Paolo Mainardi

Preclinical activity profile of α-lactoalbumin, a whey protein rich in tryptophan, in rodent models of seizures and epilepsy

PURPOSE To evaluate the potential anticonvulsant activity of α-lactalbumin (ALAC), a whey protein rich in tryptophan (TRP) relative to other large neutral amino acids (LNAAs), in rodent models of seizures and epilepsy. METHODS The effects of ALAC administered per os were evaluated by standard protocols against audiogenic seizures in Genetic Epilepsy Prone Rats (GEPR-9 rats), maximal electroshock (MES)-induced seizures in rats, pilocarpine-induced seizures in mice, spontaneous chronic seizures in mice exposed to pilocarpine-induced status epilepticus (SE), and absence seizures in WAG/Rij rats. In some models, carbamazepine (CBZ) was included as an active control. Plasma TRP/LNAAs ratios were measured by GC-MS. RESULTS Single doses of ALAC up to 500 or 6000 mg/kg were devoid of anticonvulsant activity in all models tested. Conversely, 5- and 12-day treatment with ALAC (250-1000 mg/kg/day) in GEPR rats reduced dose-dependently seizure scores and prolonged latency to clonus onset, with full persistence of the effect for up to 12h. ALAC (125-500 mg/kg/day for 15 days) protected against seizures induced by 250 mg/kg pilocarpine, but was less effective against higher pilocarpine doses. Similarly to CBZ, ALAC (125-500 mg/kg/day for 15 days) was also effective against spontaneous seizures in the post-pilocarpine SE model. ALAC (up to 6000 mg/kg/day for 12 days) did not prevent MES-induced seizures, although it reduced the duration of tonic extension at doses between 250 and 1000 mg/kg/day. Absence seizures in WAG/Rij rats were not significantly affected by ALAC. Plasma TRP/LNAAS ratios increased 2- to 3-fold after dosing with ALAC (250 mg/kg/day) for 7 and 14 days, respectively. CONCLUSIONS ALAC exerts significant protective activity against seizures in animal models, the effect being especially prominent against audiogenic seizures in GEPR-9 rats, seizures induced by low-dose pilocarpine in mice, and spontaneous seizures in mice exposed to pilocarpine-induced SE. This action is likely to be mediated by increased availability of TRP in the brain, with a consequent increase in 5-HT mediated transmission.

Protective activity of α-lactoalbumin (ALAC), a whey protein rich in tryptophan, in rodent models of epileptogenesis

The aim of the present work was to evaluate the potential activity of α-lactoalbumin (ALAC), a whey protein rich in tryptophan (TRP), in two rodent models of epileptogenesis and we explored a possible mechanism of action. The effects of ALAC (oral administration) were tested in two standard epileptogenesis protocols, namely the pilocarpine post-status epilepticus model in mice and the WAG/Rij rat model of absence epileptogenesis. The mechanism of action was investigated by assessing the effects of ALAC in two seizure models (N-methyl-d-aspartate (NMDA) and pentylenetetrazol (PTZ) -induced seizures) including d-serine co-administration. ALAC showed protecting properties in both models of epileptogenesis, reducing spontaneous seizures development. In acute seizure models, ALAC possessed antiseizure properties at some of the doses tested (PTZ-seizures: >50% seizure-reduction between 250 and 375 mg/kg; NMDA-seizures: >90% reduction at 250 and 500 mg/kg). When a dose of d-serine ineffective per se was co-administered with ALAC, ALAC effects were significantly reversed in both models. ALAC is active in experimental models of seizure and epileptogenesis. Its effects are likely mediated by the inhibition of NMDA receptors at the glycine binding site, possibly secondarily to the in vivo enzymatic conversion of ALAC-generated tryptophan to kynurenic acid. However, other mechanisms of action contributing to ALAC effects cannot be excluded.

Liposome‐Entrapped γ‐Aminobutyric Acid Inhibits Isoniazid‐Induced Epileptogenic Activity in Rats

Summary: Sprague—Dawley rats with interictal and ictal spike activity induced by intraperitoneally injected isoniazid (INH) were treated, 5 min before or 30 min later, with liposome‐entrapped γ‐aminobutyric acid (GABA) (LEG) or GABA or phosphatidylserine. Crossover injections were given in random sequence and INH alone was also injected in every animal as a control. LEG inhibited either seizures or interictal spikes in both groups. No decrease of epileptogenic activity was seen after GABA or phosphatidylserine treatment alone. It is suggested that LEG could contribute to the reconstitution of the GABA pool decreased by INH.

The excitatory amino acid antagonist amino-phosphono-valeric acid (APV) provides protection against penicillin-induced epileptic activity in the rat

The effects of intraperitoneal injection of 2-amino-5-phosphono-valeric acid (APV) on EEG-monitored penicillin-induced epileptic activity in rats were evaluated. A significant decrease in the frequency of spikes occurred with low APV dosages (10 and 20 mg/kg), while an almost complete disappearance of spike activity was observed at higher APV doses (40 and 160 mg/kg). Our data suggest that excitatory amino acids play a relevant role in penicillin-induced epileptic activity in rats.

Effect of nitric oxide donors on GABA uptake by rat brain synaptosomes

The effect of nitric oxide donors and L-arginine on the uptake of GABA was studied in synaptosomes purified from rat brain. The neurotransmitter uptake was significantly reduced by S-nitrosoacetylpenicillamine and by sodium nitroprusside, although in this case to a lesser extent. A slight inhibitory effect was found preincubating rat brain synaptosomes with 1 mM L-arginine as well. The S-nitrosoacetylpenicillamine effect gradually disappeared with decomposition of the substance by exposure to light. The nitric oxide effect appears to be mainly due to a decrease in the V for synaptosomal GABA uptake and seems to be related to a partial collapse of nerve endings ionic gradients. Functionally, it could result over time in a reduced availability of GABA at the synapses involved.

Evaluation of the Mechanisms by Which Gamma-Amino- Butyric Acid in Association with Phosphatidylserine Exerts an Antiepileptic Effect in the Rat

The i.p. injection in rats of GABA (740 mg/Kg) after sonication with an equal amount of phosphatidylserine (PS) has an antiepileptic effect. The injection of plain GABA has no such an effect. Blood, brain and synaptosomal accumulation of exogenous labeled GABA under the two circumstances are evaluated. In the case of GABA/PS injection there is a higher passage of the exogenous labeled neurotransmitter into the blood and brain nerve endings (synaptosomes). A higher synaptosomal accumulation of the exogenous labeled neurotransmitter is found even when GABA and PS are injected separately. Since these accumulation increases occur at a time when there is the antiepileptic effect, they seem relevant to it. Our interpretation of the chain of the events resulting in the antiepileptic action is that the phospholipid facilitates from the beginning the first passage of the exogenous neurotransmitter form the peritoneum to the blood. Then a higher passage to the brain tissue and eventually to the GABA-ergic nerve endings ensues. The brisker accumulation of the exogenous neurotransmitter in the nerve endings could be at the basis of a more efficient GABA-ergic inhibitory control in the brain.

Accumulation of labeled gamma-aminobutyric acid into rat brain and brain synaptosomes after i.p. injection

The accumulation of labeled GABA into brain and brain nerve endings was studied in the adult rat after i.p. injection of large doses of neurotransmitter (740 mg/Kg). In the first 5–30 minutes after the injection the exogenous neurotransmitter reaches a stable plasma level of around 5 mM. The accumulation of radioactive GABA into the brain presents a latency of a few minutes from the time of the injection. Thereafter, the accumulation of the neurotransmitter is almost linear with time. Once in the brain tissue labeled GABA is in part broken down. The exogenous neurotransmitter is taken up in GABA-ergic nerve endings with a steep increase between 20 and 30 minutes after the injection. From a quantitative point of view, the data show that the brain accumulation of labeled GABA at 30 minutes post injection is minimal in the respect of the steady state average concentration of the endogenous neurotransmitter (0.014%). However, the amount of radioactive GABA which accumulates in the nerve endings, at the same post injection time, is around 7% of the endogenous neurotransmitter in that comparment. The data thus show a selective enrichment of exogenous systemic GABA in a physiologically important compartment of the brain.

Adsorption of γ-aminobutyric acid to phosphatidylserine membranes

The interaction of the negatively-charged phosphatidylserine (PS) and γ-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles.GABA does not permeate through PS membranes and, in concentrations of 10−5-10−4 M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of14C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.

From the Ancient Diets to the Recent Acquisitions on the Role of Brain Inflammation in Epilepsy, Are there Any Links?

Recent studies put in evidence the role of brain inflammation in the pathogenetic mechanisms of seizures. It has been reported that an intestinal inflammation may be able to migrate to the brain, thus an intestinal inflammation could be the original cause of epilepsy, as osteopaths believed in the ‘20s. We attempted to demonstrate the role of gut-brain axis in epilepsy on the basis of recent acquisitions.

Sudden death in Unverricht-Lundborg patients: is serotonin the key?

Sudden unexpected death in epilepsy (SUDEP) has an incidence of 1–2.5 per 1,000 patient-years and accounts for up 30% of deaths in people with epilepsy [1, 2]. Although knowledge about mechanisms for SUDEP still remains largely unknown, it is well known that risk factors for SUDEP include drug-resistant seizures, symptomatic epilepsy, young age, male gender, long duration of epilepsy, and poor compliance with antiepileptic treatment [1, 2]. Khiari and colleagues [3] recently reported an unusual elevated rate of unexpected deaths in a series of patients with Unverricht–Lundborg disease (EPM1), the most common form of progressive myoclonus epilepsy, associated with mutations in the Cystatin B (CSTB) gene. In particular, 6 out of 19 (31.5%) EPM1 subjects died from SUDEP, independent of seizure control and antiepileptic treatment. However, no specific explanation for this association was provided. In the last 10 years, we observed two cases (1 male, 1 female) of SUDEP among six EPM1 followed-up at our Epilepsy Center (Striano P., unpublished data). This percentage (33%) is strikingly similar to that reported by Khiari and colleagues [3]. We hypothesize that a dysfunction of the serotoninergic system may be a potential mechanism explaining the higher risk of SUDEP in EPM1 patients. Several lines of evidence suggest that an abnormal serotoninergic transmission may be responsible for the myoclonic/seizure phenotype in EPM1 [4, 5]. CSTBdeficient mice show constitutively altered concentration of tryptophan, serotonin, and 5-hydroxyindolacetic acid (5HIAA) in the cerebral cortex and cerebellum [4]. In addition, a significant reduction in the levels of tryptophan and its metabolites along serotonin and kynurenine pathways has been found in the serum of both CSTB-deficient mice animal models and EPM1 patients [5]. Experimental and clinical data support that a reduced serotonergic activity is a potential crucial neurochemical change playing a role in animal models of audiogenic seizures (DBA/2 and DBA/1 mice), probably through control of central respiration [6]. Moreover, SUDEP in this animal model may be prevented by the administration of serotonin re-uptake inhibitors (SSRI) ([6], Faingold, personal communication, American Epilepsy Society 2008). Notably, cerebrospinal monoamine metabolites, in particular 5HIAA, have been recently found also decreased in a baboon model of epilepsy and SUDEP [7, 8]. In conclusion, although our hypothesis is merely speculative, data from the literature suggest that the potential link between the serotonergic system and pathophysiology of SUDEP merits to be further investigated by future specific studies. P. Striano (&) F. Zara Muscular and Neurodegenerative Diseases Unit, G. Gaslini Institute, University of Genova, Genova, Italy e-mail: pstriano@email.it