Article Figures & Data
Figures
- Figure 1.
Immunocytochemical detection of CSPG and stub antigen/core protein adjacent to a dorsal quadrant lesion 5 d after treatment with saline (control) or ChABC. The lesion cavity is positioned at the top left, with intact tissue to the right and below the dotted line. After ChABC treatment (B), CSPG-immunoreactivity is condensed right at the lesion relative to the more diffuse labeling observed adjacent to saline-treated lesions (A). In contrast, immunoreactive staining for core protein greatly increased after ChABC treatment (D) compared with the saline-treated lesion (C). Scale bar, 100 μm.
- Figure 2.
Evidence for axonal growth from a PN graft into the spinal cord. A, This diagram depicts the positioning of the graft (PNG) between the C3 Hx (shaded area) and C5 DQ lesion. Regenerated axons (dotted lines) are shown to extend from several regions of the ventrolateral white matter at C3 into the intermediate gray of the C5 spinal cord ventral to the lesion site. B, Many BDA-labeled axons are found in the graft (PNG) close to the interface (dotted line) with the spinal cord, with some crossing into the ventral gray matter (VGM). CC, Central canal. Thionin counterstain. C, Most BDA-labeled axons (arrows) failed to cross the PN graft–spinal cord interface when the lesion was treated with saline. Several axons with a bend at the terminal end appear to be repulsed from the interface. Occasionally, short segments of axons appear in the spinal cord adjacent to the interface (arrowheads). D, Treatment of the C5 lesion with ChABC was sufficient to allow the extension of many axons (arrows) across the PN graft–spinal cord interface. Scale bars: B, 250 μm; C, D, 100 μm.
- Figure 3.
BDA-labeled axons have extended from a PN graft (top left out of frame) into spinal cord gray matter independent from Schwann cells labeled with an antibody to p75 receptor (green in B). Overlap of the images (C) indicates that most Schwann cells remain within the PN graft, disengaged from the growth of regenerated axons into the spinal cord.
- Figure 4.
Relationship of BDA-labeled axons with spinal cord neurons. A, Spinal cord tissue ventral to the PN graft (top of image) contained many regenerated axons (arrows), with some extending toward a motoneuron pool in the medial ventral horn (bottom left). B, C, Higher-magnification images from boxes outlined in A show branching of BDA-labeled axons and multiple varicosities along their length. Possible anatomical contact between regenerated axons and thionin-stained spinal cord neurons is evident (arrows). Scale bars: A, 250 μm; B, C, 50 μm.
- Figure 5.
Examples of possible anatomical contact between regenerated axons and spinal cord neurons. BDA-labeled axons form multiple branches and punctuate endings that appear adjacent to (or in contact with) spinal cord neurons (arrows) of the intermediate gray matter (A, B) ventral to an apposed PN graft (out of frame but would be at the top of A and B) and with ventral motoneurons (C, D). In C, several motoneurons are intimately associated with labeled rubrospinal and/or reticulospinal axons in which there appears to be heightened branching by axons closest to individual motoneurons. In D, punctuate terminals of regenerated axons are prominent on the surface of motoneurons. Scale bars: A–C, 50 μm; D, 25 μm.
- Figure 6.
Indications of synaptic contact between regenerated axons and spinal cord neurons. A, The distribution of BDA-labeled axons is depicted in A, and the distribution of synaptophysin (SYN)-IR in the same field is presented in B. Merging of these images (C) provides an indication of the presence of synaptic contacts associated with regenerated axons (arrows). Multiple synaptic sites are found along the length of regenerated axons as well as on the cluster of fibers found in the bottom center of the image (highlighted in the inset).
- Figure 7.
Reconstruction of axonal outgrowth from PN grafts exposed to BDA. The entire PN graft (PNG) appears filled with BDA-labeled axons after exposing the cut end of the middle of the graft to BDA. The distribution of regenerating axons within the spinal cord is presented in tracings next to their respective montage of images. Several retrogradely filled spinal cord neurons (arrows) close to the graft–host interface are indicative of neurons that grew an axon into the PN graft.
- Figure 8.
Examples of forelimb use at rest and during rope walking. Some of the images are from a mirror facing the camera on the far side of the testing platform. Some animals treated with ChABC (A, D) before placement of the distal PN graft demonstrated the ability to use the affected forelimb for balance during grooming and movement along a suspended rope compared with the absence of such use in saline-treated animals (C, F). Forelimb use that was recovered after ChABC treatment (A, D) was absent for at least 24 h after severing the PN graft (B, E). R indicates the injury-affected right forelimb.
- Figure 9.
Animals in both groups exhibited a significant decrease in the range of motion immediately after creation of the C5 DQ lesion (compare Pre-injury with week 1 scores). Over the next 3 weeks, there was a steady improvement in forelimb swing in ChABC-treated animals compared with additional decline in saline-treated animals. At 4 weeks, there was a significant improvement in range of motion of the affected forelimb of ChABC-treated animals that continued through week 7 after distal graft insertion. Behavioral assessment 1 d after cutting of the PN graft indicated a decline in forelimb use by ChABC-treated animals that was indistinguishable from saline-treated animals. At some point during the 7 week period for behavioral analysis, three of seven ChABC-treated rats achieved a score of 4, whereas the maximum for the five saline-treated rats did not rise above a score of 2.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Table 1. Description of experimental groups and schedule of surgical procedures Table 2. Raw data scores for Range of Forelimb movement over a 7 week period and after cutting the PN graft. Objective scoring of the extent of axonal outgrowth is provided for each animal.
- supplemental material - Figure 1. Diagrammatic representation of the placement of the osmotic pump (1) and infusion catheter (2) for delivery of chondroitinase into a cervical (C) level 5 spinal cord injury site. Spacers (cross hatched) seated beneath the catheter were secured to the dorsal surface of vertebral bodies 4 and 6 with cyanoacrylate. A variable number of spacers were used to adjust the depth of the cannula (3) to limit damage of the spinal cord beyond creation of the dorsal quadrant lesion cavity. The region of the C3 hemisection (C3HX) is indicated by the dotted area rostral to C4.
- supplemental material - Figure 2. Densitometric measures of CSPG, core protein, GFAP and collagen IV immunoreactivity 5 days after saline or chondroitinase treatment. Region 1 is immediately ventral to the lesion site while regions 2 and 3 are progressively more distal to the cavity. No change in GFAP or collagen IV immunoreactivity was detected in any of the regions after saline or ChABC treatment. In all regions there was a decrease in CSPG immunoreactivity with a concomitant increase in core protein or stub antigen immunoreactivity I regions 1 and 2.
- supplemental material - Figure 3. Histogram of the average number of brain stem and propriospinal neurons that regenerated their axon into a peripheral nerve graft, as detected by retrograde labeling with True Blue. The greatest number of regenerated brain stem neurons was located in the reticular formation, with good contribution by neurons of the raphe and locus coeruleus. There also was a considerable contribution by propriospinal neurons.
- supplemental material - Movie Clip 1 � Saline Treated, 1 week after distal nerve end was apposed to C5DQ site. This animal is unable to use the right forelimb for support or for locomotion. The forelimb remains at less than 900, digits are curled and the animal has difficulty ambulating around the cage.
- supplemental material - Movie Clip 2 � Chondroitinase Treated, 1 week after distal nerve end was apposed to C5DQ site. The forelimb alternates between less than 900 and being perpendicular to the body trunk. The digits are curled, there is dorsal flexion of the paw and very little weight placed upon the affected limb. This animal is slightly less impaired than the saline-treated animal of clip 1.
- supplemental material - Movie Clip 3 - Saline Treated, 8 weeks after distal nerve end was apposed to C5DQ site. This animal has a persisting deficit in forelimb movement similar to what was seen at 1 week post graft apposition.
- supplemental material - Movie Clip 4 - Chondroitinase Treated, 8 weeks after the distal nerve end was apposed to C5DQ site. At this time point the animal exhibits clear forward swing of the right forelimb with apparent weight support during stepping. The right forepaw exhibits some lateral rotation during placement but it is important that this animal exhibits plantar stepping.
- supplemental material - Movie Clip 5 - Chondroitinase Treated, 8 weeks after distal nerve end was apposed to C5DQ site, 1 day after cutting the PN graft. There is a dramatic change in the ability of this animal to use the right forelimb for weight support and stepping. Forelimb swing is reduced to about 900 and the forepaw now is curled whereas there was distinct plantar stepping prior to transection of the PN graft.
