Anna Binaschi

Localized delivery of fibroblast growth factor–2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model

A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor–2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.

On the role of somatostatin in seizure control: clues from the hippocampus.

The role of the hippocampal somatostatin (somatotropin release-inhibiting factor, SRIF) system in the control of partial complex seizures is discussed in this review. The SRIF system plays a role in the inhibitory modulation of hippocampal circuitries under normal conditions: 1) SRIF neurons in the dentate gyrus are part of a negative feedback circuit modulating the firing rate of granule cells; 2) SRIF released in CA3 interacts both with presynaptic receptors located on associational/commissural terminals and with postsynaptic receptors located on pyramidal cell dendrites, reducing excitability of pyramidal neurons; 3) in CA1, SRIF exerts a feedback inhibition and reduces the excitatory drive on pyramidal neurons. Significant changes in the hippocampal SRIF system have been documented in experimental models of temporal lobe epilepsy (TLE), in particular in the kindling and in the kainate models. SRIF biosynthesis and release are increased in the kindled hippocampus, especially in the dentate gyrus. This hyper-function may be instrumental to control the latent hyperexcitability of the kindled brain, preventing excessive discharge of the principal neurons and the occurrence of spontaneous seizures. In contrast, the hippocampal SRIF system undergoes damage in the dentate gyrus following kainate-induced status epilepticus. Although surviving SRIF neurons appear to hyperfunction, the loss of hilar SRIF interneurons may compromise inhibitory mechanisms in the dentate gyrus, facilitating the occurrence of spontaneous seizures. In keeping with these data, pharmacological activation of SRIF1 (sst2) receptors, i.e. of the prominent receptor subtype on granule cells, exerts antiseizure effects. Taken together, the data presented suggest that the hippocampal SRIF system plays a role in the control of partial complex seizures and, therefore, that it may be proposed as a therapeutic target for TLE.

MicroRNA profiles in hippocampal granule cells and plasma of rats with pilocarpine-induced epilepsy – comparison with human epileptic samples

The identification of biomarkers of the transformation of normal to epileptic tissue would help to stratify patients at risk of epilepsy following brain injury, and inform new treatment strategies. MicroRNAs (miRNAs) are an attractive option in this direction. In this study, miRNA microarrays were performed on laser-microdissected hippocampal granule cell layer (GCL) and on plasma, at different time points in the development of pilocarpine-induced epilepsy in the rat: latency, first spontaneous seizure and chronic epileptic phase. Sixty-three miRNAs were differentially expressed in the GCL when considering all time points. Three main clusters were identified that separated the control and chronic phase groups from the latency group and from the first spontaneous seizure group. MiRNAs from rats in the chronic phase were compared to those obtained from the laser-microdissected GCL of epileptic patients, identifying several miRNAs (miR-21-5p, miR-23a-5p, miR-146a-5p and miR-181c-5p) that were up-regulated in both human and rat epileptic tissue. Analysis of plasma samples revealed different levels between control and pilocarpine-treated animals for 27 miRNAs. Two main clusters were identified that segregated controls from all other groups. Those miRNAs that are altered in plasma before the first spontaneous seizure, like miR-9a-3p, may be proposed as putative biomarkers of epileptogenesis.

FGF-2 Overexpression Increases Excitability and Seizure Susceptibility but Decreases Seizure-Induced Cell Loss

Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.

Kainate seizures increase nociceptin/orphanin FQ release in the rat hippocampus and thalamus: a microdialysis study

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been suggested to play a facilitatory role in kainate seizure expression. Furthermore, mRNA levels for the N/OFQ precursor are increased following kainate seizures, while its receptor (NOP) density is decreased. These data suggest increased N/OFQ release. To obtain direct evidence that this is the case, we have developed a microdialysis technique, coupled with a sensitive radioimmunoassay, that allows measurement of N/OFQ release from the hippocampus and thalamus of awake, freely moving animals. In both these brain areas, the spontaneous N/OFQ efflux decreased by approximately 50% and 65% when Ca2+ was omitted and when tetrodotoxin was added to the perfusion medium, respectively. Perfusion of the dialysis probe with high K+ increased N/OFQ release (approximately threefold) in a Ca2+‐dependent and tetrodotoxin‐sensitive manner. Kainate seizures caused a twofold increase in N/OFQ release followed, within 3 h, by a return to baseline levels. Approximately 5 h after kainate, a late increase in N/OFQ release was observed. On the following day, when animals were having only low grade seizures, N/OFQ release was not significantly different from normal. These phenomena were observed with similar patterns in the hippocampus and in the thalamus. The present data indicate that acute limbic seizures are associated with increased N/OFQ release, which may prime the molecular changes described above, i.e. cause down‐regulation of NOP receptors and activation of N/OFQ biosynthesis.

Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy

The alterations in GABA release have not yet been systematically measured along the natural course of temporal lobe epilepsy. In this work, we analyzed GABA extracellular concentrations (using in vivo microdialysis under basal and high K(+)-evoked conditions) and loss of two GABA interneuron populations (parvalbumin and somatostatin neurons) in the ventral hippocampus at different time-points after pilocarpine-induced status epilepticus in the rat, i.e. during development and progression of epilepsy. We found that (i) during the latent period between the epileptogenic insult, status epilepticus, and the first spontaneous seizure, basal GABA outflow was reduced to about one third of control values while the number of parvalbumin-positive cells was reduced by about 50% and that of somatostatin-positive cells by about 25%; nonetheless, high K(+) stimulation increased extracellular GABA in a proportionally greater manner during latency than under control conditions; (ii) at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made in humans) this increased responsiveness to stimulation disappeared, i.e. there was no longer any compensation for GABA cell loss; (iii) thereafter, this dysfunction remained constant until a late phase of the disease. These data suggest that a GABAergic hyper-responsiveness can compensate for GABA cell loss and protect from occurrence of seizures during latency, whereas impaired extracellular GABA levels can favor the occurrence of spontaneous recurrent seizures and the maintenance of an epileptic state.

Induction of B1 bradykinin receptors in the kindled hippocampus increases extracellular glutamate levels: A microdialysis study

A link between temporal lobe epilepsy (the most common epileptic syndrome in adults) and neuropeptides has been established. Among neuropeptides, the possible involvement of bradykinin has recently received attention. An autoradiographic analysis has shown that B1 receptors, which are physiologically absent, are expressed at high levels in the rat brain after completion of kindling, a model of temporal lobe epilepsy. Thus, the present work aimed at investigating the functional implications of this observation, by studying the effect of B1 receptor activation on extracellular glutamate levels in the kindled hippocampus. Microdialysis experiments have been performed in two groups of rats, control and kindled. Glutamate outflow has been measured under basal conditions and after chemical stimulation with high K+ (100 mM in the dialysis solution). Basal glutamate outflow in kindled animals was significantly higher than in controls. High K+-evoked glutamate outflow was also more pronounced in kindled animals, consistent with the latent hyperexcitability of the epileptic tissue. The B1 receptor agonist Lys-des-Arg9-BK induced an increase of basal and high K+-evoked glutamate outflow in kindled but not in control rats, and the selective B1 receptor antagonist R-715 prevented both these effects. Furthermore, R-715 significantly reduced high K+-evoked glutamate outflow when applied alone. These data suggest that the bradykinin system contributes to the modulation of epileptic neuronal excitability through B1 receptors.

Increased extracellular levels of glutamate in the hippocampus of chronically epileptic rats

An increase in the release of excitatory amino acids has consistently been observed in the hippocampus during seizures, both in humans and animals. However, very little or nothing is known about the extracellular levels of glutamate and aspartate during epileptogenesis and in the interictal chronic period of established epilepsy. The aim of this study was to systematically evaluate the relationship between seizure activity and changes in hippocampal glutamate and aspartate extracellular levels under basal and high K(+)-evoked conditions, at various time-points in the natural history of experimental temporal lobe epilepsy, using in vivo microdialysis. Hippocampal extracellular glutamate and aspartate levels were evaluated: 24h after pilocarpine-induced status epilepticus (SE); during the latency period preceding spontaneous seizures; immediately after the first spontaneous seizure; in the chronic (epileptic) period. We found that (i) basal (spontaneous) glutamate outflow is increased in the interictal phases of the chronic period, whereas basal aspartate outflow remains stable for the entire course of the disease; (ii) high K(+) perfusion increased glutamate and aspartate outflow in both control and pilocarpine-treated animals, and the overflow of glutamate was clearly increased in the chronic group. Our data suggest that the glutamatergic signaling is preserved and even potentiated in the hippocampus of epileptic rats, and thus may favor the occurrence of spontaneous recurrent seizures. Together with an impairment of GABA signaling (Soukupova et al., 2014), these data suggest that a shift toward excitation occurs in the excitation/inhibition balance in the chronic epileptic state.

Bradykinin B2 receptors increase hippocampal excitability and susceptibility to seizures in mice

Bradykinin (BK) and its receptors (B1 and B2) may exert a role in the pathophysiology of certain CNS diseases, including epilepsy. In healthy tissues, B2 receptors are constitutively and widely expressed and B1 receptors are absent or expressed at very low levels, but both receptors, particularly B1, are up-regulated under many pathological conditions. Available data support the notion that up-regulation of B1 receptors in brain areas like the amygdala, hippocampus and entorhinal cortex favors the development and maintenance of an epileptic condition. The role of B2 receptors, instead, is still unclear. In this study, we used two different models to investigate the susceptibility to seizures of B1 knockout (KO) and B2 KO mice. We found that B1 KO are more susceptible to seizures compared with wild-type (WT) mice, and that this may depend on B2 receptors, in that (i) B2 receptors are overexpressed in limbic areas of B1 KO mice, including the hippocampus and the piriform cortex; (ii) hippocampal slices prepared from B1 KO mice are more excitable than those prepared from WT controls, and this phenomenon is B2 receptor-dependent, being abolished by B2 antagonists; (iii) kainate seizure severity is attenuated by pretreatment with a non-peptide B2 antagonist in WT and (more effectively) in B1 KO mice. These data highlight the possibility that B2 receptors may have a role in the responsiveness to epileptogenic insults and/or in the early period of epileptogenesis, that is, in the onset of the molecular and cellular events that lead to the transformation of a normal brain into an epileptic one.

Bystander effect on brain tissue of mesoangioblasts producing neurotrophins

Neurotrophic factors (NTFs) are involved in the regulation of neuronal survival and function and, thus, may be used to treat neurological diseases associated with neuronal death. A major hurdle for their clinical application is the delivery mode. We describe here a new strategy based on the use of progenitor cells called mesoangioblasts (MABs). MABs can be isolated from postnatal mesoderm tissues and, because of a high adhesin-dependent migratory capacity, can reach perivascular targets especially in damaged areas. We generated genetically modified MABs producing nerve growth factor (MABs-NGF) or brain-derived neurotrophic factor (MABs-BDNF) and assessed their bystander effects in vitro using PC12 cells, primary cultures, and organotypic cultures of adult hippocampal slices. MABs-NGF-conditioned medium induced differentiation of PC12 cells, while MABs-BDNF-conditioned medium increased viability of cultured neurons and slices. Slices cultured with MABs-BDNF medium also better retained their morphology and functional connections, and all these effects were abolished by the TrkB kinase blocker K252a or the BDNF scavenger TrkB-IgG. Interestingly, the amount of BDNF released by MABs-BDNF produced greater effects than an identical amount of recombinant BDNF, suggesting that other NTFs produced by MABs synergize with BDNF. Thus, MABs can be an effective vehicle for NTF delivery, promoting differentiation, survival, and functionality of neurons. In summary, MABs hold distinct advantages over other currently evaluated approaches for NTF delivery in the CNS, including synergy of MAB-produced NTF with the neurotrophins. Since MABs may be capable of homing into damaged brain areas, they represent a conceptually novel, promising therapeutic approach to treat neurodegenerative diseases.