Inhibition of the Sur1-Trpm4 channel reduces neuroinflammation and cognitive impairment in subarachnoid hemorrhage

Abstract

Background and purpose: Subarachnoid hemorrhage (SAH) can leave patients with memory impairments that may not recover fully. Molecular mechanisms are poorly understood, and no treatment is available. The sulfonylurea receptor 1-transient receptor potential melastatin 4 (Sur1-Trpm4) channel plays an important role in acute central nervous system injury. We evaluated upregulation of Sur1-Trpm4 in humans with SAH and, in rat models of SAH, we examined Sur1-Trpm4 upregulation, its role in barrier dysfunction and neuroinflammation, and its consequences on spatial learning.

Methods: We used Förster resonance energy transfer to detect coassociated Sur1 and Trpm4 in human autopsy brains with SAH. We studied rat models of SAH involving filament puncture of the internal carotid artery or injection of blood into the subarachnoid space of the entorhinal cortex. In rats, we used Förster resonance energy transfer and coimmunoprecipitation to detect coassociated Sur1 and Trpm4, we measured immunoglobulin G extravasation and tumor necrosis α overexpression as measures of barrier dysfunction and neuroinflammation, and we assessed spatial learning and memory on days 7 to 19.

Results: Sur1-Trpm4 channels were upregulated in humans and rats with SAH. In rats, inhibiting Sur1 using antisense or the selective Sur1 inhibitor glibenclamide reduced SAH-induced immunoglobulin G extravasation and tumor necrosis α overexpression. In models with entorhinal SAH, rats treated with glibenclamide for 7 days after SAH exhibited better platform search strategies and better performance on incremental and rapid spatial learning than vehicle-treated controls.

Conclusions: Sur1-Trpm4 channels are upregulated in humans and rats with SAH. Channel inhibition with glibenclamide may reduce neuroinflammation and the severity of cognitive deficits after SAH.

Keywords: cognition; memory; subarachnoid hemorrhage; sulfonylurea receptor; transient receptor potential melastatin 4.

Conflict of interest statement

Disclosures

Dr Simard holds a US patent (7,285,574), a novel nonselective cation-channel in neural cells and methods for treating brain swelling. Dr Simard is a member of the scientific advisory board and holds shares in Remedy Pharmaceuticals. No support, direct or indirect, was provided to Dr Simard, or for this project by Remedy Pharmaceuticals.

The other authors report no conflicts.

Figures

Figure 1

Sur1-Trpm4 channel in human subarachnoid hemorrhage (SAH) shown by Förster resonance energy transfer (FRET). A, H&E-stained section showing SAH (left) and adjacent section immunolabeled for Sur1 (right; inverse black and white images shown for clarity); arrow: arteriole shown in B; asterisk: neurons shown in B. B, Immunolabeled cortex shows expression of Sur1 and Trpm4 in elongated microvessels and pyramidal neurons. C, Coimmunolabeled cortex shows coexpression of Sur1 and Trpm4 and FRET in elongated microvessels and pyramindal neurons (left); the bar graph (right) shows FRET efficiency for (1) COS-7 cells coexpressing Sur1 and Trpm4, immunolabeled with anti-Trpm4 antibody directed against a cytoplasmic loop and coimmunolabeled with anti-Sur1 antibody directed against either a cytoplasmic loop (Sur1-Trpm4; positive control) or against the extracellular N-terminus (Sur1-Trpm4#; negative control); (2) human pancreas immunolabeled with anti-Sur1 and anti-Kir6.2 antibodies; (3) human brains with SAH immunolabeled with anti-Sur1 and anti-Trpm4 antibodies as in B and C; n=58 to 64 measurements for COS-7 cells; n=28 to 34 for human pancreas and SAH cortex.

Figure 2

Sur1-Trpm4 channel in rat subarachnoid hemorrhage (SAH) shown by Förster resonance energy transfer (FRET) and coimmunoprecipitation. A–C, H&E-stained section (A) and immunolabeled sections (B and C) of rat entorhinal SAH; coimmunolabeled cortex shows coexpression of Sur1 and Trpm4 and FRET in elongated microvessels and astrocytes; representative of findings in 5 rats. D, Immunoblot (top) and densitometric analyses of immunoblots (bottom) for Trpm4 in rat entorhinal SAH; the immunoblot shows Trpm4 in total lysate (lanes 2, 3) and in the fraction immunoisolated using anti-Sur1 antibody (lanes 4, 5); lane 1, molecular mass marker; the bar graphs give values for 4 replicates. *, P<0.05; **, P<0.01.

Figure 3

Antisense against Abcc8 and glib-enclamide reduce immunoglobulin G (IgG) extravasation at 24 hours. A and B, Cortex adjacent to subarachnoid hemorrhage (SAH) immunolabeled for Sur1 (A) or IgG (B) from rats administered Scr-ODN or AS-ODN; box plots of quantitative analyses are shown; *, P<0.05; **, P<0.01; 3 rats per group; filament puncture model. C, Cortex adjacent to SAH immunolabeled for IgG from rats administered vehicle (Veh) or glibenclamide (Glib); box plots of quantitative analyses are shown; **, P<0.01; 8 rats per group; entorhinal cortex injection model.

Figure 4

Antisense against Abcc8 and glibenclamide reduce tumor necrosis α (TNFα) overexpression at 24 hours. A, Cortex adjacent to subarachnoid hemorrhage (SAH) immunolabeled for TNFα from rats administered Scr-ODN or AS-ODN; box plots of quantitative analyses are shown; **, P<0.01; same rats as in Figure 3A and 3B. B, Cortex adjacent to SAH immunolabeled for immunoglobulin G (IgG) from rats administered vehicle (Veh) or glibenclamide (Glib); box plots of quantitative analyses are shown; **, P<0.01; the same rats as in Figure 3C.

Figure 5

Glibenclamide reduces impairments in spatial learning and memory in rats with bilateral entorhinal SAH. A and B, Time spent searching for the submerged platform (A) and the percentage time spent in 1 of 3 platform search strategies (B) during incremental spatial learning in uninjured naïve controls (7 rats), and in rats with bilateral entorhinal SAH administered vehicle or glibenclamide (9 rats per group). C and D, Time spent in the correct quadrant during the memory probe (C) and during the test of rapid learning (D) in the same rats. *, P<0.05; **, P<0.01 comparing naïve with vehicle, or naïve with glibenclamide.

Figure 6

Glibenclamide reduces hippocampal tissue injury in rats with bilateral entorhinal subarachnoid hemorrhage (SAH). A–C, Hippocampi stained with Black Gold II (A), Fluoro-Jade C (B) or DAPI (C) from uninjured control rats (CTR), and rats with bilateral entorhinal SAH administered vehicle (Veh) or glibenclamide (Glib); in (A), the region of interest that was analyzed encompassing the perforant pathway (PP) is outlined; DG, dentate gyrus. D, Box plots of quantitative analyses of white matter labeling with Black Gold II in the PP, of cellular injury in the PP identified by Fluoro-Jade C staining, and of pyknotic nuclei in the DG of control rats and rats with entorhinal SAH administered vehicle or glibenclamide; 4 to 5 rats/group; *, P<0.05; **, P<0.01.