Journal: CNS Neuroscience & Therapeutics

Article Title: KCC2 Dysfunction Mediated by Microglial BDNF / TrkB Signaling Exacerbates Early Post‐Stroke Seizure Susceptibility

doi: 10.1002/cns.70795

Figure Lengend Snippet: Suppression of the BDNF/TrkB signaling pathway by K252a can partly reverse the KCC2 downregulation and dysfunction after hypoxia injury. (A) NeuN and KCC2 immunolabeling in the hippocampal CA1 region of MCAO‐R mice, and those pretreated with K252a are depicted in confocal scanning images. Scale bars: 50 μm. (B) Quantitative assessment of membrane KCC2 labeling density in fluorescently labeled cells relative to NeuN (Sham: N = 4, MCAO‐R N = 7, MCAO‐ R + K252a: N = 7). (C) Confocal microscopy images illustrate KCC2 (green) and NeuN (blue‐violet) expression in cultured hippocampal neurons exposed to OGD/R with or without K252a. (Control: N = 23 cells; OGD/R: N = 21 cells; OGD/ R + K252a: N = 32 cells, all from N = 5 cultures). Scale bar: 5 μm. (D) Quantitative assessment of membrane KCC2 in cultured hippocampal neurons. (E) Experimental determination of E GABA recorded by a small‐tip whole‐cell patch clamp is depicted in I‐V plots. (F) Statistical analysis indicates a significant positive shift in E GABA after MCAO‐R, which is partially reversed by K252a (MCAO‐ R + K252a: −55.0 ± 4.2 mV, N = 3, n = 21 vs. MCAO‐R: −52.1 ± 3.8 mV, N = 3, n = 15, p = 0.08; MCAO‐ R + K252a vs. Sham: −57.5 ± 3.6 mV, N = 4, n = 19, p = 0.12). (G) Patch clamp recordings using a low‐chloride electrode solution demonstrate that both the amplitude (H) and frequency (I) of mIPSCs, which are KCC2‐dependent, are significantly reduced by MCAO‐R, with only the amplitude being partially rescued by pretreatment with K252a (MCAO‐R: 17.0 ± 3.4 pA, N = 3, n = 17 vs. MCAO‐ R + K252a:19.3 ± 2.8 pA, N = 3, n = 18, p = 0.13; MCAO‐R + K252a vs. Sham:21.3 ± 3.3 pA, N = 3, n = 15). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the Sham group; ## p < 0.01 and ### p < 0.001 compared to the MCAO‐R group or OGD/R group.

Article Snippet: FUR was purchased from Sigma‐Aldrich, and minocycline, K252a, and CLP290 were from MedChemExpress.

Techniques: Immunolabeling, Membrane, Labeling, Confocal Microscopy, Expressing, Cell Culture, Control, Patch Clamp

Journal: CNS Neuroscience & Therapeutics

Article Title: KCC2 Dysfunction Mediated by Microglial BDNF / TrkB Signaling Exacerbates Early Post‐Stroke Seizure Susceptibility

doi: 10.1002/cns.70795

Figure Lengend Snippet: Alleviating the severity of MCAO‐R‐induced seizures by blocking the BDNF/TrkB signaling and suppressing microglial activation. (A) Representative traces demonstrate the effective suppression of epileptiform burst firing induced by OGD/R in cultured hippocampal neurons by K252a. (B–D) Bar graphs present quantitative analysis, indicating that blocking the BDNF/TrkB signaling pathway with K252a significantly reduces the percentage of neurons exhibiting epileptiform bursting firing (B), the bursting frequency (C), and the total number of APs during 10 min (D) compared to control neurons (Control: N = 18 cells, OGD/R: N = 24 cells; FUR+OGD/R: N = 11 cells, all from N = 3 cultures). (E, F) Pretreatment with K252a or minocycline significantly decreases the occurrence of Racine IV‐V seizures at 40 mg/kg (E) and 50 mg/kg (F) cumulative PTZ dose. The distribution of seizure scores among mice is represented by the R2, R3, R4, and R5. (G‐H) Line charts reveal that K252a or minocycline reduced the percentage of MCAO‐R mice with Racine IV‐V seizures (G) and the average seizure score (H) at a cumulative PTZ dose of 40 and 50 mg/kg. (I) The cumulative PTZ dose required to induce epileptic behaviors with a Racine IV‐V is increased by blocking the BDNF/TrkB signaling pathway and suppressing microglial activation (Sham: N = 10, MCAO‐R: N = 13, MCAO‐ R + K252a: N = 11, MCAO‐R + Mino: N = 10). (J) Simplified schematic summarizing that MCAO‐R induces microglial activation (↑Iba1) and BDNF release, leading to TrkB activation, KCC2 downregulation, a depolarizing shift in E GABA and increased seizure risk, whereas minocycline and K252a act upstream at microglia and TrkB, respectively, to ameliorate this cascade. * p < 0.05 and ** p < 0.01 compared to the Sham group; # p < 0.05 and ## p < 0.01 compared to the MCAO‐R group.

Article Snippet: FUR was purchased from Sigma‐Aldrich, and minocycline, K252a, and CLP290 were from MedChemExpress.

Techniques: Blocking Assay, Activation Assay, Cell Culture, Control