Delayed expression of osteopontin after focal stroke in the rat

Abstract

Focal brain ischemia induces inflammation, extracellular matrix remodeling, gliosis, and neovascularization. Osteopontin (OPN) is a secreted glycoprotein that has been implicated in vascular injury by promoting cell adhesion, migration, and chemotaxis. To investigate the possible involvement of OPN in brain matrix remodeling after focal stroke, we examined the expression of OPN in ischemic cortex after permanent or temporary occlusion of the middle cerebral artery (MCAO) of the rat. OPN mRNA and protein levels in nonischemic cortex were not detected consistently, although significant induction of OPN was observed in the ischemic cortex. OPN mRNA increased 3.5-fold at 12 hr and reached peak levels 5 d (49.5-fold; p < 0.001) after permanent MCAO. The profile of OPN mRNA induction after transient MCAO (160 min) with reperfusion was essentially the same as that of permanent MCAO. In situ hybridization and immunohistochemical studies demonstrated strong induction of OPN in the ischemic cortex, which was localized primarily in a subset of ED-1-positive macrophages that accumulated in the ischemic zone. Moreover, OPN immunoreactivity was detected in the matrix of ischemic brain, suggesting a functional role of the newly deposited matrix protein in cell-matrix interactions and remodeling. Indeed, using a modified Boyden chamber, we demonstrated a dose-dependent chemotactic activity of OPN in C6 astroglia cells and normal human astrocytes. Taken together, these data suggest that the upregulation of OPN after focal brain ischemia may play a role in cellular (glia, macrophage) migration/activation and matrix remodeling that provides for new matrix-cell interaction.

Fig. 1.

Northern blot analysis of OPN mRNA expression in rat ischemic cortex after permanent MCAO. A, Represent Northern blot for OPN and rpL32 cDNA probes to the samples from spontaneously hypertensive rats (SHR) after permanent MCAO. Total cellular RNA (40 μg/lane) was resolved by electrophoresis, transferred to a nylon membrane, and hybridized to the indicated cDNA probe. Ipsilateral and contralateral cortex samples (denoted by +) from individual rats of sham surgery (S; 5 d) or after 1, 3, 6, 12, and 24 hr and 2, 5, 10, and 15 d of permanent MCAO are depicted. B, Quantitative Northern blot data (n = 4) for OPN mRNA expression after focal stroke. The data were generated by PhosphorImager analysis and were displayed graphically after being normalized with rpL32 mRNA signals. *p < 0.05; ***p < 0.001, as compared with sham-operated animals.

Fig. 2.

Northern analysis of OPN mRNA expression in rat ischemic cortex after temporary MCAO with reperfusion. This figure is illustrated as described in the Figure 1 legend except that temporary occlusion (160 min) of the middle cerebral artery was used. The indicated time points refer to the time of reperfusion. The hybridization was performed in the presence of OPN and rpL32 probes simultaneously.

Fig. 3.

Northern blot analysis of OPN mRNA expression in SHR and WKY at 12 hr and 5 d after permanent MCAO.A, Total cellular RNA was isolated from the ischemic (Ipsilateral) and nonischemic (Contralateral) cortex of hypertensive (SHR) and normotensive (WKY) rats at 12 hr and 5 d after permanent MCAO. This representative Northern blot was performed by using an OPN cDNA probe, as described in Materials and Methods and in the legend to Figure 1A. B, Quantitative analysis of OPN mRNA expression in hypertensive (SHR) and normotensive (WKY) rats after permanent MCAO, as described in the legend to Figure 1B. The data are presented as the mean values of four animals (n= 4) from each strain after the samples loaded in each lane with rpL32 were normalized. ***p < 0.001, as compared with WKY at the same time point.

Fig. 4.

In situ localization of OPN mRNA expression in the ischemic cortex. Shown is film autoradiography of OPN mRNA expression 5 d after permanent MCAO (A). OPN mRNA-expressing cells are located abundantly throughout the entire infarcted region. The ED-1 antibody and OPN mRNA colocalized in many, but not all, of the macrophages in the infarct (B). Morphologically, the ED-1-positive and OPN-positive cells (B,inset) have an ameboid appearance indicative of their activated phagocytic state. Scale bars: in B, 20 μm; in inset, 10 μm.

Fig. 5.

Histological and immunohistochemical staining of OPN expression in ischemic cortex. A, Hematoxylin and eosin-stained (H&E) section of normal cerebral cortex (sham-operated animals at day 5). B, H&E-stained focal ischemic zone at 24 hr after permanent MCAO. C, Focal ischemic zone at 5 d (H&E). D, Immunohistochemistry showing numerous OPN-positive macrophages in this 5 d lesion. E, Focal ischemic cortex at 15 d (H&E). F, Immunohistochemistry for OPN in focal ischemic lesion at 15 d.

Fig. 6.

Immunohistochemical detection of temporal OPN expression in ischemic cortex after permanent MCAO. A, Immunostaining for GFAP at the boundary of the ischemic lesion 5 d after permanent MCAO. Note the strong immunostaining of GFAP in astrocytes of the nonischemic lesion. B, Immunostaining for GFAP 15 d after MCAO. The GFAP expression pattern is similar to day 5, but the signal is stronger. C, Immunostaining for OPN 5 d after MCAO. Note the diffuse cellular staining and the large number of positive-staining cells in the middle of the lesion. See the inset for a detailed view of OPN in the cells (arrow) and extracellular matrix (arrowhead). D, Immunostaining for OPN in the ischemic lesion 15 d after MCAO. Most of the cells are negative for OPN. E, Ischemic lesion 5 d after MCAO, filled with a large number of ED-1-positive cells that indicate macrophage/monocyte lineage. F, Ischemic 15 d lesion with ED-1-positive cells. Note the organization of this lesion.

Fig. 7.

Effects of OPN on C6 astroglia (A) and normal human astrocyte (B) cell migration. The migration assay was performed in the Transwell cell culture chamber, as described in Materials and Methods. The indicated concentration of OPN was coated on the lower surface of the membrane. The cell suspension (containing 6 × 10⁵ C6 cells or 4 × 10⁴ human astrocytes in a vol of 0.2 μl) was added in the upper compartment and incubated for 24 hr. The relative number of migrated cells (on the lower surface of the filters) was measured by optical density at 640 nm for C6 cells or determined microscopically by counting four high-power fields per filter for human astrocytes. In the concentration range of 0–0.24 μm OPN, ∼0.5–4% of C6 cells or 0.7–3% of human astrocytes were migrated. The data are the mean ± SE of three experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001, as compared with the control.