Rats subjected to structural brain damage induced by sustained convulsions triggered by systemic administration of pilocarpine (PILO) are a useful model for investigation of the mechanisms essential for seizure generation and spread in rodents. After PILO administration, three distinct phases are observed: (a) an acute period of 1-2 days' duration corresponding to a pattern of repetitive limbic seizures and status epilepticus; (b) a seizure-free (silent) period characterized by a progressive return to normal EEG and behavior of 4-44 days' duration; and (c) a period of spontaneous recurrent seizures (SRS) starting 5-45 days after PILO administration and lasting throughout the animal's life. PILO (320-350 mg/kg intraperitoneally, i.p.) was administered to rats, and the content of hippocampal monoamines and amino acids was measured in the acute, silent, and SRS periods by liquid chromatography. Norepinephrine (NE) level was decreased during all periods whereas dopamine (DA) content was increased. Serotonin (5-hydroxytryptamine, 5-HT) was increased only in the acute period. Utilization rate measurement of monoamines showed increased NE consumption and decreased DA consumption during all phases. 5-HT utilization rate was increased only in the acute period. Amino acid content showed a decrease in aspartate (ASP) and glutamate (GLU) concentrations associated with increased gamma-aminobutyric acid (GABA) level during the acute period. The silent phase was characterized by a decrease in glycine (GLY) and GABA levels and an increase in GLU concentration. The SRS period showed an increase in all amino acid concentrations. These findings show important neurochemical changes in the course of establishment of an epileptic focus after brain damage induced by status epilepticus triggered by pilocarpine.

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In order to follow the spatial and temporal evolution of neuronal damage, cellular activation and stress responses subsequent to lithium-pilocarpine seizures of various durations in the adult rat, we analyzed the expression of Fos protein and local cerebral glucose utilization as markers of cellular activation, HSP72 immunoreactivity and acid fuchsin staining as indicators of cellular stress and injury, and Cresyl violet staining for the assessment of neuronal damage. The expression of Fos appeared very early, 2-30 min after the onset of polyspikes and intensified during the following 4 h. Fos immunoreactivity was especially high in the hippocampus, cerebral cortex, amygdala and anterior olfactory nuclei. Local cerebral glucose utilization measured during the second hour of seizures was largely increased (350-580%) over control levels in cortical areas, amygdala, dentate gyrus, caudate nucleus and mediodorsal thalamus. HSP72 immunoreactivity never appeared earlier than 40-50 min after the onset of polyspikes, and was most prominent in hippocampal CA3 area, cerebral cortex (except the piriform cortex) and anterior olfactory nuclei. Acid fuchsin staining was maximal in the piriform cortex and the polymorphic layer of the dentate gyrus. Staining was moderate in the sensorimotor cortex and the amygdala. Neuronal damage was extensive in the piriform and entorhinal cortices, the hippocampal CA3 area and the polymorphic layer of the dentate gyrus, basal amygdala, mediodorsal thalamus and anterior olfactory nuclei. In conclusion, the present study shows that brain regions with the highest expression of Fos and the largest metabolic activation were also highly stained with acid fuchsin and most heavily damaged. Conversely, there is no clear relationship between HSP72 expression, cellular activation and neuronal damage.

Patch-clamp recordings indicated the presence of P2X7 receptors at neural progenitor cells (NPCs) in the subgranular zone of the dentate gyrus in hippocampal brain slices prepared from transgenic nestin reporter mice. The activation of these receptors caused inward current near the resting membrane potential of the NPCs, while P2Y1 receptor activation initiated outward current near the reversal potential of the P2X7 receptor current. Both receptors were identified by biophysical/pharmacological methods. When the brain slices were prepared from mice which underwent a pilocarpine-induced status epilepticus or when brain slices were incubated in pilocarpine-containing external medium, the sensitivity of P2X7 and P2Y1 receptors was invariably increased. Confocal microscopy confirmed the localization of P2X7 and P2Y1 receptor-immunopositivity at nestin-positive NPCs. A one-time status epilepticus in rats caused after a latency of about 5 days recurrent epileptic fits. The blockade of central P2X7 receptors increased the number of seizures and their severity. It is hypothesized that P2Y1 receptors after a status epilepticus may increase the ATP-induced proliferation/ectopic migration of NPCs; the P2X7 receptor-mediated necrosis/apoptosis might counteract these effects, which would otherwise lead to a chronic manifestation of recurrent epileptic fits.

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Summary:Purpose: This study was performed to study the role of adenosine triphosphate (ATP) in the brain of pilocarpine-induced chronic epileptic rats.Methods: ATP-mediated changes in intracellular calcium were studied by the fura-2 method. Immunohistochemistry and Western blotting methods were used to localize and quantify P2X7 receptors in these animals.Results: The fluorometric study in chronic rats revealed a biphasic response indicating the presence of P2X7 receptors.The Western blotting study showed an increase of 80% of P2X7 expression in chronic rats compared with the control group. P2X7 immunoreactivity resembled mossy fiber sprouting at the dentate gyrus of epileptic animals.Conclusions: These results suggest that purinergic receptors may participate in the pathophysiology of temporal lobe epilepsy. Key Words: Epilepsy-ATP-P2X7-PilocarpineRat.The role of adenosine triphosphate (ATP) as a peripheral and central neurotransmitter is mediated by P2 purinoceptors (1). The response of ATP as a fast neurotransmitter is mediated by ionotropic P2X receptors that have a high permeability to calcium ions. Seven subtypes of these receptors have been cloned (P2X1-7), and some of them (P2X4, P2X6, and P2X7) are abundant in hippocampus, amygdala, thalamus, and cortex (2). In contrast to other subtypes, P2X7 can form a 900-Da pore when activated by ATP, and this pore can dialyze the cytoplasmatic content, producing a large change in the intracellular ion homeostasis (3). Moreover, in vitro study in hippocampal slices showed that ATP released by electrical stimulation of Schaffer collateralcommissural afferents take part in excitatory synaptic transmission (4). ATP released from nerve endings stimulates an increase in intracellular Ca 2+ in rat brain and can activate the microglia that are able to release several cytotoxic and protective substances (5,6). However, there is no evidence that ATP-mediated calcium entry plays a pathophysiologic role in epilepsy. This study was performed to study the ATP role in temporal lobe epilepsy (TLE) using pilocarpine-induced epileptic rats. Thus, we studied hippocampal ATP-mediated calcium influx, and we localized and quantified purinergic P2X7 receptors. METHODSChronic epileptic animals were obtained 60 days after status epilepticus induced by pilocarpine (380 mg/kg, i.p.). For the control group, a saline solution was used instead of pilocarpine. The intracellular calcium response mediated by ATP was measured with fluorometry. P2X7 expression was studied by immunohistochemistry (IHC) and quantified by Western blotting.For the fluorometric study, hippocampi slices were submitted to the fura-2 method described by Grynkiewicz et al. (7). In brief, rats from epileptic (n ‫ס‬ 5) and control (n ‫ס‬ 5) groups were killed, their brains removed and placed in cold artificial cerebrospinal fluid (aCSF) containing (in mM): 130 NaCl, 24 NaHCO 3 , 10 D-glucose, 1.3 NaH 2 PO 4 , 3.5 KCl, 5 MgCl, and 0.2 CaCl 2 . Afterward, the brains were sliced (500 m) in a vibratome. Hippocampal slices were inc...

Our data demonstrated that the IFN-alpha was able to induce Fas-mediated apoptosis together with a reduction in the tumor size; such an observation may suggest the importance to investigate still unexplored mechanisms to be exploited in craniopharyngioma therapy.

Exercise stimulates the release of beta-endorphin and other endogenous opioid peptides that are believed to be responsible for changes in mood, perception of pain and also performance. Although the vast majority of literature data support the role of physical exercise in increasing beta-endorphin levels, indirect measures such as increased endorphin levels in peripheral blood do not reflect opioid levels in the central nervous system. The purpose of the present study was to verify whether acute and chronic exercise using both voluntary and forced exercise procedures could modify the expression of μ-opioid receptors (MOR) in rat hippocampal formation. Immunoblotting analysis showed significantly enhanced MOR expression in the hippocampal formation in the acute (forced and voluntary) exercise groups when compared to the control group. Conversely, a significant reduction of MOR expression was noted in the chronic forced and chronic voluntary exercise groups compared to the acute forced and voluntary groups respectively. MOR expression was not significantly different in rats trained using both acute or chronic exercise. Immunohistochemistry analysis showed a higher number of MOR-positive cells for acute forced and voluntary exercise groups in the CA1, CA3, hilus and dentate gyrus regions compared to the control group. Our findings indicate that acute and chronic exercise modulates MOR expression in the hippocampal formation of rats.