Sulfonylurea receptor 1 expression in human cerebral infarcts

Abstract

In animal models of stroke, sulfonylurea receptor 1 (Sur1), a member of the adenosine triphosphate binding cassette transporter gene family, is transcriptionally upregulated in neural and vascular cells in which it plays a leading role in edema formation and necrotic cell death. To date, expression of Sur1 in the brains of humans with cerebral infarcts has not been systematically evaluated. We examined Sur1 expression in postmortem specimens obtained from 13 patients within the first 31 days after focal infarcts, 5 patients with lacunar infarcts, and 6 normal control brains using immunohistochemistry. Elevated immunoreactivity for Sur1 was detected in all cases of focal infarcts, with 3 distinct temporal patterns of expression: 1) neurons and endothelium showed the greatest elevation during the first week, after which levels declined; 2) astrocytes and microglia/macrophages showed progressive increases during the first 31 days; and 3) neutrophils near the infarct showed prominent immunoreactivity that did not change over time. Upregulation of Sur1 was corroborated using in situ hybridization for Abcc8 mRNA. Sulfonylurea receptor 1 immunoreactivity in lacunar infarcts was less prominent and more sporadic than in nonlacunar infarcts. In conjunction with previous studies, these data suggest that Sur1 may be a promising treatment target in patients with acute cerebral infarction.

FIGURE 1

Sulfonylurea receptor 1 (Sur1)–positive controls. (A) A hematoxylin and eosin–stained section of pancreas showing pancreatic islet cells with adjacent fat and serous glands. (B–D) Immunohistochemistry using primary antibodies against Sur1 protein shows labeling of pancreatic islet cells (B), which colocalizes with insulin in β cells (C); merged fluorescent image is shown in (D). (E, F) Immunohistochemistry using primary antibodies against Sur1 shows labeling of dentate granule cells (E) or pyramidal cells (F) of the hippocampus; nuclei are stained with DAPI. In (F), the tissues were double immunolabeled using anti-NeuN antibody, with the merged image shown. Scale bars = (D) 100 μm (for A–D); (E, F) 10 μm. Red/CY3, Sur1; green/FITC, insulin and NeuN; blue, DAPI.

FIGURE 2

Sulfonylurea receptor 1 (Sur1) immunoreactivity is upregulated in neural and vascular cells in acute ischemic infarcts. (A) Hematoxylin and eosin–stained section of an acute ischemic infarct showing scattered reperfusion microhemorrhages. (B, C) By immunohistochemistry with diaminobenzidine chromogen, upregulated Sur1 immunoreactivity is observed in all neural and vascular cells (B), relative to contralateral cortex (C). (D–L) With fluorescent double labeling, greater Sur1 immunolabeling is observed within CD31-positive capillaries (D), myeloperoxidase (MPO)-positive neutrophils (F, H), NeuN-positive neurons (J), and glial fibrillary acidic protein (GFAP)–positive astrocytes (L) in the ischemic tissues (D, F, H, J, L) versus contralateral control tissues (E, G, I, K, M). Merged images of double labeling are shown in the third and fourth columns. Original magnifications = (A–C) 10×; (D–L) and inset and (B) 40×. Scale bars = (B, C) 100 μm; (D, F, H, J, L) 10 μm. Red/CY3, Sur1; green/FITC, PECAM-1 (CD31), MPO, NeuN, and GFAP; blue, DAPI. The figures are from cases 3 and 6.

FIGURE 3

In situ hybridization shows upregulated Abcc8 mRNA in acute ischemic infarcts. (AYH) Tissues from recently infarcted cortex were hybridized with antisense probe directed against Abcc8 and immunolabeled for sulfonylurea receptor 1 (Sur1); greater dual labeling for Abcc8 mRNA and Sur1 protein was identified within endothelial cells in capillaries (A), arterioles (C), and venules (E), and in scattered neutrophils (A, C) and neurons (G) in the ischemic tissues (A, C, E, G) versus contralateral control tissues (B, D, F, H); no counterstain was used. Original magnification = (AYH) 20. Scale bar = 10 Km. These figures are from cases 3 and 6.

FIGURE 4

Sulfonylurea receptor 1 (Sur1) immunoreactivity is upregulated in subacute ischemic infarcts only in inflammatory cells. (A) Hematoxylin and eosinYstained section of subacute ischemic infarct, showing abundant microglia/macrophages with neovascularization. Sur1 protein is upregulated in scattered cells within a subacute infarct (B) but not in the contralateral cortex (C). Fluorescent double labeling of subacute infarcts shows colocalization of Sur1 with myeloperoxidase (MPO)-positive neutrophils (D), CD68-positive microglia/macrophages (E), and glial fibrillary acidic protein (GFAP)Ypositive astrocytes (F). There was essentially no staining in CD31-positive endothelial cells or NeuN-positive neurons (data not shown). Merged images are shown in the right column; nuclei were counterstained with DAPI. Original magnifications = (AYC) 10; (DYF) 40. Scale bar = 10 Km. Red/CY3, Sur1; green/FITC, PECAM-1 (CD31), NeuN, MPO, CD68, and GFAP; blue, DAPI. Figures are from cases 11 and 13a.

FIGURE 5

Semiquantitative analysis of the time course of sulfonylurea receptor 1 (Sur1) immunoreactivity in acute and subacute infarcts. (A) The abundance of Sur1 in NeuN-positive neurons from the ischemic region (red circles) and from contralateral cortex (black x) is plotted against the postmortem interval (PMI) (left panel) and against the postischemic interval (PII), the time between the clinical presentation of stroke and death (right panel); Spearman correlation coefficient (ρ) is given; *** p < 0.001. (B–D) The abundance of Sur1 immunoreactivity in CD31-positive endothelium of capillaries, venules, and arterioles (B); in glial fibrillary acidic protein (GFAP)–positive astrocytes and CD68-positive microglia/macrophages (C); and in myeloperoxidase (MPO)-positive neutrophils (D) from the ischemic region (red circles) and from contralateral cortex (black x) is plotted against the postischemic interval (PII).

FIGURE 6

Oncosis and cell blebbing in sulfonylurea receptor 1 (Sur1)–immunopositive endothelial cells and neurons in acute cerebral infarcts. (A, B) Recently infarcted cortex shows degenerating capillaries with CD31-positive endothelial cells (A) that immunolabel intensely for Sur1 (merged image on the right); vessels in the contralateral control tissues are negative for Sur1 (B). (C, D) Recently infarcted cortex shows degenerating neurons with variable labeling intensity for NeuN (C) and marked immunoreactivity for Sur1; neurons in the contralateral control tissues are negative for Sur1 (D); merged images are shown in the right columns of (A, C) and in (B, D). Note the cytoplasmic swelling with membrane irregularities, that is, features of osmotic necrosis (blebbing), within Sur1-positive endothelial cells and neurons (A, C); nuclei are counterstained with DAPI. Original magnification = (A–D) 100×. Scale bar = 10 μm. Red/CY3, Sur1; green/FITC, PECAM-1 (CD31) and NeuN; blue, DAPI. The figures are from cases 3 and 6.

FIGURE 7

Variable upregulation of sulfonylurea receptor 1 (Sur1) immunoreactivity within lacunar infarcts. (A) Hematoxylin and eosin–stained section of a lacunar infarct showing abundant microglia/macrophages, with neovascularization and evolving gliosis. (B, C) Sur1 protein immunoreactivity is upregulated in several cells in the region of the infarct (B) but is absent in a contralateral control subcortical nucleus (C). (D–H) Fluorescent double labeling of lacunar infarcts shows punctate staining within CD31-positive capillaries (D) and NeuN-positive neurons (E) and marked labeling of inflammatory cells, including myeloperoxidase (MPO)-positive neutrophils, CD68-positive microglia/macrophages, and glial fibrillary acidic protein (GFAP)–positive astrocytes (F–H, respectively); merged images are shown in the right column; nuclei are counterstained with DAPI. Original magnifications = (A–C, H) 10×; (D–G) 40×. Scale bar = 10 μm. Red/CY3, Sur1; green/FITC, PECAM-1/CD31, MPO, NeuN, CD68, and GFAP; blue, DAPI. The figures are from cases 16a and 16b.