Epilepsy Research, 12 (1992) 151-156 0920-1211/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
151
EPIRES 00487
Polyamine metabolism in epileptic cortex
J. L a s c h e t a'b, S. T r o t t i e r c, T. G r i s a r a a n d V. Leviel b aLaboratory of Comparative and General Biochemistry and Department of Neurology, University of Liege, Liege (Belgium), bLaboratoire de Physiologie Nerveuse LPN1, CNRS de Gif-sur-Yvette and cINSERM JF 90-12, CHR de Rennes, Rennes (France) (Accepted 10 April 1992)
Key words." Human; Temporal lobe epilepsy; Polyamines; Hippocampus; Temporal cortex; Plasticity
Polyamine (tissue) concentrations have been studied in hippocampus and temporal neocortex from patients with temporal lobe epilepsy. Depth electrode recordings demonstrated hippocampal origin of the seizures, the temporal neocortex being involved during the discharge propagation. Neuropathological examination of excised tissues showed glial proliferation or glioma in Ammon's horn (CA), whereas the temporal neocortex did not exhibit any histological abnormality. Polyamine (putrescine or PUT, spermidine or SPD, spermine or SPM) concentrations were determined on surgical samples from the hippocampus and various areas of temporal neocortex. Human post-mortem tissue from temporal lobe regions was used for controls. In post-mortem controls and temporal neocortex specimens from epileptic patients, polyamine levels were similar (in nmol/g wet weight: PUT = 40-100; SPD = 200-350; SPM = 100-200). In CA, polyamine levels exhibited striking changes: SPD content was significantly increased (350-700 nmol/g) while SPM was lowered (50-100). PUT was only increased in CA invaded by the tumoral process (100-180). Accordingly, a very high SPD/SPM molar ratio in the abnormal CA region was observed, indicating an acceleration of polyamine neosynthesis which is usually related to ornithine decarboxylase induction. Metabolic changes in polyamines appear to be selective of human epileptic hippocampus. A relationship between glial proliferation (gliosis or neoplasia), epileptic firing and polyamines is discussed.
Introduction
Polyamines (putrescine or PUT, spermidine or SPD, spermine or SPM) are biogenic amines present in all mammalian tissues. These organic polycations are endogenous factors involved in genomic expression, by regulating nucleic acid structure and activity, especially in developing tissues or tumors where polyamine neosynthesis is increased 1°. More recently, polyamines were found to regulate Correspondence to: Dr. J. Laschet, Laboratory of General and Comparative Biochemistry, University of Liege, 17 place Delcour, B-4020 Liege, Belgium.
ionic transport or fluxes in the brain, especially in relation with intracellular Ca 2÷11'12. Moreover, a polyamine modulatory site has been discovered associated with the glutamatergic receptor-cationic channel complex (NMDA subtype) 22. In animal models, some relationships between polyamines and epilepsy have already been suggested. Intracerebral microinjection of polyamines (especially PUT) induced epileptic electrocortical activities in normal rats 5. In astrocytes from cortex of audiogenic mice, N-monoacetylputrescinedependent activation of PUT uptake, polyamine concentrations and the rate of amino acid synthesis from PUT are very much increased in compar-
152
Materials and methods
quate anticonvulsive therapy. The anatomical definition of the epileptogenic area was based on the data obtained during the stereoelectroencephalography (SEEG) session described by Talairach and Bancaud2°; this method makes it possible to distinguish the brain structures where the electrical epileptic discharges first appear (onset area) from those where they secondarily propagate (propagation area). All patients had seizures originating in the CA and propagating in the temporal neocortex. One patient (MAMO) had morphological alterations on the CT scan suggesting the presence of a tumor which was confirmed post-surgically by the morphological examination of the excised tissue as an oligodendroglioma. Details of the surgical procedure have been described elsewhere 2°. In the present study, we selected surgical brain specimens from the CA and the temporal neocortex (temporal gyri and temporal polar region). Each brain sample was cut into two parts, one for biochemical analysis, the other for neuropathological examination (Dr. Daumas-Duport, Ste Anne Hospital, Paris). Marked cellular alterations were localized in CA: dense gliosis and variable degree of neuronal loss. In one case (MAMO) an oligodendroglioma was identified in CA. The temporal neocortex (temporal gyri and pole) was histologically normal.
Temporal lobe epilepsy patients
Analysis of polyamine concentrations
Three patients (2 males, 1 female), ranging in age from 20 to 27 years at the time of neurosurgery, are presented in Table l, indicating age of onset, duration of epilepsy and etiology. Surgery was considered because of failure of ade-
Surgical specimens from epileptic patients were immediately frozen at surgery time and stored at -80°C. Gray matter was dissected and used for polyamine determinations. Perchloric extracts of those samples were dansylated by the method de-
ison to normal astrocytes from mouse brain 13. Polyamine concentrations have been studied in human brain tumors s, in brains of infants suffering malnutrition 14, in fetal brain ~4'19 as well as in adult post-mortem brain s'lS. Surgical brain specimens from epileptic patients undergoing partial lobectomy have been used for numerous biochemical analyses. Some metabolic alterations were found to be associated with the epileptogenicity (spiking versus non-spiking) of the human cortex, namely in amino acid contents 18'21, in aromatic monoamine contents 3"6, in some enzyme activities of rate-limiting neurotransmitter metabolism ~7, but also in ionic transport systems7. In temporal lobe epilepsy, cellular plasticity changes such as neuronal loss and gliosis, sprouting of mossy fibers were observed in the epileptic hippocampus 9. It was therefore of interest to investigate the polyamine metabolism in cerebral regions from epileptic patients, since polyamines are usually involved in growth processes. In our study, special attention was given to the region where spiking activity started (origin of seizures) and to the region where spiking activity secondarily appeared (propagation). The goal of the present study was to determine whether polyamine metabolism changes are associated with spiking activity
TABLE 1
Temporal lobe epilepsy patients Patient
Age at time of neurosurgery (sex)
Etiology
Age at onset of epilepsy Radiological (duration in years) examination
Neuropathological examination
STEPI
20 (M)
12 (8)
Normal
Normal
LAMA
26 (F)
Hyperthermic convulsions at 2 years Hyperthermic convulsions at 2 years
Normal
Normal
MAMO
29 (M)
Deep left temporal hypodensity
Oligodendroglioma (CA)
6 (20) 20 (9)
153
scribed by Brown et al. 4. The dansyl-polyamines were then analyzed by the HPLC method described by Bontemps et al. 2. Control specimens of temporal lobe were obtained from a patient without a history of neurological disease who died after massive intestinal hemorrhage. Two other controls (specimens of normal frontal cortex) were obtained during neurosur-
gery for deep benign lesion. Results
Polyamine contents in each analyzed temporal area are shown in Fig. 1 for patient STEPI, in Fig. 2 for patient LAMA and in Fig. 3 for patient MAMO. Table II summarizes the results of poly-
• PUT/nmol/g 1000
[ ] SPD/nmol/g 900
• SPM/nmol/g • SPD/SPM %
800 700 600 500 400 300 200 100 0
Pext
Pint
Tla
Tlm
Tlp
T2p
T3a(I)
T3a(ll)
T3p
T3
CAa
CAp
Fig. 1. Regional polyamine contents in the temporal lobe of the epileptic patient STEPI. a, m, p, int, ext: anterior, middle, posterior, internal, external.
• PUT/nmol/g 700
[ ] SPD/nmol/g
600
• SPM/nmol/g
500
• SPD/SPM %
400 300 200 100 0 Tla
Tla
(i)
(it)
Tlm
TZm
TZm
T2m
T2p
T2p
T2p
(I)
(11)
(lit)
(I)
(it)
(ill)
T2T3 T3a
T3m (It)
T3
U
CA
CA
CA
(I)
(It)
(Ill)
Fig. 2. Regional polyamine contents in the temporal lobe of the epileptic patient LAMA. a, m, p: anterior, middle, posterior.
154
• 1000
PUTlnmollg
[ ] SPD/nmollg m SPMlnmollg
900
m SPDISPM % 800 700 600 500 400 300 200 100 0
TZm (I)
T2m
(11)
T2p (I)
T2p
(11)
subLTa
CA/LT (I)
CA/LT
(11)
Fig. 3. Regional polyamine contents in the temporal lobe of the epileptic patient M A M O . a, m, p: anterior, middle, posterior.
amine contents (means+standard deviations) in the areas of onset and propagation of electro-clini-
cal seizures and control samples, i.e., CA and temporal or frontal neocortex.
T A B L E II
Polyamine contents in human brain cortex Putrescine (nmol/g wet weight)
Sperm±dine (nmol/g wet weight)
Spermine (nmot/g wet weight)
SPD/SPM (%)
Control temporal lobe Post-mortem patient Neocortex ( n - l l) A m m o n ' s horn ( n - l )
47.2 ± 4.8 43.1
278 ± 65 203
175 ± 41 124
163 ± 47 164
Control frontal lobe Two surgery patients Neocortex (n = 4)
59.3 ± 26.7
259 ± 47
232 ± 22
112 ± 12
94.6 ± 10.0 57.2 ± 6.1
213 ± 17 354 ± 2
237 + l0 77.9 4-_ 55.3
89.7 ± 5.4 605 ± 426
49.1 ± 11.0 38.8 ± 10.6
232 ± 58 430 ± 48
150 ± 22 85.5 ± 26.8
152 ± 39 530 ± 138
94.8 ± 39 140.3 ± 67
293 ± 80 581 ± 58
169 ± 27 63.3 ± 11.6
183 ± 76 925 ± 79
Epileptic temporal lobe STEPI Neocortex ( n - 1 0 ) A m m o n ' s horn ( n - 2 ) LAMA Neocortex(n-15) A m m o n ' s horn ( n - 3 ) Epileptic and tumoral temporal lobe MAMO Neocortex (n 5) A m m o n ' s horn ( n = 2 )
155
Molar ratios of SPD to SPM (SPD/SPM) were calculated. This ratio is usually considered to be a metabolic index for polyamine synthesis [an increase of the value of SPD/SPM is correlated with an increased activity of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis1°]. In controls, polyamine contents were similar within the different analyzed regions: temporal pole (P), first to third temporal gyri (T~Tz-T3), uncus (U) and CA. In the epileptic specimens, the main alterations of polyamine concentrations were restricted to CA: spermidine concentrations [SPD] were significantly elevated, [SPM] were decreased; therefore SPD/SPM indices were abnormally high (Figs. 1-3 and Table II). This suggests an induction of ornithine decarboxylase activity similar of that described in growing tissue ~°. No significant difference was observed for SPD and SPM contents, or for the SPD/SPM index in the temporal neocortex. These results indicate that the metabolic alterations of polyamines appeared to be selective for CA. Moreover, we have to note that these areas were histologically abnormal and characterized by glial or tumoral proliferation. PUT concentrations were never modified except in the case of oligodendroglioma where [PUT] was increased in all temporal structures with the highest value found in the tumor itself (Fig. 3). Similar results were reported in human astrocytoma 8.
mechanisms may be involved in glial alterations present in epileptogenic tissue. In addition, a modulatory effect of polyamines on the excitatory glutamatergic system has recently been proposed 22, by increasing the affinity for glycine at the co-agonist site resulting in a possible increased glutamatergic response. Preliminary data (unpublished) obtained in Xenopus oocytes expressing the rat NMDA receptors demonstrated that SPD increased NMDA currents whereas SPM inhibited them. Nevertheless, it has to be considered that spermine is also involved in the activation of intracellular Ca 2+ transport in the brainl 1,12. An induction of ornithine decarboxylase was described in rat brain lesions induced by glutamatergic analogs 16. A reciprocal metabolic relationship could be suggested between the N M D A glutamatergic excitatory receptor and polyamine-dependent activation of gene expression in growing cells. Such a possibility needs more evidence but could be of importance in the understanding of the plasticity changes related to synchronous firing and epileptic activity. In conclusion, these preliminary results indicate marked changes in polyamine metabolism selectively restricted to the regions of temporal lobe seizure onset. Further studies should be developed to better elucidate the role of polyamines in cellular plasticity and neurotransmitter involvement to promote epileptogenesis.
Discussion Acknowledgements The present preliminary results demonstrated that biochemical changes in polyamine metabolism are selectively found in epileptic hippocampus, the structure where all focal seizures were starting. Further studies will be necessary to prove that polyamine concentration changes were related to induction of ODC as has been demonstrated in limbic seizures, after electroshock induced convulsions in the rat ~'23. Moreover, previous reports indicated that increased levels of polyamines are associated with a number of growth processes by activating nucleic acid and protein synthesis ~°. We suggest that such
We wish to thank Drs. P. Chauvel, J.-P. Chodkiewicz and C. Daumas-Duport for their assistance in obtaining patient data and surgical tissue, Dr. B. Guibert, B. Evrard and A. Minet for their technical assistance. This investigation has been supported by the Commission of the European Communities, by the National Institute for Health and Medical Research (INSERM, France), by the National Center for Scientific Research (CNRS, France) and by the Belgian Queen Elisabeth Medical Foundation.
156
References 1 Baudry, M., Lynch, G. and Gall, C., Induction of ornithine decarboxylase as a possible mediator of seizure-elicited changes in genomic expression in rat hippocampus, J. Neurosci., 6 (1986) 3430-3435. 2 Bontemps, J., Laschet, J. and Dandrifosse, G., Analysis of dansylderivatives of di- and polyamines in mouse brain, human serum and duodenal biopsy specimens by high performance liquid chromatography on a standard reversed-phase column, J. Chromatogr., 311 (1984) 59-67. 3 Bri6re, R., Sherwin, A.L., Robitaille, Y., Olivier, A., Quesney, L.F. and Reader, T.A., ~-1 Adrenoceptors are decreased in human epileptic foci, Ann. Neurol., 19 (1986) 2630. 4 Brown, N.D., Strickler, M.P. and Whaun, J.M., Femtomolar ion-pair high-performance liquid chromatographic method for determining Dns-polyamine derivatives of red blood cell extracts utilizing an automated polyamine analyzer, J. Chromatogr., 245 (1982) 101-108. 5 De Sarro, G.B., Ascioti, C., Bagetta, G., Libri, V. and Nistic6, G., Behavioural and electrocortical changes of the polyamines after microinfusion in several areas of the rat brain.ln: G. Nistic6 et al. (Eds.), Neurotransmitters, Seizures, and Epilepsy III, Raven Press, New York, NY, 1986, pp. 423~438. 6 Goldstein, D.S., Nadi, N.S., Stull, R., Wyler, A.R. and Porter, R.J., Levels of catechols in epileptogenic and nonepileptogenic regions of the human brain, J. Neurochern., 50 (1988) 225-229. 7 Grisar, T. and Delgado-Escueta, A.V., Astroglial contribution in human temporal lobe epilepsy: K ÷ activation of (Na +,K +)-ATPase in bulk isolated glial cells and synaptosomes, Brain Res., 364 (1986) 1 11. 8 Harik, S.I. and Sutton, C.H., Putrescine as a biochemical marker of malignant brain tumors, Cancer Res., 39 (1979) 5010-5015. 9 Houser, C.R., Miyashiro, J.E., Swartz, B.E., Walsh, G.O., Rich, J.R. and Delgado-Escueta, A.V., Altered patterns of dynorphin immunoreactivity suggest mossy fiber reorganization in human hippocampal epilepsy, J. Neurosci., 10 (1990) 267-282. 10 Jfinne, J., P6s6, H. and Raina, A., Polyamines in rapid growth and cancer, Biochim. Biophys. Acta, 473 (1978) 241 293. 11 Jensen, J.R., Lynch, G. and Baudry, M., Polyamines stimulate mitochondrial calcium transport in rat brain, J. Neurochem., 48 (1987) 765-772.
12 Jensen, J.R., Lynch, G. and Baudry, M., Allosteric activation of brain mitochondrial Ca 2~ uptake by spermine and by Ca2+: brain regional differences, J. Neurochem., 53 (1989) 1182-1187. 13 Laschet, J., Grisar, T., Bureau, M. and Guillaume, D., Characteristics of the putrescine uptake and subsequent GABA formation in primary cultured astrocytes from normal C57BL/6J and epileptic DBA/2J mouse brain cortices, Neuroscience, 48 (1992) 151 157. 14 McAnulty, P.A., Yusuf, H.K.M., Dickerson, J.W,T., Hey, E.N. and Waterlow, J.C., Polyamines of the human brain during normal fetal and postnatal growth and during postnatal malnutrition, J. Neurochem., 28 (1977) 1305 1310. 15 Perry, T.L., Hansen, S. and MacDougall, L., Amines of human whole brain, J. Neurochem., 14 (1967) 775 782. 16 Reed, L.J. and Belleroche, J., Induction of ornithine decarboxylase in cerebral cortex by excitotoxin lesion of nucleus basalis: association with postsynaptic responsiveness and Nmethyl-D-aspartate receptor activation, J. Neurochem., 55 (1990) 780-787. 17 Sherwin, A., Quesney, F., Gauthier, S., Olivier, A., Robitaille, Y., McQuaid, P., Harvey, C. and Van Gelder, N., Enzyme changes in activity spiking areas of human epileptic cerebral cortex, Neurology, 34 (1984) 927~33. 18 Sherwin, A., Robitaille, Y., Quesney, F., Olivier, A., Villeinure, J., Leblanc, R., Feindel, W., Andermann, E., Gotman, J., Andermann, F., Ethier, R. and Kisch, S., Excitatory amino acids are elevated in human epileptic cerebral cortex, Neurology, 38 (1988) 920 923. 19 Sturman, J.A. and Gaull, G.E., Polyamine biosynthesis in human fetal liver and brain, Pediat. Res., 8 (1974) 231-237. 20 Talairach, J. and Bancaud, J,, Stereotaxic exploration and therapy in epilepsy. In: P.J. Vinken and G. Bruyn (Eds.), ttandbook of Clinical Neurology, Vol. 15, The Epilepsies, North Holland, Amsterdam, 1974, pp. 758-782. 21 Van Gelder, N.M., Sherwin, A.L. and Rasmussen, T., Amino acid content of epileptogenic human brain: focal versus surrounding regions, Brain Res., 40 (1972) 385-393. 22 Williams, K., Romano, C., Dichter, M.A. and Molinoff, P.B., Modulation of the NMDA receptor by polyamines, Life Sci., 48 (1991) 469498. 23 Zawia, N.H. and Bondy, S.C., Electrically stimulated rapid gene expression in the brain: ornithine decarboxylase and cfos, Mol. Brain Res., 7 (1990) 243 247.
151
EPIRES 00487
Polyamine metabolism in epileptic cortex
J. L a s c h e t a'b, S. T r o t t i e r c, T. G r i s a r a a n d V. Leviel b aLaboratory of Comparative and General Biochemistry and Department of Neurology, University of Liege, Liege (Belgium), bLaboratoire de Physiologie Nerveuse LPN1, CNRS de Gif-sur-Yvette and cINSERM JF 90-12, CHR de Rennes, Rennes (France) (Accepted 10 April 1992)
Key words." Human; Temporal lobe epilepsy; Polyamines; Hippocampus; Temporal cortex; Plasticity
Polyamine (tissue) concentrations have been studied in hippocampus and temporal neocortex from patients with temporal lobe epilepsy. Depth electrode recordings demonstrated hippocampal origin of the seizures, the temporal neocortex being involved during the discharge propagation. Neuropathological examination of excised tissues showed glial proliferation or glioma in Ammon's horn (CA), whereas the temporal neocortex did not exhibit any histological abnormality. Polyamine (putrescine or PUT, spermidine or SPD, spermine or SPM) concentrations were determined on surgical samples from the hippocampus and various areas of temporal neocortex. Human post-mortem tissue from temporal lobe regions was used for controls. In post-mortem controls and temporal neocortex specimens from epileptic patients, polyamine levels were similar (in nmol/g wet weight: PUT = 40-100; SPD = 200-350; SPM = 100-200). In CA, polyamine levels exhibited striking changes: SPD content was significantly increased (350-700 nmol/g) while SPM was lowered (50-100). PUT was only increased in CA invaded by the tumoral process (100-180). Accordingly, a very high SPD/SPM molar ratio in the abnormal CA region was observed, indicating an acceleration of polyamine neosynthesis which is usually related to ornithine decarboxylase induction. Metabolic changes in polyamines appear to be selective of human epileptic hippocampus. A relationship between glial proliferation (gliosis or neoplasia), epileptic firing and polyamines is discussed.
Introduction
Polyamines (putrescine or PUT, spermidine or SPD, spermine or SPM) are biogenic amines present in all mammalian tissues. These organic polycations are endogenous factors involved in genomic expression, by regulating nucleic acid structure and activity, especially in developing tissues or tumors where polyamine neosynthesis is increased 1°. More recently, polyamines were found to regulate Correspondence to: Dr. J. Laschet, Laboratory of General and Comparative Biochemistry, University of Liege, 17 place Delcour, B-4020 Liege, Belgium.
ionic transport or fluxes in the brain, especially in relation with intracellular Ca 2÷11'12. Moreover, a polyamine modulatory site has been discovered associated with the glutamatergic receptor-cationic channel complex (NMDA subtype) 22. In animal models, some relationships between polyamines and epilepsy have already been suggested. Intracerebral microinjection of polyamines (especially PUT) induced epileptic electrocortical activities in normal rats 5. In astrocytes from cortex of audiogenic mice, N-monoacetylputrescinedependent activation of PUT uptake, polyamine concentrations and the rate of amino acid synthesis from PUT are very much increased in compar-
152
Materials and methods
quate anticonvulsive therapy. The anatomical definition of the epileptogenic area was based on the data obtained during the stereoelectroencephalography (SEEG) session described by Talairach and Bancaud2°; this method makes it possible to distinguish the brain structures where the electrical epileptic discharges first appear (onset area) from those where they secondarily propagate (propagation area). All patients had seizures originating in the CA and propagating in the temporal neocortex. One patient (MAMO) had morphological alterations on the CT scan suggesting the presence of a tumor which was confirmed post-surgically by the morphological examination of the excised tissue as an oligodendroglioma. Details of the surgical procedure have been described elsewhere 2°. In the present study, we selected surgical brain specimens from the CA and the temporal neocortex (temporal gyri and temporal polar region). Each brain sample was cut into two parts, one for biochemical analysis, the other for neuropathological examination (Dr. Daumas-Duport, Ste Anne Hospital, Paris). Marked cellular alterations were localized in CA: dense gliosis and variable degree of neuronal loss. In one case (MAMO) an oligodendroglioma was identified in CA. The temporal neocortex (temporal gyri and pole) was histologically normal.
Temporal lobe epilepsy patients
Analysis of polyamine concentrations
Three patients (2 males, 1 female), ranging in age from 20 to 27 years at the time of neurosurgery, are presented in Table l, indicating age of onset, duration of epilepsy and etiology. Surgery was considered because of failure of ade-
Surgical specimens from epileptic patients were immediately frozen at surgery time and stored at -80°C. Gray matter was dissected and used for polyamine determinations. Perchloric extracts of those samples were dansylated by the method de-
ison to normal astrocytes from mouse brain 13. Polyamine concentrations have been studied in human brain tumors s, in brains of infants suffering malnutrition 14, in fetal brain ~4'19 as well as in adult post-mortem brain s'lS. Surgical brain specimens from epileptic patients undergoing partial lobectomy have been used for numerous biochemical analyses. Some metabolic alterations were found to be associated with the epileptogenicity (spiking versus non-spiking) of the human cortex, namely in amino acid contents 18'21, in aromatic monoamine contents 3"6, in some enzyme activities of rate-limiting neurotransmitter metabolism ~7, but also in ionic transport systems7. In temporal lobe epilepsy, cellular plasticity changes such as neuronal loss and gliosis, sprouting of mossy fibers were observed in the epileptic hippocampus 9. It was therefore of interest to investigate the polyamine metabolism in cerebral regions from epileptic patients, since polyamines are usually involved in growth processes. In our study, special attention was given to the region where spiking activity started (origin of seizures) and to the region where spiking activity secondarily appeared (propagation). The goal of the present study was to determine whether polyamine metabolism changes are associated with spiking activity
TABLE 1
Temporal lobe epilepsy patients Patient
Age at time of neurosurgery (sex)
Etiology
Age at onset of epilepsy Radiological (duration in years) examination
Neuropathological examination
STEPI
20 (M)
12 (8)
Normal
Normal
LAMA
26 (F)
Hyperthermic convulsions at 2 years Hyperthermic convulsions at 2 years
Normal
Normal
MAMO
29 (M)
Deep left temporal hypodensity
Oligodendroglioma (CA)
6 (20) 20 (9)
153
scribed by Brown et al. 4. The dansyl-polyamines were then analyzed by the HPLC method described by Bontemps et al. 2. Control specimens of temporal lobe were obtained from a patient without a history of neurological disease who died after massive intestinal hemorrhage. Two other controls (specimens of normal frontal cortex) were obtained during neurosur-
gery for deep benign lesion. Results
Polyamine contents in each analyzed temporal area are shown in Fig. 1 for patient STEPI, in Fig. 2 for patient LAMA and in Fig. 3 for patient MAMO. Table II summarizes the results of poly-
• PUT/nmol/g 1000
[ ] SPD/nmol/g 900
• SPM/nmol/g • SPD/SPM %
800 700 600 500 400 300 200 100 0
Pext
Pint
Tla
Tlm
Tlp
T2p
T3a(I)
T3a(ll)
T3p
T3
CAa
CAp
Fig. 1. Regional polyamine contents in the temporal lobe of the epileptic patient STEPI. a, m, p, int, ext: anterior, middle, posterior, internal, external.
• PUT/nmol/g 700
[ ] SPD/nmol/g
600
• SPM/nmol/g
500
• SPD/SPM %
400 300 200 100 0 Tla
Tla
(i)
(it)
Tlm
TZm
TZm
T2m
T2p
T2p
T2p
(I)
(11)
(lit)
(I)
(it)
(ill)
T2T3 T3a
T3m (It)
T3
U
CA
CA
CA
(I)
(It)
(Ill)
Fig. 2. Regional polyamine contents in the temporal lobe of the epileptic patient LAMA. a, m, p: anterior, middle, posterior.
154
• 1000
PUTlnmollg
[ ] SPD/nmollg m SPMlnmollg
900
m SPDISPM % 800 700 600 500 400 300 200 100 0
TZm (I)
T2m
(11)
T2p (I)
T2p
(11)
subLTa
CA/LT (I)
CA/LT
(11)
Fig. 3. Regional polyamine contents in the temporal lobe of the epileptic patient M A M O . a, m, p: anterior, middle, posterior.
amine contents (means+standard deviations) in the areas of onset and propagation of electro-clini-
cal seizures and control samples, i.e., CA and temporal or frontal neocortex.
T A B L E II
Polyamine contents in human brain cortex Putrescine (nmol/g wet weight)
Sperm±dine (nmol/g wet weight)
Spermine (nmot/g wet weight)
SPD/SPM (%)
Control temporal lobe Post-mortem patient Neocortex ( n - l l) A m m o n ' s horn ( n - l )
47.2 ± 4.8 43.1
278 ± 65 203
175 ± 41 124
163 ± 47 164
Control frontal lobe Two surgery patients Neocortex (n = 4)
59.3 ± 26.7
259 ± 47
232 ± 22
112 ± 12
94.6 ± 10.0 57.2 ± 6.1
213 ± 17 354 ± 2
237 + l0 77.9 4-_ 55.3
89.7 ± 5.4 605 ± 426
49.1 ± 11.0 38.8 ± 10.6
232 ± 58 430 ± 48
150 ± 22 85.5 ± 26.8
152 ± 39 530 ± 138
94.8 ± 39 140.3 ± 67
293 ± 80 581 ± 58
169 ± 27 63.3 ± 11.6
183 ± 76 925 ± 79
Epileptic temporal lobe STEPI Neocortex ( n - 1 0 ) A m m o n ' s horn ( n - 2 ) LAMA Neocortex(n-15) A m m o n ' s horn ( n - 3 ) Epileptic and tumoral temporal lobe MAMO Neocortex (n 5) A m m o n ' s horn ( n = 2 )
155
Molar ratios of SPD to SPM (SPD/SPM) were calculated. This ratio is usually considered to be a metabolic index for polyamine synthesis [an increase of the value of SPD/SPM is correlated with an increased activity of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis1°]. In controls, polyamine contents were similar within the different analyzed regions: temporal pole (P), first to third temporal gyri (T~Tz-T3), uncus (U) and CA. In the epileptic specimens, the main alterations of polyamine concentrations were restricted to CA: spermidine concentrations [SPD] were significantly elevated, [SPM] were decreased; therefore SPD/SPM indices were abnormally high (Figs. 1-3 and Table II). This suggests an induction of ornithine decarboxylase activity similar of that described in growing tissue ~°. No significant difference was observed for SPD and SPM contents, or for the SPD/SPM index in the temporal neocortex. These results indicate that the metabolic alterations of polyamines appeared to be selective for CA. Moreover, we have to note that these areas were histologically abnormal and characterized by glial or tumoral proliferation. PUT concentrations were never modified except in the case of oligodendroglioma where [PUT] was increased in all temporal structures with the highest value found in the tumor itself (Fig. 3). Similar results were reported in human astrocytoma 8.
mechanisms may be involved in glial alterations present in epileptogenic tissue. In addition, a modulatory effect of polyamines on the excitatory glutamatergic system has recently been proposed 22, by increasing the affinity for glycine at the co-agonist site resulting in a possible increased glutamatergic response. Preliminary data (unpublished) obtained in Xenopus oocytes expressing the rat NMDA receptors demonstrated that SPD increased NMDA currents whereas SPM inhibited them. Nevertheless, it has to be considered that spermine is also involved in the activation of intracellular Ca 2+ transport in the brainl 1,12. An induction of ornithine decarboxylase was described in rat brain lesions induced by glutamatergic analogs 16. A reciprocal metabolic relationship could be suggested between the N M D A glutamatergic excitatory receptor and polyamine-dependent activation of gene expression in growing cells. Such a possibility needs more evidence but could be of importance in the understanding of the plasticity changes related to synchronous firing and epileptic activity. In conclusion, these preliminary results indicate marked changes in polyamine metabolism selectively restricted to the regions of temporal lobe seizure onset. Further studies should be developed to better elucidate the role of polyamines in cellular plasticity and neurotransmitter involvement to promote epileptogenesis.
Discussion Acknowledgements The present preliminary results demonstrated that biochemical changes in polyamine metabolism are selectively found in epileptic hippocampus, the structure where all focal seizures were starting. Further studies will be necessary to prove that polyamine concentration changes were related to induction of ODC as has been demonstrated in limbic seizures, after electroshock induced convulsions in the rat ~'23. Moreover, previous reports indicated that increased levels of polyamines are associated with a number of growth processes by activating nucleic acid and protein synthesis ~°. We suggest that such
We wish to thank Drs. P. Chauvel, J.-P. Chodkiewicz and C. Daumas-Duport for their assistance in obtaining patient data and surgical tissue, Dr. B. Guibert, B. Evrard and A. Minet for their technical assistance. This investigation has been supported by the Commission of the European Communities, by the National Institute for Health and Medical Research (INSERM, France), by the National Center for Scientific Research (CNRS, France) and by the Belgian Queen Elisabeth Medical Foundation.
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