The antiserum was affinity-purified using the affinity gel attatched with the above antipeptides respectively to improve the specificity of each antibody. Western blotting Microsomal protein samples extracted from different normal rat brain regions including the frontal lobe, frontal cortex, occipital lobe, hippocampus, cerebellum, striatum, midbrain, and medulla were boiled in Laemmli buffer and resolved on 4C15% gradient Tris-glycine sodium dodecyl sulfateCpolyacrylamide gel electrophoresis (SDS-PAGE) gels. immunoreactivity increases in surrounding astrocytes, while CYP4F4 immunoreactivity shifts from endothelia of cerebral vessels to astrocytes. the omega-hydroxylation of endogenous Rabbit Polyclonal to XRCC6 eicosanoids such as leukotriene B4 (LTB4) (Jin et al., 1998; Bylund et al., 2003), arachidonic acid (Hoagland et al., 2001; Lasker et al., 2000), and prostaglandin A1 and E1 (Kawashima et al., 1997). These eicosanoids are important inflammatory molecules, and their metabolism by the CYP4F subfamilies inactivates them as inflammatory signaling molecules, indicating that CYP4Fs may play a role in the resolution of inflammation. Traumatic brain injury (TBI) is a major public health problem and can lead to lethal multiple organ dysfunction syndrome, in which acute systemic inflammation plays an essential role (Faden et al., 1989; McIntosh, 1994; Ott et al., 1994). A previous study from our laboratory (Cui et al., 2003) indicated that rat CYP4F4 and CYP4F5 gene Lupulone expression in hippocampus was modulated in a time-dependent fashion after TBI, indicating a potential role for CYP4Fs in inflammation following TBI. We hypothesize that CYP4Fs are widely distributed in the brain and that TBI can induce broader changes of CYP4Fs expression in multiple brain regions. It is important to know how CYP4Fs expression varies during brain inflammation post-TBI in order to predict the effects of brain injury on drug metabolism in the brain, identify means to enhance recovery post injury, and regulate inflammatory mediator levels by modulation of CYP4Fs activity. Methods All chemicals utilized, if not specifically defined, were of reagent grade quality or higher. Brain injury Male Sprague Dawley rats (250C300?g body wt) were purchased from Harlan Laboratories (Indianapolis, IN). All protocols were conducted in compliance with guidelines set forth in the and approved by the University of Texas Health Science Center’s Institutional Animal Care and Use Committee. Rats were initially anesthetized using 5% isoflurane with a 1:1?N2O/O2 mixture and then maintained with 2.5% isoflurane with a 1:1?N2O/O2 mixture via face mask. Animals were mounted around the Lupulone stereotaxic frame secured by ear bars and an incisor bar. Rats received a single, unilateral impact at 6?m/sec, 2.5-mm deformation performed midway between the bregma and the lambda with Lupulone the edges of craniotomies 1?mm lateral to the midline. The brains of sham-operated but uninjured rats served as controls. Core body temperature was monitored constantly by a rectal thermometer and maintained at 36C36.5C. After injury, the scalp was sutured and closed. The animals were sacrificed at different time points (24?h and 2 weeks) Lupulone after the injury. Microsome and RNA preparations After animals were sacrificed, microsomes from frontal lobe, frontal cortex, occipital lobe, hippocampus, cerebellum, striatum, midbrain and medulla were prepared by differential centrifugation as described by Saito and Strobel (1981). All procedures were carried out at 4C. The separated brain tissues were washed and homogenized in 6 volumes of homogenization buffer (20?mM potassium phosphate buffer, 1?mM EDTA, 0.25?mM sucrose, pH 7.2C7.4) with Complete Protease Inhibitor Cocktail Tablets (2 tablets/100?mL buffer, Roche Inc.). Each homogenate was centrifuged at 3,220for 20?min, and the supernatant fraction was collected. The supernatant fraction was further centrifuged at 105,000for 60?min. After centrifugation, the supernatant fraction was discarded and the pellet was homogenized and washed in homogenization buffer without protease inhibitors, and centrifuged again at 105,000for 60?min. The final pellets were resuspended in homogenization buffer without protease inhibitors and.