This protocol was evaluated and approved by the Institutional Animal care and Use Committee at Loma Linda University, Loma Linda, CA.
The SBI model
The SBI model in rodents has been described previously . Briefly, the rationale is that a controlled insult - a partial right frontal lobectomy - mimics standard surgical procedures. By using fixed coordinates (see below), the repeatability of this procedure is insured. By creating a lesion close to the right primary sensory and motor cortices, it is possible to observe impairments in neurological function. Both sensory as well as motor systems are affected and are quantifiable with established, objective tests. Brain water content measurement after sacrificing the animal assures an objective, quantitative method to evaluate edema formation.
Adult male Sprague - Dawley rats (weight 300-350 g) were used for this study. The rodent model of SBI was used as described previously . All animals were intubated and subject to general anesthesia, using 3% halothane for induction and 1% for maintenance. A square cranial window was drilled such that the left lower corner of the square was at the bregma. The dura was opened and reflected to expose the underlying right frontal lobe. Using a sharp blade, two incisions were made along the saggital and coronal planes leading away from the bregma to limit the partial lobectomy to 2 mm lateral to the saggital plane and 1mm rostral to the coronal plane. The depths of the resection extended to the base of the anterior fossa. Hemostasis was achieved by using saline irrigation, packing, and electrocautery. Sham surgery included only craniotomy and replacement of the bone flap without dural penetration. Animals were sacrificed at 24 hours. The 24 hour time point appears to be the time of peak edema formation in rodents, making it thus the prime target for any therapy aimed at edema reduction.
All animals were intubated for sixty minutes regardless of the length of the operation and group. This allows for a constant in the length of intubation and hydrogen administration. No surgery exceeded sixty minutes.
H2was administered via the endotrachial tube. A tank premixed with 2.9% hydrogen, 21% oxygen and nitrogen balance was used for the treatment group and sham (2.9%, Praxair, CA, USA). The control group received 21% oxygen and nitrogen balance. Treatment time consisted of 60 minutes for all groups. The tubing was checked for leaks and adequate concentrations of hydrogen at the endotrachial tube prior to the procedure using a Hydrogen-meter (H2Scan, CA, USA).
Brain Water Content
The animals were sacrificed under deep anesthesia at 24 hours after frontal lobe injury. The brains were removed and divided into frontal ipsilateral, frontal contralateral, parietal ipsilateral, parietal contralateral, brain stem and cerebellum on ice. These parts were weighed immediately (wet weight) and weighed again after drying in an oven at 105ºC for 48 hours (dry weight) as described previously . The percent of water content was calculated as [(wet weight - dry weight)/wet weight] × 100%. The number of animals used in each group for brain edema study was control (n = 12), sham (n = 8), hydrogen-treated (n = 12).
Methodology for the myeloperoxidase (MPO) assay has been described previously . Briefly, frozen samples (n = 18) were cut into 0.1-0.2 g pieces and homogenized with Tris-HCl and Na+buffer. The samples were then centrifuged at 14,000 g for 30 minutes. The supernatants were discarded and the samples were resuspended in 0.5%HTAB/K+buffer. Several cycles of freezing in liquid nitrogen followed by sonication at 25°C were performed, before spectrophotometrical analysis at 460 nm. The value was divided by the exact weight of the sample and expressed in MPO activity Unit/gram.
Lipid Peroxidation Assay
The method for Lipid Peroxidase (LPO) Assay has been described in detail previously . Briefly, animals (n = 18) were anesthetized and brain samples were collected at 24 hours after SBI. The level of lipid peroxidation products (malondialdehyde [MDA]) in the right cerebral cortexes was measured using a LPO-586 kit (OxisResearch; Portland, OR). Right frontal cerebral cortexes were homogenized in 20 mmol/L phosphate buffer (pH 7.4) with 0.5 M butylated hydroxytoluene in acetonitrile. The homogenates were centrifuged at 20,800 g for 10 min at 4°C and the supernatants were collected. Protein concentration was measured by DC protein assay (Bio-Rad) and the samples were reacted with a chromogenic reagent at 45°C for 60 min. After incubation, the samples were centrifuged at 20,800 g for 10 min at 4°C and supernatants were measured at 586 nm. The level of MDA was calculated as picomoles per milligram protein according to the standard curve.
We used a 21-point score adapted from the one developed for stroke by Garcia et al.  as presented in Table 1. Furthermore, we used the wire-hang and beam balance tests for assessment of balance, strength, and coordination. The wire-hang and beam balance test were assessed based on a three point scale with dependence on time and/or reaching the platform. 0 = less than 15 seconds, no movement towards platform, 1 = less than 40 seconds, no movement towards platform, 2 = less than or equal to half distance to platform within 40 seconds, and 3 = reaching platform within 40 seconds. Three trials were performed while animals were receiving a five minute rest period in between trials. The final score was the average of three trials.
All data were collected in SigmaPlot and analyzed with SigmaStat (both Systat Software, Inc, San Jose, CA). The one-way ANOVA test was used to calculate statistical significance between samples. P value below 0.05 was considered as statistically significant.