sea-0400 and Brain-Ischemia

Ischemic white matter injury has been relatively little studied despite its importance to the outcome of stroke. To aid such research a new rat model has been developed in vivo and used to assess whether blockade of the sodium/calcium exchanger is effective in protecting central axons from ischemic injury. Vasoconstrictive agent endothelin-1 was injected into the rat spinal cord to induce ischemia. KB-R7943 or SEA0400 was administered systemically to block the operation of the sodium/calcium exchanger. Endothelin-1 caused profound reduction of local blood perfusion and resulted in a prompt loss of axonal conduction. Whereas recovery of conduction following vehicle administration was only to 10.5 ± 9% of baseline (n = 8) 4.5 h after endothelin-1 injection, recovery following KB-R7943 (30 mg/kg, i.a.) administration was increased to 35 ± 9% of baseline (n = 6; P < 0.001). SEA0400 (30 mg/kg, i.a.) was also protective (33.2 ± 6% of baseline, n = 4; P < 0.001). Neither drug improved conduction by diminishing the severity of the ischemia. The protective effect of KB-R7943 persisted for at least 3 days after ischemia, as it improved axonal conduction (76.3 ± 11% for KB-R7943 vs. 51.0 ± 19% for vehicle; P < 0.01) and reduced lesion area (55.6 ± 15% for KB-R7943 vs. 77.9 ± 9% for vehicle; P < 0.01) at this time. In conclusion, a new model of white matter ischemia has been introduced suitable for both structural and functional studies in vivo. Blocking the sodium/calcium exchanger protects central axons from ischemic injury in vivo.

Topics: Aniline Compounds; Animals; Axons; Brain; Brain Ischemia; Electrophysiological Phenomena; Endothelin-1; Immunohistochemistry; Neural Conduction; Neuroprotective Agents; Phenyl Ethers; Rats; Rats, Sprague-Dawley; Sodium-Calcium Exchanger; Thiourea

The Na(+)/Ca(2+) exchanger (NCX), an ion-transporter located in the plasma membrane of neuronal cells, contributes to intracellular Ca(2+) homeostasis. Within the brain, three isoforms (NCX1, NCX2, and NCX3) are widely distributed. However, it is not clear to what extent these isoforms are involved in ischemic brain damage in mammals. We therefore used genetically altered mice and isoform-selective NCX inhibitors in a model of transient focal ischemia to investigate the role of each NCX isoform in ischemic brain damage. NCX isoform-mutant mice (NCX1(+/-), NCX2(+/-), and NCX3(+/-)) and wild-type mice were subjected to 90min of middle cerebral artery occlusion (MCAO) followed by 24h of reperfusion. One of three NCX inhibitors [SN-6, KB-R7943, or SEA0400 (3 or 10mgkg(-1), i.p.)] was administered to ddY mice at 30min before more prolonged (4-h) MCAO followed by 24h of reperfusion. After transient MCAO reperfusion, the cerebral infarcts in NCX1(+/-) mice, but not those in NCX2(+/-) or NCX3(+/-) mice, were significantly smaller than those in wild-type mice. SN-6 and SEA0400, which are more selective for the NCX1 isoform, significantly reduced the infarct volume at 10mg/kg. In contrast, KB-R7943, which is more selective for NCX3, did not. These results suggest that the NCX1 isoform may act preferentially (vs. the NCX2 and NCX3 isoforms) to exacerbate the cerebral damage caused by ischemic insult in mice, and that NCX1-selective inhibitors warrant investigation as a potential therapeutic agents for stroke.

Topics: Aniline Compounds; Animals; Apoptosis; Benzyl Compounds; Brain Infarction; Brain Ischemia; Infarction, Middle Cerebral Artery; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Phenyl Ethers; Sodium-Calcium Exchanger; Thiazolidines; Thiourea

The mechanisms of myocardial dysfunction and calcium handling disturbance underlying cerebral ischemia remain obscure. Here we for the first time report that acute cerebral ischemia significantly increased left ventricular end diastolic pressure (LVEDP), but decreased +dP/dt, -dP/dt, and left ventricular systolic pressure (LVSP). Significant increase in either the resting or KCl-induced [Ca2+](i)in ventricular myocytes was also detected by scanning confocal microscopy at 2 and 24 hours after cerebral ischemia. Verapamil as a blocker of I(Ca,L), ryanodine as a specific inhibitor of RyR, thapsigargin as a highly specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) and SEA0400 as a selective NCX inhibitor changed the area under the curve of averaged ratio of fluorescence (FI/F(0)I) induced by KCl. Cardiac expression of Ca(v)1.2 was significantly up-regulated at 2 and 24 hours after cerebral ischemia, whereas cardiac expression of SERCA2a and Na(+)-Ca(2+) exchanger (NCX) was significantly down-regulated at the same time period after cerebral ischemia. Cardiac expression of phospholamban (PLB) was significantly elevated at 2 hours after cerebral ischemia but was restored to about normal level at 24 hours after injury. These data suggest that acute cerebral ischemia may specifically disturb cardiac function and calcium homeostasis, which are related to increase of Ca(v)1.2 and decrease of through up-regulating Ca(v)1.2 and PLB, down-regulating SERCA2a and NCX, subsequently leading to Ca2+ overload by the enhancement of Ca2+ influx and inhibition of intracellular Ca2+ extrusion and cerebral ischemia-induced myocardial dysfunction.

Topics: Aniline Compounds; Animals; Brain Ischemia; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Enzyme Inhibitors; Heart Ventricles; Male; Muscle Contraction; Myocytes, Cardiac; Phenyl Ethers; Potassium Chloride; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Calcium Exchanger; Thapsigargin; Time Factors; Verapamil

The effect of the newly synthesized compound 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) on the Na+-Ca2+ exchanger (NCX) was investigated and compared against that of 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943). In addition, the effects of SEA0400 on reperfusion injury in vitro and in vivo were examined. SEA0400 was extremely more potent than KB-R7943 in inhibiting Na+-dependent Ca2+ uptake in cultured neurons, astrocytes, and microglia: IC50s of SEA0400 and KB-R7943 were 5 to 33 nM and 2 to 4 microM, respectively. SEA0400 at the concentration range that inhibited NCX exhibited negligible affinities for the Ca2+ channels, Na+ channels, K+ channels, norepinephrine transporter, and 14 receptors, and did not affect the activities of the Na+/H+ exchanger, Na+,K+-ATPase, Ca2+-ATPase, and five enzymes. SEA0400, unlike KB-R7943, did not inhibit the store-operated Ca2+ entry in cultured astrocytes. SEA0400 attenuated dose- dependently paradoxical Ca2+ challenge-induced production of reactive oxygen species, DNA ladder formation, and nuclear condensation in cultured astrocytes, whereas it did not affect thapsigargin-induced cell injury. Furthermore, administration of SEA0400 reduced infarct volumes after a transient middle cerebral artery occlusion in rat cerebral cortex and striatum. These results indicate that SEA0400 is the most potent and selective inhibitor of NCX, and suggest that the compound may exert protective effects on postischemic brain damage.

Topics: Aniline Compounds; Animals; Animals, Newborn; Astrocytes; Brain Ischemia; Calcium Signaling; Cerebral Cortex; Corpus Striatum; Ion Channels; Ion Transport; Phenyl Ethers; Rats; Rats, Wistar; Reperfusion Injury; Sodium-Calcium Exchanger; Thiourea