Very few CGRP+ axons were found at 0.5mm distal to the midline, with about 18% and 5% of CGRP+ axons in the no pathway and GFP pathway groups. corpus callosum, 2.5 mm from the midline. At the same time, a 1 mm lesion was made through the corpus callosum at the midline in an anteroposterior direction. A group of control animals received lesions and Ad-NGF injections only at the transplant and target sites, without a bridging pathway. DRG cell suspensions from postnatal day 1 or 2 2 rats were injected at the transplantation site three to four days later. Two weeks after transplantation, brain sections were stained using an anti-CGRP antibody. The CGRP-positive axons were counted at 0.5 mm and 1.5 mm from the lesion site in both hemispheres. Few axons grew past the lesion in animals with control pathways, but there was robust axon growth across the lesion site in the FGF2/NGF and NGF-expressing pathways. This study indicated that preformed NGF and combination guidance pathways support more axon growth past a lesion in the adult mammalian brain. VCA-2 the lesion rather than it. It is impossible to determine the exact longitudinal trajectory of individual fibers, but we counted CGRP+ axons in sections which clearly did not include the midline lesion and characterized these as axons growing around it (Fig 4). Again, we counted the number of CGRP+ axons at 0.5mm and 1.5mm from the midline on the transplant side and on the target side. About 24% and 4% of CGRP+ axons in the no pathway and GFP pathway groups (Fig. 4A) reached a point 0.5mm proximal to the midline. Very few CGRP+ axons were found at 0.5mm distal to the midline, with about 18% and 5% of CGRP+ axons in the no pathway and GFP pathway groups. Less than 10% of CGRP+ axons reached the target area in each of these groups. However, in both NGF and FGF/NGF expressing pathways (Fig. 4B and 4C), many CGRP+ axons grew from the transplants along the pathways and around the lesion into target area. More than 75% of CGRP+ axons were found near the midline on the transplant side in both NGF and FGF/NGF-expressing pathways. Although the number of CGRP+ axons decreased on the target site, more than 40% of CGRP+ axons reached the target area in both pathways. Open in a separate window Figure 4 CGRP+ axons have grown around the lesion into the contralateral corpus callosum by 2 weeks post-transplant. A) Representative section from an animal with GFP expression along the pathway. Very few CGRP+ fibers grew around the lesion into the contralateral corpus callosum. B and C) Representative sections from the animals with either NGF (B) or FGF/NGF (C) expression along the pathway. More CGRP+ fibers grew around the lesion into the SKF 86002 Dihydrochloride contralateral corpus callosum. D) Percentage of CGRP+ fibers that grew SKF 86002 Dihydrochloride around the lesion of the corpus callosum. This shows in a similar pattern as in Figure 3, with a higher percentage of CGRP+ axons that grew around the lesion into the contralateral side in both NGF and FGF/NGF pathways. *p 0.001, FGF/NGF, NGF vs GFP, p 0.01, FGF/NGF, NGF vs None (no pathway); p 0.05, FGF/NGF vs NGF; GFP vs None (no pathway). Data = Mean SEM. Scale bar = 200m. The growth of CGRP+ axons around the lesion showed a similar pattern as across the lesion in all pathways. There was a significant difference in NGF and FGF/NGF groups at all points compared to GFP and no-pathway groups (NGF and FGF/NGF to GFP, p 0.001, at all points; NGF and FGF/NGF to none-pathway, p 0.01, at all points). There was no significant difference between NGF and FGF/NGF groups at any point, or between GFP and no pathway at any point (Fig. 4D, NGF to FGF/NGF, p 0.05; GFP to none-pathway, p 0.05). Lesion site morphology Lesions were created in this experiment using a micro-knife to transect the corpus callosum at the midline in an anteroposterior direction. Some sections from all groups were stained for ED-1, SKF 86002 Dihydrochloride NG2, GFAP or CSPG. Immunostaining for the monocyte/macrophage marker ED-1 showed that macrophages were abundant in the lesion area (Fig. 5A, section from NGF pathway). Cells at the lesion site also expressed NG2, a SKF 86002 Dihydrochloride chondroitin sulfate proteoglycan produced by glial progenitor SKF 86002 Dihydrochloride cells (Fig. 5B, section from NGF pathway). Sections with CSPG and CGRP double staining showed that although CSPGs were up-regulated around the lesion site, CGRP+ axons still grew into that area in both NGF and.