TAURINE AND COBALT INDUCED EPILEPSY IN THE RAT: A BIOCHEMICAL AND ELECTROCORTICOGRAPHIC STUDY

Abstract– The level of taurine around the epileptic focus induced by cobalt-gelatine pellet implantation into rat brain was found to be reduced at 8-11 days post-operation, the time of maximal spike activity in the electrocorticogram. It returned towards normal at later times. This pattern was seen not only in the primary focus, but also to a lesser extent in the secondary focus in the contralateral cortex. Acute administration of taurine intraperitoneally or intraventricularly resulted in at most transient effects on epileptic spiking.
Chronic oral taurine elevated brain taurine in normal rats only after prolonged administration, but in cobalt-treated rats it prevented the fall of taurine in the secondary focus, and reduced the extent and duration of the fall in the primary focus; nonetheless chronic oral or intraventricular administration failed to modify the development of spike activity. At 11 days after implantation, chronic oral taurine did not significantly reverse the falls in transmitter amino acids in the primary focus. It is concluded that taurine is ineffective in altering the development or expression of this type of cobalt-induced epilepsy in the rat, in spite of adequate penetration to the brain. Possible reasons for the discrepancies with studies by other workers are discussed.     

Changes in the Na, K-ATPase of neuronal membranes in penicillin-induced epileptic foci in the cerebral cortex of the rat]

The relationship between electrophysiological changes and Na, K-ATPase activity of neuronal membranes in sodium penicillin-induced epileptic foci was studied. Na,K-ATPase activity is inhibited both in the primary focus and in homotopic contralateral area during latent period and in the stage of forming epileptic activity. In the stage of marked convulsive activity Na, K-ATPase is inhibited only in the primary focus. It is shown that penicillin at a concentration range of 2 x 10(-6)--2 x 10(-3) M does not influence Na,K-ATPase activity of crude synaptosomes of the rat brain cortex. It is suggested that Na,K-ATPase inactivation may serve as a pathogenetic factor in the development of convulsive process.     

A postsynaptic type nodal axon]

A node of Ranvier in which the nodal axon is post-synaptic to a terminal axon was found in the cerebral cortex of the rat near by an epileptic focus. This type of synapse is in itself a rare observation but is also worthwile considering because of its vicinity with this focus.     

Changes in the spectrum of LDH isoenzymes in foci of epileptic activity induced by penicillin in the cerebral cortex of rats

The correlation between electrophysiological changes and isozymes of LDH of the rat brain cortex was studied in seizure foci induced by application of sodium penicillin. It was discovered that activity of LDH1 was suppressed, and that of LDH5 fraction was elevated in the determinant focus, which indicates the enhanced glucose anaerobic transformation. The spectrum of LDH isozymes did not practically differ from the indicators in control animals in a homotopic region of the contralateral hemisphere prior to creation of the mirror focus. The anaerobic processes were found to be increased in the mirror focus and in the determinant one as well. Similar pattern of changes in electrophysiological and neurochemical characteristics in the determinant and dependent mirror foci attests to the formation of a pathological system out of the two epileptic foci.     

Tyrosinated alpha-tubulin changes following epileptogenic and non-epileptogenic lesions in rat brain cortex

The expression of the tyrosinated isoform of alpha-tubulin was monitored in rat frontal cortex, in order to investigate the neuronal plasticity changes occurring either in a mirror focus or in a deafferented area. A mirror focus was triggered by epidural implantation of a cobalt gelatin disk in the contralateral left somatosensory area (group one). A deafferented area was obtained by surgical removal of the left frontal cortex (group two). All animals including controls underwent EcoG recordings immediately before killing (45, 60, 90 days post surgery). The right frontal cortex was removed from all the animals and processed with Western blot method. EcoG recordings revealed a paroxysmal activity in epileptic rats, whereas in rats with frontal deafferentation and controls, EcoG activity was normal. A significant increase in tyrosinated alpha-tubulin expression was detected both in the mirror focus (group one) and the "non-epileptic" deafferented frontal cortex (group two) in comparison with controls (group three). The transcallosal deafferentation, which is involved in both epileptogenic and non-epileptogenic lesions, is supposed to play a role in the mechanism responsible for the plasticity responses recorded in the cortical areas studied.     

Electroencephalographic study of iron-induced chronic focal cortical epilepsy in rat: propagation of cortical epileptic activity to substantia nigra and thalamus.

Cortical epileptic focus was produced by an intracortical injection of FeCl3 in rat cerebral cortex using standard techniques. How after its onset in the cortical focus, the epileptiform activity evolved with time in the thalamus and substantia nigra has been determined. To study the propagation of the epileptiform activity, the local EEG and multiple unit action potentials were recorded from these structures simultaneously with the cortical epileptiform EEG. The results showed that in thalamus and substantia nigra epileptiform activity appeared simultaneously with that in the cortical focus. Intensity of epileptic activity in thalamus and substantia nigra on the whole increased in parallel with that in the cortical focus. The results suggest that the thalamic and nigral epileptiform activity may reinforce the cortical epileptiform activity.     

Effect of diazepam on the Na, K-ATPase state in a penicillin--induced hyperactivity focus in the cerebral cortex]

Intramuscular injection of diazepam to rats at doses of 0.01 and 2 mg/kg 25-30 min after penicillin application to the rat brain cortex leads to alteration of periodic appearance of epileptic seizures (ES), to changes in the seizure pattern, and to emergence of periodic acceleration of epileptiform discharges (ED). Injection of diazepam at a dose of 2 mg/kg 20 min before penicillin application results in the reduction of ED latency in the epileptogenic focus and in a decrease in their frequency before seizures as compared to the control animals without diazepam injection. ES appear irregularly, their quantity is markedly reduced while duration is increased. Diazepam injection leads to disappearance of the rat moving reaction during ER and ES. In vivo experiments diazepam (2 mg/kg) does not influence brain cortex Na, K-ATPase of crude synaptosomes. However, diazepam leads to an increase in Na, K-ATPase activity both in the primary and dependent secondary epileptogenic foci. It is suggested that the anticonvulsant action of diazepam may be underlain by its activating effect on Na, K-ATPase of neuronal membranes in the epileptogenic focus.     

The subjective auditory space of epileptic patients with lesions in both the temporal cortical area and the hippocampus

Parameters of the subjective auditory space formed under the conditions of dichotic stimulation during movement of the acoustic image in different directions were studied in 13 epileptic patients with lesions in both the temporal cortex and the hippocampus. The pattern and degree of the movement trajectory of the subjective acoustic image (SAI) were determined in two groups of patients (in seven patients with the right-side focus and six patients with the left-side focus of convulsive activity, depending on the direction of moving and the initial interauricular delay (700, 400, and 200 μs)). A significant decrease in the SAI movement trajectory was observed in patients with right-or the left-side lesions in the temporal cortex and hippocampus as compared to healthy subjects or patients with temporal cortical epilepsy in the cases when the SAI movement stopped at the side of the epileptic focus. The analysis of the parameters of the SAI movement in each hemisphere showed a highly significant difference between the movement trajectories for all values of initial delays as compared to the control group of healthy subjects (p < 0.01) and patients with a relatively isolated lesion of the right temporal cortex (p < 0.05). A significant role of the epileptic lesion of the hippocampus (which is not an auditory structure) in the impairment of spatial hearing was demonstrated. The characteristics of the functional asymmetry because of simultaneous epileptic lesion of the temporal cortex and hippocampus, which led to the impairment of the binaural effect, are discussed.     

Inhibition by 1,25 dihydroxyvitamin D3 of c-myc down-regulation and DNA fragmentation in cytosine arabinoside-induced erythroid differentiation of K562 cells.

The effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on DNA fragmentation, altered expression of the heat shock protein (hsp) 70 gene, and protooncogenes c-myc and c-myb was studied during chemical induction of erythroid differentiation in K562 cells. Preincubation of K562 cells with 1,25(OH)2D3 did not alter the concentration of hemoglobin in cells which did differentiate, but led to a reduction in the accumulation of low molecular weight DNA generated by Ara-C administration. The extent of this reduction was similar to the degree of inhibition of hemoglobin formation in the culture as the whole. Preincubation with 1,25(OH)2D3 had no effect on the increase of hsp 70 gene expression induced by a 48-hr treatment with Ara-C, but prevented the Ara-C-induced down-regulation of the protooncogene c-myc. The protooncogene c-myb was down-regulated after 15 min of treatment with Ara-C, and exposure to 1,25(OH)2D3 prior to Ara-C caused a further down-regulation of its expression. The data suggest that the events associated with erythroid differentiation may be separable into at least two groups; one of these may have an influence on the kinetics of the cell cycle traverse, and the other may be related to the expression of the erythroid phenotype.     

A specific protein abnormality associated with cobalt-induced epilepsy in mice

Density profiles of protein patterns from cortical tissue exhibit an increase in only one peak when mice are rendered epileptic by application of cobalt to the cortex. The increase and diminution in peak height, attributed to a change in the concentration of a single protein (protein 3), coincides with the severity of seizure activity; with the degree of abnormality of the cortex region affected; and with the time of onset, duration, and disappearance of the epileptic condition. Thus, the concentration of protein 3 is highest in tissue from the site of cobalt application (up to 10× normal), is increased less in the focus (up to 5× normal), while in the mirror focus (contralateral, not exposed surgically), the increase in the concentration of protein 3 is still detectable, but not as pronounced. The concentrations in these cortex regions decrease to normal in reverse order to their elevation when the epileptic signs begin to diminish. Furthermore, the increase of protein 3 in all three areas is proportional to the severity of epilepsy. The concentration of protein 3 also becomes enhanced when the cortex is injured, but no progressive increase in the concentration occurs with time, nor does the concentration reach that observed in the site of cobalt application or the focal region. These mice do not exhibit spontaneous seizures, but injection of pentelynetetrazol confirms that animals with brain injury only are more susceptible to seizures. The results of this study suggest that both the area of cortex affected and the intensity of metabolic alterations may be precipitating factors in establishing an epileptic condition. This view is in agreement with clinical observations on epilepsy.This work is in partial fulfillment of M.Sc. requirements.     

糖皮质激素的抗痫作用及其与γ-氨基丁酸的关系

为了探讨糖皮质激素的抗癫痫效应和作用机制, 本研究观察了糖皮质激素对戊四氮诱导的慢性点燃型癫痫大鼠的行为和脑电图的影响, 并应用免疫细胞化学双重染色技术探查了大脑皮质神经元内糖皮质激素受体（ＧＲ） 与γ－ 氨基丁酸（ＧＡＢＡ） 的共存情况。结果显示, 在慢性点燃型癫痫大鼠, 在点燃后的第３ 天或第１５ 天, 先经静脉给予地塞米松（４ｍ ｇ／ｋｇ）, 再经腹腔注射戊四氮（３０ｍ ｇ／ｋｇ） 可明显减弱或完全抑制癫痫发作。免疫细胞化学双重染色证明, ＧＲ和ＧＡＢＡ共存于大脑皮质部分神经元。以上结果提示, 糖皮质激素具有抗慢性癫痫的效应, 其作用机制可能与ＧＲ调节同一神经元内ＧＡＢＡ的合成有关。     

Neuroprotective action of FK-506 (tacrolimus) after seizures induced with pilocarpine: quantitative and topographic elemental analysis of brain tissue

In the present work, X-ray fluorescence microscopy with a synchrotron source for the exciting radiation was applied for topographic and quantitative elemental analysis of rat brain tissue in pilocarpine-induced epilepsy and neuroprotection with FK-506. The mass per unit area of the elements P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br, and Rb was determined in four fields of the hippocampal formation (sectors 1 and 3 of Ammon’s horn–CA1, CA3; dentate gyrus; hilus of dentate gyrus) and the parietal cortex. The results obtained for epileptic rats treated with FK-506 (SNF) were compared with data obtained previously for epileptic rats (SNS) and a control group. Many statistically significant differences in elemental composition were observed between the SNF and SNS groups. Higher mass per unit area of P was noticed in CA1 and CA3 regions of the hippocampus of SNF rats in comparison with SNS rats. A similar relation was observed for K in all five brain areas analyzed. Also, Fe in CA3 and dentate gyrus, Cu in the parietal cortex, and Zn in CA3 and in the cortex were present at a higher level in the SNF group in comparison with the SNS group. The findings obtained in the present study suggest that the neuroprotective action of FK-506 in epileptic rat brain may involve not only the inhibition of calcineurin but also blockade of the K⁺ channels.     

Electron spin resonance study of the role of NO . catalase in the activation of guanylate cyclase by NaN3 and NH2OH. Modulation of enzyme responses by heme proteins and their nitrosyl derivatives.

The role of NO . catalase in the activation of partially purified soluble guanylate cyclase of rat liver by NaN3 and NH2OH was examined by electron spin resonance (ESR) spectroscopy. Equilibration of bovine liver catalase with NO resulted in formation of a paramagnetic species exhibiting a three-line ESR spectrum similar to that of NO . catalase. This paramagnetic complex produced concentration-dependent stimulation of preparations of partially purified guanylate cyclase that were devoid of detectable endogenous heme content. The stimulation of partially purified guanylate cyclase by NO . catalase was similar to that obtained with NO . hemoglobin and with NO . cytochrome P-420 prepared by reaction of hepatic microsomes of phenobarbital-treated rats with NO. By contrast, these same enzyme preparations did not respond to NO or catalase alone. Addition of hematin or hemoglobin plus a reducing agent to purified guanylate cyclase restored enzyme responsiveness to NO and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to NaN3 or NH2OH. Responses to the latter agents were restored by catalase and potentiated by a H2O2-generating system. Formation of the NO . catalase complex was evident by ESR spectroscopy in test solutions containing NaN3 or nh2oh, catalase, and a glucose-glucose oxidase, H2O2-generating system. The presence of NO . catalase correlated well with the ability of test solutions to activate purified guanylate cyclase. These results provide evidence for catalase-dependent NO generation from NaN3 and NH2OH under conditions leading to guanylate cyclase activation. Preformed NO . hemoglobin or NO . cytochrome P-420 also activated heme-deficient partially purified guanylate cyclase. The ability of several preformed NO . heme protein complexes, but not NO, to stimulate heme-deficient guanylate cyclase supports the concept that formation of the paramagnetic nitrosyl . heme complex, mediated by either enzymatic or nonenzymatic reactions, is a common and essential step in the process by which NO or NO-forming compounds activate guanylate cyclase. In the absence of the NO ligand, both hemoglobin and catalase suppress the stimulatory effects of the corresponding NO . heme proteins on guanylate cyclase. Release of each heme protein from the NO . heme protein complex occurs more rapidly under aerobic compared to anaerobic conditions. However, hemoglobin is approximately 2000 times more effective as an inhibitor of NO . hemoglobin stimulation of guanylate cyclase than is catalase as an inhibitor of NO . catalase action. This finding may explain the more pronounced decline in the rate of cGMP generation in air in the presence of NO . hemoglobin compared to NO . catalase. The results imply that guanylate cyclase responses to activators that can form NO are determined by both the stimulatory activity of the endogenous heme acceptors of NO and the relative inhibitory effects of the unliganded heme proteins present.     

The superior colliculus, a neuronal network resistant to the Gaba withdrawal syndrome

Chronic intracortical perfusion of GABA (Gamma Amino Butyric Acid) and its subsequent withdrawal generates the GABA withdrawal syndrome (GWS). This particular epileptic model has been observed in the motor cortex of monkeys and rats. Our purpose was to study the GWS in the motor cortex (MC), dorsal hippocampus (DH), and superior colliculus (SC). Thirty chronically-implanted adult Wistar rats were separated into 3 groups of 10 (8 experimental and 2 controls). The first group received GABA in MC, the second in the DH and the third in the SC. GABA was released in doses of 10 to 60 micrograms/microliter/h for 6 days employing osmotic mini-pumps. Two control rats per group received saline solution in the above-mentioned structures. Rats perfused in the MC showed GWS after interruption of the GABA flow. The group perfused in the DH showed paroxysmal discharges and epileptic seizures during perfusion. They also later showed GWS. No epileptic effects were observed in the SC-perfused group during either the GABA perfusion or during withdrawal. None of the six control animals showed epileptic effects. Our results show that the SC offers a strong resistance to GWS. This could be explained by the particular neuronal network structure of rat SC.     

Subjective Acoustic Field of Patients with Cortical Temporal Lobe Epilepsy as Revealed Using Signals Simulating Various Directions of Sound Movement

Perception of signals simulating directional movement of a sound source was studied in two groups of patients with cortical temporal lobe epilepsy and epileptic activity foci in the right or left temporal area of the cortex. On dichotic stimulation, the character and length of the trajectories of subjective auditory images (SAIs) were determined as dependent on the direction of SAI movement and the initial interaural delay (700, 400, and 200 s). For any delay or direction examined, SAI trajectories were shorter in the patients of both groups than in healthy subjects. Regardless of the side of an epileptic focus, the shortest trajectories were detected in the hemisphere where SAI movement ended, especially at an interaural delay of 200 s. The narrowest subjective acoustic field was observed in patients with epileptic foci in the right temporal cortex. Possible mechanisms of the changes in spatial hearing are discussed. The changes in SAI perception are assumed to result from distorted binaural interactions, which manifest themselves in functional asymmetry of the two auditory centers and may be caused by a convulsive activity focus present in one temporal lobe.     

A GABA-EEG test of the blood-brain barrier near epileptic foci

The permeability of the blood-brain barrier (BBB) to gamma-aminobutyric acid (GABA) in the region of an epileptic focus may be assessed by infusing GABA and measuring a change in epileptic spike activity on the EEG. GABA does not cross the normal BBB but will suppress epileptic spike activity when it does cross where the BBB is damaged. 9 alumina-cobalt experimental epileptic foci were all initially suppressible, but 7 then became unsuppressible . When the foci were irradiated to lower the BBB, all 7 became temporarily suppressible. The experiments demonstrate that (1) epileptic foci can be equally active both with the BBB 'open' and 'closed'; (2) the intravenous GABA-EEG test can detect whether the BBB near the epileptic focus is open to GABA, and (3) anatomic tests of BBB integrity (in these experiments intravenous trypan blue) cannot determine if whether BBB near the focus is 'open' to GABA. Since the intravenous GABA-EEG test reveals the permeability of the BBB in the immediate environment of the epileptic focus, it may be very useful in the selection of a susceptible therapeutic group for inhibitory amino acid therapy.     

Anticonvulsive effect of superoxide dismutase

The influence of superoxide dismutase (SOD) on the development of focal epileptic activity (EpA) in the rat brain cortex has been investigated. Intraperitoneal administration of SOD to rats (1 mg/kg) 30 minutes before penicillin application to the sensorimotor cortex led to marked relaxation of EpA and a decrease in the concentration of lipid peroxidation (LPO) products in EpA focus. The results corroborate our earlier assumption on an important pathogenetic role of LPO disturbances in epileptogenesis and make reasonable the combination of the traditional anticonvulsive therapy with the agents activating the oxidative system.     

Effect of diazepam on electrical activity and Na,K-ATP-ase level in a penicillin-induced epileptic focus in the rat cerebral cortex

Application of penicillin solution to the motor cortex in rats evoked the appearance of interictal discharges and epileptic seizures. After administration of diazepam in a dose of 2 mg/kg, Na,K-ATPase activity in the unpurified synaptosomes fraction of the cortex in the zone of the focus was increased by practically 100% compared with the level of activity of the enzyme in the focus without diazepam. Interictal discharges and epileptic seizures underwent different changes following intramuscular injection of diazepam. The frequency and variability of amplitude of the interictal discharges increased after administration of diazepam, whereas epileptic seizures were depressed. This effect was potentiated with an increase in the dose of diazepam. It is suggested that the opposite action of diazepam on epileptic seizures and interictal discharges may be evidence that the mechanisms lying at the basis of the development of these phenomena are different.Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 4, pp. 349–357, July–August, 1980.     

Phospholipid content in "late" posttraumatic epileptic focus in rabbits

The phospholipids content in the cortex, hippocamp and stem tissues in rabbits with "late" posttraumatic epileptic focus (1 year after the light brain injury) was estimated. There was established an increase of the content of phosphatidylethanolamine, sphingomyeline, phosphatidylinosite, phosphatidic acid, non-identified phospholipids, decrease of the content of phosphatidylcholine and cardiolipine and appearance of lysophosphatidylcholine in the cortical epileptic focus tissues. The conclusion is made that the changes within the phospholipids pool may cause the epileptogenic disturbance in the neurons, i.e. the changes of functional properties of the excitable membranes and the activity of the mytochondrial phospholipid-dependent enzyme complex.     

Up-regulation of apelin in brain tissue of patients with epilepsy and an epileptic rat model

Prolonged epileptic seizures or SE can cause neuronal cell death. However, the exact role of neuroprotectant against brain injury during epileptic seizure needs to be further elucidated. The aim of this study was to investigate the expression of the apelin, a novel neuroprotective peptide, in brain tissues of the patients with temporal lobe epilepsy (TLE) and experimental rats using immunohistochemistry, immunofluorescence and Western blotting analysis and to discuss the possible role of apelin in TLE. Thirty temporal neocortical tissue samples from the patients with drug-refractory TLE underwent surgical therapy and nine histologically normal temporal lobes tissues as controls were used in our study. Fifty-six Sprague-Dawley rats were randomly divided into seven groups, including one control group and six groups with epilepsy induced by lithium-pilocarpine. Hippocampus and adjacent cortex were taken from the controls and epileptic rats at 1, 3, 7, 14, 30, and 60 days after onset of seizures. Apelin was mainly expressed in the neurons of TLE patients and controls, and was significantly increased in TLE patients compared with the controls. Apelin was also expressed in the neurons of experimental and control rats, it was gradually increased in the experimental rat post-seizure and reached a stable high level in chronic epileptic phase. Our results demonstrated that the increased expression of apelin in the brain may be involved in human TLE.