Abstracts

Rationale: Systemic application of the muscarinic agonist, pilocarpine, is commonly utilized in rodents to induce an acute status epilepticus (SE), evolving into a chronic epileptic condition. Recent findings suggest that SE induced by pilocarpine may be also triggered by changes in the blood-brain barrier (BBB) permeability (Marchi et al 2007, Epilepsia, in press). We tested the role of increased permeability of BBB in enhancing the pro-convulsant effect of pilocarpine using in vitro brain preparations.Methods: We determined, by HPLC, brain and blood levels of pilocarpine immediately before the onset of SE in rats (350 mg/Kg i.p) and guinea pigs (220 mg/Kg i.p.). Pilocarpine was always injected 30’ after scopolamine (1mg/Kg). The efficacy of pilocarpine was evaluated in the in vitro whole brain guinea pig preparation and in rat hippocampal slices applying dose identical to those measured in vivo. The role of modification of BBB permeability in contributing to pilocarpine-induced SE was mimicked in vitro by 1) perfusing the whole brain preparation with compounds that enhance BBB permeability, such as bradykinin or histamine and by 2) increasing the concentration of extracellular K+ in the hippocampal slice before the infusion of pilocarpine. Results: In guinea pig, pilocarpine was 190 ± 10 µM in brain and 580 ± 50µM in blood. Arterial perfusion of the in vitro isolated guinea pig brain with 10 to 800µM of pilocarpine did not cause epileptiform activity, but rather reduced synaptic transmission and induced steady fast (20-25 Hz) oscillatory activity in limbic cortices. These effects were reversibly blocked by co-perfusion of the muscarinic antagonist atropine sulphate (5 µM). Pilocarpine induced epileptiform discharges only when perfused with compounds that enhance BBB permeability, such as bradykinin (n=2) or histamine (n=10). This pro-epileptic effect was abolished when the BBB-impermeable muscarinic antagonist atropine methyl bromide (5 µM) was co-perfused with histamine and pilocarpine. In the absence of BBB permeability enhancing drugs, pilocarpine induced epileptiform activity only after perfusion at concentration >10 mM. In rat, pilocarpine blood and brain levels at SE were 1400 ± 200 µM and 200 ±80 µM, respectively. When applied to rat hippocampal slices, pilocarpine failed to exert significant synchronized epileptiform activity when applied at concentrations identical or higher to levels measured in vivo (10-500µM). Intense electrographic seizure-like events occurred only in the co-presence of levels of K+ (6mM) mimicking BBB leakage at relative low concentration of pilocarpine (50µM).Conclusions: We propose that acute epileptiform discharges induced by pilocarpine treatment in vitro, as well as in vivo, are mediated by an increase in BBB permeability. Early systemic events increasing BBB permeability may promote entry of co-factors into the brain leading to pilocarpine-induced SE. Supported by NIH-NS43284, NIH-HL51614, NIH-NS46513, NIH-NS049514 and NIH-NS38195