New Xymedon Analogues for Stimulation of Posttraumatic Regeneration of the Spinal Cord in Rats.

Effect of systemic administration of synthetic pyrimidine derivatives, xymedon and compounds 29D and 34D, was studied in rats with experimental dosed contusion spinal cord injury. Xymedon promoted recovery of motor function after injury. Compounds 29D and 34D more effectively restored the parameters of open-field and Rotarod tests in comparison with xymedon. Compound 29D more effectively than xymedon maintained the number of Olig2+ oligodendrocytes in the corticospinal tract and NG2 cells in all investigated areas of the white matter. In the group treated with compound 34D, the differences in the number of NG2+ cells were revealed only in the anterior funiculi, where the number of these glial cells was 2-fold higher than in the xymedon-treated group. Obtained results suggest that the studied xymedon analogs, compounds 29D and 34D, can exert their therapeutic action through different molecular and cellular pathways.

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.

SPACEFLIGHT EFFECTS ON GLIAL CELLS OF M4USE B0ONEMARROW.

Immunohistochmistrry was used to analyze lumbar enlarements in C57BL/76N mice on day 30 of a space flight with and w/o 7 day deadaptation to Earth. Space flown and non-deadpted mice had a increased number of S100B+ -cells in the cental channel zone and decreased population of GFAP astrocytes in ventral horns, Olig2+ cells in the corticospinal tract, PO+ -cells in vental horns and dorsal roots entry zone and Krox24+ -cells in ventral horns, dorsal roots entry zone and central channel zone, and reduced OSP fluorescence density in ventral channels. Elevated expression of myelin-producing Olig2, PO and Krox24 markers on day 7 of deadaptation implies recovery, significance of immunocytochemical changes in the myelin-producing cells in space flight, and suggests portentially rapid normalization of pathologic deviations in myelin fibers in consequence of sthe hypogravity motor syndrome.

Poly(ε-caprolactone) nerve conduit and local delivery of vegf and fgf2 genes stimulate neuroregeneration.

We studied regeneration of rat sciatic nerve while overcoming of a 5-mm diastasis with the aid of nanostructured conduit made of biocompatible and biodegradable poly(ε-caprolactone) and filled with fibrin hydrogel matrix. Implantation of the conduit into the nerve in combination with local delivery of the expression plasmid carrying genes encoding vascular endothelial growth factor (vegf) and fibroblast growth factor 2 (fgf2) leads to an increase in number of myelinated fibers and S-100(+) cells in the peripheral nerve stump and improved recovery of the nerve function. Under conditions of direct gene therapy, an advantage of electrospun poly(ε-caprolactone) conduit with high-porosity was revealed on the basis of these criteria in comparison with biocompatible silicon conduit.

Usage of plasmid vector carrying vegf and fgf2 genes after spinal cord injury in rats.

Using rat model of spinal cord contusion injury at TVIII, we compared the effectiveness of immediate single transplantation of human mononuclear umbilical cord blood cells transfected with pBud-VEGF-FGF2 plasmid and immediate direct injection of the same plasmid into the lesion area. The results suggest that the delivery of therapeutic genes vegf and fgf2 in cells is more effective than direct injection of plasmid DNA with the same genes (judging from the number of myelinated fibers). Better tissue preservation and motor function recovery in experiments with direct injection of plasmid pBud-VEGF-FGF2 suggest that direct gene therapy seems to be an effective additional procedure to the method of gene delivery with transfected stem and progenitor cells.

Reaction of oligoglia to spinal cord injury in rats and transplantation of human olfactory ensheathing cells.

In experiments on rats with lateral TVIII hemisection of the spinal cord and transplantation of ensheating olfactory cells, we studied structural changes at the lesion site and adjacent rostral and dorsal regions of the spinal cord. The state of oligodendrocytes and myelin fibers and motor function in experimental animal were analyzed. Open field testing (BBB test) showed that motor functions steadily increased (by 13% on average) within the interval from day 21 to day 53 after transplantation. Histological examination showed that groups of transplanted cells carrying human nuclear marker (HNu(+)cells) were still present at the lesion site 30 days after surgery. Some of these cells migrated in the rostral and caudal directions from the injection site to a distance up to 6 mm. At the initial period after hemisection, the number of oligodendrocytes (O4(+)-cells) in the immediate vicinity to the lesion site decreased 2-fold, but no significant changes in the number of neurons were found in the rostral and dorsal fragments of the spinal cord compared to the corresponding parameter in controls. Sixty days after transplantation, the cross-section area in the rostral part of the spinal cord at a distance of 3 mm from damage site increased by 15.3% compared to the control. The number of O4(+)-cells at the lesion site and in adjacent rostral and caudal parts of the spinal cord by 22.8% surpassed that in the control group. The number of remyelinated axons also increased. These findings suggest that the absence of pronounced structural changes in the rostral and caudal parts of the spinal cord compared to lesion site at early stages after damage and cell transplantation. At the same time, pronounced activation of oligodendrocytes in this region suggests their involvement together with Schwann cells into remyelination of regenerating axons, which can serve as a factor of partial restoration of motor functions after spinal cord injury.

Study of the efficiency of transplantation of human neural stem cells to rats with spinal trauma: the use of functional load tests and BBB test.

Human ensheating neural stem cells of the olfactory epithelium were transplanted to adult male rats immediately after contusion trauma of the spinal cord at T9 level rostrally and caudally to the injury. Voluntary movements (by a 21-point BBB scale), rota-rod performance, and walking along a narrowing beam were monitored weekly over 60 days. In rats receiving cell transplantation, the mean BBB score significantly increased by 11% by the end of the experiment. The mean parameters of load tests also regularly surpassed the corresponding parameters in controls. The efficiency of transplantation (percent of animals with motor function recovery parameters surpassing the corresponding mean values in the control groups) was 62% by the state of voluntary motions, 37% by the rota-rod test, and 32% by the narrowing beam test. Morphometry revealed considerable shrinking of the zone of traumatic damage in the spinal cord and activation of posttraumatic remyelination in animals receiving transplantation of human neural stem cells.

Posttraumatic survival of sensory neurons during allotransplantation of rat embryonic tissues into the nerve.

Allotransplantation of the liver, hindlimb, and bone marrow tissues from 14-day-old embryos into the sciatic nerve had a modulatory effect on survival of various populations of axotomized neurons in L5 spinal ganglion in an adult rat.

Survival and phenotypic characteristics of axotomized neurons in spinal ganglia.

Trauma to the peripheral processes of sensory neurons of different subpopulations was followed by indirect immunohistochemical analysis of the expression of Bcl-X(L) and Bax, which are, respectively, antiapoptotic and proapoptotic proteins of the Bcl-2 family, and also of the cytokine interleukin-1beta, with the aim of identifying the roles of these substances in controlling apoptosis. The survival abilities of these neurons after central and peripheral axotomy were compared by studying the expression of the high molecular weight component of the neurofilament triplet NF200 and isolectin B4 (IB4). By day 30 after central axotomy, there were no changes in the total numbers of neurons in ganglia L(IV)-L(V) in rats, though there were significant reductions in the numbers of NF200+ neurons. In spinal ganglion L(V) of mice, the proapoptotic protein was detected in the nuclei of 46% of small neurons, which account for 20% of all neurons in the ganglion. By day 30 after nerve compression, Bax was expressed in the nuclei of 30% of neurons and the cytoplasm of 20% of neurons. In intact animals, the antiapoptotic protein Bcl-X(L) was seen in the cytoplasm of 30% of small neurons, as well as in satellite cells surrounding large and intermediate neurons. By day 30 after nerve trauma, Bcl-X(L) was not expressed in spinal ganglion L(V). Interleukin-1beta was present in the cytoplasm of 17% of neurons belonging to the subpopulations of large and intermediate neurons. By day 30 after nerve compression, interleukin-1 beta+ neurons were not identified.

Post-traumatic survival of sensory neurons of different subpopulations.

Immunohistochemical studies were performed to address the expression of the high-molecular-weight component of the neurofilament triplet NF200 (a marker of neurons forming A fibers) and the binding of isolectin B4 (IB4) by neurons of the L4-5 spinal ganglia after ligation or section of the sciatic nerve in rats. A total of 15% of neurons in the ganglia of intact rats expressed NF200. By 90 days after nerve ligation, the proportion of NF200+ neurons decreased two-fold; administration to these rats of the nerve regeneration stimulator xymedone increased the number of NF200+ neurons by 50.7% compared with controls (ligation, no treatment). In intact rats, 23.6% of neurons bound IB4. The proportion decreased by 2.6% 30 days after nerve ligation and to undetectable levels by 90 days; xymedone increased the proportion of surviving IB(4)+ neurons more than eight-fold. IB(4)+ neurons were more likely to enter post-traumatic apoptosis. Ligation of the nerve was followed by survival of fewer NF200+ and IB(4)+ neurons than section of the nerve, which suggests that axon lengthening is a factor maintaining neuron survival. The pyrimidine derivative xymedone increased the survival of neurons of both subpopulations, especially IB(4)+ neurons.

Effect of stimulation of nerve regeneration on posttraumatic neuronal survival in dorsal root ganglia.

The survival of neurons is a key condition for complete posttraumatic regeneration of the peripheral nerve. In experiments on rats we studied survival capacity of different neuronal subpopulations in L(IV)-L(V) dorsal root ganglia after ligation or transection and suturing of the sciatic nerve. Experiments with nerve ligation showed that IB4+ neurons are more sensitive to the injury than NF200+ neurons. By day 90 after ligation of the sciatic nerve IB4+ neurons were virtually not detected in the dorsal root ganglia. By day 90 after nerve transection the number of surviving NF200+ and IB4+ neurons decreased by 26.1 and 21.4%, respectively, in comparison with intact animals. Treatment with xymedon, a regeneration stimulator, led to a 48.5% increase in the number of surviving NF200+ neurons by day 30 after ligation of the nerve and a 50.7% increase by day 90. The number of surviving IB4+ neurons increased more than 8-fold by this term after ligation of the nerve and drug stimulation. Xymedon had a neuroprotective effect towards both neuron subpopulations, more intensely preventing apoptosis of IB4+ neurons.