[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.

[Peptide drug cortexin inhibits brain caspase-8]. / Peptidnyi preparat korteksin ingibiruet kaspazu-8 mozga.

Cortexin, a drug containing hydrolyzed brain peptides, has long been used in clinics, but the mechanisms of its action remain obscure. We have hypothesized that cortexin-related neuroprotection is associated with the ability of the drug to inhibit brain proteases. Cortexin effectively inhibited brain caspase-8, while its effects on caspase-1, -3, -9, cathepsin B and calpain were much less pronounced or absent. In addition, we isolated a peptide fraction from cortexin holding all the inhibitory capacity of the original drug, but with a much more simple composition. Both cortexin and its fraction prevented neuronal damage in a culture model of glutamate-induced cell death. Neuroprotective effect of Cortexin may be mediated by inhibition of the initiator caspase-8 in the brain.

[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.

A secreted caspase-3-substrate-cleaving activity at low pH belongs to cathepsin B: a study on primary brain cell cultures.

The cysteine proteases caspase-3 and cathepsins are involved in both neuronal plasticity and neuropathology. Using primary neuroglial and glial cerebellar cultures, the pH dependence of cleavage of a synthetic caspase-3 substrate, Ac-DEVD-AMC, was studied. At acidic pH, cathepsin B cleaved Ac-DEVD, this activity being significantly higher than that of caspase-3 at pH 7.4. This activity is blocked by peptide inhibitors of both caspase-3 and cathepsin B. Substitution of culture medium for balanced salt solution stimulated cathepsin B secretion in both types of cultures. Ischemia (oxygen-glucose deprivation) significantly decreased secretion of cathepsin B activities into the culture medium.

Antioxidant and neuroprotective properties of N-docosahexaenoyl dopamine.

N-Docosahexaenoyl dopamine exhibited antioxidant activity in the test with a stable oxygen radical galvinoxyl. This compound produced a dose-dependent protective effect on cultured granular cells from rat cerebellum under conditions of oxidative stress. N-Docosahexaenoyl dopamine decelerated the development of symptoms of Parkinson's disease in mice receiving neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Central administration of a caspase inhibitor impairs shuttle-box performance in rats.

Recent studies suggest that caspase-3-mediated mechanisms are essential for neuronal plasticity. N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val- Asp(OMe)-fluoromethyl ketone (z-DEVD-fmk), a caspase inhibitor with predominant specificity toward caspase-3, has been shown to block long-term potentiation in hippocampal slices. Intrahippocampal infusion of a caspase-3 inhibitor to rats has been shown to significantly impair spatial memory in the water maze. The present work was designed to study whether i.c.v. administration of a caspase-3 inhibitor z-DEVD-fmk impairs learning in other tasks related to specific forms of memory in rats. The rats received bilateral injections of z-DEVD-fmk or N-benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (z-FA-fmk) ("control" peptide) at a dose of 3 nmol. Administration of z-DEVD-fmk significantly decreased the number of avoidance reactions in some blocks of trials in the active avoidance (shuttle box) learning, while z-FA-fmk had no effect as compared with intact rats. However, only a slight effect of the caspase inhibitor across the session was found. z-DEVD-fmk impaired development of some essential components of the two-way active avoidance performance, such as escape reaction, conditioned fear reaction, and inter-trial crossings. Measurement of caspase-3 activity in rat brain regions involved in active avoidance learning revealed most expressed z-DEVD-fmk-related inhibition of the enzyme activity (about 30%) in the fronto-parietal cortex. A similar effect was close to significant in the hippocampus, but not in the other cerebral structures studied. In primary cultures of cerebellar neurons z-DEVD-fmk (2-50 microM) inhibited caspase-3 activity by 60-87%. We suggest that moderate inhibition of caspase-3 resulting from the central administration of z-DEVD-fmk to rats may impair active avoidance learning. Taking into account previous data on the involvement of neuronal caspase-3 in neuroplasticity phenomena we assume that the enzyme may be important for selected forms of learning.

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.

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.

Protective effect of Biolan during ischemic damages to cultured cerebellar granular cells.

Biolan containing carnosine and delta sleep-inducing peptide produced a protective effect on cerebellar granular cells from 7-day-old rats exposed in culture to ischemia (oxygen and glucose deprivation) and cytotoxic influence of glutamate. These results indicate that Biolan holds much promise for correction of ischemic brain damages in clinical practice.

[Ischemic injury of the ultrastructure of neurons in organotypic hippocampal tissue culture]. / Ishemicheskie povrezhdeniia ul'trastruktury neironov v organotipicheskoi kul'ture tkani gippokampa.

An ultrastructural study of organotypic hippocampal explants subjected to the effect of ischemia for 30 min followed by a period of reoxygenation lasting for 20-24 hours, was performed. The data obtained indicate that ischemic injury of area CA1 neurons develops mainly as a necrosis. In addition, some neurons along with necrotic changes, demonstrate some signs of apoptosis, suggesting that the execution of its program may accompany these changes, thus leading the cells to the state of so-called apoptotic-necrotic continuum. On the other hand, the observed ultrastructural changes directed to metabolic activation and energy deficit compensation, may indicate the ability of neurons for realization of reparative processes following ischemic injury.

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.

[Ultrastructural plasticity in the hippocampus organotypic tissue culture]. / Plasticheskie perestroiki ul'trastruktury gippokampa v organotipicheskoi kul'ture tkani.

Hippocampal cross sections of 7-9 day old rats were explanted onto semipermeable microporous membranes for 14-16 days followed by electron microscopic investigation. Besides the structures typical for adult hippocampus in vivo, substantial ultrastructural signs of metabolism and synaptic connections in explants were revealed: redistribution of polysomes and cisterns of granular endoplasmic reticulum, numerous pinocytotic invaginations, growth cones, somatic filopods, dotted (perforated) contacts, new formation of synapses and vacant postsynaptic sites. The data obtained indicate the active compensatory processes of plasticity, which ensure the maintenance of structure and function of hippocampal tissue in vitro.

Hypoxic and posthypoxic neuronal injury in hippocampal cell culture: attenuation by lipophylic antioxidant U-18 and superoxide dismutase.

The neuroprotective effects of synthesized lipophylic antioxidant from hindered phenol class (U-18) and hydrophylic antioxidative enzyme superoxide dismutase (SOD) were tested on long-term mouse hippocampal cell cultures exposed to hypoxia/reoxygenation. The application of U-18 to the cultures during 6-8 hr hypoxia followed by 16-18 hr reoxygenation in the absence of antioxidant significantly reduced neuronal death. Thus, lipophylic free radical scavenger may exert a delayed neuroprotective effect, probably owing to persistent incorporation into phospholipid membranes and prevention of their lipid peroxidation by means of prolonged intramembranous free radical quenching. On the other hand, the exposure of the cultures to U-18 during 15 hr hypoxia without subsequent reoxygenation also led to significant reduction of neuronal death compared with that observed without antioxidant. These findings suggest that free radical neuronal damage may occur under conditions of prolonged restricted oxygen access to the neurons. The hypoxic/posthypoxic neuronal injury significantly decreased in the cultures exposed to hydrophylic cytoplasmic enzyme SOD (300 U/ml). The neuroprotective effects of both lipophylic U-18 and hydrophylic SOD on the cultures exposed to hypoxia/reoxygenation might reflect the damaging free radical overproduction in different morphofunctional compartments of the nerve cell.

[The role of posthypoxia reoxygenation in the destruction of cultured hippocampal neurons]. / Rol' postgipoksicheskoi reaksigenatsii v destruktsii kul'tiviruemykh neironov gippokampa.

A comparative quantitative analysis of neuronal death was performed on mice hippocampal cell cultures, which were subjected to hypoxia only or to hypoxia with subsequent reoxygenation during equal time. It was shown that the number of dead neurons significantly increased after 5-7 hs hypoxia followed by 3 hs reoxygenation period, comparatively with neuronal death observed after 8-10 hs hypoxia without subsequent reoxygenation. These results demonstrate that neurodestructive processes initiated by hypoxia develop more intensively during the posthypoxic reoxygenation than during the equal time of hypoxia and suggest the role of free radical and peroxide compound formation in neuronal death after renewal of oxygen access to nerve cells.

[Cultivation of reaggregated brain cells in rapidly rotating mini-rollers]. / Kul'tivirovanie reagregirovannykh kletok mozga v bystro vrashchaiushchikhsia mini-rollerakh.

An original method of cultivation of reaggregated brain cells with the aid of high-speed portable mini-rollers is described. The mini-roller consists of parallel rollers and an electric motor rotating the flasks at a speed of 60 to 70 rpm. The Moscona technique was used for preparing brain cell suspensions. During cultivation of the suspension of dissociated cells in high-speed mini-rollers, reaggregates with an internal organotypic structure were obtained. The method suggested provides stable and reproducible results.