9-day Neuroanatomical Study
The ability of fibrin scaffolds containing the HBDS and NT-3 to influence regeneration was evaluated at 9 days after SCI. Studies performed previously in collaboration with Martin Schwab (University of Zurich) where fibrin scaffolds were implanted after spinal cord dorsal hemisection showed that fibrin was present at 10 days but degraded by 14 days (Sakiyama and Schwab, unpublished results). Therefore, a 9-day time point was chosen in order to study the acute effects of the fibrin scaffold while it is still present in the injury environment. Previously, 100 ng/mL of NT-3 was found to be the optimal dose for promoting neurite outgrowth in vitro
]; therefore, we investigated doses surrounding this optimal in vitro
Neuronal Fiber Sprouting
Tuj1 staining was performed to assess the effect of fibrin scaffolds on neuronal fiber sprouting. GFAP staining was used as an indicator of the lesion border; the lesion size in all cords was observed to be 2-3 mm in length at 9 d. Neuronal fiber sprouting into the lesion area was dramatically different between experimental groups. In the TBS group, very few neuronal fibers sprouted into the lesion area; most fibers ended abruptly at the lesion border (). In contrast, fibrin-treated groups showed neuronal fiber infiltration of the lesion site, both at the border of the intact cord and well inside the lesion (). Fibrin remnants frequently remained inside the lesion area (as visualized by dansyl-labeled peptide in the HBDS), and neuronal fibers were observed touching the borders of fibrin remnants (not shown). In the F-DS-NT3(1000 ng/mL) group, particularly dense neuronal fiber staining was observed (inset, ). Although neuronal fibers infiltrated the lesion, neuron cell bodies (NeuN, neuronal nuclear, staining) were not observed in the lesion (all treatment groups, data not shown).
Figure 2 Effect of controlled delivery of NT-3 from fibrin scaffolds on neuronal fiber sprouting. A-D. Neuronal staining (Tuj1) in spinal cords after treatment with (A) TBS, (B) fibrin (F-only), (C) fibrin and NT-3 at 1000 ng/mL (F-NT3(1000 ng/mL)), and (D) fibrin (more ...)
Differences in neuronal fiber density between treatment groups were quantified in the whole lesion and in three equal-area regions of lesion separately (rostral, middle, and caudal thirds) (). For reference, the Tuj1 density of normal cords was measured to be ~5% in white matter, ~80% in gray matter, and 40% overall. The overall (whole lesion) neuronal fiber density in the TBS-treated lesions was 3%. For control groups containing fibrin scaffolds, overall neuronal fiber density in the lesion was ~6-7%. In the F-DS-NT3(1000 ng/mL) treatment group, the overall neuronal fiber density in the lesion was 10.4%, which represents a ~1.5-fold increase over control groups containing fibrin, and a 3-fold increase over the TBS-treated group. Overall neuronal fiber density in the F-DS-NT3(1000 ng/mL) was higher than overall neuronal fiber density in the F-NT3(1000 ng/mL) group, where no delivery system was present (7%, 1.5-fold increase). This trend was most pronounced in the rostral third of the lesion; in both the F-DS-NT3(500 ng/mL) (14%) and F-DS-NT3(1000 ng/mL) (15%) groups, rostral fiber density was greater than in the F-NT3(1000 ng/mL) group (7%), representing a 2-fold increase. These results indicate that NT-3 delivery from the HBDS enhanced neuronal fiber density relative to NT-3 delivery from unmodified fibrin scaffolds, especially at the rostral edge of the lesion, where spouting of supraspinal axons would be anticipated.
Glial Scar Formation
To assess the formation of the glial scar, astrocyte staining (using an antibody to GFAP) was performed. In the TBS group, astrocyte density was high at both the gray matter (which contains neuronal and glial cell bodies) and white matter (which contains axonal tracts) border of the lesion (). This indicates the formation of a dense astroglial scar surrounding the lesion, which poses as significant biochemical barrier to regenerating axons. However, in fibrin scaffold-treated groups, astrocyte density appeared to be reduced, particularly at the border of the white matter with the lesion (arrowheads in ). Reduced scar formation, particularly at the white matter border, is important for the regeneration of long axonal tracts, such as those of the corticospinal tract. To investigate this difference, GFAP density was quantified at the white matter and the gray matter lesion borders in five treatment groups, TBS, F-only, F-DS, F-NT3(1000 ng/mL), and F-DS-NT3(1000 ng/mL). shows that astrocyte density at the white matter border was significantly lower in fibrin scaffold-treated groups than in the TBS group. Astrocyte density at the gray matter border with the lesion did not show a difference between the TBS and fibrin scaffold-treated groups. Thus, implantation of fibrin scaffolds appears to decrease scar formation at the white matter border of the lesion.
Figure 3 Effect of fibrin scaffolds on astrocytes at the lesion border. A-D. Astrocyte staining (GFAP) of spinal cords after treatment with (A) TBS, (B) fibrin (F-only), (C) F-NT3(1000 ng/mL), and (D) F-DS-NT3(1000 ng/mL). Horizontal sections, with the rostral (more ...)
Migration into the Lesion
In order to investigate the relationship between neuronal fibers and astrocytes, we performed double staining with Tuj1 and GFAP antibodies. In TBS-treated cords, dense astrocyte staining at the border of the lesion was associated failure of Tuj1-positive neuronal fibers to extend into the lesion. In contrast, in the F-DS-NT3(1000 ng/mL) treated cords, neuronal fibers and long astrocyte processes (arrowhead) occurred along channel-like formations connecting the lesion to the intact cord (). Astrocytes were not frequently present within the lesion. Other fibrin scaffold-treated groups showed similar results (data not shown), suggesting that fibrin scaffolds may reduce scar density and encourage longitudinal regeneration of neuronal fibers and astrocytes along channel-like formations connecting the lesion with the intact cord.
Figure 4 Infiltration of (A) TBS and (B) F-DS-NT3(1000 ng/mL) treated cords by neurons and astrocytes. In all panels, blue is Hoechst nuclear stain. Horizontal sections. A, B. Neuronal fiber (Tuj1, red) and astrocyte (GFAP, green) double staining at rostral border (more ...)
The effect of fibrin scaffold treatment on macrophage/microglia infiltration was assessed as an indication of inflammation. Macrophages and microglia were present surrounding the lesion in all treatment groups (). In cords treated with fibrin scaffolds, ED-1-positive cells were present just inside the lesion in areas between neuronal fiber channel-like formations connecting the lesion to the intact cord. ED-1 staining was particularly dense at the white matter border of the lesion (, arrowhead and ), where the cells appeared vacuolated and phagocytic. Macrophages/microglia were also present around remnants of fibrin (F), where they were smaller and did not appear vacuolated (). The macrophage/microglia density inside the lesion and surrounding the lesion was compared between treatment groups (). No difference in macrophage/microglia density was observed between treatment groups, indicating that the presence of fibrin does not increase the density of macrophage/microglia inside or surrounding the lesion. In all treatment groups, the density in the white matter bordering the intact cord was greater than inside the lesion, indicating that macrophages/microglia are more attracted to surrounding white matter than to the lesion itself. Therefore, the presence of fibrin scaffolds did not increase the density of macrophage/microglia inside the lesion or in the intact cord bordering the lesion.
Figure 5 Effect of fibrin scaffolds on macrophage/microglia density. A-D. Macrophage/microglia (ED-1) staining of TBS (A) and F-DS-NT-3(1000 ng/mL) (B-D) treated cords. Horizontal sections, with the rostral cord oriented toward to top. Arrowhead indicates dense (more ...)
Sprouting of Neuronal Subpopulations
To determine the effects of fibrin scaffold treatment on different neuronal subpopulations, the regenerative responses of ascending sensory neurons (CGRP), primary spinal motoneurons (ChAT), and neurons of the raphespinal tract (5-HT) were investigated. The CGRP-positive sensory fibers were found in encroaching dorsal roots, the intact dorsal spinal cord, and inside the lesion. In the TBS-treated cords, CGRP-positive fibers were present at the border of the gray matter (GM) with the lesion (), but were rarely observed sprouting past the astroglial scar at the lesion border. In all fibrin scaffold-treated groups, CGRP-positive fibers were observed sprouting beyond the border with the lesion (). However, the density of CGRP-positive fibers was much lower than the density of Tuj1-positive fibers; therefore, sensory neuron fibers do not make up the majority of the neuronal fibers present in the lesion site and at the lesion borders.
Figure 6 Effect of fibrin scaffolds on neuronal subpopulations. Horizontal sections, with the rostral cord oriented toward to bottom. A,B. Sensory neuron staining (calcitonin gene related peptide, CGRP) of TBS (A) and F-DS-NT-3(1000 ng/mL) (B) treated cords. Caudal (more ...)
The ChAT antibody stains large neuronal cell bodies located within ventral gray matter (spinal cord alpha motoneurons). Fewer ChAT-positive fibers sprouted into the lesion in the TBS group () than in the fibrin scaffold-treated groups (, F-DS-NT3 (1000ng/mL) shown). In all fibrin scaffold-treated groups, ChAT fiber staining was evident inside the lesion at the rostral and caudal edges of the lesion and in the middle of the lesion near fibrin remnants (data not shown). The density of ChAT-positive fibers was observed to be comparable to the density of Tuj1-positive fibers, indicating that ChAT-positive fibers were a major contributor to the population of Tuj1-positive fibers in fibrin-scaffold treated cords.
The regenerative response of serotonergic neurons was investigated using 5-HT staining. Sprouting of serotonergic neurons into the lesion was not observed in the lesion of the TBS-treated cords (), whereas sprouting into the lesion was seen in all fibrin scaffold-treated groups (). Notably, this sprouting did not continue more than 500 μm into the lesion. The density of 5-HT-positive fibers was much lower than the density of Tuj1-positive fibers; therefore, serotonergic fibers did not make up the majority of the neuronal fibers present at the lesion site.
In summary, fibrin scaffolds significantly enhanced the regenerative environment by decreasing the astroglial scar formation at the white matter border of the lesion. Furthermore, the controlled release of NT-3 with the HBDS resulted in greater neuronal fiber sprouting than other fibrin scaffold treatments after 9 days.
Functional Regeneration Study (12 weeks)
The effect of the controlled release of NT-3 on functional regeneration was assessed for 12 weeks. Immediately after injury, one of three treatments was administered: TBS, F-NT3(1000 ng/mL), or F-DS-NT3(1000 ng/mL). Because of the requirement for prolonged animal care and the need for larger group sizes, a complete set of control treatments (including F-only, F-DS groups) was not performed.
In order to assess the effect of treatment on functional ability, BBB open field motor testing [21
] was performed weekly (). Immediately after injury, animals had no hindlimb function (score of 0). One week after injury, animals had regained slight or extensive movement of two or three hindlimb joints (BBB score of 2-3). At 12 weeks, most animals had regained sweeping motion or plantar paw placement of their hindlimbs (BBB score of 8). Some animals had regained plantar placement of their hindpaw with weight support or occasional weight supported plantar steps (score of 9-10). However, no differences in functional motor recovery were observed between the treatment groups.
Figure 7 Functional assessment of SCI with BBB testing in 12-week study. One week after injury, slight or extensive movement of two or three joints was regained, as indicated by a BBB score of 2-3 (a score of 21 indicates unimpaired hindlimb movement). No statistical (more ...)
Upon histological examination, the lesion in all groups was much larger at 12 weeks than at 9 days (5 mm vs. 2-3 mm in length), largely due to formation of cystic cavities at the rostral and caudal borders of the lesion (data not shown).