Article Figures & Data
Figures
- Figure 1.
Confirmation of expression of NGF and FGF2 after adenoviral injections into the corpus callosum and striatum. A , Schematic of injection protocol. Blue circles represent injection sites for Ad-FGF2 and combination Ad-FGF2/NGF or Ad-GFP (control); green circles represent Ad-NGF injections, small volume (0.2 μl) at transplant site (arrow) in the left corpus callosum and large volume (2 μl) at the target site in the right striatum. Brown circles represent transplanted DRG neurons, injected 1 week after the adenoviral pathway. B , Double immunostaining shows CGRP+ fibers (brown) growing along a pathway of FGF2 expression (blue). C , D , Higher magnification insets depict axons following needle tracks from Ad-FGF2 injections, which overshot the corpus callosum. E , Beta-galactosidase staining shows the location and density of transfected cells 7 d after injection of LacZ/Ad. F , Immunostaining for NGF shows widespread expression in the target striatum and up into the external capsule.
- Figure 2.
NGF expression at the transplant site is necessary for cell survival. A–C , Representative CGRP-immunostained sections through transplant sites. A , Transplant after 24 h with no Ad-NGF. B , Transplant after 7 d with no Ad-NGF. C , Transplant after 14 d with Ad-NGF at transplant site. Scale bars: 200 μm. D , Counts of CGRP+ cell bodies at the transplant site 1–14 d after transplantation. For each animal, cell bodies were counted in three sections separated by 150 μm and added together (semiquantitative method). At 14 d, there were no CGRP+ cell bodies found in any of the three animals lacking NGF expression at the transplant site; the small mark visible at that point in the graph is for illustrative purposes only. Error bars represent mean ± SEM at each time point. *p < 0.05.
- Figure 3.
Injection of adenovirus increases expression of CSPG along the corpus callosum. A , Injection of LacZ/Ad at six locations along the corpus callosum shows good expression of the transgene (blue) and little axon growth from CGRP+ DRG neurons (brown). B , Higher magnification of an adjacent section showing diminished CGRP+ axon growth after growing only several millimeters from transplant. C , The entire length of the corpus callosum shows high expression of CSPG, with a few cells in the gray matter along the injection site also being positive for CSPG (arrow). D , Composite image shows axons grow poorly within this environment. Scale bars: A , 0.5 mm; B–D , 100 μm.
- Figure 4.
Expression of guidance cues can either be positive or negative regulators of axonal growth within the corpus callosum. Representative CGRP-immunostained sections show nociceptive axon growth 2 weeks after DRG neuron transplants. A , LacZ expression pathway leading to an NGF striatal target supports little axon growth across the corpus callosum. B , Pathway expression of FGF2 supports robust growth of axons across the corpus callosum into the contralateral external capsule; however, only about half of these axons turn and enter the striatum. C , Higher magnification of axons growing in the direction opposite to the expression pathway (arrowhead in B ). The vast majority of these axons either stop or make hairpin turns back toward the expression pathway. D , Higher magnification of axons in B (double arrows) showing axons leaving the white matter tract and entering the striatum. E , F , Expression of the cell adhesion molecules L1 does not support growth of axons along the corpus callosum ( F ), and its coexpression with FGF2 reduces the growth supportive nature of FGF2 ( E ). TGF-β1 even with the coexpression of FGF2 does not support axon growth along the pathway, and reduces it to less than LacZ controls. Scale bars: (in A ) B , E – G , 1 mm; C , D , 200 μm
- Figure 5.
Virus-mediated expression of neurotrophic molecules along a pathway improves long-distance growth of axons toward a desired target. Representative CGRP-immunostained sections show nociceptive axon growth 3 weeks after DRG neuron transplants. A , GFP pathway, no target. There was no CGRP+ fiber growth beyond the midline in any animals from this group. B , GFP pathway, NGF target in striatum. This section is from the single animal that showed CGRP+ fiber growth beyond the midline in this group. C , FGF2 pathway, NGF target in striatum. There is ample CGRP+ fiber growth into the contralateral (target) hemisphere, to and beyond the pathway turning point within the corpus callosum. D , FGF2 + NGF (5:1) combination pathway, NGF target in striatum. There is robust CGRP+ fiber growth into the contralateral hemisphere within the corpus callosum, with more successful turning into the target striatum. E , F , Higher magnification of boxed areas in A . G , H , Higher magnification of boxed areas in B . I , J , Higher magnification of boxed areas in C . K , L , Higher magnification of boxed areas in D . Areas pictured in E , G , I , and K include the path point “midline + 1 mm”, which is quantified in the graph. Areas pictured in F , H , J , and L show the extent of CGRP+ axon growth toward the striatal target, with arrows indicating the most distal fibers. M , Higher magnification of boxed area in L . N , Average number of fibers counted at various distances along the pathway, divided by the number counted at 1 mm left of midline (ipsilateral to transplant) to adjust for any differences in transplant size or survival. Mid, midline; c.c., corpus callosum. Mid + 1 mm and Mid + 2 mm are on the right side, contralateral to the transplant. Error bars represent means ± SEM. *p < 0.05; **p < 0.01 (Kruskal-Wallis). GFP only, n = 4; GFP/NGF target, n = 3; FGF/NGF target, n = 4; FGF+NGF/NGF target, n = 8. Scale bars: A–D , 1 mm; E–L , 200 μm.
- Figure 6.
Expression of a chemorepulsive molecule adjacent to the pathway turning point increases the proportion of axons making the desired turn. A , Schematic of injection protocol, blue circles along corpus callosum represent Ad-FGF2/NGF injections, green circles represent Ad-NGF injections at the DRG transplant site (left) and striatal target (right), red circles are Ad-semaphorin injections (only in the +Sema group), and brown circles represent transplanted DRG neurons, injected 1 week after the adenoviral pathway. B , Double labeled section showing location of Sema3A expressing cells (green) relative to CGRP+ axons turning into the striatum (red). B' , Higher magnification of box insert from B . C–F , Representative sections immunostained for CGRP; without semaphorin ( C , D ), or with semaphorin ( E , F ) adjacent to the turn. D and F are higher magnifications of boxed areas in C and E . Arrows indicate mediolateral location of Ad-NGF target injection; asterisks are in the corpus callosum beyond the pathway turning point. The arrowhead in F indicates macrophages within the corpus callosum (not CGRP+ staining). Scale bars: B , C , 500 μm; D , 200 μm.
- Figure 7.
NGF overexpression in the cortex leads to axon growth along the brain surface when neurons are transplanted into the corpus callosum. A , Schematic of injection protocol with cortical target: Blue circles along corpus callosum represent Ad-FGF2/NGF or Ad-GFP (control) injections, green circles represent Ad-NGF injections at the DRG transplant site (left) and cortical target (right). Brown circles represent transplanted DRG neurons, injected 1 week after the adenoviral pathway. B–M , Representative coronal sections after 3 weeks' growth, immunostained for CGRP. B , GFP pathway, NGF target in cortex; an asterisk indicates the area where axons are found growing up toward the brain surface in a separate section, shown in E ; the arrowhead points to CGRP+ fibers growing within the meninges, which have mostly pulled away from the brain parenchyma during tissue processing. C , D , FGF2+NGF (5:1) combination pathways, NGF target in cortex. Two different animals from this treatment group are represented to illustrate variability in axon-pathfinding decisions. The arrow in D points to CGRP+ axons growing up toward the brain surface, whereas the arrowhead in D points out dense fiber growth within the meninges. E , A separate section from the same animal as B , showing where the CGRP+ axons have grown up from the transplant site toward the brain surface. F , Higher magnification of area indicated by arrowhead in B , showing CGRP+ fiber growth within the meninges. G , H , Higher magnification of boxed areas in B . I , J , Higher magnification of boxed areas in C . K , Higher magnification of area indicated by arrowhead in D , showing CGRP+ fibers within the pia and coursing down into the cortex. L , M , Higher magnification of boxed areas in D . N , Quantification of CGRP+ axon growth in control (GFP) pathways and FGF2+NGF pathways at 1 mm beyond the midline when the NGF-expressing target was either in the striatum or in the cortex. There is significantly more axon growth in FGF2+NGF pathways than in GFP pathways for both injection paradigms, but fewer axons follow the corpus callosum pathway when the target is in the cortex because they choose the alternative route along the brain surface. O , Quantification of CGRP+ axon growth into the target area, either halfway between the brain surface and the corpus callosum (pictured in H , J , and M ), or 0.5 mm below the corpus callosum in the striatum. With the striatal target, there is a significant difference between GFP pathways and FGF2+NGF pathways, but with the cortical target, axon growth along the brain surface to the target eliminates the difference between pathways. Error bars represent means ± SEM. Mann–Whitney U tests at each path point. *p < 0.05. GFP/NGF, n = 3; FGF+NGF/NGF, n = 4. Scale bars: B–D , 1 mm; E–J , 200 μm.
