In vivo and in vitro effects of pilocarpine: relevance to ictogenesis

Abstract

Objectives: A common experimental model of status epilepticus (SE) utilizes intraperitoneal administration of the cholinergic agonist pilocarpine preceded by methyl-scopolamine treatment. Currently, activation of cholinergic neurons is recognized as the only factor triggering pilocarpine SE. However, cholinergic receptors are also widely distributed systemically and pretreatment with methyl-scopolamine may not be sufficient to counteract the effects of systemically injected pilocarpine. The extent of such peripheral events and the contribution to SE are unknown and the possibility that pilocarpine also induces SE by peripheral actions is yet untested.

Methods: We measured in vivo at onset of SE: brain and blood pilocarpine levels, blood-brain barrier (BBB) permeability, T-lymphocyte activation and serum levels of IL-1beta and TNF-alpha. The effects of pilocarpine on neuronal excitability was assessed in vitro on hippocampal slices or whole guinea pig brain preparations in presence of physiologic or elevated [K+](out).

Results: Pilocarpine blood and brain levels at SE were 1400 +/- 200 microM and 200 +/- 80 microM, respectively. In vivo, after pilocarpine injection, increased serum IL-1beta, decreased CD4:CD8 T-lymphocyte ratios and focal BBB leakage were observed. In vitro, pilocarpine failed to exert significant synchronized epileptiform activity when applied at concentrations identical or higher to levels measured in vivo. Intense electrographic seizure-like events occurred only in the copresence of levels of K+ (6 mM) mimicking BBB leakage.

Conclusions: Early systemic events increasing BBB permeability may promote entry of cofactors (e. g. K+) into the brain leading to pilocarpine-induced SE. Disturbance of brain homeostasis represents an etiological factor contributing to pilocarpine seizures.

Figure 1

Relationship between postpilocarpine stages and the experimental end points. EEG recordings from frontal cortex (LF–RF) and hippocampus (LH–RH) in rats exposed to 350 mg/kg of pilocarpine after pretreatment with methyl-scopolamine (1 mg/kg). Note the progressive synchronization and spread of epileptiform activity until Stage IV. Behavioral and electrographic seizures were observed approximately 30 min after pilocarpine (bottom trace). Note the different time scales in the bottom panels. Also note that all the data presented in this paper were obtained from either naive, or Stage I, or Stage IV animals, to emphasize progressive changes that always preceded SE.

Figure 2

Modest pilocarpine permeability in naïve or Stage IV rats. (A) In naive animals (n = 3), pilocarpine permeability was significantly lower than predicted by its oil:water coefficient. Thus, pilocarpine permeability was similar to the paracellular pathway tracer sucrose. (B) Similar trans-BBB values were obtained in Stage IV animals in the regions indicated (n = 6). The asterisks indicate p < 0.05 by ANOVA; n. s., not significantly different.

Figure 3

In vitro electrophysiological recordings of pilocarpine’s action on rat hippocampal slices (A–B). C. Effect of 4 μM kainic acid perfused for 3 min via the arterial system in the guinea pig whole brain preparation (n = 5). Recordings were performed in the anterior and posterior piriform cortex (APC and PPC), in the medial and lateral entorhinal cortex (MERC and LERC) and in the CA1 subfield of the hippocampus. In C2, the effects of 800 μM pilocarpine for 30 min is shown. Note the different voltage calibrations in C and D. See text for details.

Figure 4

Expression of muscarinic receptors in white blood cells may be an early event after pilocarpine exposure. The results shown were obtained by fluorescence-based analysis of immune cells collected from blood samples immediately after pilocarpine injection, before or during SE. Note that, a significant decrease of CD4-expressing cells was observed shortly (20–30 min) after pilocarpine injection and prior to SE (n = 6).

Figure 5

The proinflammatory mediator Il-1β is greatly elevated prior to pilocarpine-induced status epilepticus (A). In contrast, TNF-α levels were unaffected (B). The asterisk indicates p < 0.05, while the double asterisk indicates p < 0.02 (n = 6). Methyl-scopolamine fails to counterbalance the effects of pilocarpine in vitro. The concentration of pilocarpine used in these experiments was 1.5 mM to match the levels measured in rat serum prior to SE (shown in Fig. 2B). The concentration of methyl-scopolamine was 4 μM, again chosen to match the dosage of in vivo experiments. We allowed 45 min between exposure to the blocker and the pilocarpine challenge. The muscarinic antagonist methyl-scopolamine failed to prevent release of Il-1β by endothelial cells in presence of white blood cells (C). Similarly, the morphological alterations induced by pilocarpine were not affected by pretreatment with methyl-scopolamine. N = 6 wells per experimental data point.

Figure 6

(A) Blood–brain barrier permeability for the hydrophilic low molecular weight tracer ¹⁴C-Sucrose is preserved after pilocarpine injection, but prior to development of SE. Note that, the brain:serum ratio in animals at stage IV was similar to the ratio measured in scopolamine-treated controls. In addition, numerous areas of hypoperfusion were seen (B), as demonstrated by the poor filling of regional vascular districts; these regions are highlighted by dashed lines. The blue fluorescent images were obtained by DAPI nuclear staining to show that the corresponding images were taken from approximately the same locations. As term of paragon, compare these micrographs with ischemia-reperfusion data presented elsewhere (Cavaglia et al., 2001). (C, D) The fluorescent protein-based microangiographic technique (in green) used to visualize regional breakdown of the blood–brain barrier revealed multiple small leaky spots in all limbic and parietal cortex regions measured.

Figure 7

(A) BBB integrity is altered by pilocarpine. FITC extravasation was photographed in selected areas to demonstrate patchy leakage spots in pilocarpine-treated animals. The blue fluorescent images were obtained by DAPI nuclear staining to show that the corresponding images were taken from approximately the same locations. The bar corresponds to 750 μm. Neuronal uptake of FITC-albumin was observed in correspondence of area of leaky vessels (B1–B2).

Figure 8

BBB integrity is altered by pilocarpine: neuronal cells take up the fluorescent albumin used for this study. (A) Examples of leaky spots in pre-SE animals. The left panel refers to a naive control. (B) Montage to show the entire hippocampal formation and the perihippocampal cortex. (C) Uptake of FITC-albumin was observed in pyramidal cell and granular cell layers. Note the overall of DAPI staining and the uptake of FITC signal.

Figure 9

The epileptogenic effects of pilocarpine are revealed using hippocampal slice (n = 10) by concomitant increases of ACSF potassium to serum levels. (A) Under low (4.35 mM) [K]_out, pilocarpine exerts little excitatory synchronous field effects at concentrations comparable to those measured in vivo brain (see Fig. 2B). However, when the same, or lower, concentrations are applied in presence of slightly elevated [K]_out, pilocarpine acts as a potent generator of epileptiform discharges.