Abcc8 (Sulfonylurea Receptor-1) Impact on Brain Atrophy after Traumatic Brain Injury Varies by Sex.

Females have been understudied in pre-clinical and clinical traumatic brain injury (TBI), despite distinct biology and worse clinical outcomes versus males. Sulfonylurea receptor 1 (SUR1) inhibition has shown promising results in predominantly male TBI. A phase II trial is ongoing. We investigated whether SUR1 inhibition effects on contusional TBI differ by sex given that this may inform clinical trial design and/or interpretation. We studied the moderating effects of sex on post-injury brain tissue loss in 142 male and female ATP-binding cassette transporter subfamily C member 8 (Abcc8) wild-type, heterozygote, and knockout mice (12-15 weeks). Monkey fibroblast-like cells and mouse brain endothelium-derived cells were used for in vitro studies. Mice were injured with controlled cortical impact and euthanized 21 days post-injury to assess contusion, brain, and hemisphere volumes (vs. genotype- and sex-matched naïves). Abcc8 knockout mice had smaller contusion volumes (p = 0.012) and larger normalized contralateral (right) hemisphere volumes (nRHV; p = 0.03) after injury versus wild type. This was moderated by sex: Contusions were smaller (p = 0.020), nRHV was higher (p = 0.001), and there was less global atrophy (p = 0.003) in male, but not female, knockout versus wild-type mice after TBI. Less atrophy occurred in males for each copy of Abcc8 lost (p = 0.023-0.002, all outcomes). In vitro, sex-determining region Y (SRY) stimulated Abcc8 promoter activity and increased Abcc8 expression. Loss of Abcc8 strongly protected against post-traumatic cerebral atrophy in male, but not female, mice. This may partly be mediated by SRY on the Y-chromosome. Sex differences may have important implications for ongoing and future trials of SUR1 blockade.

HMGB1 is a Potential Mediator of Astrocytic TLR4 Signaling Activation following Acute and Chronic Focal Cerebral Ischemia.

Limited, and underutilized, therapeutic options for acute stroke require new approaches to treatment. One such potential approach involves better understanding of innate immune response to brain injury such as acute focal cerebral ischemia. This includes understanding the temporal profile, and specificity, of Toll-like receptor 4 (TLR4) signaling in brain cell types, such as astrocytes, following focal cerebral ischemia. This study evaluated TLR4 signaling, and downstream mediators, in astrocytes, during acute and chronic phases post transient middle cerebral artery occlusion (MCAO). We also determined whether high mobility group box 1 (HMGB1), an endogenous TLR4 ligand, was sufficient to induce TLR4 signaling activation in astrocytes in vivo and in vitro. We injected HMGB1 into normal cortex, in vivo, and stimulated cultured astrocytes with HMGB1, in vitro, and determined TLR4, and downstream mediator, expression by immunohistochemistry. We found that expression of TLR4, and downstream mediators, such as inducible nitric oxide synthase (iNOS), occurs in penumbral astrocytes in acute and chronic phases after focal cerebral ischemia, but was undetectable in cortical astrocytes in the contralateral hemisphere. In addition, cortical injection of recombinant HMGB1 led to a trend towards an almost 2-fold increase in TLR4 expression in astrocytes surrounding the injection site. Consistent with these results, in vitro stimulation of the DI TNC1 astrocyte cell line, with recombinant HMGB1, led to increased TLR4 and iNOS message levels. These findings suggest that HMGB1, an endogenous TLR4 ligand, is an important physiological ligand for TLR4 signaling activation, in penumbral astrocytes, following acute and chronic ischemia and HMGB1 amplifies TLR4 signaling in astrocytes.

SUR1-TRPM4 channels, not KATP, mediate brain swelling following cerebral ischemia.

BACKGROUND: Preclinical and emerging clinical data show that glibenclamide reduces space occupying edema and brain swelling following cerebral ischemia. Glibenclamide is a potent inhibitor of numerous sulfonylurea receptor (SUR)-regulated channels, including KATP (SUR1-KIR6.2, SUR2A-KIR6.2, SUR2B-KIR6.2, SUR2B-KIR6.1) and SUR1-TRPM4. Here, we used molecularly specific oligodeoxynucleotides (ODNs) to investigate the role of various SUR-regulated ion channel subunits in post-ischemic brain swelling. METHODS: Focal cerebral ischemia was induced in adult male rats by permanent middle cerebral artery occlusion (pMCAo). We used this model to study the effects of antisense-ODNs (AS-ODNs) directed against Abcc8/SUR1, Trpm4/TRPM4, Kcnj8/KIR6.1 and Kcnj11/KIR6.2 on hemispheric swelling, with sense or scrambled ODNs used as controls. We used antibody-based Förster resonance energy transfer (immuno-FRET) and co-immunoprecipitation to study the co-assembly of SUR1-TRPM4 heteromers. RESULTS: In the combined control groups administered sense or scrambled ODNs, pMCAo resulted in uniformly large infarct volumes (mean ± SD: 57.4 ± 8.8 %; n = 34) at 24 h after onset of ischemia, with no effect of AS-ODNs on infarct size. In controls, hemispheric swelling was 23.9 ± 4.1 % (n = 34), and swelling was linearly related to infarct volume (P < 0.02). In the groups administered anti-Abcc8/SUR1 or anti-Trpm4/TRPM4 AS-ODN, hemispheric swelling was significantly less, 11.6 ± 3.9 % and 12.8 ± 5.8 % respectively (P < 0.0001), and the relationship between infarct volume and swelling was reduced and not significant. AS-ODNs directed against Kcnj8/KIR6.1 and Kcnj11/KIR6.2 had no significant effect on hemispheric swelling (23.3 ± 5.4 % and 22.9 ± 5.8 % respectively). Post-ischemic tissues showed co-assembly of SUR1-TRPM4 heteromers. CONCLUSIONS: Post-ischemic hemispheric swelling can be decoupled from infarct volume. SUR1-TRPM4 channels, not KATP, mediate post-ischemic brain swelling.

Sulfonylurea Receptor 1, Transient Receptor Potential Cation Channel Subfamily M Member 4, and KIR6.2:Role in Hemorrhagic Progression of Contusion.

In severe traumatic brain injury (TBI), contusions often are worsened by contusion expansion or hemorrhagic progression of contusion (HPC), which may double the original contusion volume and worsen outcome. In humans and rodents with contusion-TBI, sulfonylurea receptor 1 (SUR1) is upregulated in microvessels and astrocytes, and in rodent models, blockade of SUR1 with glibenclamide reduces HPC. SUR1 does not function by itself, but must co-assemble with either KIR6.2 or transient receptor potential cation channel subfamily M member 4 (TRPM4) to form KATP (SUR1-KIR6.2) or SUR1-TRPM4 channels, with the two having opposite effects on membrane potential. Both KIR6.2 and TRPM4 are reportedly upregulated in TBI, especially in astrocytes, but the identity and function of SUR1-regulated channels post-TBI is unknown. Here, we analyzed human and rat brain tissues after contusion-TBI to characterize SUR1, TRPM4, and KIR6.2 expression, and in the rat model, to examine the effects on HPC of inhibiting expression of the three subunits using intravenous antisense oligodeoxynucleotides (AS-ODN). Glial fibrillary acidic protein (GFAP) immunoreactivity was used to operationally define core versus penumbral tissues. In humans and rats, GFAP-negative core tissues contained microvessels that expressed SUR1 and TRPM4, whereas GFAP-positive penumbral tissues contained astrocytes that expressed all three subunits. Förster resonance energy transfer imaging demonstrated SUR1-TRPM4 heteromers in endothelium, and SUR1-TRPM4 and SUR1-KIR6.2 heteromers in astrocytes. In rats, glibenclamide as well as AS-ODN targeting SUR1 and TRPM4, but not KIR6.2, reduced HPC at 24 h post-TBI. Our findings demonstrate upregulation of SUR1-TRPM4 and KATP after contusion-TBI, identify SUR1-TRPM4 as the primary molecular mechanism that accounts for HPC, and indicate that SUR1-TRPM4 is a crucial target of glibenclamide.

Correction: Cerebral microbleeds in a neonatal rat model.

[This corrects the article DOI: 10.1371/journal.pone.0171163.].

SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells.

BACKGROUND: Hemorrhagic transformation is a major complication of ischemic stroke, is linked to matrix metalloproteinase-9 (MMP-9), and is exacerbated by tissue plasminogen activator (tPA). Cerebral ischemia/reperfusion is characterized by SUR1-TRPM4 (sulfonylurea receptor 1-transient receptor potential melastatin 4) channel upregulation in microvascular endothelium. In humans and rodents with cerebral ischemia/reperfusion (I/R), the SUR1 antagonist, glibenclamide, reduces hemorrhagic transformation and plasma MMP-9, but the mechanism is unknown. We hypothesized that tPA induces protease activated receptor 1 (PAR1)-mediated, Ca2+-dependent phasic secretion of MMP-9 from activated brain endothelium, and that SUR1-TRPM4 is required for this process. METHODS: Cerebral I/R, of 2 and 4 hours duration, respectively, was obtained using conventional middle cerebral artery occlusion. Immunolabeling was used to quantify p65 nuclear translocation. Murine and human brain endothelial cells (BEC) were studied in vitro, without and with NF-κB activation, using immunoblot, zymography and ELISA, patch clamp electrophysiology, and calcium imaging. Genetic and pharmacological manipulations were used to identify signaling pathways. RESULTS: Cerebral I/R caused prominent nuclear translocation of p65 in microvascular endothelium. NF-κB-activation of BEC caused de novo expression of SUR1-TRPM4 channels. In NF-κB-activated BEC: (i) tPA caused opening of SUR1-TRPM4 channels in a plasmin-, PAR1-, TRPC3- and Ca2+-dependent manner; (ii) tPA caused PAR1-dependent secretion of MMP-9; (iii) tonic secretion of MMP-9 by activated BEC was not influenced by SUR1 inhibition; (iv) phasic secretion of MMP-9 induced by tPA or the PAR1-agonist, TFLLR, required functional SUR1-TRPM4 channels, with inhibition of SUR1 decreasing tPA-induced MMP-9 secretion. CONCLUSIONS: tPA induces PAR1-mediated, SUR1-TRPM4-dependent, phasic secretion of MMP-9 from activated brain endothelium.

Salutary effects of glibenclamide during the chronic phase of murine experimental autoimmune encephalomyelitis.

BACKGROUND: In multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), inflammation is perpetuated by both infiltrating leukocytes and astrocytes. Recent work implicated SUR1-TRPM4 channels, expressed mostly by astrocytes, in murine EAE. We tested the hypothesis that pharmacological inhibition of SUR1 during the chronic phase of EAE would be beneficial. METHODS: EAE was induced in mice using myelin oligodendrocyte glycoprotein (MOG) 35-55. Glibenclamide (10 µg/day) was administered beginning 12 or 24 days later. The effects of treatment were determined by clinical scoring and tissue examination. Drug within EAE lesions was identified using bodipy-glibenclamide. The role of SUR1-TRPM4 in primary astrocytes was characterized using patch clamp and qPCR. Demyelinating lesions from MS patients were studied by immunolabeling and immunoFRET. RESULTS: Administering glibenclamide beginning 24 days after MOG35-55 immunization, well after clinical symptoms had plateaued, improved clinical scores, reduced myelin loss, inflammation (CD45, CD20, CD3, p65), and reactive astrocytosis, improved macrophage phenotype (CD163), and decreased expression of tumor necrosis factor (TNF), B-cell activating factor (BAFF), chemokine (C-C motif) ligand 2 (CCL2) and nitric oxide synthase 2 (NOS2) in lumbar spinal cord white matter. Glibenclamide accumulated within EAE lesions, and had no effect on leukocyte sequestration. In primary astrocyte cultures, activation by TNF plus IFNγ induced de novo expression of SUR1-TRPM4 channels and upregulated Tnf, Baff, Ccl2, and Nos2 mRNA, with glibenclamide blockade of SUR1-TRPM4 reducing these mRNA increases. In demyelinating lesions from MS patients, astrocytes co-expressed SUR1-TRPM4 and BAFF, CCL2, and NOS2. CONCLUSIONS: SUR1-TRPM4 may be a druggable target for disease modification in MS.

Glibenclamide pretreatment protects against chronic memory dysfunction and glial activation in rat cranial blast traumatic brain injury.

Blast traumatic brain injury (bTBI) affects both military and civilian populations, and often results in chronic deficits in cognition and memory. Chronic glial activation after bTBI has been linked with cognitive decline. Pharmacological inhibition of sulfonylurea receptor 1 (SUR1) with glibenclamide was shown previously to reduce glial activation and improve cognition in contusive models of CNS trauma, but has not been examined in bTBI. We postulated that glibenclamide would reduce chronic glial activation and improve long-term memory function after bTBI. Using a rat direct cranial model of bTBI (dc-bTBI), we evaluated the efficacy of two glibenclamide treatment paradigms: glibenclamide prophylaxis (pre-treatment), and treatment with glibenclamide starting after dc-bTBI (post-treatment). Our results show that dc-bTBI caused hippocampal astrocyte and microglial/macrophage activation that was associated with hippocampal memory dysfunction (rapid place learning paradigm) at 28days, and that glibenclamide pre-treatment, but not post-treatment, effectively protected against glial activation and memory dysfunction. We also report that a brief transient time-window of blood-brain barrier (BBB) disruption occurs after dc-bTBI, and we speculate that glibenclamide, which is mostly protein bound and does not normally traverse the intact BBB, can undergo CNS delivery only during this brief transient opening of the BBB. Together, our findings indicate that prophylactic glibenclamide treatment may help to protect against chronic cognitive sequelae of bTBI in warfighters and other at-risk populations.

Cerebral microbleeds in a neonatal rat model.

BACKGROUND: In adult humans, cerebral microbleeds play important roles in neurodegenerative diseases but in neonates, the consequences of cerebral microbleeds are unknown. In rats, a single pro-angiogenic stimulus in utero predisposes to cerebral microbleeds after birth at term, a time when late oligodendrocyte progenitors (pre-oligodendrocytes) dominate in the rat brain. We hypothesized that two independent pro-angiogenic stimuli in utero would be associated with a high likelihood of perinatal microbleeds that would be severely damaging to white matter. METHODS: Pregnant Wistar rats were subjected to intrauterine ischemia (IUI) and low-dose maternal lipopolysaccharide (mLPS) at embryonic day (E) 19. Pups were born vaginally or abdominally at E21-22. Brains were evaluated for angiogenic markers, microhemorrhages, myelination and axonal development. Neurological function was assessed out to 6 weeks. RESULTS: mRNA (Vegf, Cd31, Mmp2, Mmp9, Timp1, Timp2) and protein (CD31, MMP2, MMP9) for angiogenic markers, in situ proteolytic activity, and collagen IV immunoreactivity were altered, consistent with an angiogenic response. Vaginally delivered pups exposed to prenatal IUI+mLPS had spontaneous cerebral microbleeds, abnormal neurological function, and dysmorphic, hypomyelinated white matter and axonopathy. Pups exposed to the same pro-angiogenic stimuli in utero but delivered abdominally had minimal cerebral microbleeds, preserved myelination and axonal development, and neurological function similar to naïve controls. CONCLUSIONS: In rats, pro-angiogenic stimuli in utero can predispose to vascular fragility and lead to cerebral microbleeds. The study of microbleeds in the neonatal rat brain at full gestation may give insights into the consequences of microbleeds in human preterm infants during critical periods of white matter development.

The Sur1-Trpm4 channel regulates NOS2 transcription in TLR4-activated microglia.

BACKGROUND: Harmful effects of activated microglia are due, in part, to the formation of peroxynitrite radicals, which is attributable to the upregulation of inducible nitric oxide (NO) synthase (NOS2). Because NOS2 expression is determined by Ca(2+)-sensitive calcineurin (CN) dephosphorylating nuclear factor of activated T cells (NFAT), and because Sur1-Trpm4 channels are crucial for regulating Ca(2+) influx, we hypothesized that, in activated microglia, Sur1-Trpm4 channels play a central role in regulating CN/NFAT and downstream target genes such as Nos2. METHODS: We studied microglia in vivo and in primary culture from adult rats, and from wild type, Abcc8-/- and Trpm4-/- mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), using in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, qPCR, patch clamp electrophysiology, calcium imaging, the Griess assay, and chromatin immunoprecipitation. RESULTS: In microglia in vivo and in vitro, LPS activation of TLR4 led to de novo upregulation of Sur1-Trpm4 channels and CN/NFAT-dependent upregulation of Nos2 mRNA, NOS2 protein, and NO. Pharmacological inhibition of Sur1 (glibenclamide), Trpm4 (9-phenanthrol), or gene silencing of Abcc8 or Trpm4 reduced Nos2 upregulation. Inhibiting Sur1-Trpm4 increased the intracellular calcium concentration ([Ca(2+)]i), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca(2+)]i induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca(2+)/calmodulin protein kinase II and of CN, consistent with reduced nuclear translocation of NFAT. The regulation of NFAT by Sur1-Trpm4 was confirmed using chromatin immunoprecipitation. CONCLUSIONS: Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca(2+)-sensitive gene expression, including Nos2.

Silencing of Abcc8 or inhibition of newly upregulated Sur1-Trpm4 reduce inflammation and disease progression in experimental autoimmune encephalomyelitis.

BACKGROUND: In experimental autoimmune encephalomyelitis (EAE), deletion of transient receptor potential melastatin 4 (Trpm4) and administration of glibenclamide were found to ameliorate disease progression, prompting speculation that glibenclamide acts by directly inhibiting Trpm4. We hypothesized that in EAE, Trpm4 upregulation is accompanied by upregulation of sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which are highly sensitive to glibenclamide, and that Sur1-Trpm4 channels are required for EAE progression. METHODS: EAE was induced in wild-type (WT) and Abcc8-/- mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). Lumbar spinal cords were examined by immunohistochemistry, immuno-Förster resonance energy transfer (immunoFRET), and co-immunoprecipitation for Sur1-Trpm4. WT/EAE mice were administered with the Sur1 inhibitor, glibenclamide, beginning on post-induction day 10. Mice were evaluated for clinical function, inflammatory cells and cytokines, axonal preservation, and white matter damage. RESULTS: Sur1-Trpm4 channels were upregulated in EAE, predominantly in astrocytes. The clinical course and severity of EAE were significantly ameliorated in glibenclamide-treated WT/EAE and in Abcc8-/-/EAE mice. At 30 days, the lumbar spinal cords of glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice showed significantly fewer invading immune cells, including leukocytes (CD45), T cells (CD3), B cells (CD20) and macrophages/microglia (CD11b), and fewer cells expressing pro-inflammatory cytokines (TNF-α, IFN-γ, IL-17). In both glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice, the reduced inflammatory burden correlated with better preservation of myelin, better preservation of axons, and more numerous mature and precursor oligodendrocytes. CONCLUSIONS: Sur-Trpm4 channels are newly upregulated in EAE and may represent a novel target for disease-modifying therapy in multiple sclerosis.

Methemoglobin is an endogenous toll-like receptor 4 ligand-relevance to subarachnoid hemorrhage.

Neuroinflammation is a well-recognized consequence of subarachnoid hemorrhage (SAH), and may be responsible for important complications of SAH. Signaling by Toll-like receptor 4 (TLR4)-mediated nuclear factor κB (NFκB) in microglia plays a critical role in neuronal damage after SAH. Three molecules derived from erythrocyte breakdown have been postulated to be endogenous TLR4 ligands: methemoglobin (metHgb), heme and hemin. However, poor water solubility of heme and hemin, and lipopolysaccharide (LPS) contamination have confounded our understanding of these molecules as endogenous TLR4 ligands. We used a 5-step process to obtain highly purified LPS-free metHgb, as confirmed by Fourier Transform Ion Cyclotron Resonance mass spectrometry and by the Limulus amebocyte lysate assay. Using this preparation, we show that metHgb is a TLR4 ligand at physiologically relevant concentrations. metHgb caused time- and dose-dependent secretion of the proinflammatory cytokine, tumor necrosis factor α (TNFα), from microglial and macrophage cell lines, with secretion inhibited by siRNA directed against TLR4, by the TLR4-specific inhibitors, Rs-LPS and TAK-242, and by anti-CD14 antibodies. Injection of purified LPS-free metHgb into the rat subarachnoid space induced microglial activation and TNFα upregulation. Together, our findings support the hypothesis that, following SAH, metHgb in the subarachnoid space can promote widespread TLR4-mediated neuroinflammation.

Complex N-glycosylation stabilizes surface expression of transient receptor potential melastatin 4b protein.

N-glycosylation is important for the function and regulation of ion channels. We examined the role of N-glycosylation of transient receptor potential melastatin (Trpm) 4b, a membrane glycoprotein that regulates calcium influx. Trpm4b was expressed in vivo in all rat tissues examined. In each tissue, Trpm4b had a different molecular mass, between ∼129 and ∼141 kDa, but all reverted to ∼120 kDa following treatment with peptide:N-glycosidase F, consistent with N-glycosylation being the principal form of post-translational modification of Trpm4b in vivo. In six stable isogenic cell lines that express different levels of Trpm4b, two forms were found, high mannose, core-glycosylated and complex, highly glycosylated (HG), with HG-Trpm4b comprising 85% of the total Trpm4b expressed. For both forms, surface expression was directly proportional to the total Trpm4b expressed. Complex N-glycosylation doubled the percentage of Trpm4b at the surface, compared with high mannose N-glycosylation. Mutation of the single N-glycosylation consensus sequence at Asn-988 (Trpm4b-N988Q), located near the pore-forming loop between transmembrane helices 5 and 6, prevented glycosylation, but did not prevent surface expression, impair formation of functional membrane channels, or alter channel conductance. In transfection experiments, the time courses for appearance of HG-Trpm4b and Trpm4b-N988Q on the surface were similar. In experiments with cycloheximide inhibition of protein synthesis, the time course for disappearance of HG-Trpm4b from the surface was much slower than that for Trpm4b-N988Q. We conclude that N-glycosylation is not required for surface expression or channel function, but that complex N-glycosylation plays a crucial role in stabilizing surface expression of Trpm4b.

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

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.

The Sur1-Trpm4 Channel in Spinal Cord Injury.

Spinal cord injury (SCI) is a major unsolved challenge in medicine. Impact trauma to the spinal cord shears blood vessels, causing an immediate 'primary hemorrhage'. During the hours following trauma, the region of hemorrhage enlarges progressively, with delayed or 'secondary hemorrhage' adding to the primary hemorrhage, and effectively doubling its volume. The process responsible for the secondary hemorrhage that results in early expansion of the hemorrhagic lesion is termed 'progressive hemorrhagic necrosis' (PHN). PHN is a dynamic process of auto destruction whose molecular underpinnings are only now beginning to be elucidated. PHN results from the delayed, progressive, catastrophic failure of the structural integrity of capillaries. The resulting 'capillary fragmentation' is a unique, pathognomonic feature of PHN. Recent work has implicated the Sur1-Trpm4 channel that is newly upregulated in penumbral microvessels as being required for the development of PHN. Targeting the Sur1-Trpm4 channel by gene deletion, gene suppression, or pharmacological inhibition of either of the two channel subunits, Sur1 or Trpm4, yields exactly the same effects histologically and functionally, and exactly the same unique, pathognomonic phenotype - the prevention of capillary fragmentation. The potential advantage of inhibiting Sur1-Trpm4 channels using glibenclamide is a highly promising strategy for ameliorating the devastating sequelae of spinal cord trauma in humans.

The sulfonylurea receptor 1 (Sur1)-transient receptor potential melastatin 4 (Trpm4) channel.

The sulfonylurea receptor 1 (Sur1)-NC(Ca-ATP) channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC(Ca-ATP) channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K(ATP) channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.

Glibenclamide-10-h Treatment Window in a Clinically Relevant Model of Stroke.

Glibenclamide improves outcomes in rat models of stroke, with treatment as late as 6 h after onset of ischemia shown to be beneficial. Because the molecular target of glibenclamide, the sulfonylurea receptor 1 (Sur1)-regulated NC(Ca-ATP) channel, is upregulated de novo by a complex transcriptional mechanism, and the principal pathophysiological target, brain swelling, requires hours to develop, we hypothesized that the treatment window would exceed 6 h. We studied a clinically relevant rat model of stroke in which middle cerebral artery occlusion (75% < reduction in LDF signal ≤90%) was produced using an intra-arterial occluder. Recanalization was obtained 4.5 h later by removing the occluder. At that time, we administered recombinant tissue plasminogen activator (rtPA; 0.9 mg/kg IV over 30 min). Immunolabeling showed modest expression of Sur1 5 h after onset of ischemia, with expression increasing 7- to 11-fold (P < 0.01) by 24 h. Rats were administered either vehicle or glibenclamide (10 µg/kg IP loading dose plus 200 ng/h by constant subcutaneous infusion) beginning 4.5 or 10 h after onset of ischemia. In rats treated at 4.5 or 10 h, glibenclamide significantly reduced hemispheric swelling at 24 h from (mean ± SEM) 14.7 ± 1.5% to 8.1 ± 1.6% or 8.8 ± 1.1% (both P < 0.01), respectively, and significantly reduced 48-h mortality from 53% to 17% or 12% (both P < 0.01), and improved Garcia scores at 48 h from 3.8 ± 0.62 to 7.6 ± 0.70 or 8.4 ± 0.74 (both P < 0.01). We conclude that, in a clinically relevant model of stroke, the treatment window for glibenclamide extends to 10 h after onset of ischemia.

Sequential activation of hypoxia-inducible factor 1 and specificity protein 1 is required for hypoxia-induced transcriptional stimulation of Abcc8.

Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.

Comparison of experimental lung injury from acute renal failure with injury due to sepsis.

BACKGROUND: Acute renal failure (ARF) and acute respiratory distress syndrome (ARDS) coexist frequently, and the mortality rate of this combination is very high. It is well established that cytokines and chemokines play a major role in the pathogenesis of ARDS. In addition, heat shock proteins (HSPs) have been shown to be protective against ARDS. OBJECTIVES: The purpose of this study was to investigate the pathophysiology of ARDS in two different conditions, sepsis and ARF. METHODS: We examined five different rat animal models including sham-operated control, sepsis and three ARF models induced by renal ischemia/reperfusion injury, bilateral nephrectomy or bilateral ligation of renal pedicles. We analyzed pulmonary histology, pulmonary vascular permeability, cellular infiltration, and expression of cytokines, chemokines and HSPs. RESULTS: Like sepsis, the three forms of ARF led to ARDS, as manifested by increased pulmonary vascular permeability and histological changes consistent with ARDS. On the other hand, ARF and sepsis differed in that ARF was associated with markedly lower levels of pulmonary cellular infiltration. Furthermore, while pulmonary expression of tumor necrosis factor-alpha increased in sepsis, cytokine-induced neutrophil chemoattractant 2 increased in nephrectomized rats indicating that different inflammatory mediators were involved in the injury mechanism. Finally, pulmonary expression of multiple HSPs including HSP27-1, HSP70, HSP70-4, HSP70-8 and HSP90 was significantly different between the two conditions. CONCLUSIONS: We conclude that the pathophysiology of ARDS following ARF is distinct from that in sepsis. ARF-induced ARDS is characterized by a low level of cellular infiltration, induction of cytokine-induced neutrophil chemoattractant 2, and a discrete expression profile of HSPs.

Mre11-Rad50-Nbs1 complex is activated by hypertonicity.

When exposed to hypertonic conditions, cells accumulate double-strand DNA breaks (DSBs) like they are exposed to ionizing radiation. It has been proposed that inactivation of the Mre11-Rad50-Nbs1 (MRN) complex due to nuclear exit is responsible for the accumulation of DSBs as cells fail to repair DSBs produced during normal cellular activity. In this study, we examined the MRN complex in cells switched to hypertonicity. Surprisingly, we found that the MRN complex stayed in the nucleus and remained intact in response to hypertonicity. In fact, the MRN complex was dramatically activated after 4 h of switch to hypertonicity in a dose-dependent manner as shown by formation of foci. Activation of ATM and the MRN complex by hypertonicity and bleomycin was additive as was activation of their downstream targets including gammaH2AX and Chk2 indicating that the cellular response to DSB was intact in hypertonic conditions. Activation of Chk2 in response to hypertonicity was not observed in mutant cells with functionally impaired MRN complex confirming that they are in the same pathway. After 20 h of a switch to hypertonicity, MRN foci and gammaH2AX returned to a control level, suggesting that cells adapted to hypertonicity by repairing DNA. We conclude that cells respond normally to DSB and repair the DNA damages induced by hypertonicity.