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Epileptogenesis following insults to the brain may be triggered by a dysfunctional blood-brain barrier (BBB) associated with albumin extravasation and activation of astrocytes. Using ex vivo recordings from the BBB-disrupted hippocampus after neocortical photothrombotic stroke, we previously demonstrated abnormal activity-dependent accumulation of extracellular potassium with facilitated generation of seizure like events and spreading depolarizations. Similar changes could be observed after intracerebroventricular (icv) application of albumin. We hypothesized that alterations in extracellular potassium and glutamate homeostasis might lead to alterations in synaptic interactions. We therefore assessed the effects of icv albumin on homo- and heterosynaptic plasticity in hippocampal CA1, 24 h after a single injection or 7 days after continuous infusion of icv albumin. We demonstrate alterations in both homo- and heterosynaptic plasticity compared to control conditions in ex vivo slice studies. Albumin-treated tissue reveals (1) reduced long-term depression following low-frequency stimulation; (2) increased long-term potentiation of population spikes in response to 20 Hz stimulation; (3) potentiated responses to Schaffer collateral stimulation following high-frequency stimulation of the direct cortical input and low-frequency stimulation of alveus and finally, (4) TGFβ receptor II (TGFβR-II) involvement in albumin-induced homosynaptic plasticity changes. We conclude that albumin-induced network hyperexcitability is associated with abnormal homo- and heterosynaptic plasticity that could partly be reversed by interference with TGFβR-II-mediated signaling and therefore it might be an important factor in the process of epileptogenesis.

Post-injury epilepsy (PIE) is a common complication following brain insults, including ischemic, and traumatic brain injuries. At present, there are no means to identify the patients at risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood–brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures remains unknown. Here we demonstrate in vitro and in vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process and highlight the manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE.

Neuroimaging of exogenous tracer extravasation has become the technique of choice in preclinical and clinical studies of blood–brain barrier permeability. Such tracers have a larger molecular weight than small ions, neurotransmitters and many drugs. Therefore, it is assumed that tracer extravasation indicates both permeability to these and the cancelation of the electrical polarization across the barrier. Electrophysiological anomalies following intracarotideal administration of dehydrocholate, a bile salt causing extravasation of the albumin-binding tracer Evans blue, seemingly supported this. By contrast, electron microscopic studies suggested a different hierarchical pattern of blood–brain barrier dysfunction, a milder degree of impairment being characterized by increased function of the transcellular pathway and a severe degree by opening of the tight junctions. This would imply that the extravasation of macromolecules can occur before disruption of the electrical barrier. However, functional evidence for this has been lacking. Here, we further investigated the electrophysiological anomalies following intracarotideal application of dehydrocholate in rats and found that it caused focal cerebral ischemia by middle cerebral artery thrombosis, the electrophysiological recordings being characteristic of long-lasting spreading depolarization. These observations indicated that intracarotideal dehydrocholate is not a suitable model to study the isolated dysfunction of the blood–brain barrier. Second, we studied the topical application of dehydrocholate to the brain and the application of mannitol into the carotid artery. In both models, we found significant extravasation of Evans blue but no changes in either extracellular potassium or the CO2-dependent intracortical direct current deflection. The latter is assumed to depend on the proton gradient across the barrier in rats which we confirmed in additional experiments in vivo and in vitro. The stability of the extracellular potassium concentration and the CO2-dependent direct current deflection are two functional tests which indicate the integrity of the electrical barrier. Hence, our results provide functional evidence that the blood–brain barrier opening to large molecules does not necessarily imply the opening to small ions consistent with the hierarchy of damage in the previous electron microscopic studies.

Recent studies showed that spreading depolarizations (SDs) occurs abundantly in patients following ischemic stroke and experimental evidence suggests that SDs recruit tissue at risk into necrosis. We hypothesized that BBB opening with consequent alterations of the extracellular electrolyte composition and extravasation of albumin facilitates generation of SDs since albumin mediates an astrocyte transcriptional response with consequent disturbance of potassium and glutamate homeostasis. Here we show extravasation of Evans blue-albumin complex into the hippocampus following cortical photothrombotic stroke in the neighboring neocortex. Using extracellular field potential recordings and exposure to serum electrolytes we observed spontaneous SDs in 80% of hippocampal slices obtained from rats 24 h after cortical photothrombosis. Hippocampal exposure to albumin for 24 h through intraventricular application together with serum electrolytes lowered the threshold for the induction of SDs in most slices irrespective of the pathway of stimulation. Exposing acute slices from naive animals to albumin led also to a reduced SD threshold. In albumin-exposed slices the onset of SDs was usually associated with larger stimulus-induced accumulation of extracellular potassium, and preceded by epileptiform activity, which was also observed during the recovery phase of SDs. Application of ifenprodil (3 μM), an NMDA-receptor type 2 B antagonist, blocked stimulus dependent epileptiform discharges and generation of SDs in slices from animals treated with albumin in-vivo. We suggest that BBB opening facilitates the induction of peri-infarct SDs through impaired homeostasis of K+.

Disruption of the blood-brain barrier (BBB) is a characteristic finding in common neurological disorders. Human data suggest BBB disruption may underlie cerebral dysfunction. Animal experiments show the development of epileptiform activity following BBB breakdown. In the present study we investigated the neurophysiological, structural and functional consequences of BBB disruption. Adult rats underwent focal BBB disruption in the rat sensory-motor cortex using the bile salt sodium deoxycholate (DOC). Magnetic resonance imaging in-vivo showed an early BBB disruption with delayed reduction in cortical volume. This was associated with a reduced number of neurons and an increased number of astrocytes. In-vitro experiments showed that the threshold for spreading depression and the propagation velocity of the evoked epileptic potentials were increased 1 month after treatment. Furthermore, animals' motor functions deteriorated during the first few weeks following BBB disruption. Treatment with serum albumin resulted in a similar cell loss confirming that the effect of DOC was due to opening of the BBB. Our findings suggest that delayed neurodegeneration and functional impairment occur following the development of the epileptic focus in the BBB-permeable cerebral cortex.

In models of temporal lobe epilepsy, in-vitro exposure of the entorhinal cortex (EC) to low concentrations of acetylcholine (ACh) induces muscarinic-dependent seizure-like events. Potassium channels from the KCNQ/Kv7 family, which close upon activation of muscarinic receptors, are mutated in several epileptic syndromes such as benign familial neonatal convulsions (KCNQ2/KCNQ3) and sudden unexplained death in epilepsy (KCNQ1). Therefore, we tested the hypothesis whether the ictogenic effect of ACh involves alterations of KCNQ channels. In horizontal temporo-hippocampal slices from pilocarpine-treated chronically epileptic rats, field potential recordings of epileptiform activity were performed in response to the application of ACh, the KCNQ blocker linopirdine, and KCNQ agonists. In the EC of control rats, ACh (20 and 50 μM) induced nested fast activity in the range of 15–20 Hz riding on < 1 Hz slow oscillations. By contrast, in slices from pilocarpine-treated rats, 5 μM ACh was sufficient to induce interictal discharges that frequently transformed to epileptiform events at 20 μM ACh. While the non-specific KCNQ/Kv7 channel blocker linopirdine (20 and 50 μM) had no effect in control animals, in slices from epileptic rats it induced interictal discharges or seizure-like events. These could be blocked by the unspecific KCNQ/Kv7 agonist retigabine and attenuated by the Kv7.1 agonist L364–373. Immunohistochemistry revealed reduced expression of KCNQ2 and KCNQ3 in the EC and of KCNQ3-positive dendrites in the subiculum of epileptic rats. These results indicate that channels of the KCNQ family are key regulators of seizure susceptibility and their decreased availability in the epileptic tissue may reduce seizure threshold and contribute to ictogenesis.