Spatiotemporal expression of Dexras1 after spinal cord transection in rats

Abstract

Dexras1, a brain-enriched member of the Ras subfamily of GTPases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI), to specific targets to enhance NO signaling, and is strongly and rapidly induced during treatment with dexamethasone. It is unknown how the central nervous system (CNS) trauma affects the expression of Dexras1. Here we used spinal cord transection (SCT) model to detect expression of Dexras1 at mRNA and protein level in spinal cord homogenates by real-time PCR and Western blot analysis. The results showed that Dexras1 mRNA upregulated at 3 day, 5 day, and 7 day significantly (P < 0.05) that was consistent with the protein level except at 7 day. Immunofluorescence revealed that both neurons and glial cells showed Dexras1 immunoreactivivty (IR) around SCT site, but the proportion is different. Importantly, injury-induced expression of Dexras1 was co-labeled by caspase-3 (apoptotic marker) and Tau-1 (marker for pathological oligodendrocyte). Furthermore, colocalization of Dexras1, carboxy-terminal PSD95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON) and nNOS was observed in neurons and glial cells, supporting the existence of ternary complexes in this model. Thus, the results that the transient high expression of Dexras1 which localized in apoptotic neurons and pathological oligodendrocytes might provide new insight into the secondary response after SCT.

Fig. 1

Results show the change in Dexras1 at mRNA and protein level with real-time PCR and Western blot analysis in rostral and caudal spinal cord homogenate after SCT. All experiments were done at least for thrice. Statistical analyses were made with one-way ANOVA followed by Tukey’s post hoc analysis. (A), Real-time PCR for Dexras1. The results were expressed as means ± SEM relative mRNA of the samples was normalized to that of β₂-M. In the rostral side (a) of 3, 5, and 7 day SCT groups, the levels are significantly different from that of control; In the caudal side (b), the 3 and 5 day SCT groups show the significant changes compared with control. *P < 0.05. (B), Western blot analysis for Dexras1. Representative blots (a) were showed; data were expressed (b) as means ± SEM of three samples per time point. Ratios of densities of Dexras1 to β-actin protein were compared. At 3 and 5 day after SCT the protein level is significantly different from that of control in either rostral or caudal side. *P < 0.05. In (c) the results are shown with and without blocking of the antibody, and the blocking peptide used as control refers to the synthetic peptide

Fig. 2

Immunofluorescence results show the change and the distribution of Dexras1-IR after SCT. (A), The change and the distribution of Dexras1 were observed in representative photomicrographs in transverse sections of the spinal cord from sham-operated group (a) and 5 day injured group (b). Magnifield of images (c–j) for (a) and (b) showed Dexras1-IR in ventral horn (c, g), intermediate zone (d, h), dorsal horn (e, i), and white matter (f, j), respectively, in sham-operated group (c–f) and 5 day injured group (g–j). In (k, l) the results are shown with (l) and without (k) blocking of the antibody, and the blocking peptide used as control refers to the synthetic peptide. Scale bar: (a, b), 200 μm; (c–j), 50 μm; (k, l); and 100 μm. (B), Quantitative results for numbers of Dexras1-IR-positive cells. *P < 0.05 compared to sham by ANOVA followed by Tukey’s post hoc analysis (N = 18 slices from three animals for each group, six slices per animal)

Fig. 3

Coimmunofluorescence analysis of localization of Dexras1 at 5 day after SCT. (A), Colocalization of Dexras1 (a, d, g) with different cellular markers: NeuN (b), CNP (e), and OX-42 (h); Panel (c, f, i) shows the merged graphs. (B), Colocalization of Dexras1 (a, d, g, j) with GFAP in ventral horn (b), intermediate zone (e), dorsal horn (h), and white matter (k), respectively; Panel (c, f, i, l) shows the merged graphs. Scale bar: 50 μm. (C), Quantitative results summarize the proportion of different cellular markers in ventral horn, intermediate zone, dorsal horn, and white matter

Fig. 3

Coimmunofluorescence analysis of localization of Dexras1 at 5 day after SCT. (A), Colocalization of Dexras1 (a, d, g) with different cellular markers: NeuN (b), CNP (e), and OX-42 (h); Panel (c, f, i) shows the merged graphs. (B), Colocalization of Dexras1 (a, d, g, j) with GFAP in ventral horn (b), intermediate zone (e), dorsal horn (h), and white matter (k), respectively; Panel (c, f, i, l) shows the merged graphs. Scale bar: 50 μm. (C), Quantitative results summarize the proportion of different cellular markers in ventral horn, intermediate zone, dorsal horn, and white matter

Fig. 4

The expression of Dexras1 in pathological cells at 5 day following SCT. (A), Identification of apoptotic cell types. Colocalization of caspase-3 (a, d) with NeuN (b), CNP (e). Panel (c, f) shows the merged graphs. (B), Colocalization of Dexras1 (a, d, g) with caspase-3 (b, e) and Tau-1 (h). Scale bar: 50 μm. (C), The number of Dexras1 and caspase-3 positive cells in different part of transverse sections at 5 day after SCT increased significantly compared with sham-operated. Values are expressed as mean ± SEM *P < 0.05 (n = 3)

Fig. 5

Representative photomicrographs that illustrate the colocalization of Dexras1 with CAPON and nNOS in at 5 day after SCT. (A), colocalization of Dexras1 (a, d) with CAPON (d, e) in ventral horn (a–c) and white matter (d–f). (B), colocalization of Dexras1 (a, d) with nNOS (d, e) in ventral horn (a–c) and white matter (d–f). Scale bar: 50 μm