SkQR1 Reduces Neurologic Deficit Caused by Rat Brain Compression Ischemia.

The protective effect of antioxidant SkQR1 was examined on the model of left-sided compression ischemia in rat sensorimotor cortex. The special tests aimed to determine the neurologic deficit in the limbs and assess performance of the forelimbs showed that a 2.5-min ischemia produced no disturbance in the limb functions on postsurgery days 1, 3, and 7. Elevation of compression time resulted in neurologic deficit in animals, and its severity depended on this time. A single intravenous injection of SkQR1 (250 nmol/kg body weight) performed 30 min after ischemia significantly reduced the degree of neurologic deficit. In vitro model of ischemia in surviving rat hippocampal slices showed that a 15-min-long ischemia significantly inhibited the population excitatory postsynaptic potentials, which did not restore during reperfusion. Preincubation of the slices with SkQR1 did not significantly affect recovery of these potentials.

GK-2 Reduces Death of Cultured Granule Neurons in Cerebellum Induced by the Toxic Effects of Zinc Ions.

Peptide mimetic of nerve growth factor GK-2 in a dose of 1-2 mg/liter improves survival of cultured rat cerebellar granule neurons exposed to the cytotoxic effect of zinc ions, but has no protective effect against copper ion cytotoxicity. Experiments on cultured rat hippocampal slices demonstrated that GK-2 did not affect reactivity of pyramidal neurons and long-term potentiation in the hippocampal field CA1 and the probability of glutamate release from presynaptic terminals in the synapses of the CA3-CA1 fields. The results suggest that GK-2 does not affect the functional properties of synaptic transmission under normal conditions, but protects neurons from the toxic effects of zinc, which creates prerequisites for GK-12 use in the treatment of neurodegenerative diseases.

[Exosomes secretion and autophagy in long-term protection of neurons from excitotoxic damage]. / Sekretsiia ékzosom i autofagiia pri vyrabotke ustoichivosti neironov k ékzaitotoksicheskomu povrezhdeniiu.

In the model of induced neuronal resistance to the toxic effect of glutamate (deprivation of trophic factors), exosome secretion is demonstrated. Exosomes are secreted at the development of resistance during deprivation and at the first 24 h after preconditioning, as was shown by dot blot of extracellular fluid using anti-CD63 antibody. The autophagy inhibitor bafilomycin (0.01 µM) significantly reduces the quantity of the secreted exosomes at the stage of autophagy induction and at 24 h after induction. At the same time, inhibition of autophagy during the deprivation of trophic factors prevents the development of resistance, but inhibition of autophagy during the first 24 h after deprivation does not affect the development of resistance. We suggest that the long-term effects of preconditioning may be mediated by exosome secretion.

Effect of Noopept on Dynamics of Intracellular Calcium in Neurons of Cultured Rat Hippocampal Slices.

A neuroprotective and nootropic drug Noopept increased the frequency of spontaneous calcium transients in neurons of CA1 radial layer in cultured rat hippocampal slices. In contrast, the drug exerted no significant effect on intracellular calcium concentration and its dynamics in neurons of hippocampal CA1 pyramidal layer.

[Trophic factors deprivation induces long-term protection of neurons against excitotoxic damage]. / Vyrabotka dolgovremennoi ustoichivosti neironov k ékzaitotoksicheskomu povrezhdeniiu s pomoshch'iu deprivatsii troficheskikh faktorov.

One of the strategies to induce tolerance of neurons to toxic injury is preconditioning. Preconditioning is caused by a weak damage of cells, which become more resistant to subsequent, more severe damage. We found that preconditioning by deprivation of trophic factors, or deprivation of trophic factor and glucose effectively protects neurons against subsequent toxic effects of glutamate. Deprivation of trophic factors plays a decisive role in the development of resistance, regardless of whether it has been combined with glucose deprivation or not. Neuronal protection is achieved when the deprivation lasts from 30 min to two hours and is kept for a period of from one to five days. Preconditioning is accompanied neuronal secretion of cathepsin B occurs. We suggest that this phenomenon is associated with a more general process of exocytosis of lysosomes triggered by deprivation of trophic factors.

NMDA-Receptors Are Involved in Cu2+/Paraquat-Induced Death of Cultured Cerebellar Granule Neurons.

Rat cultured cerebellar granule neurons (CGNs) were not sensitive to CuCl2 (1-10 µM, 24 h), whereas paraquat (150 µM) decreased neuronal survival to 79 ± 3% of control level. Simultaneous treatment of CGNs with paraquat and CuCl2 (2, 5, or 10 µM Cu2+/paraquat) caused significant copper dose-dependent death, lowering their survival to 56 ± 4, 37 ± 3, or 16 ± 2%, respectively, and stimulating elevated production of free radicals in CGNs. Introduction of vitamin E, a non-competitive antagonist of NMDA subtype of glutamate receptors (MK-801), and also removal of glutamine from the incubation medium decreased toxicity of Cu2+/paraquat mixture. However, addition of Cu2+ into the incubation medium did not affect CGNs death caused by glutamate. These data emphasize that excessive copper in the brain may trigger oxidative stress, which in turn results in release of glutamate, overstimulation of glutamate receptors, and neuronal death.

[Adaptogenic and neuroprotective effects of lithium ascorbate]. / Adaptogennye i neiroprotektivnye svoistva askorbata litiya.

OBJECTIVE: Investigation of the neuroprotective properties of lithium ascorbate on the stress models in vivo and in vitro. MATERIAL AND METHODS: Neurocytological and behavioral studies on nerve cell culture and animal stress models. RESULTS: Significant neuroprotective effect of lithium ascorbate in neuronal cultures exposed to glutamate toxicity and adaptogenic effect of this drug in stress model in rats were shown. CONCLUSION: The results suggest lithium ascorbate has a high neuroprotective potential in stress models in vivo and in vitro.

[REORGANIZATION OF THE ULTRASTRUCTURE OF NEOCORTICAL NEURONS IN RATS TREATED WITH CELL-FREE DNA].

Neuron ultrastructure was studied in layers III-V of rat brain neocortex 24 hours after intraperitoneal (n=3) or intravenous (n=3) injection of cell-free DNA (7.7x10(-5) g/kg body weight). A plastic restructuring of nuclear chromatin, nucleolar hypertrophy, deep invaginations of nuclear envelope, hyperplasia of mito- chondria and their close contact with other organelles and the nucleus, formation of cytoplasmic tubulovesicular bodies which may promote enhanced synaptic vesicle transport to presynaptic axonal terminals, activation of astrocyte glia were found. The data obtained suggest that injection of cell-free DNA lead to pronounced ultrastructural reorganization in neocortical neurons directed to protein synthesis activation, enhancement of synaptic transmission efficiency, as well as intensification of energy metabolism, that may contribute to reparative and compensatory restorative processes in cerebral ischemic pathology.

Homocysteine toxicity in organotypic cultures of rat retina.

Effects of homocysteine in toxic concentrations on retinal neurons were studied in vitro. In organotypic roller cultures of postnatal (8-12-day-old) and adult rat retina homocysteine caused multiple damage to neurons in the outer nuclear layer, in deep compartments of the inner nuclear layer, and ganglion cell layer.

Roller organ cultures of the retina from postnatal rats.

Whole retinas of 2-14-day-old rats were cultured in a roller device for 2-14 days. Floating retinas of 7-14-day-old rats formed hole spheroid structures (spheroids) with the wall completely retaining the linear structure and layer-by-layer cellular and fibrous architecture, including the outer nuclear, outer plexiform, inner nuclear, inner plexiform layers, layers of ganglion cells and nerve fibers. The retina obtained at earlier terms of development often formed folds, with pyknotic nuclei of dead neurons in their deep compartments. In organ cultures of the retina isolated from rats at early postnatal periods, rosettes were formed in sites of local injury to the outer nuclear layer and pigmented epithelium. Roller organ cultures can be used for in vitro studies of the development and experimental diseases of the retina.

Characteristics of the development of the sensorimotor cortex in rats aged 7-8 days in roller cultures of free-floating brain slices.

The development and formation of the neuronal architectonics of organotypic structures of the sensorimotor cortex in rats aged 7-8 days were studied in roller cultures. Free-floating slices were cultured for 2-3 weeks. Serial paraffin sections of cultured tissue were stained with fast cresyl violet as described by Nissl. Initially planar sections of the cortex were found to change their configuration during the process of cultivation and were transformed into spherical structures, retaining the major histotypic features of cortical formations. Radially orientated pyramidal cells and fusiform neurons formed a cortical rudiment, not discriminated into layers, over the whole surface of the spherical tissue structures. In free-floating slices of the sensorimotor cortex of rats aged 7-8 days in roller cultivation, histogenetic processes continued, leading to the formation of histotypic cortical structures similar to the phylogenetically more ancient allocortical formations of the forebrain.

Organotypic cultures of free-floating slices of human embryo medulla oblongata.

The aim of the present work was to produce organotypic cultures of human embryo brain tissues, as needed for investigation of the effects of serum factors on mental diseases and their pharmacological treatment. Cultures were made using slices of medulla oblongata from human embryos aged 9-10 weeks, obtained from medical abortions. Free-floating slices were cultured using a modified roller method for four weeks. Light and electron microscopy, along with immunocytochemistry, demonstrated that cultured slices showed differentiation and growth of neurons, astro- and microgliocytes, with formation of the synaptic contacts and glioneuronal interactions typical of organotypic cultures. However, along with differentiated neurons and glial cells, there was some persistence of undifferentiated cellular elements with signs of stem cells and neuron and gliocyte precursor cells. These experiments showed that organotypic roller cultivation of free-floating slices of human embryo brain tissue can be used to study the processes of neuron and gliocyte development, the mechanisms of neurotoxicity and neuroprotection using a variety of morphological (including stereological) and biochemical study methods.

Roller organotypic cultures of postnatal rat retina.

Floating retinal sections from 7-12-day-old rats form ball-shaped retinal bodies during roller culturing. Histological studies of serial sections of retinal bodies showed that their outer surface is formed by the retina completely retaining organotypic cytoarchitectonics. Some retinal bodies have laminar structure consisting of several layers of the retina. At the initial stages of culturing some retinal bodies contain a cavity, which later is completely obliterated due to the growth of axons of ganglion cells and migration of glial cells and fibroblasts. This study demonstrated the possibility of long-term survival, differentiation, and in vitro axonal regeneration of ganglion cells, the main retinal efferent neurons, which can provide the basis for investigation of pathology and drug correction of injuries and stimulation of regeneration of these cells in experimental glaucoma models.

[Developmental characteristics of the sensorimotor cortex of 7-8 days old rats in the roller cultures of free-floating brain slices]. / Osobennosti razvitiia sensomotornoi kory 7-8-dnevnykh krys v rollernykh kul'turakh svobodnoplavaiushchikh srezov mozga.

Development and formation of neuronal architectonic of organotypic structures of sensomotor cortex of 7-8-day-old rats was studied in roller cultures. Free-floating cortical slices were cultured for 2-3 weeks. Serial paraffin sections of cultured tissue were stained with cresyl violet fast using Nissl's method. It was shown that during cultivation cortical slices changed their initial flat configuration and transformed into spherical bodies that retained main histiotypic features of cortical formations. Radially oriented pyramidal and fusiform neurons formed cortical structure that was not subdivided into individual layers and covered the whole surface of spherical tissue bodies. It is concluded that histogenetical processes were continuing in free-floating slices of sensomotor cortex of 7-8-day-old rats during roller cultivation. They result in formation of histiotypic cortical structure similar to phylogenetically more ancient allocortical formations of the forebrain.

[Organotypic cultures of free-floating sections of human embryonic medulla oblongata]. / Organotipicheskie kul'tury svobodnoplavaiushchikh srezov prodolgovatogo mozga émbrionov cheloveka.

The aim of the present study was the development of organotypic culture of embryonic nerve tissue of human brain, which is necessary for the study of the effect of blood serum factors in mental diseases and their medicinal treatment. For the cultivation the sections of medulla oblongata were taken from human 9-10 week embryos obtained at legal medical abortion. Free-floating sections were cultured using a modified roller method for 4 weeks. By using the methods of light and electron microscopy, as well as of immunocytochemistry, it was demonstrated that within the cultured sections the differentiation and growth of neurons, astrocytes and microgliocytes took place accompanied by the formation of synaptic contacts and the establishment of glial-neuronal relationships, characteristic to the organotypical cultures. However, along with the differentiated neurons and glial cells, undifferentiated cellular elements were partially retained, that had the signs of neuronal and glial stem cells and progenitor cells. The studies performed have demonstrated that the method of organotypic roller culture of free-floating sections of human embryonic brain tissue could be applied for the study of the processes of neuronal and gliocyte development, mechanisms of neurotoxicity and neuroprotection using various morphological (including stereological) and biochemical research methods.

Histogenesis of hippocampus and neocortex isolated from postnatal rats in organotypic roller tube cultures of floating brain sections.

Floating sections of the hippocampus and neocortex isolated from 6-10-day-old rats retained their spatial cell organization after 2-week roller-tube culturing. Cell structure in CA1, Ca2, and CA3 fields, polymorphic layer of the fascia dentata and its medial and lateral limbs were revealed in sections of the dorsal hippocampus. In neocortical sections, cortical neurons and subcortical structure were preserved, however, some cortical fragments changed their configuration and formed spherical structures, where cortical neurons were located in the external layer without forming typical of neocortex 6-layer structure.

A modified roller method for organotypic brain cultures: free-floating slices of postnatal rat hippocampus.

We describe a novel procedure for organotypic cultivation of free-floating brain sections of postnatal rats with a modified roller technique. Three hundred to 350-microm-thick sections of hippocampus are cultured for 13-15 days at 35.5 degrees C in 10-15 ml of feeding medium in 50-100 ml bottles under constant rotation on a horizontal high-speed mini-roller (60 rpm). Histological analysis (paraffin sections, Nissl Cresyl Violet and Hematoxylin/Eosin staining) demonstrates good survival of neuronal and glial cells and complete preservation of the neuronal organization of cultivated hippocampus with minimal central necrosis. This novel protocol permits not only survival and development of long-term three-dimensional organotypic postnatal brain tissue but also allows simultaneous cultivation of any number of brain sections in one bottle (up to 50 and even more) and therefore is useful for high throughput study of neurocytotoxic and hypoxic/ischemic neuronal damage with subsequent histological, immunocytochemical, biochemical, and molecular analysis.