Figure 1.
Gene expression analysis in the acute and subacute phases of spinal cord injury.
Semi-quantitative RT-PCR was employed to evaluate mRNA expression of several genes involved in destructive and reparative processes following SCI (A). The relative expression of genes was determined by measuring the band intensity and using cyclophilin as housekeeping (relative density). Overall, at 3 days after SCI few differences were observed among treatment (4G-BMHP1) and both control (saline and SCI control) groups (Bi, Ci, Di); NT3, BDNF and NGF were undetectable in all groups. At 7 days after SCI there was a general upregulation of inflammatory genes in both injected groups (biomaterial or saline) in comparison with SCI control group (Bii), while a general mRNA overexpression for GAP-43, neurotrophins, growth factors (Cii) and ECM remodelling proteins (Dii) was observed only in the treatment group in comparison with one or both control groups; NGF was undetectable in all groups. Values represent means ± SEM. Significance symbols: * p<0.05, ** p<0.001.
Table 1.
Characteristics of gene-specific primers for semi-quantitative RT-PCR.
Figure 2.
Quantitative histological analysis in the chronic phase of SCI.
(A): lesion size was quantified on spinal cord longitudinal sections stained with hematoxylin/eosin (Ai) and it was reported as cumulative area (mm2). No significant differences among groups were found when measuring both the whole cyst area (Aii) and the cavities into the cyst area (Aiii). Scale bar = 800 µm. (B): GAP-43 positive fibers (Bi and Biii, red) were measured on six longitudinal sections after immunofluorescence staining and the values were expressed as percentage of the total area of the cyst. The GAP-43 immunopositive area was significantly higher in biomaterial-treated group (4G-BMHP1) than both control groups (saline and SCI control) (Bii). In Biii the positive GAP-43 signal is showed at higher magnification (asterisk and dotted line indicate the cyst and its border, respectively). Scale bar = 400 µm in Bi, 50 µm in Biii. (C): CD68 positive cells (Ci and Ciii, green) were counted on three longitudinal sections after immunofluorescence staining and reported as cumulative number per mm2. Nuclei were counterstained with DAPI. Macrophage infiltration was observed in the tissue surrounding the cyst and into the cavities of all groups (Cii). In Ciii, at higher magnification, we reported an image representative of the CD68 positive cells (arrows) we observed in all groups. Scale bar = 400 µm in Bi, 100 µm in Biii. Values represent means ± SEM. Significance symbols: * p<0.05, 4G-BMHP1 vs SCI control; ° p<0.05, 4G-BMHP1 vs saline.
Figure 3.
Evaluation of mature and immature nerve fibers infiltrating the cyst.
Images show the cyst cavity in SCI control, saline injected and 4G-BMHP1 treated animals at 8 weeks after lesion. Asterisks indicate the scaffold. Immunolabelling for GAP-43 (red) coupled with SMI-31 (green) or SMI-32 (green) was made on longitudinal sections. Nuclei were counterstained with DAPI. Within the cyst, the majority of GAP-43+ sprouting/growing axons consisted of immature fibers expressing the non-phosphorylated neurofilament H (SMI-32+), even if also GAP-43+ mature fibers, staining for the phosphorylated neurofilament H (SMI-31+), were observed. The percentage of GAP-43+ fibers expressing either SMI-32 or SMI-32 appeared similar in all groups. Scale bar = 200 µm.
Figure 4.
Evaluation of gliotic and angiogenic processes within the lesion area.
From the left to the right, arrows indicate blood vessels (vWF+, red) infiltrating the lesion area, collagen IV deposition (red) within the cyst and the glial scar (GFAP+, green) surrounding the lesion site. Asterisks indicate the scaffold. Nuclei were counterstained with DAPI. Blood vessels infiltration appeared similar in all groups, but in the treatment group (4G-BMHP1) a greater deposition of type IV collagen occurred. Several GFAP+ astrocytes surrounding the cyst margins and infiltrating the lesion were observed in all groups but they were not detected within the scaffolds. Scale bar = 400 µm. 2
Figure 5.
Immunofluorescence analysis of cellular and non-cellular infiltrates into the injected scaffold.
(A) and (B): arrows indicate collagen IV+ cells (A, red) and NG2+ cells (B, red), whereas arrowheads indicate GFAP+ glial cells (A and B, green). (C): arrows show IBA+ microglia cells (red) and arrowheads CD68+ macrophages (green). (D) and (E): arrows indicate β-TubIII+ axons (green) surrounded by the basement membrane, composed by collagen IV (D, red) and laminin (E, red); arrowheads show collagen IV+ (D) and laminin+ (E) cells. Asterisks indicate the scaffold. Nuclei were counterstained with DAPI. A remarkable deposition of type IV collagen and laminin was observed into the biomaterial and specifically along axons. The non-neuronal cellular infiltrate was mainly represented by Iba-I+ microglia cells and NG2+ oligodendrocyte precursor cells, whereas GFAP+ astrocytes and CD68+ macrophages mostly surrounded the implant. Into the scaffold we also observed cells showing type IV collagen and laminin immunopositivity, strictly coupled to axons, likely ascribable to Schwann cells. Scale bar = 100 µm.
Figure 6.
Immunofluorescence analysis of neuronal elements and nerve fibers within the scaffold.
(A): the scaffold was infiltrated by neuronal cells (β-TubIII+, green) and sprouting/regenerating nerve fibers, that were positive for both GAP-43 (red) and β-TubIII (green). Merge of two stainings is reported at higher magnification on the right, arrows indicate some axons expressing both GAP-43 and β-TubIII. Scale bar = 100 µm for images on the left and 50 µm for image on the right. (B): co-staining for β-TubIII (red) and MBP (green) revealed that some sprouting/regenerating fibers were myelinated, as seen on the right in the merge or below in the high-magnified inserts, where myelin surrounding some GAP-43+ axons is visible (Bi, arrows). Scale bar = 100 µm for images on the left, 50 µm for image on the right and 20 µm for images below. Nuclei were counterstained with DAPI.
Figure 7.
Evaluation of locomotor recovery over 8 weeks after SCI.
BBB rating scale ranges from a minimum of 0 (no observable hindlimbs movements) to a maximum of 21 (presence of consistent plantar stepping, coordination and trunk stability). At 7.5 weeks after injury, in the treatment group (4G-BMHP1) we observed a better improvement of hindlimbs motor recovery compared to both control groups (saline and SCI control). This statistically relevant difference among scores implies that all groups had frequent or consistent plantar stepping but in the treatment group occurred also an occasional forelimbs-hindlimbs coordination. Values represent means ± SEM. Significance symbols: * p<0.05, 4G-BMHP1 vs SCI control; ° p<0.05, 4G-BMHP1 vs saline.
Figure 8.
Delivery of the biomaterial into the damaged rat spinal cord.
Rat spinal cord was exposed at T9-T10 level and subjected to moderate contusion using MASCIS impactor (Ai), then animals were injected with the biomaterial using an Hamilton syringe fixed at a micromanipulator (Aii, arrow indicates the needle penetrating into the injury site). The needle was inserted until reaching the ventral surface of the spinal cord then, starting from this position, the biomaterial was delivered into the lesion epicenter with multiple injections equally spaced of approximately 500 µm, for a total dose of 3 µl (Aiii).