Impact of very old age on the expression of cervical spinal cord cell markers in rats

Abstract

Aging is a process associated with both anatomical changes and loss of expression of some cell markers. Intermediate filaments are known to impart mechanical stability to cells and tissues. Some of them are present in different cell populations of the central nervous system. In order to explore the impact of extreme age we immunohistochemically characterized the changes in intermediate filaments and other cellular markers present in cells populating the gray matter cervical spinal cord of very old rats (28 months) taking young (5 months) counterparts as a reference. The spinal cord weight of the senile animals (12.6+/-1.1 g) was significantly higher (P<0.001) than that of the young animals (8.4+/-1.1 g). Spinal cord length also increased significantly (P<0.05) with age (7.9+/-0.3 cm vs. 8.28+/-0.1 cm for young and senile, respectively). An increase in both neurofilament staining area and density was observed in senile rats in comparison to young animals. A significant (P<0.05) age-related increment in the mean area of the cervical segments was observed. Vimentin expression in the ependymal zone decreased in area and intensity during aging. Our data show that there are some significant changes in the morphological and histochemical patterns of the cervical spinal cord in senile rats. However, they do not necessarily represent a pathologic situation and may rather reflect plastic reorganization.

Fig. 1. Anatomical changes of different cervical segments during aging

An increase in the whole size of each cervical segment was observed during aging. C5 segments of young animals (A) (n = 5) showed a round shaped structure with a round central canal while senile rats (B) (n = 5) showed a more elongated section shape and central canal. An evident change in the gray matter ventral horn shape is also observed during aging. Such differences were also observed throughout the eight cervical segments of both analysed groups (C). In all the segments differences in whole area were significant. A significant increase in the white matter as well as in the gray matter was observed in senile rats (E) in comparison with young animals (D). Nevertheless, that increase was more pronounced in the gray matter as judge by the significant reduction in the ratio between both substances (F). *Significant difference. Bar = 1 mm.

Fig. 2. NF expression during aging

(A) and (B) Show a low magnification view of a hemi C6 segment of a young (A) and a senile (B) rats. It can be seen that NF are distributed throughout the laminas except at the level of laminas I and II. At higher magnification it can be seen that although the MOD of each filament is quite similar either in young rats (C) as well as in senile animal (D) the staining intensity (NF density) of the latter is increased. Differences were statistically significant when we compared the NF density as well as the percentage of NF in relation to the gray matter area (E). (F) A fluorescence staining of NF in a senile C5 segment ventral horn where in the graymatter are seen as thin filaments an in the white matter are points indicating a cross-section of axons. Bar for (A, B, F) = 0.2 mm. Bar for (C, D) = 50 µm.

Fig. 3. GFAP expression during aging

There was a clear increase in the MOD of astrocyte immunostaining for GFAP in young (A) as compared to senile animals (B). No ependymal cells area stained. Graph (C) shows that the GFAP area increased significantly only in C5 and that there was a reduction in the percentage of GFAP stained area in relation to the entire gray matter with age. *Significant difference. Bar for (A, B) = 50 µm.

Fig. 4. Vimentin expression during aging

The expression of vimentin was mainly localized in the central canal, although it was also observed in some glial cells and surrounding the blood vessels either in young (A) and senile (C) rats. It can be seen that the density of staining and the MOD decrease in the latter. These differences were statistically significant (E). Fluorescence images at both ages (B, D) show the same pattern as with IHC for the central canal. Bar for (A–D) = 25 µm.

Fig. 5

S100 expression during aging. Panels (A) and (B) show the ependymal region and lamina X of young and senile rats, respectively, immunostained for the S100 marker. In both cases it can be seen that glial cells of the gray and white matter are stained. Also, in both cases some ependymal cells are stained as well. Panel (A) also shows a neuron and its stained nucleus (encircled cell). This pattern was also observed in some motoneurons both in young (C, D) and senile (F, G) rats. Some neurons of the sensory ganglia are also stained (E). The number of S100+ cells varied in the different segments (H) showing significant differences (*) at C2 and C8. In neither case differences in the MOD were significant (I). Bar for (A–G) = 50 µm.