Contribution of protease-activated receptor 1 in status epilepticus-induced epileptogenesis

Abstract

Clinical observations and studies on different animal models of acquired epilepsy consistently demonstrate that blood-brain barrier (BBB) leakage can be an important risk factor for developing recurrent seizures. However, the involved signaling pathways remain largely unclear. Given the important role of thrombin and its major receptor in the brain, protease-activated receptor 1 (PAR1), in the pathophysiology of neurological injury, we hypothesized that PAR1 may contribute to status epilepticus (SE)-induced epileptogenesis and that its inhibition shortly after SE will have neuroprotective and antiepileptogenic effects. Adult rats subjected to lithium-pilocarpine SE were administrated with SCH79797 (a PAR1 selective antagonist) after SE termination. Thrombin and PAR1 levels and neuronal cell survival were evaluated 48h following SE. The effect of PAR1 inhibition on animal survival, interictal spikes (IIS) and electrographic seizures during the first two weeks after SE and behavioral seizures during the chronic period was evaluated. SE resulted in a high mortality rate and incidence of IIS and seizures in the surviving animals. There was a marked increase in thrombin, decrease in PAR1 immunoreactivity and hippocampal cell loss in the SE-treated rats. Inhibition of PAR1 following SE resulted in a decrease in mortality and morbidity, increase in neuronal cell survival in the hippocampus and suppression of IIS, electrographic and behavioral seizures following SE. These data suggest that the PAR1 signaling pathway contributes to epileptogenesis following SE. Because breakdown of the BBB occurs frequently in brain injuries, PAR1 inhibition may have beneficial effects in a variety of acquired injuries leading to epilepsy.

Keywords: Epileptogenesis; Hippocampus; Pilocarpine; Protease-activated receptor; Status epilepticus; Thrombin.

Figure 1

Localization of PAR1 in the CA1 region of the hippocampus. (A) The region of interest for the histological analysis is marked by a white square on the phase contrast image (scale bar 500 μm). (B) Confocal image of hippocampal cells double stained for anti-PAR1 polyclonal antibody (red) and anti-NeuN monoclonal antibody (green) in the control group revealed that PAR1 immunoreactivity is predominant in neurons. (C) Immunohistochemistry revealed a weak PAR1 immunoreactivity in cells expressing specific astrocyte marker GFAP (green). Scale bars for B and C = 50 μm.

Figure 2

Effect of PAR1 inhibition on thrombin and PAR1 immunostaining and thionin staining in the CA1 region of the hippocampus following SE. Staining of hippocampal sections with DAPI (blue), anti-thrombin polyclonal antibody (A, red, scale bar 50 μm), anti-PAR1 polyclonal antibody (B, red, scale bar 50 μm) and thionin staining (C, scale bar 200 μm) in the control group (1), 48 hr after SE in the vehicle-treated (2) and the SCH-treated (3) groups. CA1 nuclei were visualized with DAPI staining. (A4) SE-induced increase in thrombin immunoreactivity was not affected by ip injection of the PAR1 inhibitor (averaged data from the CA1 area). (B4) PAR1 inhibition restored the SE-induced decrease of PAR1 immunoreactivity in the CA1 area. (C4) Thionin staining revealed significant cell loss in the CA1 pyramidal layer 48 hr following SE. Treatment with SCH 79797 resulted in the decrease of SE-induced cell loss. ***p<0.001, **p<0.01, *p<0.05 compared to control; #p<0.01 compared to SE+vehicle group.

Figure 3

Effect of PAR1 inhibition on survival rate and recovery from weight loss after SE. (A) Injection of PAR1 antagonist resulted in a substantial decrease in the delayed mortality rate following SE. (B) Effect of PAR1 antagonist on rat weights following SE. Number of animals used for analysis is provided in the parentheses. *p<0.05.

Figure 4

Injection of the PAR1 antagonist produced a significant suppression of IIS in the CA1 region of hippocampus during the first two weeks after SE. Extracellular recording of field potentials from the CA1 region of the dorsal part of the hippocampus in rats experienced SE. (A1) IIS recorded on the second day after SE in a SE-vehicle treated rat. (A2) Example of IIS cluster recorded on the 7^th day after SE in the same rat. (B) Cumulative data from IIS recordings collected over the first two weeks following SE. Repetitive injection of PAR1 antagonist after SE decreased the probability of IIS recording. Number of animals used for analysis shown in parentheses. ***p<0.005

Figure 5

Effect of the PAR1 antagonist on spontaneous seizures after SE. Extracellular recording of field potentials from the CA1 region of the dorsal part of the hippocampus during the first two weeks after SE. (A1) Prolonged low-frequency oscillations recorded on the 8^th day after SE in the SE+SCH treated rat. (A2) Electrographic seizures (ES) recorded on the 9^th day after SE in the SE+vehicle treated rat. Tonic (a) and clonic (b) activity shown on an expended time scale (right). (B) PAR1 inhibition decreased the probability of ES recording during the first two weeks after SE. (C) Summary plot shows the effect of PAR1 inhibition on the occurrence of behavioral seizures (BS) during the chronic period after pilocarpine-induced SE. Mean ± SE.