Subarachnoid hemorrhage (SAH) causes secondary brain injury due to vasospasm and inflammation. Here, we studied a rat model of mild-to-moderate SAH intended to minimize ischemia/hypoxia to examine the role of sulfonylurea receptor 1 (SUR1) in the inflammatory response induced by SAH. mRNA for Abcc8, which encodes SUR1, and SUR1 protein were abundantly upregulated in cortex adjacent to SAH, where tumor-necrosis factor-α (TNFα) and nuclear factor (NF)κB signaling were prominent. In vitro experiments confirmed that Abcc8 transcription is stimulated by TNFα. To investigate the functional consequences of SUR1 expression after SAH, we studied the effect of the potent, selective SUR1 inhibitor, glibenclamide. We examined barrier permeability (immunoglobulin G, IgG extravasation), and its correlate, the localization of the tight junction protein, zona occludens 1 (ZO-1). SAH caused a large increase in barrier permeability and disrupted the normal junctional localization of ZO-1, with glibenclamide significantly reducing both effects. In addition, SAH caused large increases in markers of inflammation, including TNFα and NFκB, and markers of cell injury or cell death, including IgG endocytosis and caspase-3 activation, with glibenclamide significantly reducing these effects. We conclude that block of SUR1 by glibenclamide may ameliorate several pathologic effects associated with inflammation that lead to cortical dysfunction after SAH.

An important but poorly understood feature of traumatic brain injury (TBI) is the clinically serious problem of spatiotemporal progression ("blossoming") of a hemorrhagic contusion, a phenomenon we term progressive secondary hemorrhage (PSH). Molecular mechanisms of PSH are unknown and efforts to reduce it by promoting coagulation have met with equivocal results. We hypothesized that PSH might be due to upregulation and activation of sulfonylurea receptor 1 (SUR1)-regulated NC(Ca-ATP) channels in capillary endothelial cells, predisposing to oncotic death of endothelial cells and catastrophic failure of capillary integrity. Anesthetized adult male rats underwent left parietal craniectomy for induction of a focal cortical contusion. The regulatory subunit of the channel, SUR1, was prominently upregulated in capillaries of penumbral tissues surrounding the contusion. In untreated rats, PSH was characterized by progressive enlargement of the contusion deep into the site of cortical impact, including corpus callosum, hippocampus, and thalamus, by progressive accumulation of extravasated blood, with a doubling of the volume during the first 12 h after injury, and by capillary fragmentation in penumbral tissues. Block of SUR1 using low-dose (non-hypoglycemogenic) glibenclamide largely eliminated PSH and capillary fragmentation, and was associated with a significant reduction in the size of the necrotic lesion and in preservation of neurobehavioral function. Antisense oligodeoxynucleotide against SUR1, administered after injury, reduced both SUR1 expression and PSH, consistent with a requirement for transcriptional upregulation of SUR1. Our findings provide novel insights into molecular mechanisms responsible for PSH associated with hemorrhagic contusions, and point to SUR1 as a potential therapeutic target in TBI.

Spinal cord injury (SCI) is typically complicated by progressive hemorrhagic necrosis, an autodestructive process of secondary injury characterized by progressive enlargement of a hemorrhagic contusion during the first several hours after trauma. We assessed the role of Abcc8, which encodes sulfonylurea receptor 1 (SUR1), in progressive hemorrhagic necrosis. After SCI, humans and rodents exhibited similar regional and cellular patterns of up-regulation of SUR1 and Abcc8 messenger RNA. Elimination of SUR1 in Abcc8 −/− mice and in rats given antisense oligodeoxynucleotide against Abcc8 prevented progressive hemorrhagic necrosis, yielded significantly better neurological function, and resulted in lesions that were one-fourth to one-third the size of those in control animals. The beneficial effects of Abcc8 suppression were associated with prevention of oncotic (necrotic) death of capillary endothelial cells. Suppression of Abcc8 with antisense oligodeoxynucleotide after SCI presents an opportunity for reducing the devastating sequelae of SCI.* To whom correspondence should be addressed. msimard@smail.umaryland.edu.Author contributions: J.M.S. conceived the study, participated in the analysis and interpretation of the data, and wrote the manuscript. S.K.W. designed and developed the custom antibody to SUR1 and designed in situ hybridization probes for Abcc8. M.D.N. and D.L. provided human specimens and aided in analyzing histopathology. C.T. performed immunolabeling and analysis for capillary fragmentation. Z.C. isolated and cultured microvascular endothelial cells from wild-type and SUR1-null mice and performed cell death experiments. S.I. performed most of the immunolabeling and all of the measurements of lesion volume after SCI. O.T. performed all of the surgical procedures for SCI and aided in evaluating neurobehavioral function of mice and rats. J.B. supplied SUR1-null mice. V.G. aided in the design of the experiments, analysis and interpretation of the data, and neurobehavioral assessment of mice and rats and contributed to writing the final version of the manuscript.Competing interests: J.M.S. holds a U.S. patent (number 7,285,574), "A novel non-selective cation channel in neural cells and methods for treating brain swelling." J.M.S. is a member of the scientific advisory board and holds shares in Remedy Pharmaceuticals, a company that is developing small-molecule drugs targeting NC Ca-ATP channels in acute CNS injury, including traumatic brain injury, stroke, and SCI. No support, direct or indirect, was provided to J.M.S

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.

Traumatic brain injury resulting from an explosive blast is one of the most serious wounds suffered by warfighters, yet the effects of explosive blast overpressure directly impacting the head are poorly understood. We developed a rodent model of direct cranial blast injury (dcBI), in which a blast overpressure could be delivered exclusively to the head, precluding indirect brain injury via thoracic transmission of the blast wave. We constructed and validated a Cranium Only Blast Injury Apparatus (COBIA) to deliver blast overpressures generated by detonating .22 caliber cartridges of smokeless powder. Blast waveforms generated by COBIA replicated those recorded within armored vehicles penetrated by munitions. Lethal dcBI (LD(50) ∼ 515 kPa) was associated with: (1) apparent brainstem failure, characterized by immediate opisthotonus and apnea leading to cardiac arrest that could not be overcome by cardiopulmonary resuscitation; (2) widespread subarachnoid hemorrhages without cortical contusions or intracerebral or intraventricular hemorrhages; and (3) no pulmonary abnormalities. Sub-lethal dcBI was associated with: (1) apnea lasting up to 15 sec, with transient abnormalities in oxygen saturation; (2) very few delayed deaths; (3) subarachnoid hemorrhages, especially in the path of the blast wave; (4) abnormal immunolabeling for IgG, cleaved caspase-3, and β-amyloid precursor protein (β-APP), and staining for Fluoro-Jade C, all in deep brain regions away from the subarachnoid hemorrhages, but in the path of the blast wave; and (5) abnormalities on the accelerating Rotarod that persisted for the 1 week period of observation. We conclude that exposure of the head alone to severe explosive blast predisposes to significant neurological dysfunction.

Frontal impact, closed head trauma is a frequent cause of traumatic brain injury (TBI) in motor vehicle and sports accidents. Diffuse axonal injury (DAI) is common in humans and experimental animals, and results from shearing forces that develop within the anisotropic brain. Because the specific anisotropic properties of the brain are axis-dependent, the anatomical site where force is applied as well as the resultant acceleration, be it linear, rotational, or some combination, are important determinants of the resulting pattern of brain injury. Available rodent models of closed head injury do not reproduce the frontal impact commonly encountered in humans. Here we describe a new rat model of closed head injury that is a modification of the impact-acceleration model of Marmarou. In our model (the Maryland model), the impact force is applied to the anterior part of the cranium and produces TBI by causing anterior-posterior plus sagittal rotational acceleration of the brain inside the intact cranium. Skull fractures, prolonged apnea, and mortality were absent. The animals exhibited petechial hemorrhages, DAI marked by a bead-like pattern of beta-amyloid precursor protein (beta-APP) in damaged axons, and widespread upregulation of beta-APP in neurons, with regions affected including the orbitofrontal cortex (coup), corpus callosum, caudate, putamen, thalamus, cerebellum, and brainstem. Activated caspase-3 was prominent in hippocampal neurons and Purkinje cells at the grey-white matter junction of the cerebellum. Neurobehavioral dysfunction, manifesting as reduced spontaneous exploration, lasted more than 1 week. We conclude that the Maryland model produces diffuse injuries that may be relevant to human brain injury.

In animal models of stroke, spinal cord injury, and subarachnoid hemorrhage, transient receptor potential melastatin 4 (Trpm4), a non-selective monovalent cation channel, is transcriptionally upregulated in neural and vascular cells. In these contexts, Trpm4 has been shown to co-associate with sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which play a critical role in cytotoxic edema, accidental necrotic (oncotic) cell death, blood-brain barrier (BBB) breakdown and formation of vasogenic edema. To date, the expression and molecular interactions of Trpm4 within human cerebral infarcts have not been systematically evaluated. In this study, we examined Trpm4 expression in postmortem specimens obtained from 15 patients within the first 31 days after onset of focal cerebral ischemia. Significant upregulation of Trpm4 protein was found in all cases, relative to controls. De novo transcriptional upregulation of Trpm4 protein was confirmed using in situ hybridization for Trpm4 mRNA. Trpm4 co-localized and co-associated with Sur1 within ischemic endothelial cells and neurons which exhibited membrane thickening and irregularities characteristic of necrotic cell death. Sur1 and Trpm4 co-expression in abnormal endothelial cells also was associated with vasogenic edema, as evidenced by upregulated perivascular TNFα, perivascular extravasation of serum immunoglobulin G and associated inflammation. Upregulated Trpm4 protein persisted up to one month post onset of cerebral ischemia. Furthermore, pharmacological channel blockade by glibenclamide, a selective inhibitor of sulfonylurea receptor, was found to mitigate perivascular TNFα labeling in a rat middle cerebral artery occlusion (MCAo) stroke model. We conclude that the Sur1-Trpm4 channel is upregulated and associated with BBB disruption and cerebral edema formation in human cerebral infarcts. These data suggest that pharmacological targeting of this channel may represent a promising therapeutic strategy for clinical management of ischemic stroke.

Subarachnoid hemorrhage (SAH) can lead to disabling motor, cognitive, and neuropsychological abnormalities. Part of the secondary injury to cerebral tissues associated with SAH is attributable to the neuroinflammatory response induced by blood. Heparin is a pleiotropic compound that reduces inflammatory responses in conditions outside the central nervous system. Using a model of SAH devoid of global insult, we evaluated the effect of delayed intravenous (IV) infusion of heparin, at a dose that does not produce therapeutic anticoagulation, on neuroinflammation, myelin preservation, and apoptosis. Adult male rats underwent bilateral stereotactic injections of autologous blood (50 μL) into the subarachnoid space of the entorhinal cortex. The rats were implanted with mini-osmotic pumps that delivered either vehicle or unfractionated heparin (10 U/kg/h IV) beginning 12 h after SAH. No mechanical or hemorrhagic injury was observed in the hippocampus. In vehicle controls assessed at 48 h, SAH was associated with robust neuroinflammation in the adjacent cortex [neutrophils, activated phagocytic microglia, nuclear factor-kappa B, tumor necrosis factor-alpha, and interleukin-1beta] and neurodegeneration (Fluoro-Jade C staining and loss of NeuN). In the hippocampus, a muted neuroinflammatory response was indicated by Iba1-positive, ED1-negative microglia exhibiting an activated morphology. The perforant pathway showed Fluoro-Jade C staining and demyelination, and granule cells of the dentate gyrus had pyknotic nuclei, labeled with Fluoro-Jade C and showed upregulation of cleaved caspase-3, consistent with transsynaptic apoptosis. Administration of heparin significantly reduced neuroinflammation, demyelination, and transsynaptic apoptosis. We conclude that delayed IV infusion of low-dose unfractionated heparin may attenuate adverse neuroinflammatory effects of SAH.