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Bumetanide augments the neuroprotective efficacy of phenobarbital plus hypothermia in a neonatal hypoxia–ischemia model YiQing Liu1, Yu Shangguan1, John D.E. Barks1 and Faye S. Silverstein1,2 INTRODUCTION: The NaKCl cotransporter NKCC1 facilitates intraneuronal chloride accumulation in the developing brain. Bumetanide (BUM), a clinically available diuretic, inhibits this chloride transporter and augments the antiepileptic effects of phenobarbital (PB) in neonatal rodents. In a neonatal cerebral hypoxia–ischemia (HI) model, elicited by right carotid ligation, followed by 90 min 8% O2 exposure in 7-d-old (P7) rats, PB increases the neuroprotective efficacy of hypothermia (HT). We evaluated whether BUM influenced the neuroprotective efficacy of combination treatment with PB and HT. METHODS: P7 rats underwent HI lesioning; 15 min later, all received PB (30 mg/kg), and 10 min later, half received BUM (10 mg/kg, PB-HT+BUM) and half received saline (PB-HT+SAL). One hour after HI, all were cooled (30 °C, 3 h). Contralateral forepaw sensorimotor function and brain damage were evaluated 1–4 wk later. RESULTS: Forepaw functional measures were close to normal in the PB-HT+BUM group, whereas deficits persisted in PB-HT+SAL controls; there were corresponding reductions in right cerebral hemisphere damage (at P35, % damage: PB-HT+BUM, 21 ± 16 vs. 38 ± 20 in controls). DISCUSSION: These results provide evidence that NKCC1 inhibition amplifies PB bioactivity in the immature brain and suggest that coadministration of PB and BUM may represent a clinically feasible therapy to augment the neuroprotective efficacy of therapeutic HT in asphyxiated neonates.
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n neonates with hypoxic–ischemic encephalopathy, therapeutic hypothermia (HT) (initiated within the first 6 h of life) is associated with reductions in death and neurological impairment at 18 mo (1). However, more than 40% of treated infants have poor neurodevelopmental outcomes, and there is an urgent need to identify interventions that can effectively supplement HT. In experimental models of neonatal hypoxic–ischemic (HI) brain injury, a broad range of therapeutic agents including phenobarbital (PB) augment hypothermic neuroprotection (2,3). In a well-characterized model of neonatal HI brain injury, elicited by unilateral carotid artery ligation, followed by 90 min 8% oxygen exposure, in 7-d-old (P7) rats, early post-HI treatment
with PB improved the neuroprotective efficacy of delayed onset brief moderate HT. This combination therapy resulted in sustained improvements in sensorimotor function and a greater than 50% reduction in brain damage, in comparison with saline (SAL)-injected HT-treated controls (3). PB, a γ-amino-butyric acid agonist, is the antiepileptic drug used most frequently to treat neonatal seizures, although its efficacy is limited (4). Recent studies have provided important insights into the mechanisms underlying its limited anticonvulsant efficacy in neonates and have suggested pharmacological strategies to overcome them (5–7). In mature neurons, γ-amino-butyric acid triggers membrane hyperpolarization and neuronal inhibition because of the passive influx of chloride down its electrochemical gradient. In neonatal cortex (rodent and human), the developmentally regulated chloride transporter NKCC1 is expressed, and it facilitates intraneuronal chloride accumulation. Because immature neurons have high chloride concentrations, γ-amino-butyric acid triggers chloride efflux and membrane depolarization. Moreover, NKCC1 expression may be upregulated both by neonatal HI (8) and by seizures (7). Bumetanide (BUM), a clinically available loop diuretic, inhibits NKCC1 and augments the antiepileptic effects of PB in a neonatal rat seizure model (6). A pilot clinical study of BUM as add-on treatment after PB administration for newborn seizures is underway (Clinical trials.gov, NCT00830531). This study evaluated the impact of BUM on the neuroprotective efficacy of combination therapy with PB and HT in the neonatal HI model. We found that BUM improved the neuroprotective efficacy of treatment with PB and HT, and that HT was essential to achieve optimal benefit from combination drug therapy. Results Physiological Measures
Among animals allocated to Protocols 1–4, 152/154 survived until P14; in Protocol 5, 35/36 animals survived until P35. In the first day after lesioning, BUM-treated animals gained less weight than controls (0.01 ± 1 vs. 0.85 ± 0.9 g), but weights did not differ between BUM- and SAL-treated animals at P14 or P35.
1 Department of Pediatrics, University of Michigan, Ann Arbor, Michigan; 2Department of Neurology, University of Michigan, Ann Arbor, Michigan. Correspondence: Faye S. Silverstein (
[email protected])
Received 15 July 2011; accepted 08 December 2011; advance online publication 7 March 2012. doi:10.1038/pr.2012.7 Copyright © 2012 International Pediatric Research Foundation, Inc.
Volume 71 | Number 5 | May 2012 Pediatric Research
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Articles
Liu et al.
Table 1 summarizes sequential temperature measurements for all protocols. In the first two sets of experiments that evaluated combination treatment with BUM and HT (Protocols 1 and 2), during HT mean body temperatures were slightly lower in BUM-treated than in SAL control groups (range: −0.3 to −0.9 °C, P < 0.05, repeated-measures ANOVA). In the third set of experiments, which evaluated combination treatment with two doses of BUM (Protocol 3), temperatures did not differ. In the fourth set of experiments (Protocol 4), the only variable was post-HI temperature management, and mean temperatures during this intervention differed substantially (range: −4.4 to −6 °C, P < 0.001, ANOVA) between groups. Of note, when Protocol 2 was replicated to assess late outcomes (Protocol 5), there were no significant treatment-related temperature differences. In two Protocol 5 experiments, measurements were also obtained 60 min after the end of cooling, when pups were recovering with their dams, and their temperatures did not differ.
with 27 ± 17% in animals that received SAL instead of BUM, P < 0.04, Mann–Whitney test) (Protocol 2, Figure 1b). In the next group of experiments (Protocol 3, Figure 1c), damage was again attenuated by combination treatment with PB, BUM (10 mg/kg), and HT; in contrast, treatment effects were lost with a lower dose of BUM (2.5 mg/kg) (P < 0.01, Mann– Whitney test). To determine if HT contributed to neuroprotection conferred by the combination of PB and BUM, in Protocol 4 all animals were treated with PB and BUM and half underwent HT (n = 24/group; Figure 1d). Right cerebral hemisphere damage was lower in the HT-treated group (15 ± 17% vs. 27 ± 20%, P
