Orest Tsymbalyuk

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.

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.

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.

Spinal cord injury with unilateral versus bilateral primary hemorrhage — Effects of glibenclamide

In spinal cord injury (SCI), block of Sur1-regulated NC(Ca-ATP) channels by glibenclamide protects penumbral capillaries from delayed fragmentation, resulting in reduced secondary hemorrhage, smaller lesions and better neurological function. All published experiments demonstrating a beneficial effect of glibenclamide in rat models of SCI have used a cervical hemicord impact calibrated to produce primary hemorrhage located exclusively ipsilateral to the site of impact. Here, we tested the hypothesis that glibenclamide also would be protective in a model with more extensive, bilateral primary hemorrhage. We studied the effect of glibenclamide in 2 rat cervical hemicord contusion models with identical impact force (10 g, 25 mm), one with the impactor positioned laterally to yield unilateral primary hemorrhage (UPH), and the other with the impactor positioned more medially, yielding larger, bilateral primary hemorrhages (BPH) and 6-week lesion volumes that were 45% larger. Functional outcome measures included: modified (unilateral) Basso, Beattie, and Bresnahan scores, angled plane performance, and rearing times. In the UPH model, the effects of glibenclamide were similar to previous observations, including a functional benefit as early as 24h after injury and 6-week lesion volumes that were 57% smaller than controls. In the BPH model, glibenclamide exerted a significant benefit over controls, but the functional benefit was smaller than in the UPH model and 6-week lesion volumes were 33% smaller than controls. We conclude that glibenclamide is beneficial in different models of cervical SCI, with the magnitude of the benefit depending on the magnitude and extent of primary hemorrhage.

Adenylate cyclase 5 and KCa1.1 channel are required for EGFR up-regulation of PCNA in native contractile rat basilar artery smooth muscle

In synthetic phenotype vascular smooth muscle cells (VSMC), activation of epidermal growth factor (EGF) receptor (EGFR) induces a sustained increase in intermediate conductance KCa (int‐KCa; KCa3.1) channels that is essential for proliferation. However, a comparable mechanism has not been identified in native contractile phenotype VSMC, which express large conductance KCa (maxi‐KCa; KCa1.1) channels, not int‐KCa channels. Using patch clamp of freshly isolated contractile VSMC from rat basilar artery, we found that EGF (100 ng ml−1) caused hyperpolarization (7.9 ± 3.9 mV) due to activation of iberiotoxin‐sensitive, maxi‐KCa channels. The EGFR ligands EGF (100 ng ml−1), transforming growth factor α (0.4 ng ml−1) and heparin‐binding EGF (100 ng ml−1) all caused a 20% increase in maxi‐KCa channel current that was blocked by AG‐1478 or by knock‐down of EGFR expression using cisterna magna infusion of antisense oligodeoxynucleotide (AS‐ODN). In controls, EGFR knock‐down, and EGFR gain‐of‐expression (angiotensin II hypertension), the increase in maxi‐KCa current correlated with the abundance of EGFR protein expressed. The EGFR‐mediated increase in maxi‐KCa channel activity was blocked by inhibiting cAMP‐dependent protein kinase (cAK) using KT‐5720 or Rp‐cAMP, or by inhibiting adenylate cyclase type 5 (AC‐5) using 2′,5′‐dideoxyadenosine or knock‐down of AC‐5 expression by intracisternal AS‐ODN. Direct infusion of EGF into cisterna magna caused up‐regulation of proliferating cell nuclear antigen (PCNA) in VSMC that was prevented by coinfusion of iberiotoxin or of AG‐1478. Our data, which are consistent with the hypothesis that hyperpolarization is critical for a proliferative response, are the first to implicate AC‐5 and maxi‐KCa channels in gene activation related to EGFR signalling in native contractile VSMC.

SUR1-TRPM4 and AQP4 form a heteromultimeric complex that amplifies ion/water osmotic coupling and drives astrocyte swelling

Astrocyte swelling occurs after central nervous system injury and contributes to brain swelling, which can increase mortality. Mechanisms proffered to explain astrocyte swelling emphasize the importance of either aquaporin‐4 (AQP4), an astrocyte water channel, or of Na+‐permeable channels, which mediate cellular osmolyte influx. However, the spatio‐temporal functional interactions between AQP4 and Na+‐permeable channels that drive swelling are poorly understood. We hypothesized that astrocyte swelling after injury is linked to an interaction between AQP4 and Na+‐permeable channels that are newly upregulated. Here, using co‐immunoprecipitation and Förster resonance energy transfer, we report that AQP4 physically co‐assembles with the sulfonylurea receptor 1—transient receptor potential melastatin 4 (SUR1‐TRPM4) monovalent cation channel to form a novel heteromultimeric water/ion channel complex. In vitro cell‐swelling studies using calcein fluorescence imaging of COS‐7 cells expressing various combinations of AQP4, SUR1, and TRPM4 showed that the full tripartite complex, comprised of SUR1‐TRPM4‐AQP4, was required for fast, high‐capacity transmembrane water transport that drives cell swelling, with these findings corroborated in cultured primary astrocytes. In a murine model of brain edema involving cold‐injury to the cerebellum, we found that astrocytes newly upregulate SUR1‐TRPM4, that AQP4 co‐associates with SUR1‐TRPM4, and that genetic inactivation of the solute pore of the SUR1‐TRPM4‐AQP4 complex blocked in vivo astrocyte swelling measured by diolistic labeling, thereby corroborating our in vitro functional studies. Together, these findings demonstrate a novel molecular mechanism involving the SUR1‐TRPM4‐AQP4 complex to account for bulk water influx during astrocyte swelling. These findings have broad implications for the understanding and treatment of AQP4‐mediated pathological conditions.

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

Abstract 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.