Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats

Abstract

Objective: Accumulated evidence suggests that the primary cause of poor outcome after subarachnoid hemorrhage is not only cerebral arterial narrowing but also early brain injury. Our objective was to determine the effect of recombinant osteopontin, a pleiotropic extracellular matrix glycoprotein, on early brain injury after subarachnoid hemorrhage in rats.

Design: Controlled in vivo laboratory study.

Setting: Animal research laboratory.

Subjects: One hundred seventy-seven male adult Sprague-Dawley rats weighing 300 to 370 g.

Interventions: The endovascular perforation model of subarachnoid hemorrhage was produced. Subarachnoid hemorrhage or sham-operated rats were treated with an equal volume (1 microL) of pre-subarachnoid hemorrhage intracerebroventricular administration of two dosages (0.02 and 0.1 microg) of recombinant osteopontin, albumin, or vehicle. Body weight, neurologic scores, brain edema, and blood-brain barrier disruption were evaluated, and Western blot analyses were performed to determine the effect of recombinant osteopontin on matrix metalloproteinase-9, substrates of matrix metalloproteinase-9 (zona occludens-1, laminin), tissue inhibitor of matrix metalloproteinase-1, inflammation (interleukin-1beta), and nuclear factor-kappaB signaling pathways.

Measurements and main results: Treatment with recombinant osteopontin prevented a significant loss in body weight, neurologic impairment, brain edema, and blood-brain barrier disruption after subarachnoid hemorrhage. These effects were associated with the deactivation of nuclear factor-kappaB activity, inhibition of matrix metalloproteinase-9 induction, the maintenance of tissue inhibitor of matrix metalloproteinase-1, the consequent preservation of the cerebral microvessel basal lamina protein laminin, and the tight junction protein zona occludens-1.

Conclusions: These results demonstrate that recombinant osteopontin treatment is effective for early brain injury after subarachnoid hemorrhage.

Figure 1

Effects of recombinant osteopontin (OPN) treatment on body weight (A; mean±SD) and neurological score (B; median±25th–75th percentiles) after subarachnoid hemorrhage (SAH). Sham, Sham-operated rats treated with saline or OPN, n=40 (saline, 23; OPN, 17; pre-SAH, 24 hrs post-SAH) or 6 (saline, 6; 48, 72 hrs post-SAH); SAH, SAH rats treated with vehicle or bovine serum albumin (BSA), n=29 (saline, 23; BSA, 6; pre-SAH, 24 hrs post-SAH) or 6 (saline, 6; 48, 72 hrs post-SAH); SAH-OPN0.02, SAH rats treated with 0.02μg of OPN, n=23 (pre-SAH, 24 hrs post-SAH) or 6 (48, 72 hrs post-SAH); SAH-OPN0.1, SAH rats treated with 0.1μg of OPN, n=23 (pre-SAH, 24 hrs post-SAH) or 6 (48, 72 hrs post-SAH); *p<0.0005, **p<0.01, ^#p<0.05 vs. SAH group; ^ap<0.01, ^bp<0.05 vs. sham group.

Figure 2

Effects of recombinant osteopontin (OPN) treatment on Evans blue dye extravasation in the brain after subarachnoid hemorrhage (SAH). B 0.1, O 0.02, O 0.1, S, rats treated with bovine serum albumin (0.1μg), OPN (0.02μg), OPN (0.1μg) or saline, respectively; n=6 rats per group; *p<0.0001, ^#p<0.001, **p<0.005, ^†p<0.05 vs. sham-operated rats treated with saline or OPN at 24 hours post-surgery; ^§p<0.025, ^¶p<0.05 vs. sham-operated rats treated with saline at 72 hours post-surgery; error bar, SD.

Figure 3

Effects of recombinant osteopontin (OPN) treatment on brain water content at 24 hours after subarachnoid hemorrhage (SAH). N=5 rats per group; error bar, SD.

Figure 4

Effects of recombinant osteopontin (OPN) treatment on matrix metalloproteinase (MMP)-9 induction and tissue inhibitor of MMP (TIMP)-1 reduction in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). Western blots for MMP-9 (A), TIMP-1 (B), laminin (C) and zona occludens (ZO)-1 (D). Expression levels of each protein in Western blot are expressed as a ratio of β-actin levels for normalization. N=6 rats per group; error bar, SD.

Figure 5

Western blot for cleaved interleukin (IL)-1β in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). The expression levels are expressed as a ratio of β-actin levels for normalization. N=6 rats per group; error bar, SD.

Figure 6

Effects of recombinant osteopontin (OPN) treatment on nuclear factor (NF)-κB activation in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). Western blots for phosphorylated inhibitor of NF-kB (IκB) kinase (IKK) α/β (p-IKKα/β; A), phosphorylated IκB-α (p-IκB-α; B) and IκB-α (C). Expression levels of each protein in Western blot are expressed as a ratio of β-actin levels for normalization. N=6 rats per group; error bar, SD.

Figure 7

Effects of recombinant osteopontin (OPN) treatment on endogenous OPN induction in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). The expression levels are expressed as a ratio of β-actin levels for normalization. N=6 rats per group; error bar, SD.