The KCa3.1 Blocker TRAM-34 Reduces Infarction and Neurological Deficit in a Rat Model of Ischemia/Reperfusion Stroke

Abstract

Supplementary Figure 1 TRAM-34 treatment with 10 mg/kg initiated 2 h after reperfusion and continued for 7 days twice daily noticeably reduced infarct area following similar reductions in cerebral blood flow (control: 51.2 ± 8.2% flux reduction, n = 10; TRAM-34: 49.0 ± 7.4% flux reduction, n = 11). (A) Analysis of each affected brain slice from 2 mm to 16 mm from the frontal pole of the brain, revealed a marked reduction of TTC-defined lesion areas in all slices, which was statistically significant in the 6, 8, 10, 12 and 14-mm slices. (B) The mean infarct area was 18.8 ± 3.5% of the ipsolateral TTC + hemisphere area (n = 10) in controls and 6.6 ± 1.9% in TRAM-34 treated animals (n = 11; mean ± SEM, P = 0.007). (C) TRAM-34 treatment reduces microglia activation. Shown are means (± SEM) of ED1 + hemisphere area from the 8 and 10-mm slices from 5 animals of each group. Effects of Chronic TRAM-34 TreatmentAdult (9-11 week old) male and female rats were purchased from Charles River (Wilmington, MA) and injected i.p. for 28 days with either 20 mg/kg/d of TRAM-34 divided into two daily doses or with vehicle (n = 6 per group with 3 male and 3 female). At the end of the trial, rats were sacrificed and complete blood chemistry, hematology and necropsy performed by the Comparative Pathology Laboratory of the University of California, Davis as follows: 1. Rat were euthanized with CO 2 . 2. Whole animal weight (to mg) determined using an Ohaus analytical scale.3. Blood was collected via cardiac puncture for hematological profile (Complete blood count and differential) into EDTA coated samplette tubes. The rest of the blood will was placed in a serum separator samplette tube and separated by centrifugation at 3500 rpm. 4. The hematological profile was performed by the Comparative pathology Laboratory using a Drew Mascot 850 automated cell counter. Morphology of blood cells and WBC differential was also evaluated manually. 5. Serum Chemistry was performed on a Cobas Mira Plus chemistry analyzer. 6. Necropsies were performed and any gross lesions/changes were documented. 7. Pelage and cecal/intestinal contents was sampled and examined microscopically for parasites. 8. Liver, both kidneys, spleen, GI tract, pancreas, lung, heart, brain, bone marrow, proximal and distal lymph nodes, thymus, gonads and any lesioned organs were collected and fixed in 10% buffered formalin. Tissues were sectioned and processed to paraffin blocks. Hematoxylin and eosin stained sections from each block were examined for histologic changes by trained pathologist and any changes noted. 1) 28-Day Toxicity StudyVehicle (n = 6; 3 male, 3 female) All animals showed extra-medullary hematopoiesis in the red pulp and prominent marginal zones in the white pulp. This is a normal finding in adult rats. ‡ TRAM-34The unilateral hydronephrosis in 2 female control rats and 1 female TRAM-34 treated rat is most likely congenital in origin and has been previously seen in female rats from the same vendor. 2) 6-Months Toxicity StudyIn another trial male and female rats ...

The potassium channel KCa3.1 constitutes a pharmacological target for neuroinflammation associated with ischemia/reperfusion stroke

“…We hypothesized that KCa3.1 channels are mechanistically involved in microglia/macrophage-mediated neuronal damage and demonstrate that both genetic deletion and pharmacological inhibition of KCa3.1 reduce infarct area and improve neurological deficit by reducing microglia/macrophagemediated neuronal killing and inflammatory cytokine production. These results significantly expand our previous findings in a rat model 14 by (1) À/À mice (Mouse Biology Program, UC Davis) were acclimatized to the new vivarium for 5 to 7 days before surgery. Animals were anesthetized using box induction with 5% isoflurane in medical grade oxygen and then maintained on 0.5 to 1.5% isoflurane via a facemask.…”

“…17 Neurological deficits were scored according to a 4-score test 18 and a tactile and/or proprioceptive limb-placing test 19 as previously described. 14 …”

Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke

“…Reversible focal cerebral ischemia was induced by occlusion of the left middle cerebral artery (MCA) according to Zea Longa 24. Briefly, the left common carotid artery was exposed, the external carotid artery ligated distally from the common carotid artery, and a silicone rubber‐coated nylon monofilament with a tip diameter of 0.21 ± 0.02 mm for mice and of 0.43 ± 0.02 mm for rats (Doccol Corp.) inserted into the external carotid artery and advanced into the internal carotid artery to block the origin of the MCA 24, 25. The filament was kept in place for 60 min (mice) or 90 min (rats) and then withdrawn.…”

“…It can be autoclaved and allows for the dissolution of lipophilic compounds like PAP‐1 at high concentrations (5 and 20 mg/mL for this study). Neurological deficits were scored according to a tactile and proprioceptive limb‐placing test26 as previously described 25. The investigator performing the drug administration and neurological scoring was blinded to the treatments.…”

“…Bound primary antibodies were detected with Alexa‐Fluor®546‐ or Alexa‐Fluor®647‐conjugated secondary antibodies (Life Technologies). The IHC staining for rat CD68 in Figure 7A was performed as described 25…”