Eur. Neurol. 14: 97-107 (1976)

Effect of Convulsions and Anticonvulsive Drugs on Cerebrospinal Fluid Cyclic AMP in Rabbits V. V. M yllyla Departments of Neurology and Pharmacology, University of Oulu, Oulu

Key Words. Cyclic AMP • Cerebrospinal fluid • Convulsions • Anticonvulsive drugs Abstract. Cyclic adenosine 3\5'-monophosphate (cAMP) concentration and as­ partate amino transferase (ASAT or GOT) activity were measured from cerebro­ spinal fluid (CSF) of rabbits before and after electrically induced convulsions. The convulsions caused a significant increase in CSF cAMP concentration. The treat­ ment with phénobarbital, diphenylhydantoin and carbamazepinc for 7 days de­ creased the basal cAMP values in CSF and partly inhibited the rise after the convul­ sions. The alterations in ASAT activity were parallel, but less striking. The present results suggest the involvement of cAMP in epileptic discharge and in the mecha­ nism of action of anticonvulsants.

Introduction

Received: June 6, 1975; accepted: June 6, 1975.

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Cyclic adenosine 3',5'-monophosphate (cAMP) and the enzymes synthetizing and degrading it are found in large amounts in brain suggesting that this nucleotide has an important role in central nervous system (CNS) [Sutherland et al., 1962; K lainer et al., 1962; B utcher and S utherland 1962; B reckenridge , 1972]. The involvement of cAMP in synaptic transmission has been confirmed by many investigators [M arx , 1972; V apaatalo , 1974], Abnormalities in transmitter function are char­ acteristic to epilepsy: disturbances at presynaptic and postsynaptic sites of neurons are suggested to be responsible for the initiation of abnormal ex­ cessive neuronal activity within epileptic focus, while subsequent develop-

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ment and spread of paroxysmal activity involves synaptic processes [C ur ­ 1969]. Electrical stimulation has been used for generating seizures to mimick epileptic attacks in man and animals. Metabolic alterations during seizures have been widely studied. B rodersen et al. [1973] have reported that cerebral blood flow, oxygen and glucose uptake are doubled during electrically induced seizures in man. Lactacidosis within brain is associat­ ed with seizures even when hypoxemia is avoided [M ei .drum and B rier lev , 1973]. In animals prolonged epileptic seizures can also lead to is­ chemic cell changes and brain damage [M eldrum and B rierley , 1973]. Some metabolic changes are reflecting on extracellular space, e.g. potas­ sium concentration can rise around the active neurons and its increase can also be found in cerebrospinal fluid (CSF) [G rossman , 1973]. The glial tissue is supposed to be essential for the restoration of the normal sit­ uation in extracellular space [G rossman , 1973], S himizu and D aly [1972] have shown that depolarizing agents such as potassium cause an accumulation of cAMP in incubated slices of cerebral cortex, which can be blocked by membrane stabilizers. G oldberg et al. [1970] have dem­ onstrated that electroconvulsive shock leads to a striking increase in cAMP levels of brain of experimental animals. Epileptogenic lesions of rat cerebral cortex made by freezing increase the adenyl cyclase activity and decrease phosphodiesterase activity resulting in an increase of cAMP level [W alker et al., 1973], Recently we have described elevated cAMP values in CSF of epileptic patients shortly after an attack [H eikkinen et al., 1974; M yllyla et al., 1975], Cerebral and cerebellar tissues are rich in enzymes, e.g. aspartate ami­ no transferase (ASAT of GOT) [G reen et al., 1959]. Attempts have been made to correlate the activities of these enzymes in CSF with various neurologcial disorders [H ildebrand and L evin , 1973]. Although there are reports of elevated enzyme activities in CSF after epileptic convulsions and electroconvulsive shock [M iyazaki, 1958; M ann et al., I960], in our previous work no uniform alterations in enzyme activities could be found in man [M yllyla et al., 1975]. The purpose of this study was to get in­ formation about the effects of anticonvulsive drugs and electrical convul­ sions on CSF cAMP concentration of animals and to compare the results with ASAT activity. tis ,

The investigation was carried out on adult rabbits (2.5-3 kg) of both sexes. CSF samples of about 0.5 ml were obtained by occipital puncture under light ether anes­

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Materials and Methods

Effect of Convulsions ami Anticonvulsive Drugs on CSF cAMP in Rabbits

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thesia 24 h before the convulsions and at various intervals after it. Samples contami­ nated by blood were excluded. Convulsions lasting about 30 sec were electrically in­ duced by Siemens convulsator (1.0 sec, 3 J) under ether anesthesia. In the first part of the study the samples were taken at 15 min, 1, 3, 6, 12, 24 and 48 h after the convulsions. The effect of diurnal variation of CSF cAMP was studied on 4 animals by sampling at 2 and 8 a.m. and 2 and 8 p.m. The effect of successive punctures on CSF cAMP concentration was studied on 7 animals, the samples were obtained as in the second part of the study. The CSF ASAT activity was measured from the same samples (test kit from Baker Diagnostic Reagents, Baker Chemicals, Deventer, Netherlands). In the second part of the study the animals were divided into four groups (table I) according to the treatment they received. In the first group, which served as a control, physiological saline 0.25 ml/kg twice a day was given subcutaneously (s.c.) for 7 days. Also the anticonvulsive drugs were given for 7 days, divided into two daily doses (table I). In all groups the basal samples were taken 24 h before the con­ vulsions. In the first group, half of the animals were punctured 15 min, 2, 4 and 6 h after the convulsions and the other half after 1, 3 and 5 h, respectively. In the other groups the animals were punctured at 1, 3 and 6 h after the convulsions. The con­ centrations of various anticonvulsants were measured in serum and CSF by a gas chromatographic method (Sampson et at. [1971], slightly modified for phénobarbital and carbamazepine). For measurement of cAMP concentration 0.2 ml of fresh CSF was taken from the samples. Proteins were precipitated by adding 20/d of 55% trichloroacetic acid (the final concentration 5%). The samples were stored 1-3 weeks at -20 °C before determination of cAMP, during which period no significant alteration in cAMP concentration was observed. The cAMP concentrations were measured by the pro­ tein-binding method of G ilman [1970|. The values are expressed as nanomoles/liter of CSF (nmol/1). From the rest of the samples the ASAT activity was determined (test kit from Baker Diagnostic Reagents, Baker Chemicals). The values are ex­ pressed as international units/Iiter of CSF (IU/1) at 25 °C. All the chemical and drugs were obtained commercially and they were of analyt­ ical grade. Diphenylhydantoin used was Epanutin® (Parke Davis, Hounslow, England). Carbamazepine was kindly donated by Geigy (Geigy, Helsinki, Finland). Student’s t test was used for statistical treatment of the results.

In the first part of the study it was observed that the most prominent alteration in CSF cAMP concentration was within 6 h after convulsions. The levels were approaching the initial ones in 12 h. The effects of diurnal variation in CSF cAMP concentration are shown in figure 1. The values in the morning tended to be higher but the difference was insignificant. The mean concentration of all samples ob­ tained from the seven rabbits being punctured successively but not un-

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Results

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Time of sampling

Fig. 1. Diurnal variation of CSF cAMP concentration. Means + SE (ninol/1).

Table /. The groups of animals according to the drug therapy. The daily doses of various anticonvulsants and concentrations in serum and CSF (means ±SE) after 7 days therapy Group

Number of animals

Daily Drug concentration Drug concentration in CSF, /ig/ml dose1 in serum, /ig/ml ml/kg s.c.

Physiol, saline Phénobarbital Diphenylhydantoin Carbamazepine

II 6 6 5

0.5 5 10 502





12.5 ±2.2 1.2 ±0.3 0.8 ±0.2

5.7 ± 1.0

Effect of convulsions and anticonvulsive drugs on cerebrospinal fluid cyclic AMP in rabbits.

Eur. Neurol. 14: 97-107 (1976) Effect of Convulsions and Anticonvulsive Drugs on Cerebrospinal Fluid Cyclic AMP in Rabbits V. V. M yllyla Departments...
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