY/PRINCIPAL FINDINGS: We used the cationic plastoquinone derivative, SkQR1, as a mitochondria-targeted antioxidant to alleviate the deleterious consequences of stroke. A single injection of SkQR1 before cerebral ischemia in a dose-dependent manner reduces infarction and improves functional recovery. Concomitantly, an increase in the levels of erythropoietin in urine and phosphorylated glycogen synthase kinase-3β (GSK-3β) in the brain was detected 24 h after SkQR1 injection. However, protective effects of SkQR1 were not observed in rats with bilateral nephrectomy and in those treated with the nephrotoxic antibiotic gentamicin, indicating the protective role of humoral factor(s) which are released from functional kidneys. Renal preconditioning also induced brain protection in rats accompanied by an increased erythropoietin level in urine and kidney tissue and P-GSK-3β in brain. Co-cultivation of SkQR1-treated kidney cells with cortical neurons resulted in enchanced phosphorylation of GSK-3β in neuronal cells. CONCLUSION: The results indicate that renal preconditioning and SkQR1-induced brain protection may be mediated through the release of EPO from the kidney.

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C 4 R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C 12 R1). Surprisingly, C 12 R1 increased H + conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C 4 R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C 4 R1. In experiments with HeLa cells, C 4 R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C 4 R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C 12 R1. Thus, C 4 R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.