Phenobarbital augments hypothermic neuroprotection

Abstract

Seizures are associated with adverse outcome in infants with hypoxic-ischemic encephalopathy. We hypothesized that early administration of the anticonvulsant phenobarbital after cerebral hypoxia ischemia could enhance the neuroprotective efficacy of delayed-onset hypothermia. We tested this hypothesis in a neonatal rodent model. Seven-d-old rats (n = 104) underwent right carotid ligation, followed by 90 min 8% O2 exposure; 15 min later, they received injections of phenobarbital (40 mg/kg) or saline. One or 3 h later, all were treated with hypothermia (30 degrees C, 3 h). Function and neuropathology were evaluated after 7 d (early outcomes) or 1 mo (late outcomes). Early outcome assessment demonstrated better sensorimotor performance and less cortical damage in phenobarbital-treated groups; there were no differences between groups in which the hypothermia delay was shortened from 3 to 1 h. Late outcome assessment confirmed sustained benefits of phenobarbital + hypothermia treatment; sensorimotor performance was better (persistent attenuation of contralateral forepaw placing deficits and absence of contralateral forepaw neglect); neuropathology scores were lower (median, phenobarbital 2 and saline 8.5, p < 0.05); and less ipsilateral cerebral hemisphere %Damage (mean +/- SD, 11 +/- 17 versus 28 +/- 22, p < 0.05). These results suggest that early posthypoxia-ischemia administration of phenobarbital may augment the neuroprotective efficacy of therapeutic hypothermia.

Figure 1. Phenobarbital + delayed hypothermia attenuates early forepaw placing deficit

Postnatal-day-7 (P7) rats (n=68) underwent unilateral cerebral hypoxia-ischemia and then received i.p. injections of phenobarbital 40 mg/kg or an equivalent volume of saline, recovered in a 37°C incubator for 1h (n=32) or 3h (n=36), then were cooled for 3h (30°C incubator). Lateral vibrissae-stimulated forepaw placing (10 trials/side) was evaluated on P14. Saline+delayed hypothermia (dHT)-treated animals had contralateral placing deficits; this deficit was attenuated in the group treated with phenobarbital prior to delayed HT (PB+dHT). Results were the same whether the delay to initiation of HT was 3h or 1h (*p<0.005 and †p<0.05, Mann-Whitney, vs. corresponding saline+dHT controls).

Figure 2. Persistent attenuation of contralateral forepaw placing deficit

P7 rats (n=36) underwent unilateral cerebral hypoxia-ischemia and then received injections of saline (SAL+3hdHT, solid bars) or phenobarbital 40 mg/kg (PB+3hdHT, hatched bars), with normothermia for 3h, then 3h hypothermia (30°C incubator). Rats underwent bilateral vibrissae-stimulated forepaw placing testing (10 trials/side) weekly from P14 to P35. The phenobarbital-treated group had a sustained advantage over the controls for several weeks (*p<0.0001, repeated-measures ANOVA). There was a significant interaction (p<0.005) between group and testing age; performance of the SAL+3hdHT controls converged towards the PB+3hdHT group. Values are means ±standard deviations.

Figure 3. Phenobarbital potentiates hypothermic neuroprotection: late outcome

Injury severity was scored (0–4) in 7 brain regions, with substantial variation in scores in both groups. Total scores, providing an overall measure of damage severity, were lower in the phenobarbital-treated group (PB+dHT) (p<0.05, Mann-Whitney, panel A). Panel B includes scores in each region; anterior cortex at the level of striatum, posterior cortex at the level of hippocampus, striatum, thalamus, and hippocampal CA1, CA3 and dentate gyrus (DG). Data in box plots are: median (horizontal bar), IQR (25^th–75^th percentile, box) and range (whiskers). Regional scores were significantly lower in the PB+dHT group in striatum, thalamus and hippocampus CA3 (Mann-Whitney, p <0.05).