Iron mediates endothelial cell damage and blood-brain barrier opening in the hippocampus after transient forebrain ischemia in rats

Abstract

Blood cells are transported into the brain and are thought to participate in neurodegenerative processes following hypoxic ischemic injury. We examined the possibility that transient forebrain ischemia (TFI) causes the blood-brain barrier (BBB) to become permeable to blood cells, possibly via dysfunction and degeneration of endothelial cells in rats. Extravasation of Evans blue and immunoglobulin G (IgG) was observed in the hippocampal CA1-2 areas within 8 h after TFI, and peaked at 48 h. This extravasation was accompanied by loss of tight junction proteins, occludin, and zonula occludens-1, and degeneration of endothelial cells in the CA1-2 areas. Iron overload and mitochondrial free radical production were evident in the microvessel endothelium of the hippocampus before endothelial cell damage occurred. Administration of deferoxamine (DFO), an iron chelator, or Neu2000, an antioxidant, blocked free radical production and endothelial cell degeneration. Our findings suggest that iron overload and iron-mediated free radical production cause loss of tight junction proteins and degeneration of endothelial cells, opening of the BBB after TFI.

Figure 1

Temporal and spatial patterns of BBB opening in the hippocampus following TFI. (A, B) Western blot analysis of IgG showing increase in the light and heavy chains of IgG after sham operation (Ctrl) or TFI. Levels of IgG were measured at indicated points of time after TFI, normalized to those of β-actin, and compared to control (n = 4-5 rats per group). (C) Fluorescent photomicrographs showing EBD-albumin extravasation and cerebral endothelium stained with EBA, in the hippocampal formation, 48 h after Ctrl or TFI. Note the dramatic increase in EBD extravasation in the endothelium (yellow), primarily in the alveus (Alv), stratum oriens (SO), and stratum pyramidale (SP) of the CA1-2 areas. Abbreviations: DG, dentate gyrus; H, hilus, SR, stratum radiatum. Scale bar: 1.0 mm.

Figure 2

Loss of occludin and ZO-1 and degeneration of CECs in the hippocampus after TFI. (A) Western blot analysis of occludin and ZO-1. Levels of occludin and ZO-1 were measured 8, 24, and 48 h after TFI, normalized to those of β-actin, and compared to control (n = 3-4 rats per group). ^*, P < 0.05 compared to control. (B) Fluorescence photomicrographs of the hippocampal CA1 area immunolabeled with occludin 48 h after control (Ctrl) or TFI. Scale bar, 50 µm. (C) Brightfield photomicrographs of adjacent hippocampal CA1 sections showing DNA fragmentation after TUNEL staining (top panel) and endothelial cells after immunolabeling with EBA (bottom panel) 48 h after Ctrl or TFI. Arrowheads show degeneration of endothelial cells. Abbreviations: Alv, alveus; Endo, endothelium; SO, stratum oriens. Scale bar, 100 µm.

Figure 3

Iron overload and free radical production in the CECs after TFI. (A) Brightfield photomicrographs of the hippocampal CA1 area after Perls iron staining following sham operation (Ctrl) or 0.5, 2, and 8 h following TFI. Scale bar, 50 µm. (B) Brightfield and fluorescence photomicrographs of adjacent hippocampal CA1 sections after Perls staining (top panel) or immunolabeling with EBA (bottom panel) 0.5 h after TFI. Arrowheads indicate iron overload in endothelial cells. Scale bar, 50 µm. (C) Superimposed images of fluorescence photomicrographs of hippocampal sections labeled with EBA and MitoTracker Red CM-H2XROs (MT, red), a mitochondrial free radical probe, 2 h after Ctrl or TFI, alone or with administration of DFO (200 mg/kg, s.c.) or Neu2000 (50 mg/kg; i.p.) immediately after reperfusion. Abbreviations: Endo, endothelium; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar, 50 µm.

Figure 4

DFO and Neu2000 protect cerebral endothelial cells against TFI. (A) Western blot analysis of IgG and TJ proteins 48 h after Ctrl or TFI, alone or with administration of DFO (200 mg/kg, s.c.) or Neu2000 (50 mg/kg; i.p.) immediately after reperfusion (n = 3-4 rats per group). ^*P < 0.05 compared to Ctrl; ^#P < 0.05 compared to TFI. (B) Fluorescent and brightfield photomicrographs of hippocampal CA1 sections labeled with EBD-albumin (red, top panel) or TUNEL and methyl green, a chromatin staining dye (bottom panel) 48 h after Ctrl or TFI, alone or with administration of DFO (200 mg/kg, s.c.) or Neu2000 (50 mg/kg; i.p.) immediately after reperfusion. Arrowheads marked the endothelium and CECs of CA1-2 areas. Abbreviations: Alv, alveus; Endo, endothelium; SO, stratum oriens; SP, stratum pyramidale. Scale bar, 50 µm.