Svetlana Ivanova

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.

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.

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.

Glibenclamide Is Superior to Decompressive Craniectomy in a Rat Model of Malignant Stroke

Background and Purpose— Treating patients with malignant cerebral infarctions remains a major unsolved problem in medicine. Decompressive craniectomy (DC) improves the bleak outlook but is suboptimal. Using a rat model of severe ischemia/reperfusion with very high mortality due to malignant cerebral edema, we tested the hypothesis that blocking of sulfonylurea receptor 1–regulated NCCa-ATP channels with glibenclamide would compare favorably to DC when reperfusion and treatment were begun 6 hours after onset of ischemia. Methods— Male Wistar rats underwent filament occlusion of the middle cerebral artery to reduce laser Doppler flowmetry perfusion signals by >75%, with filament removal plus treatment 6 hours later. In rats treated with vehicle versus glibenclamide (10 &mgr;g/kg IP plus 200 ng/h SC), we compared mortality, neurologic function, and brain swelling at 24 hours. In rats treated with DC versus glibenclamide, we compared neurologic function for 2 weeks and histologic outcomes. Results— Compared with vehicle, glibenclamide treatment reduced 24-hour mortality from 67% to 5% and reduced hemispheric swelling at 24 hours from 21% to 8%. DC eliminated 24-hour mortality, but neurologic function during the next 2 weeks was significantly better with glibenclamide compared with DC. Watershed cortex and deep white matter were significantly better preserved with glibenclamide compared with DC. Conclusions— In a rat model of severe ischemia/reperfusion, with reperfusion and treatment beginning 6 hours after onset of ischemia, glibenclamide is as effective as DC in preventing death from malignant cerebral edema but is superior to DC in preserving neurologic function and the integrity of watershed cortex and deep white matter.

Rodent model of direct cranial blast injury.

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.

Brief Suppression of Abcc8 Prevents Autodestruction of Spinal Cord After Trauma

Secondary injury that occurs after trauma to the spinal cord can be prevented by inhibiting expression of the gene that regulates a cation transporter. Tackling Spinal Cord Injury Damage to the brain has a way of spreading. The initial injury often sparks a secondary wave of destruction that enlarges the damaged area and increases the ultimate disability of the patient. This process presents a tempting target for therapeutic intervention and, indeed, numerous agents interfere with secondary injury in brain-damaged animals. But none of these potential drugs have proved effective in humans. Simard and his colleagues now hope to bypass these previous dead ends and successfully interfere with secondary damage by basing their animal work on data taken from human victims of spinal cord injury. These authors examined brain tissue from seven patients who had died shortly after traumatic injury to the spinal cord and show that one prominent sequel of local damage is that the surrounding tissues show higher than normal concentrations of messenger RNA (mRNA) and protein for the sulfonylurea receptor 1 (SUR1). Their data from rat and mouse show the same thing. This receptor associates with pores in cell membranes to form ion channels, one of which causes cell depolarization and ultimately cell death, creating the wave of secondary damage to the cord. Simard et al. then report that mice in which SUR1 had been genetically removed suffer much less damage to the spinal cord after injury, a result of a less robust wave of spreading damage. Treatment of rats, a better model of human spinal cord injury than mice, with antisense nucleotides that inhibit SUR1 mRNA or with glibenclamide, a nonspecific inhibitor of the whole class of SUR-like proteins, both protected against secondary injury. The capillaries in the cord surrounding the injury were intact rather than fragmented as they are in untreated rats, and the treated rats performed better on a battery of behavioral tests, showing their superior neurological function. Upon later examination, the size of the lesion in the treated animals was only one-quarter the size of the lesion in control animals. SUR1, therefore, may be a critical element in causing the secondary damage of brain trauma, in humans and rodents. Therapeutic agents that interfere with its injury-induced stimulation of ion channels should be tested in injured patients to determine whether the devastating disability that often results from spinal cord injury can be minimized. 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.

Mislocalization of eNOS and Upregulation of Cerebral Vascular Ca2+ Channel Activity in Angiotensin-Hypertension

Abstract—We tested the hypothesis that endothelial dysfunction induced by angiotensin II (Ang-hypertension) would impair regulatory control of vascular smooth muscle L-type Ca2+ channels by endothelial nitric oxide synthase (eNOS). We studied cerebral lenticulostriate arterioles (LSAs) from control rats, from rats infused with Ang (240 &mgr;g · kg−1 · h−1 SQ ×4 days), which were normotensive, and from Ang-hypertensive rats (AHR; 240 &mgr;g · kg−1 · h−1 ×28 days). Patch-clamp measurements on isolated LSA smooth muscle cells (SMCs) showed a significant increase in Ca2+ channel availability with 4- and 28-day infusions versus controls (0.47±0.03 and 0.66±0.05 vs 0.36±0.03 pS/pF, respectively;P <0.01), with Western blots showing no change in channel protein expression, consistent with altered channel regulation. In LSAs from 28-day AHR, 4,5-diaminofluorescein diacetate imaging showed diminished NO production in response to acetylcholine stimulation in vivo, and inhibition of eNOS with NG-nitro-l-arginine methyl ester failed to increase Ca2+ channel availability in isolated SMCs, indicating an abnormality with the eNOS/NO-signaling pathway regulating the channel. Immunofluorescence imaging showed that in 1 of 53, 33 of 109, and 53 of 62 LSAs from controls and from rats with 4- and 28-day infusions, respectively, eNOS was absent from its normal location at the abluminal border and was mislocalized to perinuclear Golgi. Ca2+ channel availability in LSA SMCs from controls and from rats with 4- and 28-day infusions was proportional to the fraction of LSAs showing eNOS mislocalization, but not blood pressure. These data provide the first evidence linking Ang-induced eNOS mislocalization, eNOS dysfunction, and Ca2+ channel upregulation, and they provide novel mechanistic insights into pathological changes in LSAs associated with stroke.

Alternative Splicing of cGMP-Dependent Protein Kinase I in Angiotensin-Hypertension: Novel Mechanism for Nitrate Tolerance in Vascular Smooth Muscle

Abstract— Nitrate tolerance (NT) in hypertension is attributed to reduced activity of soluble guanylyl cyclase (sGC). We examined NT in basilar artery vascular smooth muscle cells (VSMCs) from control rats, rats infused with angiotensin II (Ang; 240 &mgr;g/kg per hour for 4 days), which were normotensive, and Ang-hypertensive rats (AHR; 240 &mgr;g/kg per hour for 28 days). Ca2+-activated K+ (Maxi-K) channels in VSMCs from AHR showed reduced activation by NO donor, consistent with NT. The concentration-response relationship for 8-Br-cGMP was shifted 2.5-fold to the right, indicating that abnormal sGC alone could not account for NT. Inside-out patches from AHR showed normal activation with exogenous cGMP-dependent protein kinase I (cGKI), suggesting no abnormality downstream of cGKI. We hypothesized that the reduction in apparent affinity of 8-Br-cGMP for cGKI in AHR might be due to a change in relative amounts of cGKI&agr; versus cGKI&bgr;, since cGKI&bgr; is less sensitive to cGMP activators than cGKI&agr;. This was substantiated by showing the following in AHR: (1) reduced effect of the cGKI&agr;-selective activator 8-APT-cGMP; (2) reduced total cGKI protein (both isoforms), but an increase in cGKI&bgr; protein in quantitative immunofluorescence and Western blots; (3) similar changes in cGKI isoforms immunoisolated with Maxi-K channels; and (4) a large increase in cGKI&bgr; mRNA and a decrease in cGKI&agr; mRNA in real-time PCR and Northern blots. Upregulation of cytosolic cGKI&bgr; was evident 4 days after Ang infusion, before development of hypertension. Our data identify a functional role for cGKI&bgr; in VSMCs previously ascribed exclusively to cGKI&agr;. Ang-induced alternative splicing of cGKI represents a novel mechanism for reducing sensitivity to NO/cGMP.

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.