Antiepileptic drugs and apoptotic neurodegeneration in the developing brain

Abstract

A seizure is a sudden change in behavior caused by synchronous, rhythmic firing of neurons in the brain. Between 2% and 4% of all children in Europe and the United States experience at least one seizure before the age of 5 years (1). Epilepsy, a brain disorder characterized by recurrent seizures, affects 1–2% of humans worldwide and shows its highest incidence in the first year of life (1).

Antiepileptic drugs (AEDs) are used to prevent or interrupt seizures. They act via three mechanisms: (i) limitation of sustained repetitive neuronal firing via blockade of voltage-dependent sodium channels; (ii) enhancement of γ-aminobutyric acid (GABA)-mediated inhibition; and (iii) blockade of glutamatergic excitatory neurotransmission (2–5). Phenytoin decreases neuronal firing through use-dependent blockade of voltage-gated sodium channels. Barbiturates and benzodiazepines enhance inhibition in the brain by allosterically modulating permeability of the chloride channel coupled to the GABA type A receptor. Vigabatrin decreases GABA breakdown by blocking the GABA-degrading enzyme GABA transaminase, and valproate influences GABA synthesis and breakdown, leading to an increase of GABA concentrations in the brain. Valproate also interferes with glutamate-mediated excitation and limits sustained repetitive neuronal firing through voltage- and use-dependent blockade of sodium channels (2–5).

AEDs are among the most common causes of fetal malformations, developmental delay, and microcephaly (6–11). Increasing maternal blood levels and combinations of AEDs impose an increased risk for harm to human infants (11). AEDs may also exert unfavorable effects on human intellect when given to treat seizures in infants and toddlers. Therapy with barbiturates during the first 3 years of life may cause cognitive impairment that persists into adulthood (12–16). Although neurotoxic effects of AEDs have been recognized since the 1970s, the underlying mechanisms are not understood.

In the immature rodent brain, suppression of synaptic neurotransmission via blockade of glutamate N-methyl-d-aspartate receptors or activation of GABA type A receptors may trigger apoptotic neurodegeneration (17, 18). Because depression of synaptic neurotransmission is the common denominator in the action of AEDs, we investigated whether common AEDs may cause apoptotic neurodegeneration in the developing brain and what the underlying pathogenetic mechanisms are. Furthermore, we attempted to identify measures that will prevent AED neurotoxicity.

Rats received vehicle, phenytoin (50 mg/kg), phenobarbital (75 mg/kg), or valproate (400 mg/kg) and were killed 24 h later on P8. Using an optical disector method the numerical densities of total cells (cells per mm^{) and degenerating cells (degenerating cells per mm) in 16 brain regions of vehicle- (}n = 6), phenytoin- (n = 6), phenobarbital- (n = 6), and valproate-treated rats (n = 6) were estimated. In column 2, mean numerical total cell densities ± SEM in 8-day-old rats are shown. The extent of apoptosis in vehicle-, phenytoin-, phenobarbital-, and valproate-treated rats is shown as the ratio of degenerating cell density to total cell density and is presented as % ± SEM.

At these ages rat pups received vehicle, 75 mg/kg phenobarbital i.p., or 400 mg/kg valproate at 0 h and were killed at 24 h. Vigabatrin was administered at the dose of 400 mg/kg on 3 subsequent days, starting on P5, and the pups were perfused on P8 (24 h after the last dose). Using an optical disector method (20), the numerical densities of degenerating neurons (degenerating neurons per mm^{) in 14 brain regions of vehicle and drug-treated pups were estimated. Shown are mean numerical densities of degenerating neurons ± SEM (}n = 6). Effects of phenobarbital, valproate, and vigabatrin on the rate of apoptosis at each age are presented as ratios of mean numerical densities of degenerating cells in drug-treated vs. age-matched vehicle-treated rats (expressed as fold ↑). Note that both phenobarbital and valproate do not trigger apoptosis in brain regions displaying no physiological apoptosis and that vulnerability to their proapoptotic action depends on developmental age.

Acknowledgments

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