Lovastatin decreases the synthesis of inflammatory mediators during epileptogenesis in the hippocampus of rats submitted to pilocarpine-induced epilepsy.

Statins may act on inflammatory responses, decreasing oxidative stress and also reducing brain inflammation in several brain disorders. Epileptogenesis is a process in which a healthy brain becomes abnormal and predisposed to generating spontaneous seizures. We previously reported that lovastatin could prevent neuroinflammation in pilocarpine-induced status epilepticus (SE). In this context, this study investigated the long-lasting effects of lovastatin on mRNA expression of proinflammatory cytokines (interleukin-1ß, tumor necrosis factor α, interleukin-6) and the antiinflammatory cytokine IL-10 in the hippocampus during epileptogenesis by immunohistochemistry and real time polymerase chain reaction (RT-PCR) during the latent and chronic phases in the epilepsy model induced by pilocarpine in rats. For these purposes, four groups of rats were employed: saline (CONTROL), lovastatin (LOVA), pilocarpine (PILO), and pilocarpine plus lovastatin (PILO+LOVA). After pilocarpine injection (350mg/kg, i.p.), the rats were treated with 20mg/kg of lovastatin via an esophagic probe 2h after SE onset. All surviving rats were continuously treated during 15days, twice/day. The pilocarpine plus lovastatin group showed a significant decrease in the levels of IL-1ß, TNF-α, and IL-6 during the latent phase and a decreased expression of IL-1ß and TNF-α in the chronic phase when compared with the PILO group. Moreover, lovastatin treatment also induced an increased expression of the antiinflammatory cytokine, IL-10, in the PILO+LOVA group when compared with the PILO group in the chronic phase. Thus, our data suggest that lovastin may reduce excitotoxicity during epileptogenesis induced by pilocarpine by increasing the synthesis of IL-10 and decreasing proinflammatory cytokines in the hippocampus.

Kinin B1 receptors facilitate the development of temporal lobe epilepsy in mice.

Kinins may play a relevant role in epilepsy. In the present study, we evaluated the hippocampal expression of the remaining kinin receptor in B1 (B1KO) and B2 (B2KO) knockout mice strains during the development of pilocarpine epilepsy model. After pilocarpine injection, animals had their behavior parameters monitored to determine different phases of temporal lobe epilepsy (TLE) progression. Hippocampal mRNA expression was evaluated using specific primers for kinin receptors by Real Time-PCR. B1KO hippocampus from acute, silent and chronic phases showed no differences in B2 receptor mRNA expression when compared to control. An increased B1 receptor mRNA expression in treated B2KO hippocampus (0.97+/-0.12, acute; 0.86+/-0.09, silent; and 0.94+/-0.11, chronic phase; p<0,001) when compared to control (0.12+/-0.03) was observed. Behavioral and neurochemistry parameters suggest that kinin B1 receptor is fundamental to development of epilepsy on pilocarpine-induced model.

Chondroitin sulfate and kallikrein in saliva: markers for glossodynia.

Glossodynia or burning mouth syndrome is a multifunctional disorder. The oral mucosa is apparently normal but patients report burning and dried mouth and painful tongue and lips. The present study reports biochemical and physiological markers in saliva of patients presenting glossodynia compared to normal subjects. Saliva-buffering capacity and contents of protein and hyaluronic (HA) acid were similar in both groups. In contrast, chondroitin sulfate (CS) concentration was decreased in the saliva of patients with glossodynia when compared to control group (p=0.0036). On the other hand glandular kallikrein showed increased activity in the saliva of patients compared to normal subjects (p<0.0001). The data suggest involvement of the kinin system, possibly related to the low levels of CS. Depression could explain the low level of serotonin in patient serum (p=0.0478).

Expression of nestin in the hippocampal formation of rats submitted to the pilocarpine model of epilepsy.

Nestin is an embryonic intermediate filament component protein, transiently expressed by the immediate precursor cells of neurons and glia, during brain development. We studied the nestin distribution in the hippocampal formation of rats submitted to pilocarpine model of epilepsy. Animals were studied during the acute, silent and chronic phases. Rats from control and acute groups presented absence of nestin-immunoreactivity (IR) in the hippocampal cells. In contrast, cells from this region presented strong nestin IR during the silent phase (3 and 7 days after status epilepticus (SE) onset), disappearing 14 days after SE. Nestin IR cells were scattered expressed in all hippocampal formation during the chronic phase. Almost all nestin IR cells exhibited glial fibrillary acidic protein (GFAP), which seems to revert to a more primitive glial form, as part of an adaptive response, transiently re-expressing phenotypic features typical of earlier stages of glial development. The re-expression of this developmental protein in the damaged cerebral tissue suggests that nestin may play an important role in the reconstruction of the glial cytoskeleton and/or remodeling events occurring in the pilocarpine model of epilepsy. Understanding how astrocytes influence network function in the injured hippocampus may, therefore, provide insight into epileptogenic mechanisms.

Growth-associated phosphoprotein expression is increased in the supragranular regions of the dentate gyrus following pilocarpine-induced seizures in rats.

Neuroplasticity has been investigated considering the neuronal growth-associated phosphoprotein as a marker of neuronal adaptive capabilities. In the present work, studying the hippocampal reorganization observed in the epilepsy model induced by pilocarpine, we carried out quantitative western blotting associated with immunohistochemistry to determine the distribution of growth-associated phosphoprotein in the hippocampus of rats in acute, silent and chronic periods of this epilepsy model. The fibers and punctate elements from the inner molecular layer of the dentate gyrus were strongly immunostained in animals killed 5 h after status epilepticus, compared with the same region in control animals. Rats presenting partial seizures showed no alterations in the immunostaining pattern compared with saline-treated animals. The hippocampal dentate gyrus of animals during the seizure-free period and presenting spontaneous recurrent seizures was also characterized by strong growth-associated phosphoprotein immunostaining of fibers and punctate elements in the inner molecular layer, contrasting with the control group. As determined by western blotting analysis, growth-associated phosphoprotein levels increased following status epilepticus and remained elevated at the later time-points, both during the silent period and during the period of chronic recurring seizures. Pilocarpine-treated animals, which did not develop status epilepticus, showed no change in growth-associated phosphoprotein levels, indicating that status epilepticus is important to induce growth-associated phosphoprotein overexpression. The measurement of this overexpression could represent one of the early signals of hippocampal reorganization due to status epilepticus-induced damage.

Profile of prostaglandin levels in the rat hippocampus in pilocarpine model of epilepsy.

Pilocarpine (PILO) administered to rats acutely induces status epilepticus (acute period), which is followed by a transient seizure-free period (silent period), and finally by a chronic phase of spontaneous recurrent seizures (chronic period, SRS) that lasts for the rest of animal's life. Hippocampal neurochemical changes following PILO administration include alteration in monoamines and amino acids content during all phases of this epilepsy model. The present work was delineated to study the content of prostaglandins (PG) levels in hippocampus during the three phases of this model. The levels of PG E2, PG F2 alpha and PG D2 were measured by radioimmunoassay 1 h after PILO, 5 h after PILO, during the silent period, and interictally into the chronic period. The results show, in hippocampus of rats, increase of PG F2 alpha and PG D2 during status epilepticus, increase of PG D2 during the silent period and increase of PG E2 and PG D2 during the chronic phase, when compared with control group. These changes match previously reported alteration in monoamines and amino acid levels, showing that altered neurotransmission is accompanied by changes in second messengers and enzyme activity related to PG production during all phases of the epilepsy model.

Na+K+ ATPase activity in the rat hippocampus: a study in the pilocarpine model of epilepsy.

Biochemical abnormalities have been implicated in possible mechanisms underlying the epileptic phenomena. Some of these alterations include changes in the activity of several enzymes present in epileptic tissues. Systemic administration of pilocarpine in rats induces electrographic and behavioral limbic seizures and status epilepticus, that is followed by a transient seizure-free period (silent period). Finally a chronic phase ensues, characterized by spontaneous and recurrent seizures (chronic period), that last for the rest of the animal's life. The present work aimed to study the activity of the enzyme Na+K+ ATPase, in rat hippocampus, during the three phases of this epilepsy model. The enzyme activity was determined at different time points from pilocarpine administration (1 and 24 h of status epilepticus, during the silent and chronic period) using a spectrophotometric assay previously described by Mishra and Delivoria-Papadopoulos [Neurochem. Res. (1988) 13, 765-770]. The results showed decreased enzyme activities during the acute and silent periods and increased Na+K+ ATPase activity during the chronic phase. These data show that changes in Na+K+ ATPase activity could be involved in the appearance of spontaneous and recurrent seizures following brain damage induced by pilocarpine injection.

Mitogen-activated protein kinase is increased in the limbic structures of the rat brain during the early stages of status epilepticus.

Systemic administration of pilocarpine (PILO) in adult rat produces acute limbic seizures leading to status epilepticus. Recent studies have shown the activation of mitogen-activated protein kinase (MAPK) cascades during experimentally induced seizures. MAPK activation may be triggered by glutamatergic stimulation and may play a key role in signal transduction pathways. In the present study, immunocytochemistry was used to analyze the spatiotemporal distribution pattern of the MAPK protein and its active form (A-MAPK) following PILO-induced status epilepticus. MAPK and A-MAPK immunoreactivities exhibited different patterns of distribution in the brain of normal and epileptic rats. The saline-treated rats, as well as the animals that received PILO but did not evolve to status epilepticus, showed a weak but selective MAPK immunoreactivity, detected in the hippocampal pyramidal neurons, dentate gyrus, hilus, CA3, CA1, and entorhinal, piriform, and cingulate cortices. A-MAPK immunoreactivity was instead observed only in neurites of the CA3 and hilus and in cells of the entorhinal and piriform cortices. In PILO-treated rats, between 30 and 60 min after status epilepticus there was an increase of the immunoreactivity to both antibodies, which were differently distributed throughout several structures of the limbic system. The immunostaining showed a slight decrease after 5 h of status epilepticus. However, MAPK and A-MAPK immunopositivities decreased markedly after 12 h of status epilepticus, returning almost to the basal expression. These findings are consistent with a spatial and time-dependent MAPK expression in selected limbic structures, and its activation could represent an initial trigger for neuronal modifications that may take part in the mechanism underlying acute epileptogenesis and in long-lasting neuropathological changes of the PILO model of epilepsy.

Tyrosine phosphorylation is increased in the rat hippocampus during the status epilepticus induced by pilocarpine.

Phosphorylation of tyrosine residue in proteins is an important modulatory process for membrane transduction and cell signaling and for several cellular functions. The concentration and distribution of phosphotyrosine proteins were analyzed in the hippocampi of rats in the model of epilepsy induced by pilocarpine using Western blotting and immunohistochemistry. The concentration of several phosphotyrosine proteins increased during status epilepticus. During the seizure-free period and the chronic period of this epilepsy model, the hippocampi of rats did not exhibit changes in the expression of these proteins. Immunohistochemistry showed an increased immunoreactivity throughout the hippocampal formation of rats 1 h after status epilepticus that was acutely induced by pilocarpine. Animals killed after 3 h of status epilepticus showed an increased expression of phosphotyrosine in the hippocampal hilus and CA3 regions. After 5 h of status epilepticus, phosphotyrosine immunoreactivity persisted only in the CA3 region. After 12 h of status epilepticus, the hippocampal formation exhibited a normal phosphotyrosine immunostaining, showing that the increased expression of these proteins is related to the acute phase and that several intracellular events could undergo modifications during the status epilepticus induced by pilocarpine.

Selective alterations of glycosaminoglycans synthesis and proteoglycan expression in rat cortex and hippocampus in pilocarpine-induced epilepsy.

Proteoglycans and glycosaminoglycans are elements of matrix. In the nervous system, glycosaminoglycans modulate neurite outgrowth and are co-receptors for growth factors playing a crucial role in cell differentiation and synaptogenesis. The receptor of protein tyrosine phosphatase beta (RPTPbeta) is a chondroitin sulphate proteoglycan which plays an important role in neural morphogenesis and axon guidance mechanisms. Pilocarpine-treated rats present status epilepticus, which is followed by a seizure-free period (silent), by a period of spontaneous recurrent seizures (chronic), and the hippocampus of these animals exhibits cell loss and mossy fiber sprouting. Thus, the synthesis of heparan sulphate and chondroitin sulphate and the time course of RPTPbeta immunoreactivity were studied in the hippocampus and cerebral cortex during these phases of pilocarpine-induced epilepsy. The results showed decreased synthesis of heparan sulphate during the acute phase and an increased synthesis of chondroitin sulphate during the silent period in the cortex and hippocampus. In control rats RPTPbeta immunoreactivity was detected only in glial cells. After 6 h of status epilepticus the RPTPbeta immunoreactivity was no longer detectable in the glial cells in both tissues and intense staining became evident in the matrix, surrounding CA3 and dentate gyrus and piriform cortex neurones. In the silent and chronic periods RPTPbeta immunoreactivity was mainly detected in neuronal somata and fibers of neurones of hippocampus and cortex. These changes show a selective variation of synthesis and expression of glycosaminoglycans and RPTPbeta in relation to epilepsy suggesting a molecular interplay between glia and neurones during seizures.

Glycosaminoglycan levels and proteoglycan expression are altered in the hippocampus of patients with mesial temporal lobe epilepsy.

Extracellular matrix proteoglycans (PGs) and glycosaminoglycans (GAGs) play a crucial role in cell differentiation and synaptogenesis by modulating neurite outgrowth. The chondroitin sulfate (CS)-rich PG, the receptor protein tyrosine phosphatase zeta/beta (RPTP zeta/beta), has been related to neural morphogenesis and axon guidance. Hippocampal sclerosis is the most frequent pathologic finding in patients with intractable mesial temporal lobe epilepsy (MTLE), which is associated with neuron loss, reactive gliosis, and mossy fiber sprouting. In the present study, we investigated the concentration of CS, heparan sulfate (HS) and hyaluronic acid (HA) in the hippocampus and temporal neocortex as well as RPTP zeta/beta expression in the hippocampus of patients with MTLE. Compared to autopsy control tissue, epileptic hippocampi showed a significantly increased concentration of CS (224%; p=0.0109) and HA (146%; p=0.039). HS was instead similar to control values. No differences were found in the concentration of CS, HS, or HA in the temporal neocortex of epileptic patients when compared to control values. In contrast, RPTP zeta/beta immunoreactivity was induced in astrocytes of the inner molecular layer of the dentate gyrus of the sclerotic hippocampus. Because matrix compounds have been associated with tissue injury and repair, the present findings suggest that changes in PGs and GAGs might be related to damage-induced gliosis and neuronal reorganization in the hippocampus of MTLE patients.

Monoamines and their metabolites in cerebrospinal fluid and temporal cortex of epileptic patients.

The involvement of monoamines in the initiation or maintenance of epileptic phenomena has been extensively studied in cerebral tissues and in cerebrospinal fluid. The present work was undertaken to study monoamines and their metabolites in human spiking and non-spiking temporal cortex excised from patients with complex partial seizures unresponsive to available anticonvulsants. The same substances were also analyzed by HPLC-ED in cerebrospinal fluid obtained 24 h before the surgical procedure and compared with those from patients with chronic headache and normal neurological evaluation. The results show increased 5-HT, 5-HIAA and HVA levels in spiking compared with non-spiking cortex. Cerebrospinal fluid levels of 5-HIAA and HVA are concomitantly increased in epileptic compared with headache patients.

Castration in female rats modifies the development of the pilocarpine model of epilepsy.

Previous studies have shown that the susceptibility to pilocarpine-induced status epilepticus (SE) in female rats changes according to estrous cycle phases. These studies have also shown that following pilocarpine administration changes occur in gonadal, hypophyseal and hypothalamic hormones that could contribute for the sequence of the epileptic events. Accordingly, the present work aimed to investigate the role of sexual hormones withdrawal on the development of the pilocarpine model of epilepsy in female rats. With this purpose, castrated and non-castrated adult female Wistar rats were injected with pilocarpine and some characteristic parameters of the experimental model were observed. The results showed increased mortality after pilocarpine injection in the castrated rats when compared with non-castrated females. The latency period for SE onset and for the first spontaneous seizure was decreased in castrated when compared with non-castrated animals. The mossy fiber sprouting measured by neo-Timm scale during the chronic period, reached grade 3 for castrated epileptic rats while the non-castrated epileptic rats showed grade 2. Our results indicate that castration interferes with the epileptogenesis in the pilocarpine model of epilepsy suggesting that female sexual hormones could have protective effects against pilocarpine-induced SE.

Superoxide dismutase, glutathione peroxidase activities and the hydroperoxide concentration are modified in the hippocampus of epileptic rats.

The relationship between free radical and scavenger enzymes has been found in the epileptic phenomena and reactive oxygen species have been implicated in seizure-induced neurodegeneration. Using the epilepsy model obtained by systemic administration of pilocarpine (PILO) in rats, we investigated the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities as well as the hydroperoxide (HPx) concentration in the hippocampus of rats during status epilepticus (SE), silent and chronic periods. The enzyme activities as well as the HPx concentration were measured using spectrophotometric methods and the results compared to values obtained from saline-treated animals. The SOD activity decreased after long-lasting SE period and during the chronic phase. In addition, HPx levels increased in same periods whereas the GPx activity increased only in the hippocampus of animals submitted to 1 h of SE. Animals presenting partial seizures, those submitted to 5 h of SE and animals from the silent period (seizure free) showed normal levels of SOD, GPx and HPx. These results show a direct evidence of lipid peroxidation during seizure activity that could be responsible for neuronal damage in the hippocampus of rats, during the establishment of PILO model of epilepsy.

Increased serotonin levels in physically trained men.

It has been postulated that exercise training influences monoaminergic systems. The purpose of the present study was to determine the basal level of serum serotonin (5HT) in track and field-trained men (N = 15) and in untrained subjects matched by age, weight and height (N = 15). Serum serotonin levels were determined in blood drawn into dry tubes after a 12-h fast by high performance liquid chromatography utilizing electrochemical detection. Mean (+/- SD) serum serotonin levels were: 141.32 +/- 38.77 ng/ml for trained subjects and 97.77 +/- 30.53 ng/ml for untrained subjects (P < 0.01, Student t-test). These data show that basal serum serotonin levels are increased by exercise training.

Effect of amygdaloid kindled seizures during pregnancy on neonatal brain biogenic amines.

The effects of amygdaloid kindled seizures during pregnancy on the concentrations of noradrenaline (NE), dopamine (DA) and serotonin (5HT) and of their respective metabolites, 3-methoxy-4-hydroxyphenylglycol (MHPG), normetanephrine (NMN), homovanillic acid (HVA) and 5-hydroxy-indoleacetic acid (5HIAA), have been studied in the cerebral cortex, brain stem and cerebellum of rat offspring at birth. The levels of DA and NE were increased and those of HVA and MHPG were not modified in the cortex. The levels of DA, NE, 5HT, MHPG and 5HIAA were increased in the cerebellum. The brain stem presented a decrease in DA and 5HT levels, but increased MHPG and HVA levels. It is suggested that, in order to investigate possible changes in the biogenic amine levels on the postnatal period, carefully planned prospective studies are needed.

Effect of an aerobic exercise program on blood pressure and catecholamines in normotensive and hypertensive subjects.

Interrelations between physical exercise, monoamines and hypertension are postulated by various investigators. The purpose of the present study was to determine and compare catecholamine levels at rest and after a 12-week aerobic exercise program in 11 sedentary normotensive (N) and 8 hypertensive (H) men. Plasma catecholamines were determined by high performance liquid chromatography with electrochemical detection. A significant post-exercise increase in plasma noradrenaline was observed in the N and H groups (P < 0.01) both before and after the aerobic exercise program. The hypertensive group showed a significant reduction of the sum of 7 skinfold thickness scores after the aerobic exercise program (from 178.7 +/- 65.6 to 144.0 +/- 47.4 mm) although no significant difference was observed when the body mass index was compared. A significant reduction in diastolic blood pressure at rest was observed in the H group after the aerobic exercise program (from 99.2 +/- 2.0 to 85.0 +/- 5.5). There were no significant differences in catecholamine concentrations between groups before and after the 12-week aerobic exercise program at rest and post-exercise. These data show a relationship between physical exercise and hypertension that was not related to changes in plasma catecholamine levels.

Catecholamine response to exercise in individuals with different levels of paraplegia.

The purpose of this study was to investigate the effect of the level of injury on the serum level of norepinephrine (Nor) and epinephrine (Epi) at rest and after maximal exercise in individuals with paraplegia. Twenty-six male spinal cord-injured subjects with complete paraplegia for at least 9 months were divided into two groups of 13 subjects each according to the level of injury, i.e., T1-T6 and T7-T12. Serum Nor and Epi concentrations were measured by HPLC-ECD, at rest (PRE) and immediately after a maximal ergospirometric test (POST). Statistical analysis was performed using parametric and non-parametric tests. Maximal heart rate, peak oxygen uptake, and PRE and POST Nor were lower in the T1-T6 than in the T7-T12 group (166 +/- 28 vs 188 +/- 10 bpm; 18.0 +/- 6.0 vs 25.8 +/- 4.1 ml kg(-1) min(-1); 0.54 +/- 0.26 vs 0.99 +/- 0.47 nM; 1.48 +/- 1. 65 vs 3.07 +/- 1.44 nM). Both groups presented a significant increase in Nor level after exercise, while only the T7-T12 group showed a significant increase in Epi after exercise (T1-T6: 0.98 +/- 0.72 vs 1.11 +/- 1.19 nM; T7-T12: 1.24 +/- 1.02 vs 1.89 +/- 1.57 nM). These data show that individuals with paraplegia above T6 have an attentuated catecholamine release at rest and response to exercise as compared to subjects with injuries below T6, which might prevent a better exercise performance in the former group.

Serum serotonin levels of normal and autistic children.

1. Serum serotonin (5HT) was determined in normal and autistic children by high performance liquid chromatography with electrochemical detection (HPLC-ED), after serum deproteinization. 2. The sample deproteinization was carried out by the addition of 3.4 M HCLO4 to a small volume of the serum, followed by freezing, centrifugation, dilution and injection of sample into the HPLC. 3. Serum 5HT concentration was significantly increased in autistic children (303 +/- 92 ng/ml) (N = 19) when compared to that of normal children (215 +/- 67 ng/ml) (N = 46). The data of normal children analyzed by ANOVA for sex or age showed no difference. 4. The method employed in this study showed high resolution, good sensitivity and can be used for routine determination of serum 5HT in the clinical investigation of hyperserotonemia in autism.

Carbamazepine inhibits angiotensin I-converting enzyme, linking it to the pathogenesis of temporal lobe epilepsy.

We find that a common mutation that increases angiotensin I-converting enzyme activity occurs with higher frequency in male patients suffering from refractory temporal lobe epilepsy. However, in their brains, the activity of the enzyme is downregulated. As an explanation, we surprisingly find that carbamazepine, commonly used to treat epilepsy, is an inhibitor of the enzyme, thus providing a direct link between epilepsy and the renin-angiotensin and kallikrein-kinin systems.