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. 2008 Apr 2;28(14):3814-23.
doi: 10.1523/JNEUROSCI.0143-08.2008.

Self-assembling nanofibers inhibit glial scar formation and promote axon elongation after spinal cord injury

Vicki M Tysseling-Mattiace  1 , Vibhu Sahni, Krista L Niece, Derin Birch, Catherine Czeisler, Michael G Fehlings, Samuel I Stupp, John A Kessler
Affiliations

Self-assembling nanofibers inhibit glial scar formation and promote axon elongation after spinal cord injury

Vicki M Tysseling-Mattiace et al. J Neurosci. .

Abstract

Peptide amphiphile (PA) molecules that self-assemble in vivo into supramolecular nanofibers were used as a therapy in a mouse model of spinal cord injury (SCI). Because self-assembly of these molecules is triggered by the ionic strength of the in vivo environment, nanoscale structures can be created within the extracellular spaces of the spinal cord by simply injecting a liquid. The molecules are designed to form cylindrical nanofibers that display to cells in the spinal cord the laminin epitope IKVAV at nearly van der Waals density. IKVAV PA nanofibers are known to inhibit glial differentiation of cultured neural stem cells and to promote neurite outgrowth from cultured neurons. In this work, in vivo treatment with the PA after SCI reduced astrogliosis, reduced cell death, and increased the number of oligodendroglia at the site of injury. Furthermore, the nanofibers promoted regeneration of both descending motor fibers and ascending sensory fibers through the lesion site. Treatment with the PA also resulted in significant behavioral improvement. These observations demonstrate that it is possible to inhibit glial scar formation and to facilitate regeneration after SCI using bioactive three-dimensional nanostructures displaying high densities of neuroactive epitopes on their surfaces.

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Figures

Figure 1.
Figure 1.
Structure of IKVAV PA. a, Schematic representation showing individual PA molecules assembled into a bundle of nanofibers interwoven to produce the IKVAV PA. b, Scanning electron micrograph image shows the network of nanofibers in vitro. Scale bar, 200 nm.
Figure 2.
Figure 2.
IKVAV PA solution self-assembles in vivo. a, Phase (left) and fluorescent (right) images showing the fluorescent IKVAV PA in the injured spinal cord 24 h after injection (dorsal, left; rostral, top). b–e, Longitudinal sections of spinal cord showing the fluorescent IKVAV PA is present at 2 weeks after injection only in IKVAV PA-injected animals (c) versus control (uninjected; b), but by 4 weeks, the IKVAV PA has mostly biodegraded, as seen in the IKVAV PA-injected animals (e) compared with uninjected animals (d). Scale bars: a, 100 μm, b–e, 50 μm.
Figure 3.
Figure 3.
IKVAV PA promotes functional recovery as analyzed by the BBB scale. a, Graph shows mean mouse BBB locomotor scores at 9 weeks after SCI for animals receiving injections of glucose, EQS PA, IKVAV PA, or sham injection. The IKVAV PA group differed from all others at p < 0.045. n = 7. Because there was no difference among these controls, glucose was used in the subsequent experiment depicted in c. b, IKVAV peptide was injected at the same manner as the IKVAV PA. There were no significant differences in the BBB scores of animals injected with IKVAV peptide compared with sham controls. c, The graph shows mean mouse BBB locomotor scores between IKVAV PA and glucose injections after SCI. The IKVAV (n = 15) and glucose (n = 14) groups differ from each other at p < 0.04 by ANOVA with repeated measures. *Tukey's HSD post hoc t tests showed that scores differed at p < 0.045 at every time point 5 weeks after SCI and thereafter.
Figure 4.
Figure 4.
IKVAV PA attenuates astrogliosis in vivo after SCI. a–c, Representative confocal Z-stacks of injured areas stained with GFAP in control and IKVAV PA-injected animals. The lesion is defined as the area marked by dense infiltration (Okada et al., 2006). The two groups do not differ at 4 d (a), but at 5 weeks (b) and 11 weeks (c), there is significantly less glial scarring in the IKVAV PA-injected animals. d, GFAP immunofluorescence levels (expressed as fold increases over uninjured areas) in the IKVAV PA-injected animals are significantly reduced compared with control animals at 5 and 11 weeks (*p < 0.02, **p < 0.04 by t test). Scale bar, 20 μm.
Figure 5.
Figure 5.
Apoptotic cell death is reduced in IKVAV PA-injected animals. a, b, Collapsed confocal Z-stacks of 20-μm-thick spinal cord sections (red, activated caspase-3; blue, Hoechst nuclear stain). The IKVAV PA-injected animals had fewer cleaved caspase-3-positive cells in every 20-μm-thick section in the lesion (defined as the area marked by dense infiltration; b, d; **p < 0.008 by t test) and as far as 400 μm rostral and caudal to the lesion (a, d; *p < 0.001 by t test). c, Confocal Z-stacks of 20-μm-thick sections within 400 μm of the lesion (red, CC1; blue, Hoechst) reveals an increased density of OLs in the IKVAV PA-injected animals. See text for quantitation. Scale bar, 20 μm.
Figure 6.
Figure 6.
IKVAV PA promotes regeneration of motor axons after SCI. a, b, Representative Neurolucida tracings of BDA-labeled descending motor fibers within a distance of 500 μm rostral of the lesion in vehicle-injected (a) and IKVAV PA-injected (b) animals. The dotted lines demarcate the borders of the lesion. c–f, Bright-field images of BDA-labeled tracts in lesion (c, e) and caudal to lesion (d, f) used for Neurolucida tracings in an IKVAV PA-injected spinal cord (a, b). g, h, Bar graphs show the extent to which labeled corticospinal axons penetrated the lesion. *The groups representing three control and three IKVAV PA mice and the tracing of 130 individual axons differ from each other at p < 0.03 by the Wilcoxon rank test. R, Rostral; C, caudal; D, dorsal; V, ventral. Scale bars: a–d, 100 μm; e–f, 25 μm.
Figure 7.
Figure 7.
IKVAV PA promotes regeneration of sensory axons after SCI. a, b, Representative Neurolucida tracings of BDA-labeled ascending sensory fibers within a distance of 500 μm of the lesion epicenter in vehicle-injected (a) and IKVAV PA-injected (b) animals. The dotted lines demarcate the borders of the lesion. c–f, Bright-field images of BDA-labeled tracts in lesion (c, e) and rostral to lesion (d, f) used for Neurolucida tracings in an IKVAV PA-injected spinal cord (a, b). The top arrowhead points to an axon tip that morphologically resembles a growth cone. g, h, Bar graphs showing the extent to which labeled dorsal column axons entered and grew through the lesion. **The groups (representing 4 control and 4 IKVAV PA mice and the tracing of 185 individual axons) differ from each other at p < 0.05 by the Wilcoxon rank test. R, Rostral; C, caudal; D, dorsal; V, ventral. Scale bars: a–d, 100 μm; e–f, 25 μm.
Figure 8.
Figure 8.
Montage of spinal cord sections illustrates the third dimension of axon regeneration. The Neurolucida drawings in Figures 6 and 7 are two-dimensional reconstructions of a three-dimensional phenomenon, and they therefore do not fully demonstrate the very unusual courses and morphologies of axons passing through the lesioned area. To further demonstrate the type of path taken by axons in the IKVAV PA-treated lesions, seven bright-field sections containing a representative axon from Figure 7 were assembled (for orientation, see supplemental Fig. 7, available at www.jneurosci.org as supplemental material). These seven sections represent only a small part of the total of 94 sections for this spinal cord. Note the meandering, unusual course of the fiber through the injured tissue and that it migrates out of and back into individual sections and across different sections. Also note that the fiber terminates rostral to the lesion after passing through the lesion site. The numbers label the individual images of the combined montage in Figure 9 and in supplemental Figure 7 (available at www.jneurosci.org as supplemental material).
Figure 9.
Figure 9.
Combined montage flattens the representative axon to two dimensions to demonstrate the unusual morphology of the axon as it passes through the lesion. The Neurolucida tracings in Figures 6 and 7 follow the course of individual axons in three dimensions by tracing them from section to section and displaying their courses in a usable two-dimensional form. The drawing does not reflect the unusual morphologies of the axons. To demonstrate this, the separate sections of Figure 8 were aligned in a montage to display the course of a single axon as it starts at the caudal end of the lesion, passes through the lesion, and terminates rostral to the lesion. Note the unusual course and morphology, and that the axon terminates within normal tissue rostral to the lesion.
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