Pictorial representation of experimental procedure. The figure depicts the several time points (at day 1 and day 10) in the corresponding experimental groups where treatments were performed with VC, Pilo, PTZ, DZP and three doses of mAb. In day 1 and day 10, there is a recording of locomotor behavior for 15 min for each experimental group. On day 10, in the DZP groups and mAbs groups, pre-treatment was done 30 min prior to PTZ administration. Starting from day 10, T-maze test were performed for each group at the interval of 0h, 3h and 24 h. After 24 h T-maze test, all the fish were euthanized, and brain samples were collected which were further subjected to biochemical investigations. D1, Day 1; D10, Day 10, D11, Day 11; VC, Vehicle control (10% DMSO, I.P.); DMSO, dimethyl sulfoxide; I.P., Intraperitoneal; Pilo, Pilocarpine (400 mg/kg, I.P.); PTZ, Pentylenetetrazol (80 mg/kg, I.P.); DZP, Diazepam (1.25 mg/kg, I.P.); mAb 1, Anti-HMGB1 mAb (1 mg/kg, I.P.); mAb 2.5, Anti-HMGB1 mAb (2.5 mg/kg, I.P.); mAb 5, Anti-HMGB1 mAb (5 mg/kg, I.P.).

Representative diagram of T-maze tank. Zebrafish is kept at the dropping point and recorded the time taken to reach the deeper/bigger chamber. The blue dotted line denotes the representative swimming pattern of zebrafish that begins from the dropping point.

Representative swimming patterns for the corresponding experimental groups (n = 12) at day 10. Represents the swimming pattern of VC, Pilo, PTZ, second hit PTZ, DZP, mAb 1, mAb 2.5, and mAb 5 groups.

Mean locomotor parameters of the experimental groups (n = 12) at day 10. Mean locomotion parameters of the experimental groups representing the mean total distance traveled (cm) (A), mean time spent in upper half of the tank in (seconds) (B) and the mean time spent in lower half of the tank (seconds) (C). Data are represented as mean ± SEM, n = 12, and statistically analyzed by one-way ANOVA followed by Sidak’s multiple comparison test. ^&&&p < 0.001 and ^&&p < 0.01 represent the significance level when second hit PTZ group is compared with VC group. ***p < 0.001 and **p < 0.01 represent the significance level when second hit PTZ group is compared with Pilo group. ^$$p < 0.01 and ^$p < 0.05 represents the significance level when DZP, mAb 1, mAb 2.5, and mAb 5 treated groups is compared with second hit PTZ group.

Anti-epileptic effect of mAb against second hit PTZ-induced seizures. Seizure scores of corresponding experimental groups are demonstrated for day 10. Data are represented as mean ± SEM, n = 12, and statistically analyzed by one-way ANOVA followed by Sidak’s multiple comparison test. ^&&&&p < 0.0001, ****p < 0.0001 and ^##p < 0.01 represent the significance level when second hit PTZ group is compared with VC group, Pilo group and PTZ group, respectively. ^$$$p < 0.001 and ^$$p < 0.01 represents the significance level when DZP, mAb 1, mAb 2.5, and mAb 5 treated groups is compared with second hit PTZ group.

T-maze tracking pattern (24 h trial) of locomotor behavior for the corresponding experimental groups (n = 12). Represents the swimming pattern of VC, Pilo, PTZ, second hit PTZ, DZP, mAb 1, mAb 2.5, and mAb 5 groups.

Graph plot of the inflection ratio at the 3 h and 24 h T-maze trial of the corresponding experimental groups. Inflection ratio at the 3 h (A) and 24 h (B) T-maze trial of the corresponding experimental groups Data was represented as mean ± SEM, n = 12, and statistically analyzed by one-way ANOVA followed by Sidak’s multiple comparison test. ^&p < 0.05 and ^#p < 0.05 represents the significance level when second hit PTZ group is compared with VC group and PTZ group respectively. ^$p < 0.05 represents the significance level when DZP, mAb 1, mAb 2.5, and mAb 5 treated groups is compared with second hit PTZ group.

Analysis of Neurotransmitters level in epileptic zebrafish brains after pre-treatment with anti-HMGB1 mAb. Concentration of GABA (A) and Glutamate (B) and GABA to Glutamate ratio (C) in the corresponding experimental groups. Data are represented as mean ± SEM, n = 6, and statistically analyzed by one-way ANOVA followed by Sidak’s multiple comparison test. ^&&&p < 0.001 represents the significance level when second hit PTZ group is compared with VC group. ***p < 0.001 and *p < 0.05 represents the significance level when second hit PTZ group is compared with Pilo group. ^###p < 0.001 and ^#p < 0.05 represents the significance level when second hit PTZ group is compared with PTZ group. ^$$$p < 0.001, ^$$p < 0.01 and ^$p < 0.05 represent the significance level when DZP, mAb 1, mAb 2.5, and mAb 5 treated groups is compared with second hit PTZ group.

Modulation of inflammatory mediator (HMGB1, TLR4, NF-?B, TNF-?), transcription factor (CREB-1) and neuropeptide (NPY). The graph plot is represented as a fold changes in mRNA expression of HMGB1 (A), TLR4 (B), NF-?B (C), TNF-? (D), CREB-1 (E) and NPY (F). Data are represented as mean ± SEM, n = 6, and statistically analyzed by one-way ANOVA followed by Sidak?s multiple comparison test. &&&p < 0.001 and &p < 0.05 represents the significance level when second hit PTZ group is compared with VC group. ***p < 0.001 and *p < 0.05 represents the significance level when second hit PTZ group is compared with Pilo group. ###p < 0.001 and ##p < 0.01 represents the significance level when second hit PTZ group is compared with PTZ group. $$$p < 0.001, $$p < 0.01 and $p < 0.05 represent the significance level when DZP, mAb 1, mAb 2.5, and mAb 5 treated groups is compared with second hit PTZ group.