Figure 5.
Ecm29 KO mice exhibit increased seizure susceptibility. (A) Ecm29 homozygous KO mice exhibit increased epileptogenesis. (A1) Experimental paradigm used to assess seizure activity following the flurothyl kindling epileptogenesis model. (A2) Latency to myoclonic in wild-type (Ecm29+/+; n = 15), Ecm29 heterozygous (Ecm29+/−; n = 15), and Ecm29 null (Ecm29−/−; n = 15) mice after flurothyl treatment (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001 compared with wild-type group; multiple t tests). (A3) Changes in myoclonic seizure latency of the first (D1) and second (D2) flurothyl kindling traits indicate increased susceptibility of Ecm29 KO (Ecm29−/−) mice. *, P < 0.05; ns, not significant by unpaired t test. (A4) Shorter latency from myoclonic to generalized seizure of the first kindling traits (Day 1) reveals a lower convulsive threshold in Ecm29 KO (Ecm29−/−) mice (**, P < 0.01; ns, not significant by unpaired t test). (A5) Survival of Ecm29 homozygous KO mice was significantly lower than that of wild-type or Ecm29 heterozygotes over eight seizure traits. *, P < 0.05 based on log-rank test. (A6) Paradigm shown in top panel depicts time courses of daily DMSO (2% in 100 µl saline) or BUM (0.2 mg/kg body weight) administration at perinatal ages (from P7 to P14), followed by flurothyl exposure once daily for 8 d from P35 (red bars). Histograms of latency from myoclonic to generalized seizure of the first kindling traits (middle panel) and susceptibility index (bottom panel) show that daily BUM administration (0.2 mg/kg body weight) at perinatal ages (P7–P14) reduces severity of flurothyl-induced seizures in Ecm29 KO juvenile mice. Data points represent mean ± SEM; n = 11–15 animals per group; *, P < 0.05, two-way ANOVA followed by Tukey's multiple comparison test, comparing data to the Ecm29+/+ group and corresponding DMSO (2% in 100 µl saline) control groups. (B1) Ecm29 homozygous KO mice (Ecm29−/−) exhibit increased susceptibility to PTZ-induced seizures (60 mg/kg; i.p.) relative to wild-type mice. Hypoactivity (HYPO; phase 1); PC (phase 2); GC (phase 3); GTC (phase 4). Data points represent mean ± SEM; n = 20 animals per group; *, P < 0.05; **, P < 0.01, t test comparing data to the Ecm29+/+ group. (B2) Higher number of MCs and GC/GTCs reveals greater seizure severity in Ecm29 KO (Ecm29−/−) mice. Data points represent mean ± SEM; n = 20 animals per group; *, P < 0.05, t test comparing data to the Ecm29+/+ group. (B3) 60 mg/kg was not the lethal dose for most test mice, based on log-rank test. (C) NKCC1 inhibition rescues Ecm29−/− juvenile mice from hypersensitivity to PTZ-induced convulsion. (C1 and C2) Paradigm shown in top panel depicts time courses of daily DMSO (2% in 100 µl saline) or BUM (0.2 mg/kg body weight) administration at perinatal ages (P7–P14, C1) or at late juvenile stages (P50–P57, C2), followed by 60 mg/kg PTZ i.p. challenge at P60 (red dot). Histograms of number of PC/GTC episodes (middle and bottom panels, C1) and susceptibility scores (middle and bottom panels, C2) show that BUM administration at P7 reduces severity of PTZ-induced seizures in Ecm29 KO mice. Data points represent mean ± SEM; n = 11–22 animals per group; *, P < 0.05; **, P < 0.01; ns, not significant by t test comparing data to the Ecm29+/+ group and corresponding DMSO control groups.

Ecm29 KO mice exhibit increased seizure susceptibility. (A) Ecm29 homozygous KO mice exhibit increased epileptogenesis. (A1) Experimental paradigm used to assess seizure activity following the flurothyl kindling epileptogenesis model. (A2) Latency to myoclonic in wild-type (Ecm29+/+; n = 15), Ecm29 heterozygous (Ecm29+/−; n = 15), and Ecm29 null (Ecm29−/−; n = 15) mice after flurothyl treatment (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001 compared with wild-type group; multiple t tests). (A3) Changes in myoclonic seizure latency of the first (D1) and second (D2) flurothyl kindling traits indicate increased susceptibility of Ecm29 KO (Ecm29−/−) mice. *, P < 0.05; ns, not significant by unpaired t test. (A4) Shorter latency from myoclonic to generalized seizure of the first kindling traits (Day 1) reveals a lower convulsive threshold in Ecm29 KO (Ecm29−/−) mice (**, P < 0.01; ns, not significant by unpaired t test). (A5) Survival of Ecm29 homozygous KO mice was significantly lower than that of wild-type or Ecm29 heterozygotes over eight seizure traits. *, P < 0.05 based on log-rank test. (A6) Paradigm shown in top panel depicts time courses of daily DMSO (2% in 100 µl saline) or BUM (0.2 mg/kg body weight) administration at perinatal ages (from P7 to P14), followed by flurothyl exposure once daily for 8 d from P35 (red bars). Histograms of latency from myoclonic to generalized seizure of the first kindling traits (middle panel) and susceptibility index (bottom panel) show that daily BUM administration (0.2 mg/kg body weight) at perinatal ages (P7–P14) reduces severity of flurothyl-induced seizures in Ecm29 KO juvenile mice. Data points represent mean ± SEM; n = 11–15 animals per group; *, P < 0.05, two-way ANOVA followed by Tukey's multiple comparison test, comparing data to the Ecm29+/+ group and corresponding DMSO (2% in 100 µl saline) control groups. (B1) Ecm29 homozygous KO mice (Ecm29−/−) exhibit increased susceptibility to PTZ-induced seizures (60 mg/kg; i.p.) relative to wild-type mice. Hypoactivity (HYPO; phase 1); PC (phase 2); GC (phase 3); GTC (phase 4). Data points represent mean ± SEM; n = 20 animals per group; *, P < 0.05; **, P < 0.01, t test comparing data to the Ecm29+/+ group. (B2) Higher number of MCs and GC/GTCs reveals greater seizure severity in Ecm29 KO (Ecm29−/−) mice. Data points represent mean ± SEM; n = 20 animals per group; *, P < 0.05, t test comparing data to the Ecm29+/+ group. (B3) 60 mg/kg was not the lethal dose for most test mice, based on log-rank test. (C) NKCC1 inhibition rescues Ecm29−/− juvenile mice from hypersensitivity to PTZ-induced convulsion. (C1 and C2) Paradigm shown in top panel depicts time courses of daily DMSO (2% in 100 µl saline) or BUM (0.2 mg/kg body weight) administration at perinatal ages (P7–P14, C1) or at late juvenile stages (P50–P57, C2), followed by 60 mg/kg PTZ i.p. challenge at P60 (red dot). Histograms of number of PC/GTC episodes (middle and bottom panels, C1) and susceptibility scores (middle and bottom panels, C2) show that BUM administration at P7 reduces severity of PTZ-induced seizures in Ecm29 KO mice. Data points represent mean ± SEM; n = 11–22 animals per group; *, P < 0.05; **, P < 0.01; ns, not significant by t test comparing data to the Ecm29+/+ group and corresponding DMSO control groups.

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