Consequences of pilocarpine-induced status epilepticus in immunodeficient mice.

Systemic injection of pilocarpine in rodents induces status epilepticus (SE) and reproduces the main characteristics of temporal lobe epilepsy (TLE). Different mechanisms are activated by SE contributing to cell death and immune system activation. We used BALB/c nude mice, a mutant that is severely immunocompromised, to characterize seizure pattern, neurochemical changes, cell death and c-Fos activation secondarily to pilocarpine-induced SE. The behavioral seizures were less severe in BALB/c nude than in BALB/c wild type mice. However, nude mice presented more tonic-clonic episodes and higher mortality rate during SE. The c-Fos expression was most prominent in the caudate-putamen, CA3 (p<0.05), dentate gyrus, entorhinal cortex (p<0.001), basolateral nucleus of amygdala (p<0.01) and piriform cortex (p<0.05) of BALB/c nude mice than of BALB/c. Besides, nude mice subjected to SE presented high number of Fluorojade-B (FJB) stained cells in the piriform cortex, amygdala (p<0.05) and hilus (p<0.001) in comparison with BALB/c mice. A significant increase in the level of glutamate and GABA was found in the hippocampus and cortex of BALB/c mice presenting SE in comparison to controls. However, the level of glutamate was higher in the brains of BALB nude mice than in the brains of BALB/c wild type mice, while the levels of GABA were unchanged. These results indicate that the brains of immunodeficient nude mice are more vulnerable to the deleterious effects of pilocarpine-induced SE as they present intense activation, increased glutamate levels and more cell death.

Neuroglobin is up-regulated in the cerebellum of pups exposed to maternal epileptic seizures.

To evaluate a potential insult in the cerebellum of pups exposed to maternal epileptic seizures during intrauterine life, female rats were subjected to pilocarpine-induced epilepsy. Pups from different litters were sacrificed at 1, 3, 7 and 14 post-natal days (PN) and neuroglobin (Ngb) and gliosis were analyzed in the cerebellum by Western blotting (WB) and RT-PCR. (14)C-l-leucine-[(14)C-Leu] incorporation was used to analyze protein synthesis at PN1. Nitric Oxide (NO) and thiobarbituric acid-reactive substances (TBARS) levels were also measured. Pups from naive mothers were used as controls. The mRNA level of Ngb was increased in experimental animals at PN1 ((**)p ≤ 0.001) and PN3 ((**)p ≤ 0.001), at PN7 ((***)p ≤ 0.0001) and at PN14 ((**)p ≤ 0.001) compared to the respective controls. The protein level of Ngb increased significantly in the experimental pups at PN1 ((*)p ≤ 0.05) and at PN3 ((**)p ≤ 0.001), when compared to the control pups at PN1 and PN3. At PN7 and PN14 no difference was found. The mRNA level of GFAP increased significantly about two times at PN3 ((*)p ≤ 0.05) and PN7 ((*)p ≤ 0.05) in the experimental pups when compared to the respective controls, but was unchanged in the other studied ages. Data showed that experimental pups at PN1 exhibited reduced (about 2 times, (*)p ≤ 0.05) total protein synthesis in the cerebellum when compared to control. No differences were found in the NO and TBARS levels. Our data support the hypothesis that an up-regulation of Ngb could be a compensatory mechanism in response to the hypoxic-ischemic insults caused by seizures in pups during intrauterine life.

The A1 receptor agonist R-Pia reduces the imbalance between cerebral glucose metabolism and blood flow during status epilepticus: could this mechanism be involved with neuroprotection?

It is well known that the uncoupling between local cerebral glucose utilization (LCGU) and local cerebral blood flow (LCBF), i.e. decrease in LCBF rates with high LCGU, is frequently associated with seizure-induced neuronal damage. This study was performed to assess if the neuroprotective effect of the adenosinergic A(1) receptor agonist R-N-phenylisopropyladenosine (R-Pia) injected prior to pilocarpine is able to reduce the uncoupling between LCGU and LCBF during status epilepticus (SE). Four groups of rats were studied: Saline, Pilo, R-Pia+Saline and R-Pia+Pilo. For LCGU and LCBF studies, rats were subjected to autoradiography using [(14)C]-2-deoxyglucose and [(14)C]-iodoantypirine, respectively. Radioligands were injected 4 h after SE onset. Neuronal loss was evaluated by Fluorojade-B (FJB) at two time points after SE onset (24 h and 7 days). The results showed a significant increase in LCGU in almost all brain regions studied in the Pilo and R-Pia+Pilo groups compared to controls. However, in R-Pia pretreated rats, the uncoupling between LCGU and LCBF was moderated in a limited number of structures as substantia nigra pars reticulata and hippocampal formation rather in favor of hyperperfusion. Significant increases in LCBF were observed in the entorhinal cortex, thalamic nuclei, mammillary body, red nucleus, zona incerta, pontine nucleus and visual cortex. The neuroprotective effect of R-Pia assessed by FJB showed a lower density of degenerating cells in the hippocampal formation, piriform cortex and basolateral amygdala. In conclusion our data shows that the neuroprotective effect of R-Pia was accompanied by a compensatory metabolic input in brain areas involved with seizures generation.

Adenosina e neuroproteção na epilepsia do lobo temporal: da ativação do receptor A1 ao bloqueio do receptor A2A / Adenosine and neuroprotection in the temporal lobe epilepsy: from A1 receptor activation to A2A receptor blockade

OBJETIVO: Caracterizar o efeito do bloqueio do receptor A2A pelo SCH58261 na modulação da crise e neuroproteção de áreas cerebrais vulneráveis à lesão por pilocarpina. O efeito do SCH58261 foi também analisado em combinação com a ativação dos receptores A1 por R-Pia. MÉTODOS: Oito grupos foram estudados: Pilo, SCH+Pilo, R-Pia+Pilo, R-Pia+SCH+Pilo, e seus respectivos controles. O número de animais em status epilepticus (SE), a latência para o início do SE e a taxa de mortalidade foram avaliados. O método de Fluoro Jade B (FJB) foi realizado 24 horas e sete dias após SE. RESULTADOS: O pré-tratamento com SCH58261, R-Pia e R-Pia+ SCH58261 reduziu o número de animais em SE, aumentou a latência para o SE e diminuiu a taxa de mortalidade, comparado ao tratamento com pilocarpina. Os grupos R-Pia e R-Pia+SCH58261 apresentaram uma redução no número de células marcadas com FJB em CA3 e hilo, 24 horas e sete dias após SE, e no córtex piriforme apenas 24 horas após SE, comparado ao grupo Pilo. CONCLUSÃO: O antagonista A2A demonstrou um potente efeito anticonvulsivante, enquanto o agonista A1 teve um papel crucial na modulação da crise e promoveu significante neuroproteção.

OBJECTIVE: To characterize the effect of the A2A receptor blockage by the SCH58261 in the seizure modulation and neuroprotection of the brain areas vulnerable to injury by pilocarpine. The effect of SCH58261 was also analyzed in combination with the activation of the A1 receptors by R-Pia. METHODS: Eight groups were studied: Pilo, SCH+Pilo, R-Pia+Pilo, R-Pia+SCH+Pilo, and respective controls. The number of animals in status epilepticus (SE), the latency to the SE onset and the mortality rate were evaluated. The Fluoro Jade B (FJB) method was performed 24 hours and seven days after SE. RESULTS: The pretreatment with SCH58261, R-Pia and R-Pia+SCH58261 reduced the number of animals in SE, increased the latency to the SE and decreased the mortality rate, compared to pilocarpine treatment. The R-Pia and R-Pia+SCH58261 groups exhibited a reduction in the number of FJB stained cells in CA3 and hilus, 24 hours and seven days after SE, and in the piriform cortex only 24 hours after SE, compared to Pilo group. CONCLUSION: The A2A antagonist demonstrated a potent anticonvulsant effect, while the A1 agonist had a crucial role in the seizure modulation and promoted significant neuroprotection.

Epilepsia e neuroproteção: o papel do agonista adenosinérgico A1(RPia) na modulação da crise induzida por pilocarpina / Epilepsy and neuroprotection: employment of the RPia during status epilepticus induced by pilocarpine

OBJETIVO: O objetivo desse estudo foi caracterizar a neuroproteção do RPia em ratos submetidos ao status epilepticus (SE) induzido pela pilocarpina (Pilo). MÉTODOS: Avaliou-se o balanço entre utilização local da glicose cerebral (ULGC) e fluxo sanguíneo cerebral local (FSCL) após 4 horas de SE, e a marcação por Fluoro Jade-B (FJB), 24 horas e 90 dias após SE. Quatro grupos foram avaliados: Salina, Pilo, RPia+Salina e RPia+Pilo. RESULTADOS E CONCLUSÃO: Aumentos significantes na ULGC foram observados na maioria das regiões avaliadas nos grupos Pilo e RPia+Pilo quando comparados ao controle. Entretanto, redução significante na ULGC ocorreu na substância negra pars reticulata e giro denteado do grupo RPia+Pilo versus Pilo. Houve aumento significante do FSCL em todas as áreas estudadas, comparando-se os grupos Pilo e RPia+Pilo com o controle. Foi observado um aumento significante do FSCL durante SE em CA2, CA3, giro denteado, córtex entorrinal, corpo mamilar, núcleos talâmicos, núcleo rubro, zona incerta, núcleo oral da ponte e córtex visual, no grupo pré-tratado com RPia comparado ao tratado somente com Pilo. Grande número de células marcadas com FJB foi observado no grupo Pilo e o pré-tratamento com RPia reduziu essa marcação na formação hipocampal, córtex piriforme, amígdala basolateral e substância negra pars compacta.

OBJECTIVE: The aim of this study was to characterize the neuroprotection of the RPia in rats subjected to status epilepticus (SE) induced by pilocarpine (Pilo). METHODS: We evaluated the mismatch between local cerebral glucose utilisation (LCGU) and local cerebral blood flow (LCBF) 4 hours after SE induction. Neuronal loss was evaluated by Fluoro Jade-B (FJB) 24 hours and 90 days after SE. Four groups were studied: Saline, Pilo, RPia+Saline and RPia+Pilo. RESULTS AND CONCLUSIONS: Significant increases in the LCGU were observed in the almost all brain regions of Pilo and RPia+Pilo groups compared to control. However, significant reduction in the LCGU occurred in the substantia nigra pars reticulata and hippocampal formation of RPia+Pilo group versus Pilo. There was significant increase of the LCBF in all the studied areas, comparing the Pilo and RPia+Pilo groups with the control. The increases of LCBF was more intense in rats from RPia+Pilo compared to Pilo, and located mainly in CA2, CA3, dentate gyrus, entorhinal cortex, thalamic nuclei, mammillary body, red nucleus, zone incerta, pontine nucleus and visual cortex. A great number FJB stained cells was observed in the Pilo group and RPia pretreatment reduced the staining in the hippocampal formation, piriform cortex, basolateral amygdala and substantia nigra pars compacta.

Protective effect of the organotelluroxetane RF-07 in pilocarpine-induced status epilepticus.

Temporal lobe epilepsy is the most common form of epilepsy in humans. Caspase activation is a mechanism of cell death induced by seizures. Tellurium (IV) compounds present antitumoral, immunomodulatory and neuroprotective effects due to their ability to inhibit cysteine proteases. We studied the activity of caspase-1, -3 and -8 in the hippocampus of rats exhibiting status epilepticus induced by pilocarpine. All three caspases were activated. Tellurium (IV) compounds RF-07, RF-03 and AS-101 inhibited caspases in vitro, showing high second-order inhibition rate constants. The intraperitoneal injection of RF-07 prior to pilocarpine suppressed the behavioral and electroencephalographic seizure occurrence. According to our results, the caspases are activated as early as 90 min following SE. Tellurium (IV) compounds exerted anticonvulsant effects associated with the inhibition of caspases. These results suggest a promising therapeutic potential of organotellurium (IV) compounds as antiepileptogenic agents.