M. V. Gulyaev

Neuroprotective Effects of Mitochondria-Targeted Plastoquinone and Thymoquinone in a Rat Model of Brain Ischemia/Reperfusion Injury

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.

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector

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 (C4R1) 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 (C12R1). Surprisingly, C12R1 increased H(+) conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C4R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C4R1. In experiments with HeLa cells, C4R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C4R1 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 C12R1. Thus, C4R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.

Neuroprotective effect of glutamate-substituted analog of gramicidin A is mediated by the uncoupling of mitochondria

BACKGROUND Reactive oxygen species are grossly produced in the brain after cerebral ischemia and reperfusion causing neuronal cell death. Mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150mV. Therefore, limited uncoupling of oxidative phosphorylation could be beneficial for cells exposed to deleterious oxidative stress-associated conditions by preventing excessive generation of reactive oxygen species. METHODS Protonophoric and uncoupling activities of different peptides were measured using pyranine-loaded liposomes and isolated mitochondria. To evaluate the effect of glutamate-substituted analog of gramicidin A ([Glu1]gA) administration on the brain ischemic damage, we employed the in vitro model of neuronal hypoxia using primary neuronal cell cultures and the in vivo model of cerebral ischemia induced in rats by the middle cerebral artery occlusion. RESULTS [Glu1]gA was the most effective in proton-transferring activity among several N-terminally substituted analogs of gramicidin A tested in liposomes and rat brain and liver mitochondria. The peptides were found to be protective against ischemia-induced neuronal cell death and they lowered mitochondrial membrane potential in cultured neurons and diminished reactive oxygen species production in isolated brain mitochondria. The intranasal administration of [Glu1]gA remarkably diminished the infarct size indicated in MR-images of a brain at day 1 after the middle cerebral artery occlusion. In [Glu1]gA-treated rats, the ischemia-induced brain swelling and behavioral dysfunction were significantly suppressed. CONCLUSIONS The glutamate-substituted analogs of gramicidin A displaying protonophoric and uncoupling activities protect neural cells and the brain from the injury caused by ischemia/reperfusion. GENERAL SIGNIFICANCE [Glu1]gA may be potentially used as a therapeutic agent to prevent neuron damage after stroke.

Antitumor effects of monoclonal antibodies to connexin 43 extracellular fragment in induced low-differentiated glioma.

We studied the effect of intravenous administration of monoclonal antibodies to the second extracellular loop of connexin 43 (MAbE2Cx43) on the dynamics of glioma growth and survival of experimental animals. Morphometric analysis of magnetic resonance imaging data showed that weekly intravenous administration of MAbE2Cx43 in a dose of 5 mg/kg significantly reduced glioma volume starting from day 21 after tumor implantation. By day 29, the mean volume of glioma in the experimental group (therapy with specific antibodies) was 2-fold lower than in controls. Deceleration of glioma growth in rats receiving MAbE2Cx43 was accompanied by a significant prolongation of rat lifespan (according to Kaplan−Meier test) and even led to complete recovery without delayed relapses in 19.23% animals. The mechanism of tumor-suppressing effects of antibodies can be related to inhibition of specific functions of connexin 43 in glioma cells in the peritumoral zone.

Ginseng extract attenuates early MRI changes after status epilepticus and decreases subsequent reduction of hippocampal volume in the rat brain

Prolonged epileptic seizures during status epilepticus (SE) are known to cause neuronal death and lead to brain damage. Lesions in various brain regions can result in memory and cognitive impairment, thus searching of new neuroprotective drugs is important. We evaluated effects of single and chronic administration of ginseng extract on early and late changes in MRI measurements in the rat brain after lithium-pilocarpine SE. Butanol extract of ginseng root cell culture DAN-25 was administered after termination of SE. MRI study of the rat brain was performed 2, 7, and 30 days after SE. High-resolution T2-weighed images and T2-maps were obtained, and total damaged area, hippocampal volume, and T2 relaxation time in several brain structures were calculated. Single administration of ginseng extract attenuated early changes in brain structures found on day 2 after SE. Both single and chronic treatment with ginseng extract decreased brain damage on day 7 after SE. An increase in T2-relaxation time in the hippocampus on day 2 after SE was less prominent in ginseng-treated rats than in saline-treated rats. 30 days after SE, the reduction of hippocampal volume was found both in saline-treated and ginseng-treated rats; however, it was less pronounced in ginseng-treated rats. We conclude that administration of ginseng extract after SE termination reduced brain damage caused by prolonged seizures. Ginseng extract was effective during early period after SE and had a specific protective effect on the hippocampus.

Protection of Neurovascular Unit Cells with Lithium Chloride and Sodium Valproate Prevents Brain Damage in Neonatal Ischemia/Hypoxia.

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.

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.

Assessment of Long-Term Sensorimotor Deficit after Cerebral Ischemia/Hypoxia in Neonatal Rats

MRI studies showed that cerebral ischemia/hypoxia in neonatal rat pups applied using the Levine–Rice method induced unilateral lesions in the cerebral cortex, striatum, and hippocampus. Unilateral ischemic lesions led to long-term sensorimotor and behavioral impairments at 90–115 days after surgery on testing the animals in a battery of tests consisting of the cylinder, tapering beam, Montoya, and paw-placing tests. This combination of tests, along with MRI studies, provided reliable evaluation of long-term sensorimotor impairments in adult animals subjected to trauma in the neonatal period.

Magnetohydrodynamic Thermochemotherapy and MRI of Malignant Tumorigenesis

Citric-ferrite sol (CFS) has been synthesized and successfully tested as MRI negative contrast preparation for revealing primary tumors, and also invasions and metastases at the tumor relapses. Magnetohydrodynamic thermochemotherapy (MTC) by Cysplatin (CP), Mitoxantron (MX), Melphalan (MP) combined with CFS, which were carefully monitoring in vivo and quantified by an electronic detector, improved results of cancer treatment. At various stages of oncogenesis, MTC with CFS at a dose of 62 mg Fe/kg combined with chemotherapeutic drugs was performed at +48º C for 30 min. While such MTC, treatment of ~30 mm3 B 16 melanoma tumor by AC magnetic field led to tumor regression in female mice up to 40 %, and an increase of life span up to 300 % was achieved.

The registration of signals from the nuclei other than protons at 0.5 T MRI scanner

The practical aspects of the adaptation of the medical MRI scanner for multinuclear applications are considered. Examples of high resolution NMR spectra for nuclei 19F, 31P, 23Na, 11B, 13C, 2H, and also NQR spectrum for 35Cl are given. Possibilities of MRI for nuclei 19F, 31P, 23Na, 11B are shown. Experiments on registration of signals 19F from the fluorocarbons injected in laboratory animals are described.