Abstracts

Rationale: Recently, we described a pathological angiogenesis associated with blood-brain barrier (BBB) disruption in human and animal epileptic focus (Rigau, Morin et al, 2007). Using organotypic hippocampal cultures (OHCs), we demonstrated that in vitro seizures induced neuronal and astroglial expression of the vascular endothelial growth factor (VEGF). The secreted VEGF binds VEGFR-2 receptor which triggers an intense angiogenesis in OHCs (Morin et al. submitted). Moreover, VEGFR-2 promotes BBB degradation, which contributes to epileptogenesis (Ivens et al. 2007, Marchi et al 2006). Nevertheless, anti-angiogenic drugs which inhibit VEGF/VEGFR-2 signalling could not be used to maintain the BBB integrity, because VEGF exerts important protective effects on neurons after seizures (Nicoletti et al, 2008). Therefore, we focused on other compounds able to protect or consolidate selectively the BBB, such as angiopoietins. Methods: OHCs were cultivated according to Stoppini et al (1991). At 10 DIV, OHCs were treated with kainate (25µM) or bicuculline (10µM) to trigger seizures with or without lesions, respectively. At different time-points post-seizure (PS), we carried out immunohistochemistry or western blot. In control and treated OHCs, we compared the expression of three main proteins of tight junctions: ZO-1, occludin and claudin-5 with those of markers of blood vessel (Glut-1 in Western blot, laminin in immunohistochemistry). To test angiopoietins, Ang1 or Ang2 (200, 400 or 800 ng.ml-1) were added in culture medium at 4h PS. 24h later, we checked the expression of tight junction proteins by western blot and immunohistochemistry. Results: 1- Kinetics of BBB impairment after seizures. The expression of ZO-1, occludin and claudin-5 was significantly decreased 1h PS. At 4h PS, occludin and claudin-5 levels were similar to control levels. At 24h PS, ZO-1 expression was still decreased in kainate model, whereas it was identical to controls after bicuculline. Immunofluorescence revealed the loss of ZO-1 staining in pre-existing or immature microvessels. 2- BBB protection. OHC treatment with Ang1, applied 4h PS (kainate), induced at 400ng/ml an increase of ZO-1 expression at 24h PS, whereas Ang2 decreased ZO-1 expression. These primary results are consistent with recent data showing that Ang1 stabilizes vascularisation to the opposite of Ang2 (Shim et al, 2008) Conclusions: Here we showed that BBB is rapidly disrupted after seizures, whatever the severity of lesions. Nevertheless, tight junctions were still degraded 24h PS only in case of neuronal damage, probably due to inflammatory processes known to impair the BBB. Then, we observed that Ang1, applied after kainate-induced seizures, rebuilt the tight junctions. This in vitro model is pertinent to mimic seizure-induced angiogenesis and BBB disruption. It may be useful to screen compounds that reinforce the BBB, bearing in mind that BBB integrity could reduce epileptogenicity