tram-34 and Reperfusion-Injury

Microglia and brain infiltrating macrophages significantly contribute to the secondary inflammatory damage in the wake of ischemic stroke. Here, we investigated whether inhibition of KCa3.1 (IKCa1/KCNN4), a calcium-activated K(+) channel that is involved in microglia and macrophage activation and expression of which increases on microglia in the infarcted area, has beneficial effects in a rat model of ischemic stroke. Using an HPLC/MS assay, we first confirmed that our small molecule KCa3.1 blocker TRAM-34 effectively penetrates into the brain and achieves micromolar plasma and brain concentrations after intraperitoneal injection. Then, we subjected male Wistar rats to 90 minutes of middle cerebral artery occlusion (MCAO) and administered either vehicle or TRAM-34 (10 or 40 mg/kg intraperitoneally twice daily) for 7 days starting 12 hours after reperfusion. Both compound doses reduced infarct area by ≈ 50% as determined by hematoxylin & eosin staining on day 7 and the higher dose also significantly improved neurological deficit. We further observed a significant reduction in ED1(+)-activated microglia and TUNEL-positive neurons as well as increases in NeuN(+) neurons in the infarcted hemisphere. Our findings suggest that KCa3.1 blockade constitutes an attractive approach for the treatment of ischemic stroke because it is still effective when initiated 12 hours after the insult.

Topics: Animals; Blood Proteins; Brain; Chromatography, High Pressure Liquid; Immunohistochemistry; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Intermediate-Conductance Calcium-Activated Potassium Channels; Macrophage Activation; Macrophages; Male; Mass Spectrometry; Microglia; Nervous System Diseases; Neuroprotective Agents; Permeability; Protein Binding; Pyrazoles; Rats; Rats, Sprague-Dawley; Rats, Wistar; Reperfusion Injury

The role of SK(Ca) and IK(Ca) channels in myogenic tone and endothelium-derived hyperpolarizing factor (EDHF) responsiveness was investigated under control conditions and after ischemia and reperfusion in parenchymal arterioles (PA) versus middle cerebral arteries (MCA).. MCA and PA were dissected from male Wistar rats that were ischemic for 1 hour with 24 hours of reperfusion (n=12) or sham controls (n=12). Basal tone and reactivity to apamin (300 nmol/L), TRAM-34 (1.0 micromol/L), and nitro-L-arginine (0.1 mmol/L) were compared in PA and MCA pressurized to 40 mm Hg and 75 mm Hg, respectively. SK(Ca) and IK(Ca) channel mRNA expression was measured using real-time PCR.. PA developed greater basal tone than MCA (42+/-4% versus 19+/-3%; P<0.01). Addition of apamin and TRAM-34 increased tone of PA by 25+/-3% and 16+/-2%, respectively, whereas MCA had no response to either inhibitor. After ischemia and reperfusion, the response to nitric oxide synthase inhibition (NOS) was diminished in PA, whereas EDHF responsiveness was preserved. In addition, stimulated EDHF dilation was partially reversed by apamin and completely reversed by TRAM-34 in both control and ischemic PA. SK(Ca) and IK(Ca) channel mRNA expression was similar in PA and MCA and not altered by ischemia and reperfusion. However, IK(Ca) channel mRNA expression was 4- to 5-fold greater than SK(Ca) channels.. It appears that SK(Ca) and IK(Ca) channel activity diminishes basal tone of PA, but not MCA. The preservation of EDHF responsiveness of PA after ischemia and reperfusion suggests an important role for this vasodilator under conditions when NOS is inhibited.

Topics: Animals; Apamin; Arterioles; Biological Factors; Brain Ischemia; Calcimycin; Disease Models, Animal; Enzyme Inhibitors; Intermediate-Conductance Calcium-Activated Potassium Channels; Ionophores; Male; Middle Cerebral Artery; Nitric Oxide; Nitroarginine; Pyrazoles; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Small-Conductance Calcium-Activated Potassium Channels; Vasodilation