Table 1.
Primers used for quantitative real-time PCR.
Fig 1.
Astrocytes appear earlier in the developing spinal cord compared to brain and express higher levels of GFAP in adulthood.
Photomicrographs show immunofluorescence for GFAP, ALDH1L1 and DAPI in (A) the corpus callosum of the brain, and (B) the dorsal column white matter (WM) of the spinal cord from P0 to adulthood (P45). Histograms (C and D) show the percent area stained for GFAP or ALDH1L1 in white matter of the brain (corpus callosum (CC), or in white matter of the spinal cord (dorsal column). Histograms (F and G) show the percent area stained for GFAP or ALDH1L1 in the gray matter (GM) of the brain (cingulate cortex) or gray matter of the spinal cord (ventral horn). (E) and (H) show the ratio of GFAP to ALDH1L1 immunoreactivity in each case. ALDH1L1-immunopositive astrocytes appear earlier in the spinal cord compared to brain and express higher levels of GFAP in adulthood. (*P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, NK, scale bar = 50 μm).
Fig 2.
Astrocytes are more abundant in the spinal cord relative to brain in the early postnatal period and express higher levels of GFAP, STAT3 and IL-6 from P7 to adulthood.
Western blots (A) and associated histograms (B to E) illustrate that (C) ALDH1L1 protein, a marker of astrocytes appears earlier developmentally in the spinal cord relative to brain with higher levels at birth and at P7. In addition, GFAP protein levels (B) and the ratio of GFAP/ALDH1L1 (D) are higher in the spinal cord from P7 through P45. (E) STAT3 levels are also elevated in the spinal cord relative to brain on P21 and P45. P-STAT3 was not detected in the brain or spinal cord under the conditions of this study (blot not shown). ROD readings in each case were normalized to actin on the same membrane to control for loading. (F) GFAP RNA transcription increased after P0 in both the brain and spinal cord, although increases in the spinal cord reached significantly higher levels compared to brain at all time points examined. (G) ALDH1L1 RNA was higher in the spinal cord compared to brain at P0 through P45. (H) The ratio of GFAP/ALDH1L1 was increased during the postnatal period in both brain and spinal cord although such increases were significantly higher in the spinal cord compared to brain at all postnatal ages. (I) STAT3 and (J) IL-6 RNA were higher in the spinal cord compared to brain at birth on P21 and in adulthood. (*P < 0.05, **P ≤ 0.01, ***P ≤ 0.001 NK).
Fig 3.
GFAP and IL-6 are elevated in cultures of spinal cord compared to cortical astrocytes.
(A) Photomicrographs show GFAP and ALDH1L1 co-immunofluorescence in primary cultures of astrocytes purified from brain or spinal cord. (B-F) Histograms show counts of GFAP+ or ALDH1+ cells expressed as a ratio to the total number of DAPI+ cells in the same microscopic fields. The number of GFAP+ and GFAP/ALDH1L1+ cell was elevated in spinal cord astrocyte cultures compared to those derived from brain. (G, H) Analysis of protein homogenates from brain or spinal cord astrocyte cultures also demonstrated higher levels of GFAP expression, but no differences in ALDH1L1 (I), GFAP/ALDH1L1 ratios (J) or pSTAT3/STAT3 ratios. (L) IL-6 was higher in the supernatants of spinal cord compared to brain astrocyte cultures (P ≤ 0.001), while no differences in STAT3 were observed (K). (*P < 0.05, ***P ≤ 0.001, Students t-test). Scale bar = 50μm.
Fig 4.
Increases in GFAP after demyelinating injury are greater in the spinal cord compared to brain.
Photomicrographs show immunoreactivity for GFAP or ALDH1L1 in (A) the corpus callosum, or (B) the dorsal column white matter of adult mice at base line, and at 14 d after microinjection of the demyelinating agent lysolecithin. Histograms show the percent area of GFAP immunofluorescence, and expression of GFAP/ALDH1L1+ astrocyte, was significantly greater in the spinal cord compared to the corpus callosum 14d post-lysolecithin lesion. (*P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, Students t-test), Scale bar = 50μm.