Multinuclear MRI Research.

The presented data clearly demonstrate that multinuclear MRI has a great potential in research and clinical fields. The biomedical value of many heavy nuclei that are part of drugs, contrast agents, and molecular complexes in live tissues emphasizes the need for creating and using MRI scanners tuned to the Larmor frequencies of such nuclei. This article discusses a number of specific problems solved with the use of multinuclear MRI. Since the sensitivity of the MRI on heavy nuclei, as a rule, is insufficient for obtaining images of satisfactory quality, it is proposed to use hyperpolarization techniques for preparing objects for the MRI diagnostics. However, bearing in mind the high cost of hyperpolarization techniques, alternative approaches using contrast agents normally absent in tissues can be more suitable in certain situations. For instance, due to the absence of fluorine in the body, 19F MRI allows for successful diagnostics of the respiratory tract and lungs filled with fluorine-containing gas, detecting the location of the blood substitute Perftoran® in the body, monitoring the transport along the gastrointestinal tract of a capsule filled with a preparation containing fluorine-19 nuclei, etc. The possibility of non-invasive local measurements of the concentration of sodium-23 in the body, which, in case of a deviation from the norm, is linked to such pathologies as diabetes, hypertension, renal failure, and many others, is also demonstrated. Reported as well is the creation of transmitting and receiving infrastructure that makes possible effective MRI measurements at low fields (of about 0.5 T) at 10 Larmor frequencies corresponding to the nuclei of hydrogen, deuterium, fluorine, boron, chlorine, sodium, carbon, phosphorus, silicon and oxygen.

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-3ß, 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-3ß. 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.

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.

The Use of Technetium-99m for Intravital Tracing of Transplanted Multipotent Stromal Cells.

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.

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.

CHANGES IN THE NUMBER OF NEURONS, ASTROCYTES AND MICROGLIA IN THE BRAIN AFTER ISCHEMIC STROKE ASSESSED BY IMMUNOHISTOCHEMISTRY AND IMMUNOBLOTTING.

It is known that the mechanisms of damage in the brain after stroke are regulated by combination of several types of cells, primarily of neurons, astrocytes, endothelium and microglia. Ischemic exposure disrupts the balance in the cellular composition of the brain; in the lesion, cells die by necrosis while in tissue surrounding ischemic zone the delayed induction of apoptosis occurs, and namely the ratio of death of different cells determines the clinical outcome of the disease. Thus, the assessment of death of various cell types of the neurovascular unit is an important part of fundamental studies of the mechanisms of brain damage and pre-clinical studies of potential neuroprotective drugs. In this line, we have conducted a comparative study of the two most often used methods: immunohistochemical staining of brain sections, allowing to determine the number and localization of specific cells in the tissue among other types of cells, and immunoblotting that detects specific proteins in the tissue homogenate. We have found that, depending on the type of cells, changes in their number and composition after stroke can be diffuse or localized, which imposes restrictions on the use of any method of estimation of the number of cells in brain tissue. In general, the most preferable is the use of immunohistochemistry, however, with certain limitations, immunoblotting can be used in estimating amounts of astroglia and microglia.

Effects of activated protein C on the size of modeled ischemic focus and morphometric parameters of neurons and neuroglia in its perifocal zone.

The effects of activated protein C (APC) on the quantitative parameters of neurons and neuroglia in the perifocal zone of infarction induced in the left hemispheric cortex were studied in two groups of rats. Group 1 animals served as control (control infarction). Group 2 rats were injected with APC (50 µg/kg) in the right lateral cerebral ventricle 3 h after infarction was induced, and after 72 h the infarction size was evaluated and the neurons and neuroglia in the perifocal zone were counted. APC reduced the infarction size 2.5 times in comparison with the control and reduced by 16% the neuronal death in the perifocal zone layer V, causing no appreciable changes in layer III, and did not change the size of neuronal bodies but increased (by 11%) the size of neuronal nuclei in layer III. The protein maintained the sharply increased count of gliocytes in the perifocal zone of infarction and promoted their growth. Hence, APC protected the neurons from death in the ischemic focus by increasing the gliocyte count and stimulating the compensatory reparative processes.

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.

Neuroprotective effect of GK-2, a dipeptide mimetic of nerve growth factor, during experimental focal ischemia in middle cerebral artery basin.

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 deficit and decreased the infarction area.

MRI morphometry of the cerebral ventricles in patients with attention deficit hyperactivity disorder.

A total of 27 right-handed patients aged 7-30 years with diagnoses of attention deficit hyperactivity disorder were studied using standard MRI scans. Of these, 14 were aged below 13 years. The volumes of the lateral ventricles were measured using T1-weighted MRI images of sagittal sections of the brain to a precision of 3 mm3. External head sizes were also measured to allow ventricle volumes to be normalized. All patients underwent complex neuropsychological investigations. Memory was assessed, along with visual, auditory, tactile, and spatial recognition functions and the motor and speech spheres. Test data were assessed in terms of the severity of impairments associated with one brain structure or another on a tenpoint scale. Assessment points were summed for each hemisphere, for the "first area" (cortical structures), and all structures for statistical analysis. Neuropsychological testing revealed functional impairments predominantly of the frontal areas of the hemispheres, the hippocampus, and the reticular formation. Neuropsychological deficits were least linked with alterations in the postcentral and parietal areas of the cortex. Statistical analysis demonstrated a significant positive correlation between the normalized left lateral ventricle volume and the degree of neuropsychological impairments (r = 0.5127 at p = 0.0063) for the whole study group. The correlation was more marked on comparison of the normalized left ventricular volume and the severity of neuropsychological impairments related to the left hemisphere (r = 0.6303 at p = 0.0004). A relationship was seen between the volume of the intraventricular space and cortical functional impairments (r = 0.5071 at p = 0.0069) in patients less than 13 years old. A relationship between ventricular volume and linear head size was confirmed (r = 0.5759 at p = 0.0017), which was more marked in subjects less than 13 years old (r = 0.6833 at p = 0.01).

Comparative evaluation of two methods for studies of experimental focal ischemia: magnetic resonance tomography and triphenyltetrazoleum detection of brain injuries.

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.

Mechanisms of positive effects of transplantation of human placental mesenchymal stem cells on recovery of rats after experimental ischemic stroke.

Mesenchymal stem cells isolated from human placenta and in vitro labeled with fluorescent magnetic microparticles were intravenously injected to rats 2 days after induction of focal cerebral ischemia (endovascular model). According to MRT findings, transplantation of mesenchymal stem cells led to an appreciable reduction of the volume of ischemic focus in the brain. Two or three weeks after transplantation, labeled cells accumulated near and inside the ischemic focus, in the hippocampus, and in the subventricular zone of both hemispheres. Only few human mesenchymal stem cells populating the zone adjacent to the ischemic focus started expressing astroglial and neuronal markers. On the other hand, transplantation of mesenchymal stem cells stimulated proliferation of stem and progenitor cells in the subventricular zone and migration of these cells into the ischemic zone. Positive effects of transplantation of these cells to rats with experimental ischemic stroke are presumably explained by stimulation of proliferation of resident stem and progenitor cells of animal brain and their migration into the ischemic tissue and adjacent areas. Replacement of damaged rat neurons and glial cells by transplanted human cells, if it does take place, is quite negligible.