PC12 cell cultures were divided randomly into four groups: Group 1 served as a control without either RSI or TPEN treatment; Group 2 was treated only with TPEN; Group 3 received only RSI; and Group 4 received both RSI and treatment with TPEN. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, Texas. Male Sprague-Dawley rats weighing 400–500 g were anesthetized, intubated and mechanically ventilated with 1.5–2.0% isoflurane in air:oxygen (70:30) and prepared for parasagittal fluid percussion TBI (Mathew et al.
Briefly, the rats were placed in a stereotaxic frame and a craniotomy was performed 1 mm lateral right to the sagittal suture, midway between the bregma and lambda sutures. A modified LuerLok syringe hub was placed in the craniotomy site and anchored in place with dental hygienic acrylic resin. Rats were then connected to the trauma device and subjected to either a sham injury or moderate (2.0 atm) TBI. Rats were randomly assigned into the sham injury (n = 6 per time point) or moderate TBI (n = 6 per time point) group. The acrylic cap was removed, wounds were sutured with 4-0 prolene, the isoflurane was discontinued, and the rats were extubated and permitted to awaken from anesthesia. After survival for 0.5, 2, 4, 16 or 24 hours, rats were re-anesthetized with 4% isoflurane, decapitated and the brains were rapidly removed for dissection of the cortex and hippocampus from the ipsilateral (injured) and contralateral sides of the brain to measure MT.
The basal concentration of total MT and the MT to thionein (T) ratio were also obtained by collecting brain tissues from six rats receiving no surgery and sacrificed under anesthesia with 4% isoflurane. Fluorimetric assays allow determination of the metal load of MT (Yang et al. The fraction of the protein that is not saturated with metal ions is referred to as T. Hippocampi and cortices of Sprague-Dawley rats were dissected immediately after sacrifice for MT measurements (Yang et al. The tissues were homogenized by a Kontes electric pellet pestle (Fisher Scientific) in homogenizing buffer (0.2 M mannitol, 0.05 M sucrose, 0.01 M KCl, 0.01 M HEPES, pH 7.4) in a microliter volume that corresponds to 4 times the weight of the tissue (in mg). After centrifugation at 10,000 × g for 5 min, 10 μl supernatant was diluted in 20 mM Tris-HCl, pH 7.4 to determine total protein concentrations using the Pierce Micro BCA ™ protein assay kit. The remaining supernatant was collected and treated with 40% (v/v) acetonitrile (EM Science) for 15 min to precipitate large and hydrophobic proteins. The suspension was spun at 10,000 × g for 5 min and the supernatant was labeled immediately with 1 mM ammonium 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) (Invitrogen, Molecular Probes) in 35 mM borate buffer, pH 7.4. Tris-(2-carboxyethyl)phosphine, hydrochloride (TCEP) (Invitrogen, Molecular Probes) was added to a final concentration of 15 mM because ABD-F can label only reduced thiols. Twenty-five mM Na 2 EDTA (Sigma-Aldrich) was added for total metallothionein measurement, but omitted for T measurement. After incubation at 60 °C for 10 min, samples were separated on a reversed-phase C4 Phenomenex Jupiter 5μ column (250 mm × 4.60 mm 2 ) with a Phenomenex pre-column at a flow rate of 1 ml/min, using a Beckman Coulter System Gold HPLC system with eluent A (5 mM Tris-HCl, pH 7.4) and eluent B (50% 2-propanol in A). The condition was 90% eluent A and 10% eluent B with a double step gradient to 40% eluent B at 5 min and to 100% eluent B at 12 min. The fluorescence signal was detected with a JASCO Intelligent Fluorescence Detector Model 2020 with excitation of 384 nm and emission of 510 nm and a detector gain at ×100. All aspects of the experiment were performed at 25 °C. During analysis, however, samples were cooled at 4 °C in the auto-sampler. Results were analyzed by using one-way ANOVA to determine significance at the level of 0.05 between different time points or treatments for in vitro experiments. When ANOVA detected significance, the significance of differences was determined by the Student’s t-test with Bonferroni adjustment for multiple comparisons. Two-way ANOVA was used to analyze the effect of in vivo treatment (sham vs TBI) and time for MT/T measurements. The Student’s t-test was used to compare the difference between sham and TBI at individual time points. This work was supported by National Institutes of Health grant NS042849 (to DSP), and in part by a predoctoral fellowship (to BEH) from the National Institutes of Environmental Health Sciences training grant ES007254. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form.
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