Breakdown of the BBB is a most common feature of brain disorders, accompanied by neural and network dysfunction and degeneration (Benveniste et al., 1984; Seiffert, 2004; Tomkins et al., 2008), including epilepsy, stroke, traumatic brain injury, tumors, and neurodegenerative diseases (Brown and Davis, 2002; Davies, 2002; van Vliet et al., 2007; Stolp and Dziegielewska, 2009; Chodobski et al., 2011; Vezzani and Friedman, 2011; On et al., 2013; Wu et al., 2020). Studies on animal models of epilepsy and clinical observations among human patients reveal that the BBB has a direct role in epileptogenesis and brain damage (Mihály and Bozóky, 1984; Oby and Janigro, 2006; Friedman, 2011; Greene et al., 2022). Status epilepticus (SE) is accompanied by endothelial impairment and increased blood vessel permeability, which results in a disbalance of the neuronal environment (Obermeier et al., 2013). In particular, the ionic composition of the intercellular cerebrospinal fluid in the involved tissues is close in concentration to blood plasma (Zauner et al., 1996; Reinert et al., 2000). Such shifts in the interstitial ions can lead to changes in the impulse activity of neurons and affect the efficiency of synaptic transmission and, as a result, contribute to an increase in excitability of hippocampal neural networks (Rasmussen et al., 2020). Extravasation of blood plasma proteins into the extracellular environment of the brain in case of BBB damage also contributes to long-term hypersynchronization of neurons in the affected areas (Seiffert, 2004; van Vliet et al., 2007). Entering the brain tissue as a consequence of traumatic brain injury thrombin is able to induce seizures (Lee et al., 1997). In vitro studies have shown the enhancement of thrombin activity in the brain due to pilocarpine treatment (Golderman et al., 2019). Recent findings suggest a significant increase in thrombin level in the brain following SE (Isaev et al., 2015). Moreover, intracerebral injection of thrombin may directly induce seizures (Lee et al., 1997). In this work, we simulate certain conditions of BBB breakdown in vitro to study the effect of thrombin in blood plasma ionic media on induction of epileptiform activity in hippocampal slices. Using the classical model of TLE we have found the resemblance in the manifestation of early-life seizures in SE-treated rats compared to seizure-like activity (SLA) due to model conditions, simulating impairment of BBB in vitro.

Field potential recordings were performed from the hippocampal CA1 pyramidal layer in acute slices. We did not observe SLA appearance in any of the tested slices due to ACSF perfusion (data not shown). Bath application of blood plasma ionic media led to consistent SLA in 17 slices (Figure 1A1). This activity persisted as long as examined solution was applied. The frequency of synchronous discharges during SLA was 1.34 ± 0.11 Hz. The amplitude of SLA was at the level 0.27 ± 0.03 mV (Figure 1B). Addition of 10 U/ml thrombin alone did not lead to an increase in neuronal firing in the CA3 hippocampal region of P 6–15 rats (Isaeva et al., 2012). Thrombin has evoked SLA in the presence of 7.2 mM of K⁺ or 100 μM of glutamate in the extracellular solution (Maggio et al., 2008). In our study application of 5 U/ml thrombin together with blood plasma ionic saline induced SLA in hippocampal CA1 pyramidal layer with the frequency of 3.39 ± 0.35 Hz and amplitude 0.66 ± 0.09 mV (n = 18, Figure 1A2). These data are in agreement with our previous report when in similar conditions the epileptiform activity in cultured hippocampal neurons was significantly enhanced (Shypshyna et al., 2021). Extracellular recordings from the slices of SE-treated rats demonstrated high-frequency oscillations with the frequency of 3.31 ± 0.35 Hz and amplitude 0.51 ± 0.03 mV (n = 14, Figure 1A3). Comparative analysis of field potentials in all experimental groups reveal significant enhancement in frequency (H₍₂₎ = 26.74, P < 0.0001; post-hoc: blood plasma media vs. thrombin—P < 0.0001; blood plasma media vs. SE—P < 0.0001, Figure 1B1) and amplitude (H₍₂₎ = 20.30, P = 0.0001; post-hoc: blood plasma media vs. thrombin—P < 0.0001; blood plasma media vs. SE—P = 0.0011, Figure 1B2) of SLA due to thrombin application and SE exposure. We observed non-significant decrease in amplitude of SLA in slices obtained from Li-pilocarpine-exposed epileptic rats compared to thrombin-treated slices in blood-plasma ACSF (p = 0.99, Figure 1B2).

The main finding of our study is that TLE-treated slices shortly after SE induction produces similar significant increase in frequency and amplitude of SLA as blood plasma media together with thrombin.

Acute SE is induced by systemic application of muscarinic agonist, pilocarpine. In vitro application of pilocarpine alone did not cause epileptiform activity, but induced seizures when applied with substances that enhance BBB leakage, such as bradykinin or histamine (Uva et al., 2008). To promote an increase of BBB permeability we first perform the in vivo injection of lithium chloride. Subsequent administration of pilocarpine induced SE.

In our recent study, the application of serum-adapted solution increases the average action potentials frequency in neurons with spontaneous firing activity as well as tonic electrical activity in neurons. Increasing neuronal activity by blood plasma ACSF led to the development of epileptiform tonic activity in cultured hippocampal neurons (Shypshyna et al., 2021). Thrombin was shown to facilitate the effects of proconvulsants in the hippocampal slices from adult rats (Maggio et al., 2008).

We hypothesized the substantial role of BBB impairment in the initiation of seizures. Cerebrovascular damage in CNS disorders, including epilepsy is considered as a leading mechanism underlying epileptiform activity (Janigro, 1999; Seiffert, 2004; Marchi et al., 2007; Van Vliet et al., 2015). During intracerebral hemorrhage, blood compounds bleeding into the brain tissue and cause both an acute and a delayed effect on neuronal functioning (Friedman, 2011). Among the consequences of BBB damage are the changes in the intracerebral environment, when the ionic composition of the intercellular cerebrospinal fluid in the affected areas is close in concentration to blood plasma (Zauner et al., 1996; Reinert et al., 2000). Replacing the ACSF with blood plasma saline obviously affects the functioning of voltage-gated channels. Thus, increased concentrations of K⁺ in extracellular solution, in addition to affecting the membrane potential and synaptic transmission, potentiate the persistent Na⁺ currents in neurons (Somjen and Müller, 2000). However, such an effect contributes to the strengthening of synaptic potentials and increases the ability of neurons to recurrent synchronous discharges (Stafstrom, 2007), which we observed in our experiments. Modification of ACSF to blood plasma solution also evolves changes in Ca²⁺ and Mg²⁺ concentrations, which could neutralize the negative surface charges on the outer membrane surface and lead to the facilitation of the voltage-gated channels activation (Isaev et al., 2012). In our study, changes in the concentrations of certain ions in blood plasma media neutralized each other, which contributed to maintaining the shielding of negative charges on neural membranes at the control level. Therefore, we excluded the possibility that the epileptiform activity of the hippocampal slices in modified ACSF is related to changes in the concentrations of divalent cations.

BBB breakdown could result in penetrating and storage in the brain of toxic bloodborne molecules such as hemoglobin, albumin, thrombin, fibrinogen, iron-containing hemosiderin, plasmin, free iron, and environmental toxins (Montagne et al., 2016). Only thrombin was shown to have a potent role in the generation of early-onset SLA (Willmore et al., 1978; Lee et al., 1997; Tomkins et al., 2007). Our data are in agreement with these studies demonstrating the generation of epileptiform activity due to thrombin application in the blood plasma media. Recent findings suggest a significant increase in the thrombin level in the brain tissue caused by the enhancement of BBB permeability during pathological conditions (Woitzik et al., 2011; Isaev et al., 2015). Thrombin, through its major receptor in the neural tissue, protease-activated receptors 1 (PAR 1), produces epileptogenesis by the escalation of brain damage, induction of seizures, inflammation, and neurogenesis (Rohatgi et al., 2004).

It was shown previously that pilocarpine-induced SE may be caused by enhancement in the BBB permeability (Uva et al., 2008). It was shown the disruption of BBB shortly after SE (van Vliet et al., 2007), accompanied by the early efflux of serum proteins and disturbance in interstitial fluid homeostasis (Friedman, 2011). Later epileptic phase involved different mechanisms of propagation seizures, including activation of the innate immune system, activation of transforming growth factor beta in the response to serum albumin, extracellular accumulation of K⁺ and glutamate (Cacheaux et al., 2009; David et al., 2009), etc. In our study, we demonstrate that epileptic activity in slices of pilocarpine-treated rats obtained after SE-onset have similar features as synchronous discharges due to the application of thrombin in plasma ionic media. We propose the essential role of thrombin efflux in the acute epileptiform discharges induced by pilocarpine treatment.

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (European convention, Strasburg, 1986); the Law of Ukraine “On protection of animals from cruelty” and approved by the Animal Care Committee of Bogomoletz Institute of Physiology.

AS has designed the experiments, provided an elecrophysiological recordings, made statistical analysis and prepared the manuscript. MK performed experiments with Li-pilocarpine model of epilepsy and took part in disscusion of the results. MS has provided experiments with thrombin and participated in disscusion of manuscript. DI elaborated the idea of experiments, participated in summarizing results and making conclusions. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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