Cellular and Molecular Gradients in the Ventral Horns With Increasing Distance From the Injury Site After Spinal Cord Contusion.

To identify cellular and molecular gradients following spinal cord injury (SCI), a rat contusion model of severe SCI was used to investigate the expression of NG2 and molecules that identify astrocytes and axons of the ventral horns (VH) at different distances on 7 and 30 days post-injury (dpi). A gradient of expression of NG2+/Olig2+ cells was determined, with the highest concentrations focused close to the injury site. A decrease in NG2 mean intensity correlates with a decrease in the number of NG2+ cells more distally. Immunoelectron microscopy subsequently revealed the presence of NG2 in connection with the membrane and within the cytoplasm of NG2+ glial cells and in large amounts within myelin membranes. Analysis of the astrocyte marker GFAP showed increased expression local to injury site from 7 dpi, this increase in expression spread more distally from the injury site by 30 dpi. Paradoxically, astrocyte perisynaptic processes marker GLT-1 was only increased in expression in areas remote from the epicenter, which was traced both at 7 and 30 dpi. Confocal microscopy showed a significant decrease in the number of 5-HT+ axons at a distance from the epicenter in the caudal direction, which is consistent with a decrease in ß3-tubulin in these areas. The results indicate significant cellular and molecular reactions not only in the area of the gray matter damage but also in adjacent and remote areas, which is important for assessing the possibility of long-distance axonal growth.

The Function of NG2/CSPG4-expressing Cells in the Rat Spinal Cord Injury: An Immunoelectron Microscopy Study.

Emerging evidence supports an increased role for NG2/CSPG4-expressing cells in the process of neuroregeneration and synaptic plasticity, due to the increased production of multifunctional chondroitin sulfate proteoglycan (NG2/CSPG4). However, the response of NG2/CSPG4-expressing cells in spinal cord injury (SCI) remains to be elcudiated. Expression and distribution of NG2/CSPG4-expressing cells were studied by immunoelectron microscopy in the ventral horns (VH) of an intact and injured rat spinal cord. In the intact spinal cord, NG2/CSPG4 expression was detected on the cell membrane and in the cytoplasm of NG2 glia and was absent in neurons. Large amounts of NG2/CSPG4 were found on myelin membranes. The ability of intact astrocytes to produce NG2/CSPG4 was shown, although to a lesser extent than oligodendrocytes and NG2 glia. At 7 days after SCI at the Th8 level in the reactive glial zone of VH, the expression of NG2/CSPG4 sharply increased in NG2 glia at a distance of 3-5 mm and in reactive astrocytes were observed at all investigated distances caudally from the epicenter of injury. The obtained results indicate the presence of NG2/CSPG4-positive astrocytes in the intact spinal cord, and in the case of damage, an increase in the ability of reactive astrocytes to produce NG2/CSPG4. SCI leads to increased expression of NG2/CSPG4 by NG2 glia in the early stages after injury, which decreases with distance from the epicenter of the injury, as well as at later stages.

Spinal Cord Molecular and Cellular Changes Induced by Adenoviral Vector- and Cell-Mediated Triple Gene Therapy after Severe Contusion.

The gene therapy has been successful in treatment of spinal cord injury (SCI) in several animal models, although it still remains unavailable for clinical practice. Surprisingly, regardless the fact that multiple reports showed motor recovery with gene therapy, little is known about molecular and cellular changes in the post-traumatic spinal cord following viral vector- or cell-mediated gene therapy. In this study we evaluated the therapeutic efficacy and changes in spinal cord after treatment with the genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG), and neuronal cell adhesion molecule (NCAM) applied using both approaches. Therapeutic genes were used for viral vector- and cell-mediated gene therapy in two combinations: (1) VEGF+GDNF+NCAM and (2) VEGF+ANG+NCAM. For direct gene therapy adenoviral vectors based on serotype 5 (Ad5) were injected intrathecally and for cell-mediated gene delivery human umbilical cord blood mononuclear cells (UCB-MC) were simultaneously transduced with three Ad5 vectors and injected intrathecally 4 h after the SCI. The efficacy of both treatments was confirmed by improvement in behavioral (BBB) test. Molecular and cellular changes following post-traumatic recovery were evaluated with immunofluorescent staining using antibodies against the functional markers of motorneurons (Hsp27, synaptophysin, PSD95), astrocytes (GFAP, vimentin), oligodendrocytes (Olig2, NG2, Cx47) and microglial cells (Iba1). Our results suggest that both approaches with intrathecal delivery of therapeutic genes may support functional recovery of post-traumatic spinal cord via lowering the stress (down regulation of Hsp25) and enhancing the synaptic plasticity (up regulation of PSD95 and synaptophysin), supporting oligodendrocyte proliferation (up regulation of NG2) and myelination (up regulation of Olig2 and Cx47), modulating astrogliosis by reducing number of astrocytes (down regulation of GFAP and vimetin) and microglial cells (down regulation of Iba1).