Anticonvulsant drug-induced cell death in the developing white matter of the rodent brain

Abstract

Objective: During critical periods of brain development, both seizures and anticonvulsant medications can affect neurodevelopmental outcomes. In rodent models, many anticonvulsants trigger neuronal apoptosis. However, white matter apoptosis (WMA) has not been examined after anticonvulsant drug treatment. Herein, we sought to determine if anticonvulsant drugs induced apoptosis in the developing white matter (WM) in a rodent model.

Methods: Postnatal day (P)7 rats were treated with phenobarbital (PB-75), MK-801 (dizocilpine, 0.5), lamotrigine (LTG-20), carbamazepine (CBZ-100), phenytoin (PHT-50), levetiracetam (LEV-250), or saline; all doses are mg/kg. Brain tissue collected 24 h after treatment was stained using the terminal deoxynucleotidyl transferase dUTP nick end labeling method. The number of degenerating cells within WM, that is, anterior commissure (AC), corpus callosum, cingulum, and hippocampus-associated WM tracts, was quantified.

Results: Saline-treated rats showed low baseline level of apoptosis in developing WM on P8 in all the areas examined. PB, PHT, and MK-801 significantly increased apoptosis in all four brain areas examined. Exposure to CBZ, LTG, or LEV failed to increase apoptosis in all regions.

Significance: Commonly used anticonvulsants (PB, PHT) cause apoptosis in the developing WM in a rat model; the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 has a similar effect. These results are consistent with reports of anesthesia-induced WMA during brain development. Consistent with the lack of neuronal apoptosis caused by LTG, LEV, and CBZ, these drugs did not cause WMA. Many infants treated with anticonvulsant drugs have underlying neurologic injury, including WM damage (e.g., following intraventricular hemorrhage [IVH] or hypoxic-ischemic encephalopathy [HIE]). The degree to which anticonvulsant drug treatment will alter outcomes in the presence of underlying injury remains to be examined, but avoiding drugs (when possible) that induce WMA may be beneficial.

Keywords: Anticonvulsant; Antiepileptic drug; Apoptosis; Cell death; Neonatal.

Figure 1. Neonatal exposure to phenobarbital, phenytoin or MK-801 induces apoptosis within the anterior commissure

(A) Number of TUNEL-positive cells in the anterior commissure. Bars indicate mean + SEM. * = significantly increased white matter apoptosis above vehicle (ANOVA and Holm-Sidak test p<0.05). Numbers within the bars indicate the number of specimens analyzed. (B) Apoptosis noted in the anterior commissure as outlined by the black dotted line. Sections depicted are representative of the findings after treatment with anticonvulsants labeled. Anterior commissure is outlined in purple in the schematic in (B). Vehicle or control used is saline. White arrows denote apoptotic cells stained with TUNEL method. AC: Anterior commissure, PB: phenobarbital, PHT: phenytoin, MK: MK-801 NMDA receptor antagonist, LEV: levetiracetam, CBZ: carbamazepine, LTG: lamotrigine

Figure 2. Neonatal exposure to phenobarbital, phenytoin, or MK-801 induces apoptosis within the cingulum, corpus callosum, and hippocampal-associated white matter tracts

(A) Number of TUNEL-positive cells in the cingulum. (B) Number of TUNEL-positive cells in the corpus callosum. (C) Number of TUNEL-positive cells in the hippocampal-associated white matter. Bars indicate mean + SEM. * = significantly increased white matter apoptosis above vehicle (ANOVA and Holm-Sidak test p<0.0001). Numbers within the bars indicate the number of specimens analyzed. (D) Representative photomicrographs and schematic of regions of interest for each drug treatment. The black dotted line segregates the WM areas. In the schematic, red denotes cingulum, green color denotes corpus callosum and the yellow area depicts the hippocampus associated white matter tracts (stratum alveus/oriens, ventral hippocampal commissure, fimbria, and dorsal fornix). The sections are representative of the findings after treatment with the anticonvulsants labeled. Vehicle (control) is saline. White arrows denote apoptotic cells stained with TUNEL method. PB: phenobarbital, PHT: phenytoin, MK: MK-801 NMDA receptor antagonist, LEV: levetiracetam, CBZ: carbamazepine, LTG: lamotrigine