The protective effect of carbenoxolone on gap junction damage in the hippocampal CA1 area of a temporal lobe epilepsy rat model

Abstract

Background: Astrocytes are one of the most important types of neural cells in the central nervous system (CNS). Dysfunctional gap junction (GJ) communication could play an underlying role in epileptogenesis. Carbenoxolone (CBX) is a conventional chemical GJ blocker, and its target is connexin 43 (Cx43). Previous studies have shown that CBX can inhibit status epilepticus (SE) and spontaneous epileptic seizures (SESs). However, there is little information about the direct interaction between CBX and Cxs in temporal lobe epilepsy (TLE).

Methods: The behavior of epileptic rats was observed. Moreover, micromorphological changes in the hippocampal cornu ammonis 1 (CA1) area of epileptic rats following CBX injection were determined through transmission electron microscopy (TEM). To illustrate the possible mechanism of these changes, the Western blot method was used.

Results: After the injection of CBX, the seizure frequency, seizure duration, latency period to the first instance of SES, SESs behavioral score according to a scoring system developed by Velíšková and microstructures in the CA1 area were shown to be improved 60 days after SE by TEM. Furthermore, the dynamic expression patterns of Cx43 and Cx43 phosphorylated at Ser368 continuously declined after the injection of CBX until 60 days after SE.

Conclusions: CBX may contribute to the improvement of GJ dysfunction during epileptogenesis in the hippocampal CA1 area in a TLE rat model.

Keywords: Epilepsy; astrocyte; carbenoxolone (CBX); connexin 43 (Cx43); gap junction (GJ).

Figure 1

Characteristics of SESs in the SE-60d group and the CBX-60d group. (A) The bar graph shows the average frequency of SESs in the SE-60d group and the CBX-60d group. The average frequency was lower in the CBX-60d group; (B) the bar graph shows the average seizure duration in the SE-60d group and the CBX-60d group. The average seizure duration was lower in the CBX-60d group; (C) the bar graph shows the average seizure latency period in the SE-60d and CBX-60d groups. The average seizure duration was longer in the CBX-60d group; (D) the bar graph shows the average behavioral score in the SE-60d and CBX-60d groups. There was no difference in the average behavioral score between in the two groups. Values are the mean ± SD. Compared to SE-60d: ***, P<0.001, **, P<0.01, *, P<0.05. SES, spontaneous epileptic seizure; SE, status epilepticus; CBX, carbenoxolone; SD, standard deviation.

Figure 2

Changes in the microstructure pattern of the hippocampal CA1 area in a rat model of TLE after CBX injection. (A,B) Normal microstructure of astrocytes and GJs in the hippocampal CA1 area; (C,D,E,F,G,H) dynamic damage to astrocytes and GJs during epileptogenesis; (I,J) improvement of damaged astrocytes and GJs after the injection of CBX, a Cx43 blocker. The alphabet “A” points to “astrocyte”. The black triangle points to an astrocyte, and the long arrow points to a GJ. Scale bars have been shown in each graph. CA1, cornu ammonis 1; TLE, temporal lobe epilepsy; CBX, carbenoxolone; GJ, gap junction; Cx43, connexin 43.

Figure 3

The results of Western blot assays showing the dynamic patterns of Cx43 and p-Cx43 expression in the hippocampus of a rat model of TLE. (A) Representative results with the corresponding antibodies; (B) the bar graph shows Cx43 protein expression. The ROD for Cx43 gradually increased until 60 days after SE; (C) the bar graph shows p-Cx43 protein expression. The ROD for p-Cx43 gradually increased until 60 days after SE. n=6 in each subgroup. Compared to SE-60d: ***, P<0.001. Cx43, connexin 43; TLE, temporal lobe epilepsy; ROD, relative optical density; SE, status epilepticus.

Figure 4

The dynamic patterns of Cx43 and p-Cx43 expression after CBX injection in the hippocampus of a rat model of TLE. (A) Representative results with the corresponding antibodies; (B) the bar graph shows Cx43 protein expression after CBX injection. The ROD for Cx43 was lower in the CBX group than in the Con group; (C) the bar graph shows p-Cx43 protein expression after CBX injection. The ROD for p-Cx43 was lower in the CBX group than in the Con group. n=6 in each subgroup. Compared to SE-60d: ***, P<0.001, **, P<0.01, *, P<0.05. Cx43, connexin 43; CBX, carbenoxolone; TLE, temporal lobe epilepsy; ROD, relative optical density; Con group, control group; SE, status epilepticus.