Effect of chronic intracerebroventricluar administration of lipopolysaccharide on connexin43 protein expression in rat hippocampus

Abstract

Background: Hippocampal damages, which are accompanied by inflammation, are among the main causes of epilepsy acquisition. We previously reported that chronic intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS) modulates epileptogenesis in rats. There is a network of gap junction channels in the hippocampus that contribute to epileptogenesis. Gap junction channels are formed by oligomeric protein subunits called connexins (Cx). Astrocytic Cx43 and neuronal Cx36 are expressed in the hippocampus. In order to find out the possible role of gap junctions in seizure-modulating effect of LPS and neuroinflammation, we studied the effect of central administration of LPS on expression of Cx36 and Cx43 in rat hippocampus.

Methods: LPS, 2.5 mug/rat/day, was injected i.c.v. to male Wistar rats for 14 days. mRNA and protein abundance of Cx36, Cx43 and IL1-β were measured in rat hippocampus by real time-PCR, Western blot and ELISA techniques, at the beginning, in the middle, and at the end of the treatment period.

Results: IL1-β protein level was significantly increased 6 h after first injection of LPS. Cx36 and Cx43 mRNA expression did not alter during chronic administration of LPS. A selective decrease in Cx43 protein expression was observed after 7 injections of LPS.

Conclusion: It is suggested that Cx43 containing gap junctions in the hippocampus is down-regulated in response to chronic injection of LPS. This event can inhibit propagation of toxic and noxious molecules to neighboring cells and modulate hippocampal excitability and epileptogenesis.

Fig. 1.

(A) Melting curve analysis for Cx36, Cx43 and α-tubulin gene fragments detected by the real-time PCR assay. Each peak represents a unique PCR product in each reaction. Tm: melting temperature. (α-tubulin Tm = 81.3°C Cx36 Tm = 79.4°C Cx43 Tm = 82.6°C). (B) Amplification plots of the target and reference genes (Cx43, Cx36, α-tubulin) in the real-time PCR assay. mCT: mean threshold cycle. (α-tubulin mCT = 20.69, Cx36 mCT = 31.29 and Cx43 mCT = 25.22).

Fig. 2.

Hippocampal Cx36 (A) and Cx43 (B) gene expressions in rats after acute and chronic intracerebroventricular injection of LPS. The expression was normalized to α- tubulin. There is no significant difference between groups.

Fig. 3.

Hippocampal Cx43 (A) and Cx36 (B) protein expressions in rats after acute and chronic intracerebroventricular injection of LPS. Cx36 and Cx43 protein levels were normalized to that of α-tubulin protein. Data are expressed as means ± S.E.M (n = 6). Panel C shows representative immunoblots of Cx43 and Cx36 in LPS-treated samples. *: P<0.05 compared to control PBS group.