Computer-aided Evaluation of Polyvalent Medications' Pharmacological Potential. Multiphytoadaptogen as a Case Study.

Many human diseases including cancer, degenerative and autoimmune disorders, diabetes and others are multifactorial. Pharmaceutical agents acting on a single target do not provide their efficient curation. Multitargeted drugs exhibiting pleiotropic pharmacological effects have certain advantages due to the normalization of the complex pathological processes of different etiology. Extracts of medicinal plants (EMP) containing multiple phytocomponents are widely used in traditional medicines for multifactorial disorders' treatment. Experimental studies of pharmacological potential for multicomponent compositions are quite expensive and time-consuming. In silico evaluation of EMP the pharmacological potential may provide the basis for selecting the most promising directions of testing and for identifying potential additive/synergistic effects. Multiphytoadaptogen (MPhA) containing 70 major phytocomponents of different chemical classes from 40 medicinal plant extracts has been studied in vitro, in vivo and in clinical researches. Antiproliferative and anti-tumor activities have been shown against some tumors as well as evidence-based therapeutic effects against age-related pathologies. In addition, the neuroprotective, antioxidant, antimutagenic, radioprotective, and immunomodulatory effects of MPhA were confirmed. Analysis of the PASS profiles of the biological activity of MPhA phytocomponents showed that most of the predicted anti-tumor and anti-metastatic effects were consistent with the results of laboratory and clinical studies. Antimutagenic, immunomodulatory, radioprotective, neuroprotective and anti-Parkinsonian effects were also predicted for most of the phytocomponents. Effects associated with positive effects on the male and female reproductive systems have been identified too. Thus, PASS and PharmaExpert can be used to evaluate the pharmacological potential of complex pharmaceutical compositions containing natural products.

[The role of glial cell line-derived neurotrophic factor isoforms in human glial tumors]. / Rol' izoform neirotroficheskogo faktora glial'noi kletochnoi linii (GDNF) v glial'nykh opukholyakh cheloveka.

Glial cell line-derived neurotrophic factor (GDNF) has a wide range of actions and positively affects viability, proliferative activity and migratory ability of cells in nervous system. That is why GDNF is being considered as a therapeutic molecule in the treatment of neurodegenerative diseases, in particular Parkinson's disease. However, GDNF has the same effect on high-grade glioma cells promoting their growth, resistance to therapy and dissemination. Expression of this factor in tissues and cultures of gliomas is up to five times higher than in intact brain matter. It was revealed that epigenetic modifications in GDNF gene promoter contribute to overexpression. Target suppression of GDNF gene transcription slows down growth of glioma and decreases cell migration. This review is devoted to the effect of GDNF on glioma cells, causes and consequences of its overexpression. Further analysis of expression and function of various GDNF isoforms in glial tumors may be valuable to develop new treatment methods for these dangerous diseases.

The effect of biodegradable silk fibroin-based scaffolds containing glial cell line-derived neurotrophic factor (GDNF) on the corneal regeneration process.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies. The aim of the work was to study the effect of biodegradable silk fibroin-based scaffolds containing GDNF on the corneal regeneration process. During cultivate the highest keratocytes proliferative activity was registered with scaffolds containing 250 ng/ml and 500 ng/ml GDNF. In mice with an experimental model of epithelial-stromal damage to the cornea, silk fibroin-based scaffolds containing GDNF in various concentrations were used (in groups 1, 2 and 3 silk fibroin-based scaffolds containing GDNF in a concentration of 50 ng/ml, 250 ng/ml and 500 ng/ml, respectively; in group 4 - silk fibroin-based scaffolds without GDNF; in group 5 - a solution of GDNF with concentration of 500 ng/ml; group 6- control). The area of the corneal epithelial defect in groups 2, 3, and 5 was less than in the other groups. The most pronounced positive immunohistochemical reaction with antibodies to Bcl2, Bax, phosphoERK1/2 and phospho-JNK1/2, Ki67, Gap43 was observed in groups 2 and 3. Thus, silk fibroin-based scaffolds with GDNF stimulate the epithelialization process, proliferative activity of epithelial cells and keratocytes, accelerate the formation of the stromal nerve plexus and exhibit anti-apoptotic activity.

Audiogenic Epilepsy and Structural Features of Superior Colliculus in KM Rats.

Using immunoblotting, we showed that in rats of audiogenic epilepsy (AE) prone strain (Krushinsky- Molodkina, KM) the superior colliculus tissue (SC) contains significantly less quantity of glial neurotrophic factor (GDNF), beta-tubulin and actin in comparison to the same brain region in "0" rats, nonprone to AE. This fact led to the suggestion that the histological structure of the SC in KM rats could differ significantly from that of the "0" strain. Using neuromorphologу technique, we demonstrated that the total number of SC cells, as well as the number of neurons were significantly less in KM rats than in the "0" strain rats. Particularly strong differences were found in the deep layers of SC, the area of terminals from IC. Further studies of the midbrain structures, will help to identify the novel aspects of neural networks, involved in the genesis of AE in rats of KM strain.

Influence of "Enriched Environment" on Behavior and Neurogenesis in Mice Selected by Cognitive Trait.

Mice selected for high score in the extrapolation test (EX line) and kept under conditions of "enriched environment" for 3 months demonstrated changes in locomotor and exploratory activity and enhanced reaction to novelty. The relative brain weight was higher and neurogenesis in the hippocampal fascia dentate was more intensive in this group. In non-selected mice, the changes were similar, but insignificant in many cases.

IMMUNOHISTOCHEMICAL STUDY OF DEPIGMENTED SKIN OF VITILIGO PATIENTS.

Immunohistochemical analysis was used to study depigmented skin areas such as macular of depigmentation and skin perimakular areas in vitiligo patients. It has been shown that the cells containing melanocytic cell marker TRP1 are localized both in macular and perimakular areas. Within the macula of depigmentation all TRP1 positive cells are in close contact with the basement membrane. In perimakular areas many cells that have lost contact with the basement membrane, were localized deep in the epidermis. About 92 % of TRP1 positive perimakular cells were also vimentin positive. Vimentin positive cells were numerous in perimakular areas but missing in the macula of depigmentation. Dense groups of cells immunopositive for transcription factor Snail, known as inductor of epithelial-mesenchymal transition, were localized in perimakular areas in close proximity to the macula depigmentation border. Such cells were extremely rare within the macula of depigmentation. There is reason to assume that an intensive process, which is similar to the epithelial-mesenchymal transition, might be the cause of melanocyte death in perimakular field, and thus prevents repigmentation of depigmented areas.

Audiogenic epilepsy and GABAergic system of the colliculus inferior in Krushinsky-Molodkina rats.

GABAergic brain system is an important link in the pathological circuits of rodent audiogenic epilepsy (AE). The number of GABAergic neurons in the inferior colliculi of KM strain rats (AE prone) was not different from that of the control non-prone strain. At the same time, the glutamate decarboxylase mRNA expression (the key enzyme of GABA synthesis) was 5 times higher than in control. The data evidence that the GABAergic system does play an important role in AE proneness.

NEUROGENIC NICHE OF THE ADULT MAMMALIAN BRAIN.

At the moment, the main location of the regional areas of neural stem cells in the adult brain is considered to be subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampal formation. However, the neural stem cells are not located chaotic in these areas, and they are localized in special niches ­ structural microenvironment that enables them to maintain identity, affect its proliferation and the fate of her descendants. The components of this microenvironment are intercellular interactions, the relationship with the blood vessels, extracellular matrix and specialized areas of basement membrane. The article describes of the neurogenic niches and the mechanisms controlling cell division and differentiation of progenitor cells.

[Moving activity and wakefulness-sleep cycle changes in a mouse MPTP model of Parkinson's disease].

A group of mice with preliminary implanted (under general anesthesia) electrodes for cortical EEG and nuchal EMG was subjected to continuous baseline 24-hr video and digital polysomnographic recording with the 12/12 light/dark schedule, and then injected subcutaneously with 24 or 48 mg/kg of MPTP toxin or (the control group) saline. The recordings were continued for 2 weeks more. A significant increase in activity and the waking percentage as well as decrease in REM sleep and NREM sleep (tendency) during the dark period as compared to the baseline and control recordings was found. The effect was seen just on the 7th day following MPTP administration and became significant by the 14th day. The effect was more pronounced after 48 mg/kg injection than after 24. There were no changes during the light period. Morphological control revealed a 70% and 35% decreases in the amount of tyrosine hydroxylase positive neurons in substancia nigra/pars compacta after 48 and 24 mg/kg of MPTP, respectively, as compared to the saline group.

[Transgenic cell cultures that synthesize neurotrophic factors and the possibility of therapeutic use of its cells].

Under the leadership of Corresponding Member of the Russian Academy of Sciences L.I. Korochkin, the Laboratory of Neurogenetics and Developmental Genetics (Institute of Gene Biology, Russian Academy of Sciences) for many years has been conducting studies of nervous system development, neural cell differentiation, and application of gene and cell technology to cure neurodegenerative diseases. The results of the study initiated by L.I. Korochkin and continued by his scientific successors support the direction of allocation of transgenic neurotrofic factors and heat-shock proteins as neuroprotectors for cell therapy. Potential for usage of promoter of HSP70 heat-shock gene of Drosophila to create transgenic constructs for therapy has been shown. Further improvement of technology of nonvirus transfer for therapeutic genes, as well as production of multicomponent genetic constructs coding several therapeutic factors with synergy effect, would stimulate creation of efficient cell medicals to cure neurodegenerative diseases.

[Neonatal injections of pharmacological agents and their remote genotype-dependent effects in mice and rats].

Experimental data were reviewed which demonstrated that the neonatal injection effects of certain biologically active drugs (ACTH(4-10) fragment and its analogue Semax, piracetam, caffeine, levetiracetam, busperone, etc.) could be detected in adult animals as changes in physiological and behavioral reactions and in several morphological traits as well. Audiogenic seizures proneness, anxiety-fear and exploration behavior as well as pain sensitivity were analyzed. The remote effects discovered were either similar in direction to those applied to an adult organism, or opposite to it. Pharmacological treatments of such type presumably interfere the CNS development during early postnatal ontogeny and change the normal pattern ofbrain development. These modulatory influences could be due to changes in neurotransmitter system development and are presumably capable to induce CNS morphological deviations (numbers of neurons, adult neurogenesis).

New calretinin-positive cells with polymorphous spines in the mouse forebrain during early postnatal ontogeny.

Immunohistochemical studies of calretinin (CR) in forebrain structures adjacent to the anterior horn of the lateral ventricle in adult mice allowed us to detect a population of previously unknown mono- and bipolar cells whose bodies and processes were coated with polymorphous spines (PS) (Morfologiya, 135, No. 3, 7-19 (2009)). CR-positive spiny (CR(+)PS) cells did not contain GAD67 and were located in the white matter and layers V-VI of the frontal area of the dorsomedial cortex close to the cingulum, the rostrodorsal part of the caudate-putamen, the anterior olfactory nucleus, and the subependyma of the dorsolateral angle of the lateral ventricle. We report here studies of the distribution of these cells in seven-day-old mice. Comparative topographic analysis of definitive and early CR(+)PS cells showed that in seven-day-old mice, CR(+)PS cells were absent from the sites at which they were seen in adults, i.e., the anterior olfactory cortex, the cortical plate, and the inner part of the neostriatum. In addition, small numbers of CR(+)PS-like cells were seen at this age within the dorsal migration pathway, at the anterior margin of the neostriatum, along the dorsal border of the neostriatum with the corpus callosum, in the subependymal layer of the lateral wall of the lateral ventricle, and in the cingulum area. These data demonstrate that CR(+)PS cells may have a postnatal origin. Experiments to verify this hypothesis were performed using postnatal administration of bromodeoxyuridine (BrdU) to mice aged 2-4 days, followed by assessment of brain sections fixed at age 20 days. Double immunolabeling of sections for CR and BrdU demonstrated the presence of CR(+)PS cells containing postnatally supplied BrdU. These data provide evidence that at least some CR(+)PS cells undergo mitosis at postnatal age. In all probability, during the period from 7 to 20 days of postnatal development, CR(+)PS cells migrate to the sites that they occupy in adult animals.

A new population of calretinin-positive cells, presumptively neurons, with polymorphous spines in the mouse forebrain.

An immunohistochemical reaction was used to study the locations of calretinin-positive cells on frontal sections of the anterior part of the mouse cerebral cortex. A previously undescribed population of cells with a characteristic structure was found at the anterior horns of the lateral ventricles. These cells had small (8-10 microm) round bodies giving rise to one and occasionally two nodose processes bearing rare polymorphous spines (PS) and thickenings of irregular shape. The relatively thick primary processes branched into finer processes, which also formed thickenings and spines of different calibers and structures. Calretinin-positive cells with polymorphous spines (CR+PS) were located in the white subcortical matter, in layer VI, and, significantly more rarely, in layer V of the frontal area of the dorsomedial cortex close to the cingulum. In addition, CR+PS cells were present in the rostrodorsal part of the caudate nucleus-putamen complex, in the anterior olfactory nucleus, in the subependymal layer of the dorsolateral angle of the lateral ventricle and, more rarely, in its dorsal wall. In contrast to the situation in mice, CR+PS cells were not present in the brains of other animals (rats, rabbits, cats). CR+PS cells showed no colocalization of calretinin with GABA or other neuronal or glial markers. It is suggested that these cells represent a previously unknown, probably neuronal, type of cell in the mouse forebrain.

[Surgical treatment policy using recombinant prourokinase for submacular hemorrhages].

An immunofluorescence technique was used to study the transretinal penetration of intravitreal fibrinolytic agent Hemase (a recombinant urokinase) in an experiment on 4 rabbit eyes. Hemase (54 kD) was proved to be able to penetrate across all retinal layers 2 hours after intravitreal administration. The efficacy of Hemase was tested in the treatment of submacular hemorrhages (SMH) of various etiology. Thirteen patients with SMH associated with age-related macular degeneration or ophthalmic injury were enrolled in the study. Hemase 500 IU was intravitreally injected 24 hours before pneumatic dislocation of SMH in 10 patients. In 3 patients with massive SMH, the proposed vitrectomy procedure was employed, by epiretinally injecting the fibrinolytic agent under a gas bubble, followed by drainage of the subretinal space. The operation was ended, by administering air and Hemase, and then by putting the patient in the supine position for 3-4 hours, thereafter his/her position was recommended to be changed to the prone position to realize the mechanism of pneumatic dislocation. The studies suggest that pneumatic SMH dislocation in combination with Hemase administration permits visual acuity to be increased in 80% of cases. The efficiency of manipulation and the functional outcome of treatment depend on the etiology of hemorrhage. The proposed vitrectomy technique using Hemase reduces neuroepithelial injury, effectively drains SMH, and dislocates its remnants from the foveal area.

[A novel population of calretinin-positive, presumably non-neuronal, cells with the polymorphous spines in mouse forebrain].

Using the immunocytochemical method, the localization of calretinin-positive cells was studied in the frontal sections of anterior portion of the mouse cerebral hemispheres. The population of cells with a characteristic structure, that was not described previously, was detected in the area of anterior horns of lateral brain ventricles. These cells have small (8-10 microm) rounded perikarya which gave rise to 1 or, rarely, 2 nodose processes covered with widely spaced, polymorphous spines (PS) and irregular thickenings. Primary, relatively thick, processes divide to produce thinner processes that also formed thickenings and spines of different dimensions and structure. Calretinin-positive cells with PS (CR+PS) cells are located in the white subcortical matter, layer VI, more rarely in layer V of the frontal area of dorsomedial cortex close to the cingulum. CR+PS cells were also present in rostro-dorsal part of the caudate nucleus-putamen complex, anterior olfactory nucleus, subependymal layer of the dorsolateral angle of the lateral ventricle, and, less frequently, near its dorsal wall. In contrast to mouse brain, CR+PS cells were not found in the brain of other animals (rats, rabbits, cats).Within CR+PS cells, no co-localization of calretinin with GABA and other neuronal or glial markers was found. It is suggested that the cells described represent previously unknown, presumably non-neuronal type of the mouse forebrain.

[The 1xC3 panel of recombinant inbred mouse strains. Presence or absence of pathologic neurologic traits of one of parental strains].

Mice from the earlier developed recombinant inbred strains (RIS), which were derived by crossing 101/HY mice (carrying the mut-1 allele determining increased susceptibility to the mutagenic action of alkylating compounds) with C3H/Sn mice (lacking this trait), were tested for the presence of two neurological pathologies, audiogenic epilepsy and splitting of pyramidal cell layer of the CA3 hippocampal field (specific only to 101/HY mice). It was demonstrated that segregation of RIS relative to these traits was independent from the presence or absence of the mut-1 allele. These findings suggested the appearance of mut-1-independent mutations in the 101/HY mice, which resulted in the development of neurological pathologies. The appearance of such mutations can be the consequence of the genetic repair defects, earlier observed in the mice with the same genotype, or they can be caused by other reasons.

[Novel calretinin-positive cells with polymorphous spines in the mouse forebrain during early postnatal ontogenesis].

Using an immunocytochemical method for calretinin (CR) detection, we have earlier described (Morfologiya, 2009 v. 135. No. 3, p. 7-19) the population of previously unknown mono- and bipolar cells with polymorphous spines (PS) covering their cell bodies and processes, in adult mice forebrain structures adjacent to anterior horn of lateral ventricle. CR-positive spiny (CR+PS) cells were negative to GAD67 and were detected in the white matter and in layers V and VI of frontal area of dorsomedial cortex close to the cingulum, in in rostro-dorsal part of the caudate nucleus-putamen complex, anterior olfactory nucleus and in subependymal layer of the dorso-lateral angle of the lateral ventricle. In this work, the distribution of these cells in 7-day-old mice was studied. Comparative topographical analysis of definitive and early CR+PS cells demonstrated that in 7-day-old mice CR+PS cells were absent from the areas of their localization in adult animals - anterior olfactory nucleus, cortical plate and inner portion of neostriatum. Meanwhile, some CR+PS-like cells were detected in 7-day-old mice inside the rostral migratory route, close to neostriatum anterior boundary, along the dorsal border between neostriatum and corpus callosum, subependymal layer of lateral wall of the lateral ventricle, and in the cingulum area. These findings are indicative of the possible postnatal appearance of CR+PS cells. To test this hypothesis, the experiments were conducted in which bromodeoxyuridine (BrdU) was administered to the mice on their postnatal days 2-4 with the subsequent study of the brain sections of these animals sacrificed on their postnatal day 20. Double immunolabeling of these sections for CR and BrdU has detected the presence of CR+PS cells that contained postnatally administered BrdU. These results strongly suggest that, at least, some portion of CR+PS cells have their mitosis postnatally. It may be assumed, that CR+PS cells migrate to the sites of their distribution in adult animals from day 7 till day 20 of postnatal develpment.

[Impact of xenotransplantation of multipotent mesenchymal stem cells of the bone marrow on the recovery of the structural organization and functional activity of the retina in its damage].

Among the potential sources of cells for transplantation, the most promising autologous material for cell therapy and tissue engineering are multipotent mesenchymal stem cells (MMSC). The authors carried out an experiment dealing with MMSC xenotransplantation into the rabbit suprachoidal space on a model of laser retinal coagulation. For objective assessment of the magnitude and pattern of changes in the functional activity of different retinal cellular elements and the efficiency of MMSC transplantation, they performed electroretinographic and histological studies. The study indicated that MMSC transplantation into the suprachoroidal space is a safe and effective procedure for cell incorporation in order to treat retinal diseases. Electroretinography provided evidence that single MMSC transplantation better preserved the function of photoreceptors (a-wave). Histomorphometric studies demonstrated that the inserted cells stimulated reparative processes in the laser-induced damaged retina.