Mid-term effects of phenobarbital on apoptotic neurodegeneration and brain weight in the neonatal rat

Phenobarbital (PB) is the antiepileptic drug of choice in the treatment of seizures in the neonatal period. Bittigau et al. have reported a significantly increased apoptotic neurodegeneration in the neonatal rat brain 24 hours after a single dose of PB as well as a decreased brain weight 8 days after (PNAS 99:23, 15089–94, 2002). These results have questioned the safety of phenobarbital-treatment.

We wanted to investigate in the neonatal rat brain 1 to 14 days after a single administration of PB whether an absolute increase or merely an accelerated neuronal apoptosis and lower brain weights occurred. On postnatal day 7 (P7) 10 wistar rats each were given an intraperitoneal injection of 50 or 87,5mg PB per kg (PB-50/PB-87) or vehicle (CONTROL). On days P8, P9, P10, P11, P14 and P21 brains were removed. Evaluation of apoptotic cells was performed with TUNEL and HE stains in 10 brain regions of one brain hemisphere. In addition caspase-3 activity was measured with a fluorometric assay in four sections in the other hemisphere.

The spontaneous natural course of apoptosis was found in CONTROL. With PB-50 both TUNEL and HE stains as well as caspase 3-activity showed no significantly elevated rate of apoptosis. With PB-87 significantly higher rates of apoptotic cells were observed (e.g. TUNEL: P8: striatum, parietal cortex, thalamus; P9: thalamus, cerebellum; P11: thalamus; P14: hippocampus; P21: frontal cortex; example CONTROL vs. PB-87 on P8 in the thalamus: 8,60 vs. 18,47 mean TUNEL-positive cells/mm²).

Significantly elevated caspase 3-activities were observed with PB-87 on P8, P11 and P21 (example CONTROL vs. PB-87 on P8: 8,29 vs. 14,52 nmol AMC/min/g protein).

Both treated groups showed clinical signs of PB-treatment with significantly lower body weights from P8 to P10 with PB-50 and from P8 to P21 with PB-87 (example CONTROL vs. PB-87 on P21: 39,45 vs. 32,17g).

Brain weights were signifcantly lower with PB-50 on P10 and P14 as well as with PB-87 on P8, P11, P14 and P21 (example CONTROL vs. PB-87 on P21: 1,47 vs. 1,36g). Brain-to-body weight ratio showed significantly higher values with PB-50 on P8 and P9, and with PB-87 on P8, P10 and P11 (example CONTROL vs. PB-87 on P21: 0,037 vs. 0,042).

The results show that a therapeutic dose of PB does not increase natural apoptosis in neonatal rat brain tissue within an observation period of 14 days. Rats treated with supratherapeutic doses partly displayed significantly elevated apoptotic rates – particularly in the thalamus.

Poor feeding due to sedation led to decreased body weights in treated rats. The observed lower brain weights of treated animals were qualified by looking at the brain-to-body weight ratios wich displayed the predominant decrease in body weights.

These partially astonishing results demonstrate that an adequate PB-treatment not inevitably leads to an increased apoptosis in the brain of neonatal rats. Our hypothesis of an accelerated neuronal apoptosis could also not be clearly affirmed with supratherapeutic PB-doses. The lower brain weights of treated rats are probably rather due to poor feeding with lasting lower body weights than a loss of neuronal cells. A single administration of PB seemingly does not have effects on apoptotic neurodegeneration in every case.