Dilated Blood Vessels and Altered Nestin Expression in Endothelial and Ependymal Cells
We first analyzed the uninjured nervous system of the mutant mice. Many blood vessels appeared dilated in the brain and spinal cord of GFAP−/−vim−/− mice (Fig. ). The lumina of these structures were lined with von Willebrand factor–immunoreactive endothelial cells indicating that they were true blood vessels rather than, for example, cavities formed by degeneration (data not shown). Measurements of blood vessel diameters in these mice revealed an almost threefold increase in the number of blood vessels with a diameter >15 μm compared to wild-type mice (P < 0.0005; Fig. A). We frequently noticed that there was an unusually deep indentation in the tissue of the dorsal spinal cord in many of the mutant mice. In wild-type mice, there is often an invagination below the dorsal spinal vein, but measurements of the depth of the invagination revealed a statistically significant increase in the depth compared to wild-type mice in GFAP−/− (P < 0.005), vimentin−/− (P < 0.0001), and GFAP−/−vim−/− mice (P < 0.0001; Figs. B and 6, A–D).
Figure 1 Blood vessel dilation. Hematoxylin and eosin stained coronal brain sections from wild-type (A) and GFAP−/−vim−/− (B) mice show an increased number of large diameter blood vessels in GFAP−/−vim−/− (more ...)
Figure 2 Quantification of blood vessel dilation and the indentation below the dorsal spinal vein. (A) Average number of blood vessels with a diameter greater than 15 μm in spinal cord sections of mice of different genotypes. (B) Depth of the indentation (more ...)
Endothelial and ependymal cells in the adult CNS express both nestin (Dahlstrand et al., 1992
; Frisén et al., 1995
; Fig. A) and vimentin (Franke et al., 1979
), but not GFAP in wild-type mice. Nestin-immunoreactivity (IR) in these cells appeared normal in GFAP−/− mice (data not shown). However, in vimentin−/− mice, nestin-IR was reduced to very low levels in both endothelial and ependymal cells (Fig. B). Moreover, nestin-IR did not display a filamentous pattern but was spread diffusely throughout the cytoplasm in these cells (Fig. B). The same nestin-IR pattern was seen in GFAP−/−vim−/− mice (data not shown).
Figure 3 Altered nestin-IR in the absence of vimentin. Immunofluorescence localization of nestin in the uninjured spinal cord. (A) In a wild-type mouse, strong nestin-IR is seen in ependymal cells and endothelial cells. (B) Nestin-IR is weak and diffuse in (more ...)
Defective Glial Scar Formation after Spinal Cord Injury in GFAP−/−vim−/− Mice
We next analyzed scar formation in wild-type and mutant mice in response to an incision in the spinal cord dorsal funiculus. The mice were allowed to survive 2 d or 2 wk after the injury, and tissue sections from the site of the injury and from a spinal cord segment 10 mm rostral to the lesion were analyzed. There were no consistent differences in the histology of the scar tissue between wild-type, GFAP−/−, or vimentin−/− mice revealed by HE staining (Fig. ). However, the scar tissue was much less dense in the GFAP−/−vim−/− mice, and the scar tissue was interrupted by numerous fissures most often running in a dorso-ventral orientation (Fig. , H and L). These fissures were filled with blood, tissue fluid or debris (Fig. L). Moreover, in the GFAP−/−vim−/− animals there was a large number of red blood cells within the scar tissue, both 2 d and 2 wk after the injury, indicating more pronounced bleeding and/or defective clearance of blood from the injury site (Fig. , H and L).
Figure 4 Structure of the uninjured and injured spinal cord in wild-type and mutant mice. The micrographs show hematoxylin and eosin stained transverse sections of upper thoracic spinal cord from uninjured animals and from animals in which the dorsal funiculus (more ...)
Nestin Expression in the Injured Spinal Cord
Nestin expression is rapidly induced in astrocytes after CNS injury and serves as a sensitive marker for reactive astrocytes (Clarke et al., 1994
; Frisén et al., 1995
; Lin et al., 1995
). Nestin-IR was induced in response to spinal cord injury in mice of all genotypes (Fig. ). However, nestin-IR was more restricted in vimentin−/− or GFAP−/−vim−/− mice compared to wild-type or GFAP−/− mice (Fig. ). Whereas 2 d after the injury nestin-IR cells were seen at the site of the lesion and in the surrounding gray matter in wild-type (Frisén et al., 1995
) or GFAP−/− mice, nestin-IR was less pronounced in the gray matter in vimentin−/− or GFAP−/−vim−/− mice (Fig. ). Furthermore, 2 wk after the lesion the levels of nestin-IR in the scar were lower in vimentin−/− or GFAP−/−vim−/− mice compared to wild-type or GFAP−/− mice (Fig. ). Whereas the scar tissue appeared dense after 2 wk in wild-type, GFAP−/− or vimentin−/− mice, the labeled cells were more sparse in GFAP−/−vim−/− mice and nestin-IR was diffusely spread throughout the cytoplasm of the cells (Fig. ). Nestin-IR was induced within 2 d after the injury in segments rostral to the lesion, in the degenerating axonal tract, to a similar extent in mice of all genotypes (Fig. ). However, 2 wk after the injury the level of nestin-IR in the degenerating tract had decreased substantially in the vimentin−/− or GFAP−/−vim−/− mice, whereas it remained strong in wild-type or GFAP−/− mice (Fig. ).
Figure 5 Nestin-IR at a spinal cord lesion in wild-type and mutant mice. Nestin-IR in the uninjured spinal cord and 2 d or 2 wk after a dorsal funiculus incision. Nestin-IR is induced at the injury in mice of all genotypes, but is weaker and more diffuse in vimentin−/− (more ...)
Figure 6 Nestin-IR in degenerating axonal tracts in wild-type and mutant mice. Nestin-IR in sections taken 10 mm rostral to a dorsal funiculus incision 2 d or 2 wk after the injury. Nestin-IR is induced in astrocytes in the dorsal and central part of the dorsal (more ...)
To determine whether the reduced nestin-IR in the injured spinal cord was caused by reduced transcription of the nestin gene in vimentin−/− and GFAP−/−vim−/− mice, we performed in situ hybridization with nestin antisense riboprobes in spinal cord sections from animals that underwent a dorsal funiculus incision 4 d before. Nestin mRNA was detected in mice of all genotypes, and no reduction in nestin mRNA levels could be found in vimentin−/− and GFAP−/−vim−/− mice (Fig. ). Scattered cells in the dorsal funiculus expressed nestin mRNA and their distribution was highly reminiscent of the nestin-IR pattern. No specific hybridization was seen with the sense probe (Fig. , E and F).
Figure 7 Nestin mRNA expression in the injured spinal cord. A digoxigenin labeled riboprobe was used to localize nestin mRNA in the spinal cord 4 d after an incision in the dorsal funiculus. Nestin mRNA is expressed in scattered cells at the injury site in (more ...)
Responses to Brain Injury
Glial scar formation was also analyzed in the brain. A cortical stab wound was done with a fine needle, resulting in a much more restricted injury than the spinal cord lesion. Interestingly, 3 out of 11 GFAP−/−vim−/− mice died shortly after the injury and the necropsy showed extensive intracranial bleeding that was interpreted as the cause of death (data not shown). In the group of GFAP−/−vim−/− mice that were killed 3 d after the injury, 3 out of 5 mice showed extensive bleeding at the site of injury (Fig. D). Bleeding of comparable magnitude was not seen in mice of other genotypes. In all wild-type, GFAP−/− and vimentin−/− mice, as well as in the GFAP−/−vim−/− mice that survived the operation, the discrete injury caused by the needle did not cause any apparent clinical symptoms and was sealed within 3 wk (Fig. ). Nestin-IR was detected around the cortical lesion 3 d after the injury in mice of all genotypes (Fig. ). However, whereas nestin-IR was comparably strong and revealed distinct reactive astrocytes in both wild-type and GFAP−/− mice, it was weaker and diffuse in vimentin−/− and GFAP−/−vim−/− mice (Fig. , G and H). Nestin-IR was reduced to undetectable levels after 3 wk in mice of all genotypes (data not shown).
Figure 8 Response to cortical injury in wild-type and mutant mice. Hematoxylin and erythrosin staining and nestin-IR in adjacent sections of frontal cortex from mice 3 d or 3 wk after a fine needle cortical injury. In most of GFAP−/−vim−/− (more ...)
Ultrastructural Analysis of Scar Formation
To further characterize scar formation in the mutant mice, we analyzed the zone immediately adjacent to the central necrotic area of cortical injury by electron microscopy. This area was similar in wild-type, GFAP−/−, or vimentin−/− mice, but differed in GFAP−/−vim−/− mice. The difference was most apparent in the border zone between the severely disarranged tissue close to the wound and the more distant, compact and normal looking brain tissue. Here, the GFAP−/−vim−/− mice exhibited fragmented tissue with a high accumulation of extracellular debris. This debris was diffuse, finely granular or filamentous of moderate electron density (Fig. d). This was in contrast to mice of the other genotypes (Fig. , a–c) in which the brain tissue in the corresponding area showed easily identifiable components (e.g., myelinated and unmyelinated axons, synaptosome-like profiles), narrow extracellular spaces and virtually no extracellular debris.
Figure 9 Ultrastructural analysis of a cortical stab wound. Electron micrographs of the frontal cerebral cortex 3 d after the injury. The pictures show the border zone between the spongy tissue of the wounded area and the surrounding compact cortical tissue. (more ...)
Cell Proliferation after the Cortical Injury
To evaluate cell proliferation in the area affected by the cortical injury we have compared BrdU incorporation within a 50-h time window after the injury in wild-type, GFAP−/−, vimentin−/−, and GFAP−/−vim−/− mice. Using confocal microscopy we have counted BrdU-labeled cells within the volume of 5 × 106 μm3 that included the injury area in the middle and was reconstructed from superimposed confocal images. The number of BrdU-labeled cells within this volume ranged from 64 to 87 and no statistically significant differences were found between wild-type and mutant mice. To determine the proportion of astrocytes among the BrdU-labeled cells, S-100 positive cells were counted within the same volume. The number of S-100 positive cells ranged from 65 to 83 and no statistically significant differences were found between wild-type and mutant mice. Fig. provides a comparison between individual (Fig. , a–d) and combined (Fig. , e and f) BrdU and S-100 immunostaining of the cortical injury area in wild-type and GFAP−/−vim−/− mice. Both contain comparable numbers of BrdU positive, S-100 positive and double positive cells (some of these are indicated by arrows). Thus, the cortical injury in wild-type and mice deficient for GFAP and/or vimentin triggers a comparable induction of cell division.
Figure 10 Cell proliferation in the injury area in wild-type and mutant mice. Confocal images of BrdU labeling (A and B) and S-100 IR (C and D) in the area of brain cortical injury. No difference is apparent between wild-type and GFAP−/−vim−/− (more ...)