Abstracts

Rationale: The most widely used model of temporal lobe epilepsy consists of peripheral administration of the cholinergic muscarinic agonist pilocarpine which leads to status epilepticus and subsequent spontaneous seizures. It has been generally accepted that these effects are primarily due to cholinergic activation of limbic cortex and hippocampus. However, recent evidence pointed to an alternative mechanism of action that involved peripheral inflammation and leakage across the blood-brain barrier (1, 2).The exact mechanisms of this unexpected cerebrovascular effect of pilocarpine are not yet understood. This prompted us to further investigate the actions of pilocarpine on white blood cells and brain endothelium. Methods: The effect of pilocarpine on BBB permeability was evaluated in vitro using a Transwell system obtained with a co-culture of human derived brain endothelial cells and human astrocytes in the presence or not of monocytes. Cytokine (IL-6, TNF-alpha and IL-1beta) release from monocytes co-cultured with endothelial cells following exposure to pilocarpine was measured by ELISA test. Morphological changes of endothelial cells were evaluated by fluorescent immunocytochemistry using beta-actin staining. MMP activity was evaluated by zymography. NO production by monocyte was quantified through amperometric detection. Real time NO measurements were taken with nano-molar sensitivity in a flow-based system obtained by a co-culture of human derived brain endothelial cells with circulating mononuclear blood cells.Results: Pilocarpine increased BBB permeability to serum protein in vivo, and decreased trans-endothelial electrical resistance in vitro. Following exposure to pilocarpine (1.4 mM), we measured a rapid (minutes) release of nitric oxide, paralleled by increased levels of IL-6, but not TNF-alpha or IL-1beta. This was primarily due to activation of receptors on white blood cells occurring in spite of pretreatment with scopolamine (4 µM) and it was associated with MMP2 and MMP9 increased activity. The downstream effects of nitric oxide and cytokine/MMP release by white blood cells consisted of polymerization of endothelial cell beta-actin with subsequent loss of junctional integrity. Conclusions: Our results suggest that proinflammatory changes induced by peripherally administered pilocarpine consist of a cascade of events that include white blood cell activation and BBB damage induced by cytokine- and NO-mediated actions on endothelial cells. (Supported by NIH-NS43284, NIH-HL51614, NIH-NS46513, NIH-NS049514 and NIH-NS38195) 1)Marchi,N. et al. In vivo and in vitro effects of pilocarpine: relevance to epileptogenesis. Epilepsia in press, (2007). 2)Janigro D. et al.Adenosine-induced release of nitric oxide from cortical astrocytes. Neuroreport (1996 Jul 8)