Transtraumatic Epidural Electrostimulation of the Spinal Cord in a Pig Model.

The effect of transtraumatic epidural electrostimulation (TEES) above (T5) and below (L2) spinal cord injury in the lower thoracic region (T8-T9) in combination with treadmill exercise in pigs was evaluated using electrophysiological examination methods and behavioral tests. Two weeks after spinal cord injury, motor evoked potentials of m. soleus were recorded during electrostimulation at the level of T5 and L2 segments, which indicated activation of spinal cord structures above and below the focus of injury. After 6 weeks of TEES in combination with physical training, restoration of the characteristics of M-response and H-reflex of the soleus muscle in response to stimulation of the sciatic nerve, improvement of joint mobility, and appearance of voluntary motor activity in the hindlimbs were observed. Neuromodulation with TEES had been proven to be an effective way to stimulate posttraumatic spinal cord regeneration and can be used in the development of a neurorehabilitation protocol for patients with spinal cord injury.

Zero Gravity is a Factor that Induces Negative Changes in Myelinated Fibers of the Spinal Tracts.

The development of hypogravitational motor syndrome is an essential negative consequence of weightlessness for humans; an important role in the pathogenesis of this syndrome is played by changes in axons of the spinal tracts. Myelinated fibers and transcriptome of the spinal cord were studied in mice exposed to hypogravity during a 30-day flight on a biosatellite. Morphometric analysis of myelinated fibers of the spinal tracts showed a decrease in the thickness of the myelin sheath. Analysis of spinal cord transcriptome revealed a decrease in the expression of genes involved in the myelination of nerve fibers. These results suggest that the processes of nerve fiber myelination are involved in the development of the hypogravitational motor syndrome under weightless conditions; the 7-day readaptation period was found to be insufficient for reversion of the negative changes in the myelinated fibers of the spinal cord.

Possible Mechanisms of Axonal Transport Disturbances in Mouse Spinal Motoneurons Induced by Hypogravity.

The data obtained by transcriptome analysis of lumbar spinal cord segments, sciatic nerve, and the respiratory diaphragm of the mice performed after a space flight on board Bion-M1 biosatellite were processed by bioinformatic methods aimed at elucidation of the regularities in hypogravity-induced transcriptome changes in various compartments of motor neurons. The study revealed abnormalities of axonal transport in spinal motor neurons provoked by weightlessness. These data agree with the results of electron microscopy examination of the spinal cord in experimental animals. In space group mice sacrificed on the landing day, the content of perinuclear ribosomes in lumbar motoneurons surpassed that in control mice or in the recovery group examined 1 week after the flight. The data corroborate our hypothesis on contribution of axonal transport disturbances into pathogenesis of hypogravity motor syndrome. They can be employed as a launching pad for further study of hypogravity-triggered motor disorder mechanisms in order to elaborate the preventive therapy against the development of hypogravity motor syndrome in space flights.

Morphological Study of Myelinated Fibers of the Sciatic Nerve in Mice after Space Flight and Readaptation to the Conditions of Earth Gravity.

We revealed a decrease in the thickness of the myelin sheath and myelin delamination in the tibial nerve of C57BL/6N mice after a 30-day flight aboard the biosatellite Bion-M1. The processes of myelin degeneration continued for seven days after return of the animals to Earth and adaptation to the conditions of natural gravity. Our data add to hypothesis on the role of neurogenic component in pathogenesis of hypogravity motor syndrome.

Myelinated fibers of the mouse spinal cord after a 30-day space flight.

Myelinated fibers and myelin-forming cells in the spinal cord at the L3-L5 level were studied in C57BL/6N mice that had spent 30 days in space. Signs of destruction of myelin in different areas of white matter, reduction of the thickness of myelin sheath and axon diameter, decreased number of myelin-forming cells were detected in "flight" mice. The stay of mice in space during 30 days had a negative impact on the structure of myelinated fibers and caused reduced expression of the markers myelin-forming cells. These findings can complement the pathogenetic picture of the development of hypogravity motor syndrome.

Adenoviral vector carrying glial cell-derived neurotrophic factor for direct gene therapy in comparison with human umbilical cord blood cell-mediated therapy of spinal cord injury in rat.

STUDY DESIGN: Experimental study. OBJECTIVE: To evaluate the treatment of spinal cord injury with glial cell-derived neurotrophic factor (GDNF) delivered using an adenoviral vector (AdV-GDNF group) in comparison with treatment performed using human umbilical cord blood mononuclear cells (UCB-MCs)-transduced with an adenoviral vector carrying the GDNF gene (UCB-MCs+AdV-GDNF group) in rat. SETTING: Kazan, Russian Federation. METHODS: We examined the efficacy of AdV-GDNF and UCB-MCs+AdV-GDNF therapy by conducting behavioral tests on the animals and morphometric studies on the spinal cord, performing immunofluorescence analyses on glial cells, investigating the survival and migration potential of UCB-MCs, and evaluating the expression of the recombinant GDNF gene. RESULTS: At the 30th postoperative day, equal positive locomotor recovery was observed after both direct and cell-based GDNF therapy. However, after UCB-MCs-mediated GDNF therapy, the area of preserved tissue and the number of spared myelinated fibers were higher than those measured after direct GDNF gene therapy. Moreover, we observed distinct changes in the populations of glial cells; expression patterns of the specific markers for astrocytes (GFAP, S100B and AQP4), oligodendrocytes (PDGFαR and Cx47) and Schwann cells (P0) differed in various areas of the spinal cord of rats treated with AdV-GDNF and UCB-MCs+AdV-GDNF. CONCLUSION: The differences detected in the AdV-GDNF and UCB-MCs+AdV-GDNF groups could be partially explained by the action of UCB-MCs. We discuss the insufficiency and the advantages of these two methods of GDNF gene delivery into the spinal cord after traumatic injury.

Construction of recombinant adenovirus containing picorna-viral 2A-peptide sequence for the co-expression of neuro-protective growth factors in human umbilical cord blood cells.

STUDY DESIGN: Experimental study. OBJECTIVE: Several neuro-degenerative disorders such as Alzheimer's dementia, Parkinson's disease and amyotrophic lateral sclerosis (ALS) are associated with genetic mutations, and replacing or disrupting defective sequences might offer therapeutic benefits. Single gene delivery has so far failed to achieve significant clinical improvements in humans, leading to the advent of co-expression of multiple therapeutic genes. Co-transfection using two or more individual constructs might inadvertently result in disproportionate delivery of the products into the cells. To prevent this, and in order to rule out interference among the many promoters with varying strength, expressing multiple proteins in equimolar amounts can be achieved by linking open reading frames under the control of only one promoter. SETTING: Kazan, Russian Federation. METHODS: Here we describe a strategy for adeno-viral co-expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) interconnected through picorna-viral 2A-amino-acid sequence in transfected human umbilical cord blood mono-nuclear cells (hUCB-MCs). RESULTS: Presence of both growth factors, as well as absence of immune response to 2A-antigen, was demonstrated after 28-52 days. Following injection of hUCB-MCs into ALS transgenic mice, co-expression of VEGF and FGF2, as well as viable xeno-transplanted cells, were observed in the spinal cord after 1 month. CONCLUSION: These results suggest that recombinant adeno-virus containing 2A-sequences could serve as a promising alternative in regenerative medicine for the delivery of therapeutic molecules to treat neurodegenerative diseases, such as ALS.

Synaptosome-associated protein 25 (SNAP25) synthesis in terminal buttons of mouse motor neuron.

Previously, we formulated the hypothesis of compartmentalized protein synthesis in axons of motor neurons. In the axon hillock, along the entire length of the axon and in its ending, specific proteins are locally synthesized, which ensure the function of each compartment. In support of this hypothesis, in this work we studied the local protein synthesis in mouse motor nerve ending.

Characterization of spinal cord glial cells in a model of hindlimb unloading in mice.

Exposure to microgravity has been shown to result in damaging alterations to skeletal muscle, bones, and inner organs. In this study, we investigated the effects of microgravity by using a hindlimb unloading model (HUM) in mice. The characteristics of the lumbar spinal cords of HUM mice 30 days after hindlimb unloading were examined. Morphometric analysis showed reductions of the total area, gray matter, and white matter by 17%, 20%, and 12%, respectively. Myelinated fibers in the white matter showed prominent myelin destruction. Analysis of the number of glial fibrillary acidic protein (GFAP+)/S100 calcium-binding protein B (S100B-), GFAP+/S100B+, and GFAP-/S100B+ astrocytes in the ventral horn (VH), central channel area (CC), dorsal root entry zone (DREZ), main corticospinal tract (CST), and ventral funiculi (VF) showed that the number of GFAP+/S100B- astrocytes was increased in the DREZ and CST of HUM mice. Additionally, GFAP+/S100B+ cell numbers were significantly decreased in the VH and CST but did not differ in the CC or DREZ of HUM mice, as compared with the control. The numbers of GFAP-/S100B+ cells were significantly reduced only in the VH of HUM mice. Moreover, the number of ionized calcium-binding adaptor molecule 1 (Iba1+) microglia cells was significantly increased in the CC and DREZ of HUM mice. In control mice, homeobox protein HoxB8 (HoxB8+) cells were found only in the CC; in contrast, HoxB8+ cells were observed in all studied areas in HUM mice, with the greatest number found in the CC. Genome-wide transcriptome analysis of the lumbar spinal cords of HUM mice showed decreased expression of genes encoding myelin, extracellular matrix, cytoskeleton, and cell adhesion proteins. Real-time polymerase chain reaction (PCR) confirmed reductions in the expression of mpz, pmp2, pmp22, and prx genes, which are involved in myelination, as well as decreases in the levels of genes encoding extracellular matrix molecules, including glycoproteins (matrix gla protein (MGP), osteoglycin (OGN), microfibrillar associated protein 5 (MFAP), and collagen, type IV, alpha 1 (COL4A)), proteoglycans (perlecan (heparan sulfate proteoglycan) (HSPG)), and metalloproteinases (lysyl oxidase (LOX)). Thus, our results showed that hindlimb unloading caused decreases in gray and white matter areas, changes in gene expression, alterations in myelination, and phenotypic modifications in glial cells in the lumbar spinal cords of mice.

[Comparison of data from registries of acute coronary syndromes RECORD and RECORD-2: management of patients and its results in noninvasive hospitals].

Positive changes are declared to occur during recent years in management of hospitalized patients with acute coronary syndromes (ACS) in Russia. Most of these changes are related to availability of invasive treatment. But considerable portion of patients (pts) are still treated in hospitals without facilities for invasive myocardial revascularization (noninvasive hospitals - NIHs). Aim of this study was to compare some characteristics of management of ACS in NIHs which participated in ACS registries RECORD (2007-2008, 8 NIHs from 6 cities; n=381) and RECORD-2 (2009-2011, 3 NIHs from 3 cities, n=680). Results. Groups of pts recruited in these NIHs had similar mean age and portion of women (67.6 and 66.5 years, 51.1 and 53.1 % in RECORD-2 and RECORD, respectively, p=0.64). Time from symptoms onset to hospitalization was shorter in RECORD-2 (3.2 vs 4.1 hours for ST-elevation [STE], =0.03; 4.0 vs 6.5 hours for non ST elevation [NSTE] ACS, <0.0001). Among RECORD-2 NSTEACS pts more had ST depressions (50.6 vs 28.7%, <0.0001), high risk of death according to GRACE score (39.1 vs 20.9 %, <0.0001), but less Killip class >II (15.0 vs 21.6%, p=0.025). No such differences existed among STEACS pts. Thrombolysis was more often used in RECORD-2 (62.6 vs 34.1%, <0.0001). Both STEACS and NSTEACS RECORD-2 pts more often received clopidogrel (63.5 vs 18.8%, p<0.0001, and 41.6 vs 11.1%, <0.0001, respectively). More NSTEACS RECORD-2 pts were given parenteral anticoagulants (93.4 vs 80.4%, <0.0001), low molecular weight heparins (23.4 vs. 3.4%, <0.0001) and fondaparinux (10.4 vs 0.7%, <0.0001), but still in 20% of NSTEACS RECORD-2 pts unfractionated heparin was given subcutaneously. Twenty RECORD-2 pts (2.9%) were transferred to invasive hospital but none during first 24 hours. There were no significant differences between registers in hospital mortality (20.0 vs 21.2%, =0.84; 4.2 vs 2.7%, =0.24 in STE and NSTE ACS pts of RECORD-2 and RECORD, respectively). Conclusions. Despite some improvement in management of pts occurring in 2-3 years NIHs mortality in STEACS remained very high. Numerically higher mortality in NSTEACS could be partially attributed to higher risk of RECORD-2 pts.

[The role of spinal motoneurons in the mechanisms of hypogravitational motor syndrome development].

Studies results of gravity unloading influence on spinal control system of muscle structure and functions are summarized. It was shown that demyelization of axons due to reduction of genes expression responsible for myelin proteins synthesis, decrease in one of the key enzymes of cholinergic system--cholineacetyltransferase activity, alteration of normal kinetics of quantal and non-quantal neurotransmitter secretion, impaired autoregulation of acetylcholine secretion from motor nerve endings through presynaptic cholinergic receptors, slowing of axonal transport of substances in motor neurons that innervate postural muscles played the important role in the development of hypogravitational motor symptoms. At the same time, the evidences of neuroprotective mechanisms enclosing (increase in heat shock proteins Hsp25 and Hsp70 expression), that hinder apoptosis development in motor neurons and glial cells in the spinal cord under conditions of model hypogravity, were revealed.

[Adherence to guidelines on management of acute coronary syndrome in Russian hospitals and outcomes of hospitalization (data from the RECORD-2 Registry)].

BACKGROUND: Complete following existing guidelines for management of acute coronary syndrome (ACS) is known to be associated with better outcomes. Partly this is explained by lesser adherence to recommendations in high risk patients. Aim of our study was to assess relationship between degree of following current guidelines and in hospital outcomes independently from initial assessment of risk. METHODS: Each key recommendation from guidelines issued between 2008 and 2011 (13 for STE ACS, 12 for NSTE ACS) was given weight of 1. Sum of these units constituted index of guideline adherence (IGA). IGA was retrospectively calculated for 1656 patients included in Russian independent ACS registry RECORD-2 (7 hospitals, duration 04.2009 to 04.2011). The patients were divided into 2 groups according to quartiles of IGA distribution: 1) low adherence group (quartiles I-II); 2) high adherence group (quartiles III-IV). RESULTS: In low adherence compared with high adherence group there were significantly more patients more or equal 65 years (=0.0007), with chronic heart failure [CHF] (<0.0001), previous stroke (<0.0001), atrial fibrillation [AF] (=0.0002), Killip class more or equal II (=0.0065), high risk of death by GRACE score (=0.035). Inhospital mortality was 9.3 and 2.4% in low and high adherence group, respectively (p<0.0001). The following independent predictors of inhospital death were identified: IGA quartiles I-II (odds ratio [OR] 4.0; 95% confidence interval [CI] 2.3-7.1; <0.0001), high GRACE score (OR 3.3; 95% CI 1.8-6.0; <0.0001), admission systolic BP less or equal 100 mm Hg (OR 3.1; 95% CI 1.8-5.4; <0.0001), admission serum glucose more or equal 8 mmol/l (OR 2.9; 95% CI 1.8-4.7; <0.0001), age more or equal 65 years (OR 2.3; 95% CI 1.3-4.0; =0.005), ST elevation more or equal 1 mm on first ECG (OR 1.7; 95% CI 1.1-2.5; =0.013). From groups with low and high adherence to guidelines we selected pairs of patients (n=588) with similar (or close) age, type of ACS, GRACE score, Killip class, presence of other important risk factors (CHF, AF, previous stroke), and formed 2 equal subgroups without significant differences in important demographic, anamnestic, clinical and laboratory data. Hospital mortality was 7.8 and 2.7% in low and high adherence subgroup, respectively (p<0.0001). CONCLUSIONS: In RECORD-2 ACS registry low adherence to guidelines was more frequent among high risk patients and was independent predictor of inhospital death. Association between degree of guidelines adherence and outcomes persisted after equalizing groups by some factors of risk of mortality.

Analysis of the efficiency of gene-cell therapy in transgenic mice with amyotrophic lateral sclerosis phenotype.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by progressive death of cerebral and spinal motorneurons. Using behavioral tests we studied the efficiency of gene-cell therapy in SOD1 G93A transgenic mice receiving xenotransplantation of human umbilical cord blood mononuclear cells genetically modified with adenoviral vectors encoding vascular endothelial growth factor (VEGF) and reporter green fluorescent protein (EGFP) genes. The cells were transplanted to mice on week 27 of life (preclinical stage of the disease). Behavioral tests (open field, grip strength test) showed that transplantation of umbilical cord blood mononuclear cells expressing VEGF significantly improved the parameters of motor and explorative activity, grip strength, and animal survival. Thus, gene-cell therapy based on genetically modified mononuclear cells expressing VEGF can be efficient for the treatment of amyotrophic lateral sclerosis.

[Myelination disorders in mechanism of hypogravity motor syndrome development].

When modeling effects of hypogravitation by the method of hindlimb unloading in rats the area of cross-section in lumbar part of a spinal cord was found to reduce. The analysis of spinal cord slides showed that these changes are associated with a decrease in the area of white substance of a spinal cord. Data obtained are consistent with our previous observation of a decrease in expression of the genes encoding myelin proteins. Results of our researches give the good reasons to believe that miyelinization failure in CNS is one of the factors that underlie the development of hypogravitational motor syndrome.

[About possible specialization of different compartments of a motoneuron axon for synthesis of specific proteins].

The spontaneous quantum secretion of neurotransmitter and its regulation through the system of presynaptic acetylcholine receptors have been studied on a neuromuscular preparation of rat m. soleus of intact animals and animals in which the axonal transport was blocked via the application of colchicine to the sciatic nerve. It was shown that, after six days of colchicine application, the spontaneous quantum secretion, the reaction of presynaptic membrane, and the reaction of neurosecretory apparatus to the depolarization of nerve endings via increase of the content of potassium ions in the environment and to the activation of presynaptic receptors by carbachol are not disturbed. Keeping in mind a rather short half-life of proteins that take part in the exocytosis and its regulation, it may be concluded that their functioning does not depend on the state of the axonal transport. These data correspond to the hypothesis put forward earlier that the synthesis of some proteins performing their function in nerve terminals occurs directly at the site of their utilization but not in the perikaryon, as it has been traditionally assumed.