J. Marc Simard

Newly expressed SUR1-regulated NC(Ca-ATP) channel mediates cerebral edema after ischemic stroke.

Pathological conditions in the central nervous system, including stroke and trauma, are often exacerbated by cerebral edema. We recently identified a nonselective cation channel, the NCCa-ATP channel, in ischemic astrocytes that is regulated by sulfonylurea receptor 1 (SUR1), is opened by depletion of ATP and, when opened, causes cytotoxic edema. Here, we evaluated involvement of this channel in rodent models of stroke. SUR1 protein and mRNA were newly expressed in ischemic neurons, astrocytes and capillaries. Upregulation of SUR1 was linked to activation of the transcription factor Sp1 and was associated with expression of functional NCCa-ATP but not KATP channels. Block of SUR1 with low-dose glibenclamide reduced cerebral edema, infarct volume and mortality by 50%, with the reduction in infarct volume being associated with cortical sparing. Our findings indicate that the NCCa-ATP channel is crucially involved in development of cerebral edema, and that targeting SUR1 may provide a new therapeutic approach to stroke.

Molecular Mechanisms of Ischemic Cerebral Edema: Role of Electroneutral Ion Transport

The brain achieves homeostasis of its intracellular and extracellular fluids by precisely regulating the transport of solute and water across its major cellular barriers: endothelia of the blood-brain barrier (BBB), choroid plexus epithelia, and neuroglial cell membranes. Cerebral edema, the pathological accumulation of fluid in the brains intracellular and extracellular spaces, is a major cause of morbidity and mortality following stroke and other forms of ischemic brain injury. Until recently, mechanisms of cerebral edema formation have been obscure; consequently, its treatment has been empiric and suboptimal. Here, we provide a paradigm for understanding ischemic cerebral edema, showing that its molecular pathogenesis is a complex yet step-wise process that results largely from impaired astrocytic cell volume regulation and permeability alterations in the cerebral microvasculature, both of which arise from pathological changes in the activities of specific ion channels and transporters. Recent data has implicated the bumetanide-sensitive NKCC1, an electroneutral cotransporter expressed in astrocytes and the BBB, in cerebral edema formation in several different rodent models of stroke. Pharmacological inhibition or genetic deficiency of NKCC1 decreases ischemia-induced cell swelling, BBB breakdown, cerebral edema, and neurotoxicity. Combination pharmacological strategies that include NKCC1 as a target might thus prove beneficial for the treatment of ischemic, and potentially other types of, cerebral edema.

Glibenclamide reduces inflammation, vasogenic edema, and caspase-3 activation after subarachnoid hemorrhage

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.

Sulfonylureas Improve Outcome in Patients With Type 2 Diabetes and Acute Ischemic Stroke

Background and Purpose— The sulfonylurea receptor 1-regulated NCCa-ATP channel is upregulated in rodent models of stroke with block of the channel by the sulfonylurea, glibenclamide (glyburide), significantly reducing mortality, cerebral edema, and infarct volume. We hypothesized that patients with type 2 diabetes mellitus taking sulfonylurea agents both at the time of stroke and during hospitalization would have superior outcomes. Methods— We reviewed medical records of patients with diabetes mellitus hospitalized within 24 hours of onset of acute ischemic stroke in the Neurology Clinic, Charité Hospital, Berlin, Germany, during 1994 to 2000. After exclusions, the cohort comprised 33 patients taking a sulfonylurea at admission through discharge (treatment group) and 28 patients not on a sulfonylurea (control group). The primary outcome was a decrease in National Institutes of Health Stroke Scale of 4 points or more from admission to discharge or a discharge National Institutes of Health Stroke Scale score of 0. The secondary outcome was a discharge modified Rankin Scale score ≤2. Results— No significant differences, other than stroke subtype, were observed among baseline variables between control and treatment groups. The primary outcome was reached by 36.4% of patients in the treatment group and 7.1% in the control group (P=0.007). The secondary outcome was reached by 81.8% versus 57.1% (P=0.035). Subgroup analyses showed that improvements occurred only in patients with nonlacunar strokes and were independent of gender, previous transient ischemic attack, and blood glucose levels. Conclusion— Sulfonylureas may be beneficial for patients with diabetes mellitus with acute ischemic stroke. Further investigation of similar cohorts and a prospective randomized trial are recommended to confirm the present observations.

Endothelial sulfonylurea receptor 1–regulated NCCa-ATP channels mediate progressive hemorrhagic necrosis following spinal cord injury

Acute spinal cord injury (SCI) causes progressive hemorrhagic necrosis (PHN), a poorly understood pathological process characterized by hemorrhage and necrosis that leads to devastating loss of spinal cord tissue, cystic cavitation of the cord, and debilitating neurological dysfunction. Using a rodent model of severe cervical SCI, we tested the hypothesis that sulfonylurea receptor 1-regulated (SUR1-regulated) Ca(2+)-activated, [ATP](i)-sensitive nonspecific cation (NC(Ca-ATP)) channels are involved in PHN. In control rats, SCI caused a progressively expansive lesion with fragmentation of capillaries, hemorrhage that doubled in volume over 12 hours, tissue necrosis, and severe neurological dysfunction. SUR1 expression was upregulated in capillaries and neurons surrounding necrotic lesions. Patch clamp of cultured endothelial cells exposed to hypoxia showed that upregulation of SUR1 was associated with expression of functional SUR1-regulated NC(Ca-ATP) channels. Following SCI, block of SUR1 by glibenclamide or repaglinide or suppression of Abcc8, which encodes for SUR1 by phosphorothioated antisense oligodeoxynucleotide essentially eliminated capillary fragmentation and progressive accumulation of blood, was associated with significant sparing of white matter tracts and a 3-fold reduction in lesion volume, and resulted in marked neurobehavioral functional improvement compared with controls. We conclude that SUR1-regulated NC(Ca-ATP) channels in capillary endothelium are critical to development of PHN and constitute a major target for therapy in SCI.

Hemorrhagic Progression of a Contusion after Traumatic Brain Injury: A Review

The magnitude of damage to cerebral tissues following head trauma is determined by the primary injury, caused by the kinetic energy delivered at the time of impact, plus numerous secondary injury responses that almost inevitably worsen the primary injury. When head trauma results in a cerebral contusion, the hemorrhagic lesion often progresses during the first several hours after impact, either expanding or developing new, non-contiguous hemorrhagic lesions, a phenomenon termed hemorrhagic progression of a contusion (HPC). Because a hemorrhagic contusion marks tissues with essentially total unrecoverable loss of function, and because blood is one of the most toxic substances to which the brain can be exposed, HPC is one of the most severe types of secondary injury encountered following traumatic brain injury (TBI). Historically, HPC has been attributed to continued bleeding of microvessels fractured at the time of primary injury. This concept has given rise to the notion that continued bleeding might be due to overt or latent coagulopathy, prompting attempts to normalize coagulation with agents such as recombinant factor VIIa. Recently, a novel mechanism was postulated to account for HPC that involves delayed, progressive microvascular failure initiated by the impact. Here we review the topic of HPC, we examine data relevant to the concept of a coagulopathy, and we detail emerging data elucidating the mechanism of progressive microvascular failure that predisposes to HPC after head trauma.

De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury

The role of transient receptor potential M4 (Trpm4), an unusual member of the Trp family of ion channels, is poorly understood. Using rodent models of spinal cord injury, we studied involvement of Trpm4 in the progressive expansion of secondary hemorrhage associated with capillary fragmentation, the most destructive mechanism of secondary injury in the central nervous system. Trpm4 mRNA and protein were abundantly upregulated in capillaries preceding their fragmentation and formation of petechial hemorrhages. Trpm4 expression in vitro rendered COS-7 cells highly susceptible to oncotic swelling and oncotic death following ATP depletion. After spinal cord injury, in vivo gene suppression in rats treated with Trpm4 antisense or in Trpm4−/− mice preserved capillary structural integrity, eliminated secondary hemorrhage, yielded a threefold to fivefold reduction in lesion volume and produced a substantial improvement in neurological function. To our knowledge, this is the first example of a Trp channel that must undergo de novo expression for manifestation of central nervous system pathology.

Protective Effect of Delayed Treatment With Low-Dose Glibenclamide in Three Models of Ischemic Stroke

Background and Purpose— Ischemia/hypoxia induces de novo expression of the sulfonylurea receptor 1-regulated NC(Ca-ATP) channel. In rodent models of ischemic stroke, early postevent administration of the sulfonylurea, glibenclamide, is highly effective in reducing edema, mortality, and lesion volume, and in patients with diabetes presenting with ischemic stroke, pre-event plus postevent use of sulfonylureas is associated with better neurological outcome. However, the therapeutic window for treatment with glibenclamide has not been studied. Methods— We examined the effect of low-dose (nonhypoglycemogenic) glibenclamide in 3 rat models of ischemic stroke, all involving proximal middle cerebral artery occlusion (MCAo): a thromboembolic model, a permanent suture occlusion model, and a temporary suture occlusion model with reperfusion (105 minutes occlusion, 2-day reperfusion). Treatment was started at various times up to 6 hours post-MCAo. Lesion volumes were measured 48 hours post-MCAo using 2,3,5-triphenyltetrazolium chloride. Results— Glibenclamide reduced total lesion volume by 53% in the thromboembolic MCAo model at 6 hours, reduced corrected cortical lesion volume by 51% in the permanent MCAo model at 4 hours, and reduced corrected cortical lesion volume by 41% in the temporary MCAo model at 5.75 hours (P<0.05 for all 3). Analysis of pooled data from the permanent MCAo and temporary MCAo series indicated a sigmoidal relationship between hemispheric swelling and corrected cortical lesion volume with the half-maximum cortical lesion volume being observed with 10% hemispheric swelling. Conclusions— Low-dose glibenclamide has a strong beneficial effect on lesion volume and has a highly favorable therapeutic window in several models of ischemic stroke.

Glial fibrillary acidic protein is highly correlated with brain injury.

BACKGROUND Glial fibrillary acidic protein (GFAP) is an intermediate filament protein found in the cytoskeleton of astroglia. Recent work has indicated that GFAP may serve as a serum marker of traumatic brain injury (TBI) that is released after central nervous system cell damage. METHODS Serum from 51 critically injured trauma patients was prospectively collected on admission and on hospital day 2. All patients underwent an admission head computed tomography (CT) scan as a part of their clinical evaluation. Patients with facial fractures in the absence of documented TBI and patients with spinal cord injury were excluded. Demographic and outcome data were collected prospectively. Serum GFAP was measured in duplicate using enzyme-linked immunosorbent assay techniques. RESULTS Thirty-nine (76%) of the 51 patients had CT-documented TBI. The study cohort was 72.5% men with a mean age of 43 years and mean Injury Severity Score (ISS) of 30.2. There were no statistically significant demographic differences between the two groups. At admission day, the mean GFAP level in non-TBI patients was 0.07 pg/mL compared with 6.77 pg/mL in TBI patients (p = 0.002). On day 2 the mean GFAP level was 0.02 in non-TBI patients compared with 2.17 in TBI patients (p = 0.003). Using regression analysis to control for age, sex, and ISS, the Head Abbreviated Injury Scale was predictive of the level of GFAP on both days 1 and 2 (p values 0.006 and 0.026, respectively). Although GFAP levels were not predictive of increased hospital length of stay, intensive care unit length of stay, or ventilator days, high GFAP levels on hospital day 2 were predictive of mortality when controlling for age, sex, and ISS (odds ratio 1.45, p value 0.028). The area under the receiver operating characteristic curve for GFAP was 0.90 for day 1 and 0.88 for day 2. A GFAP cutoff point of 1 pg/mL yielded 100% specificity and 50% to 60% sensitivity for TBI. CONCLUSIONS GFAP is a serum marker of TBI, and persistent elevation on day 2 is predictive of increased mortality. Excellent specificity for CT-documented brain injury was found using a cutoff point of 1 pg/mL.

4-AMINOPYRIDINE CAUSES APOPTOSIS AND BLOCKS AN OUTWARD RECTIFIER K+ CHANNEL IN MALIGNANT ASTROCYTOMA CELL LINES

Among the ion channels and pumps activated by growth factor stimulation, K+ channels have been implicated in the growth and proliferation of several cancer cell lines. The role of these channels in central nervous system tumors, however, has not been described. This study used the malignant astrocytoma cell lines U87 and A172. 4‐Aminopyridine (4‐AP) inhibition of proliferation was dose dependent, and assessment using a TUNEL in situ assay revealed that apoptosis occurred in U87 cells with wild‐type p53 but not in A172 cells with mutant p53 (24‐hr incubation with 4 mM 4‐AP). In patch clamp experiments, we identified two types of K+ currents in both cell lines, a charybdotoxin‐sensitive Ca2+‐activated K+ channel and a 4‐AP‐sensitive outward rectifier K+ current. The outward rectifier current was blocked by 4‐AP in a dose‐dependent manner, with half‐maximal block occurring at 3.9 mM. The blocking effect of 4 mM 4‐AP was noticeable at potentials as low as −65 mV and was statistically significant at −60 mV and above, suggesting that 4‐AP‐sensitive current is active at physiological potentials. By contrast, charybdotoxin (1 μM) and tetraethylammonium · Cl (2 mM) blocked the Ca2+‐activated K+ channel in both cell lines but had no appreciable effect on cell growth. Our findings reveal that 4‐AP inhibits proliferation and the outward rectifier K+ channel in both U87 and A172 cells. More studies are needed, however, to describe the mechanism by which K+ channels influence proliferation and induce apoptosis. J. Neurosci. Res. 48:122–127, 1997.