Search

Intravenous transplantation of mesenchymal stem (stromal) cells (MSC) is a promising approach to the treatment of ischemic stroke. In the published reports of the already completed preclinical and clinical studies the dosages of transplanted MSC greatly vary. However, the optimal dosage has not been determined. The dose-dependent effect of intravenous MSC transplantation was studied, in rats with experimental cerebral infarction. To this end, 5×10

A gene-cell construct based on rat olfactory mucosa ensheathing cells transduced with an adenoviral vector encoding a mature form of brain neurotrophic factor (mBDNF) was transplanted into post-traumatic cysts of rat spinal cord. Transplantation of the gene-cell construct improved motor activity of the hind limbs and reduced the size of cysts in some animals. However, comparison of the effects of transduced and non-transduced ensheathing cells revealed no significant differences. In parallel in vitro experiments, a decrease in the proliferation of transduced cells compared to non-transduced cells was observed. It is likely that mBDNF reduces proliferation of transduced cells, which can affect their efficiency. The therapeutic efficacy of the new gene-cell construct is most likely provided by the cellular component.

Neurotrophin-3 enhances the effectiveness of human olfactory ensheathing cells in improving hind limb mobility in rats with post-traumatic cysts of the spinal cord. Transplantation of olfactory ensheathing cells into spinal cord cysts reduced their size; neurotrophin-3 did not modulate this effect. Combined preparation of human olfactory ensheathing cells and neurotrophin- 3 can be used in neurosurgery for the treatment of patients with spinal cord injuries.

Visualization of transplanted stem cells in the brain is an important issue in the study of the mechanisms of their therapeutic action. MRI allowing visualization of single transplanted cells previously labeled with superparamagnetic iron oxide particles is among the most informative methods of non-invasive intravital imaging. Verification of MRI data using pathomorphological examination at the microscopic level helps to avoid errors in data interpretation. However, making serial sections of the whole brain and searching for transplanted cells under the microscope is laborious and time-consuming. We have developed a method for 3D modeling of the distribution of transplanted cells in the brain allowing navigating through various brain structures and identifying the areas of accumulation of transplanted cells, which significantly increases the efficiency and reduces the time of histological examination.

We studied the effect of transplantation of ensheathing cells obtained from the olfactory mucosa of rats and humans on the size of posttraumatic spinal cord cysts. MRI examination showed that transplantation of these cells into experimental posttraumatic cysts of the spinal cord led to a significant decrease in cyst volume and even their complete disappearance in two animals receiving transplantation of rat or human cells. These findings attested to regenerative processes developing as a result of ensheathing cell transplantation. Further studies in this field will be aimed at elucidation of the mechanisms underlying spinal cord regeneration in the area of posttraumatic cysts after transplantation of ensheathing cells.

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.

We evaluated the effect of "sidedness" of focal stroke induced by pial blood vessel devascularization in sensorimotor cortex of right or left hemisphere on neurological deficit in rats. Animals were preliminary examined for paw preference (hemispheric "dominance") by means of food reaching test. Data of cylinder-test, vibrissae-elicited forelimb placing test and swimming task (forepaw inhibition test) were assessed to monitor functional deficit weekly for 8 weeks after stroke. No difference between rats with right or left cortical damage was observed during the experiment. Rats with ischemia in "dominant" hemisphere performed significantly better than those with "nondominant" hemisphere lesion. The revealed effect of functional brain asymmetry on the neurological rehabilitation after stroke should be considered in design of preclinical investigation of new therapeutic approaches to stroke.

An immuno-enzyme assay of NSE and MBP in the cerebrospinal fluid (CFS) and blood serum of rats during the development of acute experimental autoimmune encephalomyelitis revealed the increase of NSE, a neuronal marker, and MBP, a marker for myelin. The investigation has shown that the increased level of NSE in CSF was prior to the increased concentration of MBP. The changes of these neurospecific proteins (NSP) in the serum were not the same as in the CSF. The analysis of the ratio between NSP concentrations in CSF to serum revealed the initial marked decrease of BBB permeability to MBP but not to NSE.

Appearance of neurospecific proteins (NSP) outside the brain plays a certain pathogenetic role in the development of autosensitization occurring in many kinds of CNS injuries and diseases. Analysis of modern views of cerebrospinal fluid (CSF) exchange allows us to suppose that NSP are eliminated from the brain tissue within CSF, moving from the subarachnoid space into cranial veins, and by lymphatic way, into deep cervical lymph nodes. Elevation of NSP level in CSF indicates an actual neudegenerative process. Serum levels of NSP are determined by the balance between the elimination of NSP from the brain, on the one hand, and their metabolism and the response of the immune system to the appearance of these autoantigenes in the blood stream, on the other. Basing on their own data on the dynamics of NSP (NSE, GFAR, and MBP) concentrations and the proportions of these proteins in CSF and serum (coefficient of elimination) in rats after ischemic, hypoxic, and autoimmune cerebral lesions, the authors offer an algorithm of pathogenetic evaluation, including, on the one part, a conclusion on the presence or absence of a neurodegenerative process, and, on the other, a conclusion on a normal or lowered rate of NSP elimination (metabolism). The results of such an analysis may have a clinical significance in terms of the development of a pathogenetic therapy, including, in every individual case, not only neuroprotectors, but also pharmaceuticals directed towards correction of the functional condition of the immune system.

PEGylated (stealth) immunoliposomes covalently linked to antibodies against human gliofibrillary acidic protein (GFAP) were prepared by coupling the thiolated monoclonal anti-GFAP antibodies, D4, with a maleimide derivative of the phosphatidyl ethanolamine of the liposomal membrane. Depending on the initial protein-to-lipid ratios, the immunoliposomes prepared (with a diameter of about 70 nm) were coupled with 60 to 240 molecules of the antibodies. In vitro experiments with cultures of the embryonic rat brain astrocytes demonstrated a specific binding of these immunoliposomes, which could be inhibited by the preincubation of the cells with free non-coupled D4 but not with non-specific antibodies. Administered intravenously into rats, the immunoliposomes exhibited a kinetic behaviour typical for the PEGylated liposomes: after 24 h, approximately 20% of the doses injected were still present in the blood; the elimination rate constants were 0.05-0.09 h(-1), the elimination half-lives were 8-15 h. With such a systemic longevity, as well as with such a specificity, these immunoliposomes, non-penetrating through intact blood-brain barrier (BBB), should be useful in delivering pharmacological agents to glial brain tumours (which continue to express GFAP) or to other pathological loci in the brain with a partially disintegrated BBB.

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.

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, p0.05). 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.

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.

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.

Enzyme immunoassay showed penetration of two glia-specific antigens, glial fibrillar acid protein GFAP and specific brain glycoprotein alpha(2)GP, through the blood-brain barrier in rats treated with toxic doses of sodium barbital. The permeability of the blood-brain barrier was completely normalized 3 days after treatment. This method can be used in clinical practice for evaluation of the severity of impairment and dynamics of normalization of blood-brain barrier properties during acute intoxication with barbiturates.

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.

The role of functional state of hematoencephalic barrier (HEB) was estimated in terms of development of pathological process in coma. The quantitative determination of neurospecific protein (NSP), exactly of alpha 1 and of alpha 2 cerebral globulins (produced by astrocytes and oligodendroglial cells) was used for estimation of HEB state. The clinical examination of 138 children in comatose state caused by different factor (meningitis, encephalitis, cerebrocranial trauma, infectious toxicosis) was performed. Enzyme immuno-assay of alpha 1 and of alpha 2 cerebral globulins allowed to control HEB's functional state objectively in "brain-blood" direction: in cases of alteration of HEB's function the autoantibodies to NSP may penetrate into cerebral tissue and cause nonspecific reaction in form of edema or inflammation. The membrane stabilizing effect of Dexamethazone was established.

Hydrophobized and non-hydrophobized Fab fragments of human antibodies against gliofibrillar acid protein (GFAP) and brain specific alpha 2-glycoprotein (alpha 2GP) were used to study their penetration through the blood-brain barrier (BBB). These Fab fragments were modified by stearoyl chloride in reversed micelles of aerosol OT in octane (one or two fatty acid residues attached to protein molecule). Modified and non-modified 125I-labelled Fab fragments were intracardially administered to rats. The amount of label accumulated in brain was 55% higher than the total amount in all other organs. In contrast, non-hydrophobized Fab fragments did not penetrate through the BBB. We assume that the artificial hydrophobization of Fab fragments can increase their capability to penetrate through the cell membranes and, in particular, the BBB.

Synthesis of animal embryonic proteins in human tumors, i.e. a phenomenon of interspecific embryonal reversion is discussed. Two new interspecific embryonic antigens (IEA) with relative molecular weights of 80,000 +/- 10,000 dalton (IEA-1) and 36,000 +/- 10,000 dalton (IEA-2) were identified, using antisera obtained by immunization of rabbits with a protein fraction isolated from glycoproteins of amniotic fluid of cattle fetuses. These proteins were detectable in blood serum and amniotic fluid of cow, sheep and pig fetuses and were not in human fetus. An immunoenzymatic method was used to identify IEA-1 in blood serum in 14-20% of cancer patients and in 58% of melanoma cases. IEA-1 was not detected in blood serum of non-tumor patients. IEA-2 was identified in 21-22% of tumor and non-tumor patients.

The immunoenzyme detection systems for the measurement of the alpha-2 globulin of the brain (alpha 2M) and glial fibrillary acidic antigens (GFAP) were developed. These systems were used for the study of the penetration through hemato-encephalic barrier in rats subjected to gamma radiation. This method is recommended for the indirect evaluation of the hemato-encephalic barrier functional disorders.

EIA detection system for the measurement of alpha 2 M globulin and GFAP antigen has been developed. The limit of the sensibility was only 1 ng/ml for alpha 2M and 0.8 ng/ml for GFAP. The system was used for the studies of the penetration through the blood-brain barrier in rats with experimental acute brain ischemia. The measurement of alpha 2M and GFAP antigens by EIA technique 16-20 hours after the occlusion of the carotid artery has revealed disturbances in the blood-brain barrier permeability for specific brain proteins. The method is recommended for indirect evaluation of the blood-brain barrier functional disorders.