PMID: 24705859 DOI: 10.1007/s10072-014-1726-4
Methods
All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA). All surgical and experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications number 80-23) revised in 2011. Efforts were made to minimize the number of animals used and their suffering.

Neonatal brain hypoxia–ischemia modal
Brain HI was performed in 7-day-old male and female Sprague–Dawley rats as described previously [10, 11]. In brief, neonates were anesthetized by isoflurane and their left common carotid arteries were permanently ligated with a double 7-0 surgical silk. The procedure lasted <5 min. After surgery, neonates were returned to the cages with their mothers for 3 h. The neonates were then placed in a chamber filled with humidified 8 % oxygen–92 % nitrogen for 2 h at 37 °C. The oxygen concentration and temperature in the chamber were continuously monitored.

Drug application
The neonates were randomly divided into the following groups: (1) control, (2) brain HI, (3) brain HI and postconditioning with 1, 2 and 3 % sevoflurane, (4) brain HI and 5-HD treatment (10 mg/kg) and (5) brain HI, 5-HD treatment and postconditioning with 2 % sevoflurane. Sevoflurane postconditioning was performed by exposing neonates to various concentrations of sevoflurane in 30 % O2 for 1 h immediately after brain HI. Neonates of brain HI alone group were placed in a chamber flushed with 30 % O2 for 1 h. The mitochondrial KATP channel inhibitor 5-HD was dissolved in normal saline and administered intraperitoneally just before the start of brain HI. The dose of 5-HD was based on a previous study in which intraperitoneal injection of 10 mg/kg 5-HD blocked ischemic preconditioning-induced protection [12].

Brain injury/tissue loss quantification
After 7 days of the brain HI, rats were sacrificed under deep isoflurane anesthesia and then their brains were harvested as described previously [11, 13]. The hindbrain was removed from cerebral hemispheres and bilateral hemispheres were weighed separately. The weight ratio of left to right hemispheres was calculated.

Statistical analysis
The results are presented as mean ± SD (n ≥ 6). Statistical analysis was performed by one-way analysis of variance followed by the Tukey’s test. A P ≤ 0.05 was considered statistically significant.