Promotion of regeneration of corticospinal tract axons in rats with recombinant vascular endothelial growth factor alone and combined with adenovirus coding for this factor

Abstract

After spinal cord transection in adult rats, the axons of the corticospinal tract (CST) degenerate retrogradely and do not regenerate. This phenomenon is thought to be related to either secondary ischemia or deficiency of growth factors. To overcome the deficiency of both blood flow and growth factors, the authors added exogenous vascular endothelial growth factor (VEGF165) to the transected spinal cord either as recombinant protein alone or combined with an adenovirus coding for VEGF165. Because most growth factors are rapidly inactivated in the extracellular environment, the authors used an adenovirus coding for VEGF165 to maintain its activity for several days. In adult rats, the dorsal two thirds of the spinal cord were transected at the T-8 level. In experimental rats, either human recombinant VEGF165 or a combination of this factor and a replication-defective adenovirus coding for VEGF165 (Ad.CMV.VEGF165) was applied at the lesion site. Both recombinant VEGF165 alone and combined with Ad.CMV.VEGF165 were mixed with Matrigel, which is a reconstituted membrane basement protein extract. Control rats received Matrigel alone or Matrigel plus an adenoviral vector containing the LACZ gene (Ad.CMV.LACZ). Thirty days after spinal cord injury, the number of newly formed blood vessels was assessed in the injured area. In addition, the sensorimotor cortex was injected with anterogradely transported horseradish peroxidase (HRP) to label the CST axons in the spinal cord and to evaluate the extent of retrograde axonal degeneration and regeneration. Gene transfer was assessed using semiquantitative reverse transcription-polymerase chain reaction analysis, enzyme-linked immunosorbent assay for human VEGF and beta-galactosidase expression in injured rats treated with Matrigel plus Ad.CMV.LACZ, Matrigel plus Ad.CMV.VEGF165, and untreated injured rats. A strong gene transfer in the spinal cord tissue of adenovirus-treated rats was found from Day 3 to Day 10 postinjury, confirming infection. In the injured spinal cord area, a significant increase of blood vessels (300% over control, p < 0.005) occurred both in rats treated with recombinant VEGF165 alone and in those treated with the combination of recombinant VEGF165 and Ad.CMV.VEGF165. Also, in both of these groups of animals the retrograde degeneration of CST axons was significantly reduced compared with rats treated with Matrigel alone or Matrigel plus Ad.CMV.LACZ. Furthermore, in rats treated with recombinant VEGF165 alone or combined with Ad.CMV.VEGF165, a few HRP-labeled CST axons, which were not detectable in control rats, were seen distal to the spinal cord injury, indicating some regeneration across the injured area. These results indicate that locally applied VEGF exerts angiogenic as well as neurotrophic effects in the injured spinal cord of rats.