Journal of Neurochernistry, 1975. Vol. 24. pp. 591-592. Pergamon Press. Printed in Great Britain.
SHORT COMMUNICATION Brain gangliosides in drug-induced status epilepticus (Received 31 July 1974. Accepted 27 September 1974)
WHISLER et al. (1968) have reported that in the dog there is a decrease in the brain ganglioside concentration in status epilepticus induced by pentylenetetrazol or bemegride. In their study no data on the behaviour of the individual gangliosides under these experimental conditions were given. We have investigated the effect of a drug-induced status epilepticus on the ganglioside concentration and ganglioside pattern in the brain of another species, the rabbit, and would like to present our results briefly.
were separated by TLC, visualized by resorcinol sprays and determined quantitatively by means of a Zeiss Chromatogramm-Spektralphotometer (580 nm, light reflection; for all further details see SUNDER-PLASSMANN & BERNHEIMER, 1974). Peaks were recorded and integrated by a Goerz Servogor RE 512 recorder; the peak area obtained for each individual ganglioside was expressed as percentage of the sum of the peak areas obtained from all individual gangliosides.
MATERIALS AND METHODS The experiments were carried out in 7-9-month-old rabbits with a body weight between 1.6 and 1.8 kg. The following drugs were used: pentylenetetrazol (CardiazolR, Knoll AG, Ludwigshafen/Rh, Germany), bemegride (EukratonR, Ebewe, Unterach, Austria) and thiosemicarbazide (E. Merck, Darmstadt, Germany). Pentylenetetrazol (77-120 mgikg) and bemegride (24-42 mgikg) were administered by intermittent intravenous infusion in order to achieve a status epilepticus over a period as long as possible (10-35 and 9-51 min, revpectively). Thiosemicarbazide (75-90 mg/ kg) was given intraperitoneally as a single dose and after a latency period of about 1.5 h induced a status epilepticus of 65-75 min. For the evaluation of a status epilepticus, the occurrence of tonic and clonic fits as well as of uncoordinated movements was taken as a criterion. In the treated animals death occurred in the status epilepticus. The control animals were killed by KCI applied intravenously. Immediately after death was removed from the skull, the cerebral cortex and the caudate nucleus were prepared free from visible white matter and subjected to chemical analysis. Gangliosides were extracted from the tissues by the methods of FOLCH-PIet a!. (1957) and SUZUKI (1965) and the total ganglioside concentration was estimated by the resorcinol method (SVENNERHOLM, 1963). The individual gangliosides
RESULTS AND DISCUSSION Table 1 shows the values obtained for the brain ganglioside concentrations. From this table it may be seen that in the cerebral cortex as well as in the caudate nucleus the range of ganglioside concentrations found in the animals with a drug-induced status epilepticus was very close to that of controls. The small differences observed were not significant (P 9 0.05) as judged by a one-way analysis of variance. In particular, no significant decrease in the brain ganglioside concentration of the animals with a drug-induced status epilepticus as compared with the controls was to be found. With regard to the ganglioside pattern (Table 2), no crucial differences in the percentage distribution of the individual gangliosides between the animals with a drug-induced status epilepticus and the controls could be detected. Similar results were also obtained in an earlier preliminary experiment where a status epilepticus over a period of 2 h was induced by local application of strophanthin onto the cortex of a rabbit. Our findings of an unaltered brain ganglioside level in a status epilepticus in the rabbit induced by pentylenetetrazol, bemegride, thiosemicarbazide or strophanthin are at variance with the results of WHISLERet al. (1968), who have reported a decrease in the ganglioside levels to 70-50 per cent of the controls in the cerebral cortex of dogs with a
CONCENTRATION IN THE BRAIN TABLE1. GANGLIOSIDE
Treatment None(contro1s) Pentamethylenetetrazol Bemegride Thiosemicarbazide
n*
Cortext
6 6 3 3
1250 1183 & 1153 & 1270
+
130 205 101 135
* Number of animals. t pg Ganglioside-N-acetylneuraminic acid per g wet wt. Mean values f S.D. NI. 2413
591 L
Caudate nucleus? 1283 1242 & 1327 & 1363
+
153 236 8 7
592
Short communication TABLE 2. PERCENTAGE DISTRIBUTION OF INDIVIDUAL GANGLIOSIDES IN
THE BRAIN
Cortex Treatment None(contro1s)t Pentamethylenetetrazol Bemegride Thiosemicarbazide
n* 5
il ii
GMI 1S21 17 15
;: :: 19
13
GDI~
31-42 38 38 38 39 33 36 38 40 35 39
GDI~ 18-22 21 23 19 19 20 20 20 20 21 20
GTI 2428 27 24 24 22 28 27 28 27 27 24
GM~
16-20 18 23 18 17 23 16 17 19 16 19
Caudate nucleus GDI, GDI, 37-41 40 36 39 40 35 41 42 38 41 38
18-20 18 17 19 21 19 18 20 21 17 18
GT 1
21-28 25 24 24 22 23 25 21 22 26 25
* Number of animals. t Range and median value. status epilepticus induced by bemegride or pentylenetetra201. The discrepancy between our results and those of WHISLER et al. (1968) might possibly be explained by the different species and methods used in their investigation. On the other hand, since gangliosides are rather stable tissue components with a half-life in the order of weeks (RADINet al., 1957; BURTDN et al., 1964; SUZUKI,1967; HARZER et al., 1969)one might expect to find no gross changes of the ganglioside concentration in an acute experiment like ours. The finding of unchanged brain ganglioside levels and brain ganglioside patterns in a drug-induced status epilepticus however does not mean necessarily that gangliosides could not play a role in this state, e.g. in a neurophysiological manner.
supported by the Fonds zur Forderung der wissenschaftlichen Forschung, Austria, Grant No. 1082. Neurological Institute, E. WINTER^ University of Vienna, H. BERNHEIMER Schwarzspanierstr. 17, ~ - 1 0 9 0Vienna. Austria
REFERENCES J. M. .' M.& Fedn Am. exp. Biol. 239 230. FoLCH-PI LEES M. & SLOANE-STANLEY G. H. (1957) J . Chem. 226, 497-509. HARZER K., JArZKEW'n H. SANWFF K. (1969) Neurochem. 16, 1279-1282. RADINN. S., MARTINF. B. & BROWNJ. R. (1957) J. b i d . C 3 ~ m224, . 499-507. Acknowledgements-We are indebted to Prof. Dr. K. GLONSUNDER-PLASSMA" M. & BERNHEIMER H. (1974) Acts ING, Neurological Institute, University of Vienna, for the neuropath., Berl. 27, 289-297. statistical evaluation of the results. The investigation was SUZUKI K. (1965) J. Neurochem. 12,629-638. SUZUKI K. (1967) J. Neurochem. 14,917-925. SVENNERHOLM, L. (1963) J . Neurochem. 10,613-623. K. E., TEWSJ. K. & STONE W. E. (1968) J. NeuroPresent address: Pharmacological Institute, University WHISLER chem. 15,21S220. of Munich, German Federal Republic.
R.M . BALFoUR ~ J.7
SHORT COMMUNICATION Brain gangliosides in drug-induced status epilepticus (Received 31 July 1974. Accepted 27 September 1974)
WHISLER et al. (1968) have reported that in the dog there is a decrease in the brain ganglioside concentration in status epilepticus induced by pentylenetetrazol or bemegride. In their study no data on the behaviour of the individual gangliosides under these experimental conditions were given. We have investigated the effect of a drug-induced status epilepticus on the ganglioside concentration and ganglioside pattern in the brain of another species, the rabbit, and would like to present our results briefly.
were separated by TLC, visualized by resorcinol sprays and determined quantitatively by means of a Zeiss Chromatogramm-Spektralphotometer (580 nm, light reflection; for all further details see SUNDER-PLASSMANN & BERNHEIMER, 1974). Peaks were recorded and integrated by a Goerz Servogor RE 512 recorder; the peak area obtained for each individual ganglioside was expressed as percentage of the sum of the peak areas obtained from all individual gangliosides.
MATERIALS AND METHODS The experiments were carried out in 7-9-month-old rabbits with a body weight between 1.6 and 1.8 kg. The following drugs were used: pentylenetetrazol (CardiazolR, Knoll AG, Ludwigshafen/Rh, Germany), bemegride (EukratonR, Ebewe, Unterach, Austria) and thiosemicarbazide (E. Merck, Darmstadt, Germany). Pentylenetetrazol (77-120 mgikg) and bemegride (24-42 mgikg) were administered by intermittent intravenous infusion in order to achieve a status epilepticus over a period as long as possible (10-35 and 9-51 min, revpectively). Thiosemicarbazide (75-90 mg/ kg) was given intraperitoneally as a single dose and after a latency period of about 1.5 h induced a status epilepticus of 65-75 min. For the evaluation of a status epilepticus, the occurrence of tonic and clonic fits as well as of uncoordinated movements was taken as a criterion. In the treated animals death occurred in the status epilepticus. The control animals were killed by KCI applied intravenously. Immediately after death was removed from the skull, the cerebral cortex and the caudate nucleus were prepared free from visible white matter and subjected to chemical analysis. Gangliosides were extracted from the tissues by the methods of FOLCH-PIet a!. (1957) and SUZUKI (1965) and the total ganglioside concentration was estimated by the resorcinol method (SVENNERHOLM, 1963). The individual gangliosides
RESULTS AND DISCUSSION Table 1 shows the values obtained for the brain ganglioside concentrations. From this table it may be seen that in the cerebral cortex as well as in the caudate nucleus the range of ganglioside concentrations found in the animals with a drug-induced status epilepticus was very close to that of controls. The small differences observed were not significant (P 9 0.05) as judged by a one-way analysis of variance. In particular, no significant decrease in the brain ganglioside concentration of the animals with a drug-induced status epilepticus as compared with the controls was to be found. With regard to the ganglioside pattern (Table 2), no crucial differences in the percentage distribution of the individual gangliosides between the animals with a drug-induced status epilepticus and the controls could be detected. Similar results were also obtained in an earlier preliminary experiment where a status epilepticus over a period of 2 h was induced by local application of strophanthin onto the cortex of a rabbit. Our findings of an unaltered brain ganglioside level in a status epilepticus in the rabbit induced by pentylenetetrazol, bemegride, thiosemicarbazide or strophanthin are at variance with the results of WHISLERet al. (1968), who have reported a decrease in the ganglioside levels to 70-50 per cent of the controls in the cerebral cortex of dogs with a
CONCENTRATION IN THE BRAIN TABLE1. GANGLIOSIDE
Treatment None(contro1s) Pentamethylenetetrazol Bemegride Thiosemicarbazide
n*
Cortext
6 6 3 3
1250 1183 & 1153 & 1270
+
130 205 101 135
* Number of animals. t pg Ganglioside-N-acetylneuraminic acid per g wet wt. Mean values f S.D. NI. 2413
591 L
Caudate nucleus? 1283 1242 & 1327 & 1363
+
153 236 8 7
592
Short communication TABLE 2. PERCENTAGE DISTRIBUTION OF INDIVIDUAL GANGLIOSIDES IN
THE BRAIN
Cortex Treatment None(contro1s)t Pentamethylenetetrazol Bemegride Thiosemicarbazide
n* 5
il ii
GMI 1S21 17 15
;: :: 19
13
GDI~
31-42 38 38 38 39 33 36 38 40 35 39
GDI~ 18-22 21 23 19 19 20 20 20 20 21 20
GTI 2428 27 24 24 22 28 27 28 27 27 24
GM~
16-20 18 23 18 17 23 16 17 19 16 19
Caudate nucleus GDI, GDI, 37-41 40 36 39 40 35 41 42 38 41 38
18-20 18 17 19 21 19 18 20 21 17 18
GT 1
21-28 25 24 24 22 23 25 21 22 26 25
* Number of animals. t Range and median value. status epilepticus induced by bemegride or pentylenetetra201. The discrepancy between our results and those of WHISLER et al. (1968) might possibly be explained by the different species and methods used in their investigation. On the other hand, since gangliosides are rather stable tissue components with a half-life in the order of weeks (RADINet al., 1957; BURTDN et al., 1964; SUZUKI,1967; HARZER et al., 1969)one might expect to find no gross changes of the ganglioside concentration in an acute experiment like ours. The finding of unchanged brain ganglioside levels and brain ganglioside patterns in a drug-induced status epilepticus however does not mean necessarily that gangliosides could not play a role in this state, e.g. in a neurophysiological manner.
supported by the Fonds zur Forderung der wissenschaftlichen Forschung, Austria, Grant No. 1082. Neurological Institute, E. WINTER^ University of Vienna, H. BERNHEIMER Schwarzspanierstr. 17, ~ - 1 0 9 0Vienna. Austria
REFERENCES J. M. .' M.& Fedn Am. exp. Biol. 239 230. FoLCH-PI LEES M. & SLOANE-STANLEY G. H. (1957) J . Chem. 226, 497-509. HARZER K., JArZKEW'n H. SANWFF K. (1969) Neurochem. 16, 1279-1282. RADINN. S., MARTINF. B. & BROWNJ. R. (1957) J. b i d . C 3 ~ m224, . 499-507. Acknowledgements-We are indebted to Prof. Dr. K. GLONSUNDER-PLASSMA" M. & BERNHEIMER H. (1974) Acts ING, Neurological Institute, University of Vienna, for the neuropath., Berl. 27, 289-297. statistical evaluation of the results. The investigation was SUZUKI K. (1965) J. Neurochem. 12,629-638. SUZUKI K. (1967) J. Neurochem. 14,917-925. SVENNERHOLM, L. (1963) J . Neurochem. 10,613-623. K. E., TEWSJ. K. & STONE W. E. (1968) J. NeuroPresent address: Pharmacological Institute, University WHISLER chem. 15,21S220. of Munich, German Federal Republic.
R.M . BALFoUR ~ J.7
