Figure 1｜Self-explanatory schematic drawing indicating the point of spinal cord (SC) injury (A), the formation of a gap (A1) and of the following bridge (A2). A3 provides information on the indicative areas analyzed in the present study, as represented in cross-sections from rostral (proximal) to caudal (distal) SC spanning across the lesion. A4–A8 are the cross sections at the levels indicated by the arrows.
Previous cytological studies on the transected lumbar SC of lizards have shown the presence of differentiating glial cells, few neurons and axons in the bridge region. The bridge region is a 1–2 mm long region located between the proximal and distal stumps of the injured spinal cord segment in some cases (Alibardi, 2014b, c, 2019a; Figure 1).
Figure 2｜Histology (A–G, toluidine blue stain) and electron microscopy (H–K) of representative cross-sectioned regions of the regenerating SC at 22 days (E) and 29 days (A–D, F–K) post-injury.
The analysis of the transected SC at 22 and 34 days showed that at the time of transection, a bridge region (Figure 1A–A3) formed, which was composed of a tissue containing nervous fibers mixed to connective fibers derived from the meninges. The cross sections collected from the rostral SC stump (Figures 1A4–A5 and 2A) that moved into the bridge (Figures 1A5–A6 and 2B–G) and finally into the caudal SC stump (Figure 1A6–A8) showed that the injured SC changed in both diameter and structure. Initially, the injured SC gradually lost the demarcation between grey and white matter, the central canal enlarged into a dilated ependymal ampulla, while in the central area of the bridge, the ependymal canal disappeared leaving a glial and connective mass of tissue among empty spaces. The latter was likely derived from degenerated myelinated axons and nerve cells (Figure 2B–G). Some nerve bundles, resembling regenerating nerves, were also observed in the central regions of the bridge, suggesting regeneration at 22-34 days (Figure 2G).
In recovered SC at 34 days post-injury, the electron microscope showed presence of numerous cavities formed among sparse cells, most of which appeared as electron-dense oligodendrocytes, sometimes detected in course of initial myelination of sparse axons (Figure 2H–J). The cytoplasm of these cells, like that surrounding the myelinating axons, appeared more electron dense than the cytoplasm of neurons and astroglial cells. Other sparse cells present in the bridge were electron-pale in the cytoplasm and the nucleus was also pale although numerous heterochromatic zones were present (Figure 2K). The latter cells were recognized as astrocytes because of their content in intermediate filaments that often formed intracellular bundles inside cytoplasmic protrusions.
Figure 3｜Western blotting results from brain extracts for GAP-43 and NF200.
The immunodetection of labeled proteins extracted from the brain showed a prevalent band at 48–50 kDa, and other minor bands around 90, 42, 35 and 17 kDa (first lane on the left in Figure 3). A main and broad band at 200–240 kDa was instead reactive for NF200 (second-central lane in Figure 3). Controls, omitting the primary antibody, did not show bands (last lane on the right in Figure 3).
Figure 4｜Histology (A–D, toluidine blue stain) and immunofluorescence for GAP-43 (E–K) of regenerating SC within ~3 weeks after injury (11, 19 and 22 days post-injury).
After 11 days, the proximal stump of the SC (Figure 1A5) showed numerous degenerating axons in the grey matter of the proximal stump. SC become thinner when it moved into the bridge and consisted of a degenerating nervous tissue where no neurons were visible around the enlarged ependymal ampulla (Figures 1A5 and 4A–D). Numerous neurons, including motor neurons of the proximal SC stump at 11–22 days showed a cytoplasmic granular immunolabeling for GAP-43 (Figures 1A5 and 4E). Numerous GAP-43 labeled axons were also seen in the proximal region of the bridge (Figure 1A5) at 11, 19 and 22 days, but they were un-evenly distributed in the degenerated SC (Figure 4G–J). Although a quantitative analysis was not done in the central region of the bridge observed in cross-section, devoid of ependyma (Figure 1A6), a number of GAP-43 positive axons with variable labeling intensity were observed, and those more intensely labeled varied from 10 to 30 in the available cases. The labeling was not uniform on the entire SC section but it was only present in external areas, corresponding to the white matter, with respect to the dilated ependymal ampulla where it was present (Figure 1A5 and A7), and in sparse areas of the central bridge where the ependyma was missing. Control sections were unlabeled (Figure 4K). Few GAP-43-labeled axons were also seen in the distal stump of the SC (Figure 1A7; not shown).
Figure 5｜DAPI-nuclear fluorescence (blue; A and B) and double labeling (DAPI blue and GAP43 green C–E) at different days post-injury (11–34 days).
The single DAPI staining of cross-sectioned SC evidenced that more numerous cells were present around the enlarged ependymal ampullae in the bridge at 19, 22 and 34 days after injury in comparison to those present around the central canal in the proximal stump of the SC, as a result of the intense cell proliferation in the bridge (Figures 1A5–A7, 5A and B). The double immunostaining, for DAPI and GAP-43, confirmed that few intensely labeled axons (10–30/section) were present in the bridge regions lateral to and around the ependymal canal. In more central regions of the bridge, other labeled axons appeared irregularly distributed within the mass of the bridge tissue where the ependymal canal was missing (Figures 1A5 and 5C–E).
Figure 6｜ Immunogold (A–D) and immunogold with silver intensification (E and F) for growth associated protein 43 (GAP-43) detected at the beginning of the bridge area (level A5 in Figure 1) in injured SC at 22 days post-injury.
Figure 7｜ Immunogold with silver intensification labeling for growth associated protein 43 in nervous areas of the bridge around the dilated ependymal canal (level A5 in Figure 1) in injured spinal cord at 22 days post-injury.
To support immunofluorescence observation, some sections at 22 days of SC regeneration were also analyzed under immunogold labeling and immunogold with silver intensification to detect cell and axonal profiles (Figures 6 and 7) within the proximal regions of the bridge region (levels corresponding to A5–A6 in Figure 1). Immunogold labeling using gold particles of 10 nm in diameter showed immunolabeling details at high magnification. Using the silver enhancement procedure to enlarge gold particles it was possible to detect labeling at lower magnification of different cells and neuropilar elements. The survey was made scanning entire thin cross-sections of the bridge region under the electron microscope at high magnification (15000–40000 fold magnification for immunogold; 2000–5000 fold magnification after silver enhancement), and this procedure allowed the detection of few sparse labeled cells and axonal profiles (Figure 6A–C). Immunogold-labeled cytoplasmic regions of likely nerve cells or small axons were labeled but the limited ultrastructural preservation did not allow the safe detection of growth cones storing numerous synaptic vesicles and neuro-filaments or neurotubules (Figure 6A–C). Control sections, however, did not present any gold labeling (Figure 6D).
Figure 8｜ Immunofluorescence for NF200 in cross sections of the bridge at 19, 22, 34 days and 3 months post-injury (levels A5–A7 in Figure 1).
Figure 9｜Blue nuclear fluorescence (DAPI) and red immunofluorescence (TRITC) for NF200 observed in the bridge at 11, 22, 34 days post-injury (levels A5–A6 in Figure 1).
The immunolabeling of cross-sectioned bridge regions close to the proximal stump showed numerous intensely labeled axons in the external areas of the bridge, occupied by white matter at 19–22 days post-amputation (Figures 1A5 and 8A–C). Blood vessels exhibited a non-specific autofluorescence, yellowish for FITC and pinkish for TRITC (Figure 8A). Labeled axons were also noted around the dilated ependymal ampullae in cross sections of the bridge at 34 days post-injury, and some axons or collateral sprouting, were also noted among ependymal cells (Figure 8D). The number of immunolabeled axons or axonal sprouting increased dramatically in the bridge of lizards at 3 months after injury, in the white matter of the bridge and few were also observed around the still dilated ependymal ampullae (Figure 8E and F). This was confirmed by double labeling staining that evidenced the higher density of cells present in the bridge at 11–22 days post-injury and the sparse but numerous axons present in peripheral and peri-ependymal regions of the bridge (Figure 9A and B). Although no quantification was done, the labeled axons appeared to increase at 34 days and 3 months-post-injury (Figure 9C). Finally, no labeling for NF200 was seen in control sections of the bridge (Figure 9D).