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. 2022 May 9:15:870947.
doi: 10.3389/fnmol.2022.870947. eCollection 2022.

Anticonvulsant Effect of Carbenoxolone on Chronic Epileptic Rats and Its Mechanism Related to Connexin and High-Frequency Oscillations

Benke Liu  1   2   3 , Xiao Ran  1   2 , Yanjun Yi  1   2 , Xinyu Zhang  1   2 , Hengsheng Chen  1   2 , Yue Hu  1   2
Affiliations

Anticonvulsant Effect of Carbenoxolone on Chronic Epileptic Rats and Its Mechanism Related to Connexin and High-Frequency Oscillations

Benke Liu et al. Front Mol Neurosci. .

Abstract

Objective: This study was designed to investigate the influence and mechanism of gap junction carbenoxolone (CBX) on dynamic changes in the spectral power of ripples and fast ripples (FRs) in the hippocampus of chronic epileptic rats.

Methods: The lithium-pilocarpine (PILO) status epilepticus (SE) model (PILO group) and the CBX pretreatment model (CBX + PILO group) were established to analyze dynamic changes in the spectral power of ripples and FRs, and the dynamic expression of connexin (CX)26, CX32, CX36, and CX43 in the hippocampus of chronic epileptic rats.

Results: Within 28 days after SE, the number of spontaneous recurrent seizures (SRSs) in the PILO group was significantly higher than that in the CBX + PILO group. The average spectral power of FRs in the PILO group was significantly higher than the baseline level at 1 and 7 days after SE. The average spectral power of FRs in the PILO group was significantly higher than that in the CBX + PILO group at 1, 7, and 14 days after SE. Seizures induced an increase in CX43 expression at 1 and 7 days after SE, but had no significant effect on CX26, CX36, or CX32. CBX pretreatment did not affect the expression of CXs in the hippocampus of normal rats, but it inhibited the expression of CX43 in epileptic rats. The number of SRSs at 2 and 4 weeks after SE had the highest correlation with the average spectral power of FRs; the average spectral power of FRs was moderately correlated with the expression of CX43.

Conclusion: The results of this study indicate that the energy of FRs may be regulated by its interference with the expression of CX43, and thus, affect seizures. Blocking the expression of CX43 thereby reduces the formation of pathological high-frequency oscillations (HFOs), making it a promising strategy for the treatment of chronic epilepsy.

Keywords: carbenoxolone; chronic epilepsy; connexin; fast ripples; ripples.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Comprehensive modeling of chronic epilepsy. (A) Schematic representation of the overall protocol and various analyses performed in chronic epileptic rats. (B) The positions of the EEG recording electrodes. CBX, carbenoxolone; AP, anteroposterior; ML, mediolateral; DV, dorsoventral.
FIGURE 2
FIGURE 2
Lithium-pilocarpine produced chronic SRSs in rats. (A) Average latent phase in PILO and CBX + PILO groups. (B) Incidence curve of epileptic rats over time, showing latent phase to first seizure after SE. (C) Average number of SRSs in PILO and CBX + PILO groups during the 4 weeks after SE. (D) The number of SRSs per week after SE in PILO and CBX + PILO groups. (E,F) Bubble diagram of the relationship between the number of SRSs and the spectral power of ripples/FRs at 2 and 4 weeks after SE in PILO and CBX + PILO groups, the gathered bubbles of the two groups means weak correlation, and the separated bubbles of the two groups means strong correlation. (G) The correlation coefficient matrix of the number of SRSs and the spectral power of HFOs at 2 and 4 weeks after SE (average spectral power of FRs, r = 0.38; peak spectral power of FRs, r = 0.23; peak spectral power of ripples, r = 0.11; average spectral power of ripples, r = 0.10). *P < 0.05; SRSs, spontaneous recurrent seizures; PILO, pilocarpine; SE, status epilepticus; FRs, fast ripples; HFOs, high-frequency oscillations.
FIGURE 3
FIGURE 3
Waveforms and spectrograms features of lithium-pilocarpine-induced electrographic seizures. (A) Representative EEG traces of the baseline, epileptic seizure, and an artifact. (B–F) Waveforms and spectrograms showing the spectral and temporal characteristics of ripples (80–200 Hz) and FRs (250–500 Hz) in the PILO and CBX + PILO groups at 5 time points [1 day before SE, and 1, 7, 14, and 28 days after SE]. The waveforms were filtered with a band pass filter of 80–200 Hz and 250–500 Hz. The spectrograms reflect the accumulated time-frequency representations of the corresponding waveforms. The data demonstrate that the initiation of seizures was associated with an increase of HFOs energy, and that pretreatment with CBX could reduce HFOs energy and the degree of seizures. The HFOs are characterized by rhythmic bursts, which are significantly different from systematic artifacts, such as power line noise and its harmonics.
FIGURE 4
FIGURE 4
Expression of four kinds of CXs. (A–D) Western blot showing the expression of CX26, CX32, CX36, and CX43 in the hippocampus before and after SE in the PILO and CBX + PILO groups. (E) β-actin is used as a loading control. Each bar represents the mean ± SD of eight separate assays. *P < 0.05, **P < 0.01; CX, connexin.
FIGURE 5
FIGURE 5
Correlation analysis of the average spectral power of HFOs and the expression of CX26, CX32, CX36, and CX43. (A–D) There was no significant correlation between the average spectral power of ripples and the expression of CX26, CX32, CX36, or CX43 (P > 0.05). (E,F) There was no significant correlation between the average spectral power of FRs and the expression of CX26 or CX32 (p > 0.05). (G) The average spectral power of FRs was weakly correlated with CX36 expression (r = 0.37, p < 0.05) and (H) moderately correlated with CX43 expression (r = 0.42, p < 0.05).
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