Ante Simonić

Lithium plus pilocarpine induced status epilepticus--biochemical changes.

The aim of the study was to investigate the changes in biochemical mechanisms facilitating cellular damages in the lithium plus pilocarpine treatment and the resulting status epilepticus. The whole brain free fatty acid (FFA) level as well as the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), glutamate dehydrogenase, aspartate-aminotransferase (AST), alanine-aminotransferase, gamma-glutamoyl transferase, alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and creatine kinase in the frontal cortex, cerebellum, hippocampus and pons-medulla region of Hannover-Wistar rats were determined. The control group was intact with no previous experimental history. LiCl (125 mg/kg i.p.) was injected 20 h prior to pilocarpine (30 mg/kg i.p.) and the treated rats were sacrificed 1 or 2 1/2 h after pilocarpine administration. The results show that lithium plus pilocarpine administration and the resulting status epilepticus produced the significant increase of the brain FFA content. Decreased GPX activities were detected in the frontal cortex, cerebellum and hippocampus of the treated rats without the accompanying decrease of SOD activity. Increased AST and LDH activities were observed in the frontal cortex, increased soluble ALP activities in the frontal cortex and pons-medulla region whereas the increased activity of membrane bound ALP was detected in the hippocampus of the rats with status epilepticus. Activities of the other analysed enzymes did not change in the examined brain regions. The presented data indicate clear regional differences of biochemical changes caused by lithium plus pilocarpine treatment and the resulting status epilepticus, frontal cortex being the most affected site.

Pentylenetetrazol-induced seizures and kindling: changes in free fatty acids, superoxide dismutase, and glutathione peroxidase activity

The whole brain free fatty acid (FFA) level, as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were determined in the frontal cortex, cerebellum, hippocampus, and pons-medulla region of the single pentylenetetrazol (PZT)-treated and PZT-kindled Hannover-Wistar rats. PZT administration in the convulsive dose caused significant increase of the brain FFA content. Decreased SOD activity was detected in the frontal cortex of PZT-kindled rats, whereas decreased GPX activity was found in the frontal cortex and cerebellum of all treated rats, as well as in the hippocampus and pons-medulla of PZT-kindled rats. Kindling caused distinctive change of antioxidative defense in the frontal cortex, hippocampus, and pons-medulla region.

Altered activities of rat brain metabolic enzymes caused by pentylenetetrazol kindling and pentylenetetrazol-induced seizures

The aim of our study was to investigate amino acid and energy metabolism of pentylenetetrazol (PTZ)-kindled animals. Glutamate dehydrogenase, aspartate-aminotransferase (AST), alanine-aminotransferase, gamma-glutamyltransferase, alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and creatine kinase (CK) were determined in the frontal cortex, cerebellum, hippocampus and pons-medulla regions of Hannover-Wistar rats. The rats were randomly divided into four experimental groups: (a) control; (b) rats which received a single PTZ injection in a subconvulsive dose of 40 mg/kg i.p.; (c) rats which received a single PTZ injection in a convulsive dose of 50 mg/kg i.p.; and (d) PTZ-kindled rats. Kindling increased ALP activity throughout the brain, elevated AST as well as LDH activity in the frontal cortex and hippocampus and decreased CK activity in the frontal cortex and cerebellum. Acute seizures of the same intensity did not induce these alterations. The observed effects therefore are obviously linked to the kindling phenomenon and not to seizure activity. Changes appeared mainly in the frontal cortex and hippocampus, i.e. brain areas believed to be directly involved in kindling.

Hyperbaric oxygen treatment: the influence on the hippocampal superoxide dismutase and Na+,K+-ATPase activities in global cerebral ischemia-exposed rats

The influence of hyperbaric oxygen (HBO) treatment on the activities of superoxide dismutase (SOD) and Na(+),K(+)-ATPase was determined during different time periods of reperfusion in rats exposed to global cerebral ischemia. Ischemic animals were either sacrificed or exposed to the first HBO treatment 2, 24, 48 or 168 h after ischemic insult (for SOD activities measurement) or immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na(+),K(+)-ATPase activities measurement). Hyperbaric oxygenation procedure was repeated for seven consecutive days. The results of presented experiments demonstrated the statistically significant increase in the hippocampal SOD activity 24 and 48 h after global cerebral ischemia followed by a decrease in the enzymatic activity 168 h after ischemic insult. In the ischemic rats treated with HBO the level of hippocampal SOD activity was significantly higher after 168 h of reperfusion in comparison to the ischemic, non HBO-treated animals. In addition, it was found that global cerebral ischemia induced a statistically significant decrease of the hippocampal Na(+),K(+)-ATPase activity starting from 1 to 168 h of reperfusion. Maximal enzymatic inhibition was obtained 24 h after the ischemic damage. Decline in Na(+),K(+)-ATPase activity was prevented in the animals exposed to HBO treatment within the first 24 h of reperfusion. Our results suggest that global cerebral ischemia induces significant alterations in the hippocampal SOD and Na(+),K(+)-ATPase activities during different periods of reperfusion. Enhanced SOD activity and preserved Na(+),K(+)-ATPase activity within particular periods of reperfusion, could be indicators of a possible beneficial role of HBO treatment in severe brain ischemia.

Electroconvulsive shock in rats: changes in superoxide dismutase and glutathione peroxidase activity.

Seizures trigger a variety of biochemical processes including an influx of extracellular Ca(2+), activation of membrane phospholipases, liberation of free fatty acids, diacylglycerols, eicosanoids, lipid peroxides and free radicals. These lipid metabolites along with abnormal ion homeostasis may be involved in cell injury and cell death. The aim of this study was to determine brain antioxidant enzyme activities in rats with electroconvulsive shock (ECS)-induced seizures. ECS, single or repeated, induced a decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in various brain regions. The most prominent changes of enzymatic activities were observed in rats that received five ECSs with 24-h recovery period between them. Decreased SOD activity was observed in the frontal cortex of all treated animals except those sacrificed 24 h after single ECS, in the cerebellum of the animals that received repeated ECSs, in the hippocampus of animals that were decapitated 2 h after a single ECS and in the pons-medulla region of rats that received five daily ECSs. Decreased GPX activity was found in all examined brain regions of the rats that received five ECSs, the cortex and hippocampus of rats that were decapitated 2 h after single ECS and the cortex of those that received 10 ECSs with 48 h between them. The results show that neither 24-h nor 48-h recovery period was sufficient for the normalisation of antioxidative enzyme activities after repeated ECS treatment.

Effects of nimodipine, felodipine and amlodipine on electroconvulsive shock-induced amnesia in the rat

The effects of various doses (0.03, 0.1, 0.3 or 1.0 mg/kg) of the Ca2+ channel blockers nimodipine, felodipine and amlodipine on the learning ability of rats exposed to electroconvulsive shock were examined. The animals were trained in a passive avoidance procedure. The drugs tested were injected 30 min before the learning trial started. The electroconvulsive shock was given immediately after the learning trial response had been acquired. A passive avoidance retention test was performed 24 h later. It was found that electroconvulsive shock strongly impaired the retention of the passive avoidance response. Nimodipine, felodipine and amlodipine did not influence the passive avoidance behavior in the sham electroconvulsive shock group, but significantly improved the retention deficits in the animals exposed to electroconvulsive shock. These findings support the hypothesis that perturbations in Ca2+ homeostasis can contribute to the memory deficits associated with electroconvulsive shock. The antiamnestic effects of the substances tested make them interesting candidates for clinical trials in patients with cognitive impairment caused by electroconvulsive shock therapy.

Effect of indomethacin on motor activity and spinal cord free fatty acid content after experimental spinal cord injury in rabbits.

Study design:Determination of functional and biochemical parameters as well as the effect of specific therapies on these parameters, in the experimental model of neurotrauma in rabbits.Objective:To assess the effect of indomethacin (0.1–3.0 mg/kg for 9 days), a potent inhibitor of endogenous prostaglandin synthesis, on the motor activity and on the spinal cord tissue concentration of free palmitic, stearic, oleic, arachidonic and docosahexaenoic acids in an experimental model of a spinal cord injury in rabbits.Setting:Faculty of Medicine, University of Rijeka, Croatia.Methods:The animals were randomly divided into nine experimental groups, four sham and/or vehicle-treated and five indomethacin-treated (including one sham-operated and four injured groups). Laminectomy was followed by contusion of the spinal cord, using a modification of the technique of Albin. Motor activity was controlled daily during the course of the next nine postoperation days and scored using Tarlovs system. Spinal cord samples from the impact injury site were taken and frozen in liquid nitrogen. Total lipids were isolated and purified by a modification of the method of Folch. Free fatty acids (FFAs) were separated from the total lipid extract by preparative thin-layer chromatography, converted to the corresponding methyl esters and identified using gas chromatography, using nonadecanoic acid as the internal standard.Results:The concentrations of all analysed free fatty acids were increased in the spinal cord after neurotrauma, in comparison to control tissues. Treatment of injured rabbits with indomethacin resulted in a significant decrease in spinal cord FFAs and exerted a positive effect on neurotrauma-induced motor impairment.Conclusion:These results indicate a mechanism whereby indomethacin protects rabbits from the sequellae of neuronal damage caused by trauma, and suggests that it may be beneficial in the therapy of neurotrauma.Sponsorship:This work was supported by the Croatian Ministry of Science and Technology (project 062019).

The influence of MK-801 on the hippocampal free arachidonic acid level and Na+,K+-ATPase activity in global cerebral ischemia-exposed rats

The influence of 20 min global cerebral ischemia on the free arachidonic acid (FAA) level and Na+,K+-ATPase activity in the rat hippocampus at different time points after ischemia was examined. In addition, the effect of MK-801 on mentioned parameters was studied. Animals were exposed to 20 min global cerebral ischemia and were sacrificed immediately, 0.5, 1, 2, 6, 24, 48, 72, and 168 h after ischemic procedure. The level of the FAA and the Na+,K+-ATPase activity was measured during all reperfusion periods examined. Various doses of MK-801 (0.3, 1.0, 3.0, and 5.0 mg/kg) had been injected 30 min before ischemic procedure started. It was found that 20 min global cerebral ischemia induces a statistically significant increase of the FAA level immediately after ischemia and during the first 0.5 h of reperfusion. After a transient decrease, the level of FAA level increased again after 24 and 168 h of recirculation. Treatment with 3.0 mg/kg of MK-801 significantly prevented the FAA accumulation immediately and 0.5 h after ischemic insult while application of 5.0 mg/kg of MK-801 exerted a protective effect during the first 24 h. Global cerebral ischemia induces the significant decline in the Na+,K+-ATPase activity in the hippocampus starting from 1 to 168 h of reperfusion. Maximal inhibition was obtained 24 h after the ischemic damage. Application of 3.0 mg/kg of MK-801 exerted statistically significant protection during the first 24 h while the treatment with 5.0 mg/kg of MK-801 prevented fall in enzymatic activity during all reperfusion periods examined. Our results suggest that, in spite of different and complex pathophysiological mechanisms involved in the increase of FAA level and the decrease of the Na+,K+-ATPase activity, blockade of NMDA receptor subtype provides a very important strategy for the treatment of the postischemic excitotoxicity.

The influence of nicardipine and ifenprodil on the brain free arachidonic acid level and behavior in hypoxia-exposed rats

1. The effects of the calcium channel blockers, nicardipine and ifenprodil, on the brain free arachidonic acid level and learning ability in rats exposed to hypoxia were examined. 2. Adult rats were injected with 0.003; 0.01; 0.03; 0.1; 0.3 or 1.0 mg/kg of tested drugs i.p. Thirty min later the learning ability was tested in a passive avoidance task according to the step-through procedure. Immediately after the training trial, the animals were subjected to a period of oxygen deprivation hypoxia until the loss of the righting reflex. The retention trial was carried out 24 hr later. 3. The other groups of animals were pretreated with mentioned substances before hypoxia-exposure. Fifteen min after the loss of the righting reflex they were decapitated and brains were frozen in liquid nitrogen. The brain free arachidonic acid level was quantified by gas chromatography. 4. Both nicardipine and ifenprodil were effective in preventing a memory decline in hypoxia-exposed rats but did not prevent the accumulation of the brain free arachidonic acid in hypoxia-exposed rats. 5. The protective effects of both substances in behavioral studies during acute brain damage caused by hypoxia could not be explained by the prevention of the increase of the brain free arachidonic acid, but by some other mechanism.

Differential effects of dihydropyridine calcium channel blockers in kainic acid-induced experimental seizures in rats

The anticonvulsant effects of the dihydropyridine calcium channel blockers nifedipine, nicardipine and nimodipine were studied on experimental seizures induced by intra-hippocampal injection of kainic acid (KA) in chloralhydrate anesthetized Wistar rats. The rats were anesthetized and placed in a stereotaxic apparatus. After midline incision four screw electrodes were placed over the left and right frontal and parietal cortex and KA was injected into left dorsal hippocampus via 5-microliter Hamilton microsyringe. The changes in electroencephalograph (EEG) activity and EEG power spectra were recorded in basal conditions and 5, 10, 15, 30 and 60 min following KA injection. KA-induced excitatory changes in the surface EEG activity were associated with the marked increase in EEG power spectra in the frequency range from 14.5-22 Hz. Pretreatment with nifedipine, nicardipine and nimodipine revealed that they exerted certain differences in their anticonvulsant properties. Nimodipine significantly delayed the onset of seizures and prevented the KA-induced changes in EEG and in EEG power spectra in all recorded channels and in a dose dependent manner. Nifedipine exerted significant anticonvulsant effect only in channel four, while nicardipine was ineffective. Our results suggest that anticonvulsant action of some dihydropyridine calcium channel blockers, especially nimodipine may be in part independent of its antagonism on L-type voltage-gated calcium (Ca(2+)) channels.