Generation of recombinant extracellular fragment of vascular endothelial growth factor receptor 2 and specific monoclonal antibodies to this receptor.

cDNA extracellular Ig-like domains I-III of vascular endothelial growth factor receptor 2 (VEGFR2) was cloned in an expressing vector pET_32a. Western blotting showed immunochemical identity of recombinant VEGFR2I-III produced by prokaryotic expression system to the native receptor. BALB/c mice were immunized with VEGFR2I-III for obtaining specific antibodies to VEGFR2. Hybridomas producing monoclonal antibodies were selected by ELISA, Western blotting, and immunocytochemical assay. Thus, we obtained hybridoma producing monoclonal antibodies to VEGFR2 that selectively interact with both recombinant and native extracellular fragment of the receptor.

Activation of expression of brain-derived neurotrophic factor at the site of implantation of allogenic and xenogenic neural stem (progenitor) cells in rats with ischemic cortical stroke.

Ischemic stroke was modeled in the sensorimotor zone of the brain cortex in adult rats. Rat embryonic nervous tissue, neural stem cells from human olfactory epithelium, and rat fibroblasts (cell control) were implanted into the peri-infarction area of rats of different groups immediately after stroke modeling. Expression of BDNF mRNA was analyzed 7 days after surgery by real-time PCR. BDNF expression in cell preparation before their implantation was minimum. The expression of BDNF mRNA increased by 5-6 times in the areas of implantation of rat fibroblasts and human olfactory epithelium and by 23 times in the area of implantation of rat embryonic nervous tissue compared to periinfarction areas without cell implantation. These findings confirm the possibility of realization of the therapeutic effects of neural stem cells via expression of trophic factors.

[Influence of the neural stem cell transplantation on the restoration of CNS functions in rats with cortical stroke].

Ischemic stroke was modeled in white pedigreeless rats by the superficial blood vessel devascularization in the sensorimotor cortex. The preparations of neural progenitors--rat embryonic neural stem cells (rENSC) and human olfactory epithelium-derived neural stem cells (hOENSC) and differentiated fibroblasts ("cell control") were transplanted at the perimeter of the devascularized region. These cells marked with vital tracer stayed alive in the brain parenchyma for at least 16 days. The monitoring of contralateral forepaw motor deficit during 8 weeks demonstrated that only rats with rENSC transplantation had the stable and significant improvement of performance in cylinder test and swimming test (forepaw inhibition test) in comparison to "cell controls" and rats without cell transplantation. The maximal difference in the relative values (the efficacy) was 25% to the end of the experiment. There was no difference in the indicators of vibrissae-elicited forelimb placing test between experimental groups. The methodological approach used makes it possible to broaden the study of mechanisms of neural stem cells' therapeutic effect in stroke.

Neurological deficit and disturbances in higher nervous activity during modeling of perinatal hypoxic-ischemic damage to the central nervous system in rat pups.

Seven-day-old Wistar rat pups were subjected to unilateral occlusion of the common cerebral artery and maintained in oxygen-low atmosphere. Neurological and behavioral changes were monitored for 12 weeks. The survival rate of treated animals was 90%. Body weight gain in these rats was lower than in the control. Neurological deficit was maximum 1 week after treatment and slightly regressed by the 12th week. Locomotor activity in treated rats was higher than in controls. Administration of ketamine in subanesthetic doses caused permanent ipsilateral rotational asymmetry in animals. Spatial disorientation and cognitive deficit in rats with hypoxic-ischemic damage to the central nervous system were revealed in passive avoidance, Y-maze, and rotarod tests. The total area of the hemisphere decreased, while the area of the lateral cerebral ventricle increased at the side of occlusion over the first 4-5 weeks of postnatal development. The size of the ipsilateral hemisphere remained low in adult animals.

Monitoring of neurological deficit and disturbances in higher nervous activity in rats with focal cerebral ischemia.

Neurological, locomotor, and behavioral changes in 20 Wistar rats with permanent proximal occlusion of the middle cerebral artery and 18 control sham-operated animals were monitored for 2 months at 10-day intervals. Neurological deficit was maximum immediately after occlusion (3.0 0.6 points), then progressively decreased, but did not completely disappear (0.70 0.06 points on day 60). In control animals neurological status returned to normal on days 10-15. The degree of ketamine-induced rotational asymmetry was 4.0 0.7 rpm over 40 days after surgery and decreased to 2.5 0.6 rpm on day 60. In control rats this parameter only transiently increased to 1.00 0.03 rpm (tdelta=2.5, p<0.05). The time of stay on a rotarod and the latency of passive avoidance in rats with focal cerebral ischemia were lower than in control animals throughout the experiment. The results of complex tests can be used in the experimental search for new drugs for the therapy and rehabilitation of stroke patients.

Ketamine-induced rotational asymmetry in evaluation of motor disturbances in rats with middle cerebral artery occlusion.

Intraperitoneal injection of ketamine in subanesthetic doses to Wistar rats with unilateral occlusion of the middle cerebral artery caused ipsilateral rotation (2-10 rpm), which was recorded in an automatic rotameter. The optimal dose of ketamine was 50 mg/kg. The animals were examined in an automatic rotameter for 40 min. Motor asymmetry persisted for no less than 2 months after surgery. According to the neurological test (Menzies scale) motor asymmetry in animals with focal brain ischemia persisted for no more than 30 days. The degree of ketamine-induced motor asymmetry in intact rats was 0.10 0.03 rpm.

[Criteria of efficiency of transplantation of embryonic nervous tissue preparations in rats with 6-OHDA-impaired dopaminergic nigrostriatal system]. / Kriterii éffektivnosti transplantatsii preparatov émbrional'noi nervnoi tkani u krys s povrezhdeniem dofaminergicheskoi nigrostriarnoi sistemy 6-gidrooksidofaminom (6-OHDA).

Effectiveness of transplantation of cells from embryonal nervous tissue of the ventral mesencephalon (VM ENT) and striatum (STR ENT) by apomorphin-induced motor asymmetry (APO-test), consolidation of the transplant (the degree of glyal reaction and amount of dopaminergic neurons) and blood serum levels of GFAP was studied for 3 months in Wistar rats with 6-OHDA-impaired dopaminergic nigrostriatal system. Marked therapeutic effectiveness was registered in VM ENT transplantation in the denervated striatum and in combined transplantation of VM ENT into the lateral cerebral ventricle simultaneously with STR ENT transplantation in the striatum. Separate transplantation of VM ENT in the lateral ventricle and STR ENT in the striatum had no positive effect on recovery of the dopaminergic nigrostriatal system. A correlation was found between the degree of glial reaction of ENT transplants, severity of rotation asymmetry and serum levels of gliofibrillary protein (GFAP). GFAP in the serum for lifetime assessment of transplant consolidation and prognosis of neurotransplantation efficiency was assayed.

Comparison of the efficacy of cell preparations from embryonic ventral mesencephalon of various prenatal age transplanted intrastriatally to rats with 6-OHDA-induced Parkinsonism.

Cell preparations of ventral mesencephalon obtained from 8-, 14-, and 16-17-day rat embryos were stereotactically transplanted to homologous rats with 6-hydroxydopamine-induced hemiparkinsonism. Automated analysis of apomorphine-induced motor asymmetry for 3 months after neurotransplantation revealed higher efficacy of cell preparations from 8- and lower from 16-17-day-old embryos. These data correlated with histomorphological findings, in particular, with the size of grafts, glial reaction, and the number of dopaminergic neurons in the grafts.

Immunoenzyme assay of glial fibrillary acidic protein for evaluation of functional activity of cell grafts from embryonic ventral mesencephalon in rats with experimental hemiparkinsonism.

The relationship between the release of glial fibrillary acidic protein (GFAP) into systemic circulation and the efficacy of transplantation of embryonic nervous tissue was studied on rats with 6-OHDA-induced hemiparkinsonism. It was found that intrastriatal transplantation of cell preparations from embryonic ventral mesencephalon significantly attenuated apomorphine-induced rotation, which points to functional recovery of the dopaminergic nigrostriatal system. The degree of this recovery depends on reactive astrogliosis around the graft and survival of dopaminergic neurons. Analysis of GFAP concentration revealed significant elimination of this antigen into the circulation 7 and 14 days after transplantation. In rats with good consolidation of the graft without pronounced reactive gliosis, the concentration of GFAP reached 253.99+/-79.30 ng/ml on week 4 after transplantation and decreased to 8.2+/-3.3 ng/ml 8-12 weeks after transplantation. In rats with poor graft consolidation associated with death of transplanted neurons and gliosis in the graft and surrounding tissue the concentration GFAP increased to 476.4+/-111.0 ng/ml within 4 weeks after transplantation and remained elevated (235.0+/-44.8 ng/ml) for 12 weeks. Thus, monitoring of serum GFAP concentrations allows in vivo evaluation of the functional state of intracerebral graft and the level of reactive gliosis. This test can be used for the prognosis of transplantation efficacy.

Complex analysis of efficiency of transplantation of embryonic nerve tissue to rats with hemiparkinsonism.

Effect of transplantation of embryonic ventral mesencephalon preparation containing dopaminergic neurons on repair of the dopaminergic nigrostriatal system was studied in rats with hemiparkinsonism induced by 6-hydroxydopamine. Transplantation of embryonic ventral mesencephalon into denervated striatum led to a more than 50% decrease in apomorphine-induced rotation, recovery of dopamine and DOPAC levels in the brain, and to an increase in DOPAC excretion and the DOPAC-dopamine ratio in daily urine of rats with hemiparkinsonism. Dopaminergic neurons of the transplant survived, forming a network of tyrosine hydroxylase-positive processes growing beyond the transplant and reinnervating the adjacent compartments of the striatum. A positive correlation between urinary excretion of DOPAC and brain concentration of dopamine was revealed in denervated rats after transplantation of ventral mesencephalon. Intrastriatal transplantation of cell preparations of embryonic striatum containing no dopaminergic neurons and isolated local injury to the striatum did not affect regeneration of the denervated nigrostratal system.

[Clinical risk factors for the occurrence of tobacco dependence]. / Klinicheskie faktory riska vozniknoveniia tabachnoi zavisimosti.

In order to formulate a biosocial conception of tobacco dependence essential for fundamental grounding of smoking prevention, the authors examined 1106 subjects with smoking habits, smoking dependence and nonsmokers. A combination of internal and external factors is determined underlying the onset and maintenance of tobacco dependence. Risk factors responsible for consolidation of tobacco addiction are listed.