Temporal changes of cytochrome P450 (Cyp) and eicosanoid-related gene expression in the rat brain after traumatic brain injury

Abstract

Background: Traumatic brain injury (TBI) induces arachidonic acid (ArA) release from cell membranes. ArA metabolites form a class of over 50 bioactive eicosanoids that can induce both adaptive and/or maladaptive brain responses. The dynamic metabolism of ArA to eicosanoids, and how they affect the injured brain, is poorly understood due to their diverse activities, trace levels, and short half-lives. The eicosanoids produced in the brain postinjury depend upon the enzymes present locally at any given time. Eicosanoids are synthesized by heme-containing enzymes, including cyclooxygenases, lipoxygenases, and arachidonate monoxygenases. The latter comprise a subset of the cytochrome P450 "Cyp" gene family that metabolize fatty acids, steroids, as well as endogenous and exogenous toxicants. However, for many of these genes neither baseline neuroanatomical nor injury-related temporal expression have been studied in the brain.In a rat model of parietal cortex TBI, Cyp and eicosanoid-related mRNA levels were determined at 6 h, 24 h, 3d, and 7d postinjury in parietal cortex and hippocampus, where dynamic changes in eicosanoids have been observed. Quantitative real-time polymerase chain reaction with low density arrays were used to assay 62 rat Cyps, 37 of which metabolize ArA or other unsaturated fatty acids; 16 eicosanoid-related enzymes that metabolize ArA or its metabolites; 8 eicosanoid receptors; 5 other inflammatory- and recovery-related genes, plus 2 mouse Cyps as negative controls and 3 highly expressed "housekeeping" genes.

Results: Sixteen arachidonate monoxygenases, 17 eicosanoid-related genes, and 12 other Cyps were regulated in the brain postinjury (p < 0.05, Tukey HSD). Discrete tissue levels and distinct postinjury temporal patterns of gene expression were observed in hippocampus and parietal cortex.

Conclusions: The results suggest complex regulation of ArA and other lipid metabolism after TBI. Due to the temporal nature of brain injury-induced Cyp gene induction, manipulation of each gene (or its products) at a given time after TBI will be required to assess their contributions to secondary injury and/or recovery. Moreover, a better understanding of brain region localization and cell type-specific expression may be necessary to deduce the role of these eicosanoid-related genes in the healthy and injured brain.

Figure 1

In situ hybridization histochemistry for rat Cyp2j4 mRNA in brain 7 days after TBI. Representative fields of injured and contralateral (A) parietal cortex; (B) piriform cortex; (C) hippocampal dentate gyrus. Note the apparent loss of pyramidal neurons in the ipsilateral dentate hilar region. Little staining was observed in the corpus callosum or other white matter structures. Some microvascular profiles appeared to stain positive in these non-perfused fresh frozen brain sections. Studies were carried out starting anterior to the site of injury, staining every sixth section through the injured volume (bregma −1.8 mm to approximately −4.5 mm, according to the coordinates of Paxinos and Watson [155]). High stringency hybridization and washes were performed as described in Section In situ hybridization histochemistry.

Figure 2

In situ hybridization histochemistry for rat Cyp27a1 mRNA in brain 7 days after TBI. (A) Representative fields of injured parietal cortex with higher magnification of negative control sense probe in the same region of a different section (top inset) and neuronal staining with a large vascular profile (bottom inset). (B) Composite showing injured and contralateral hippocampal Cyp27a1 mRNA with ablation of neuronal staining, as well as heavier non-neuronal staining ipsilateral to injury. Some intense white matter staining is visible medially in the corpus callosum and the ipsilateral hippocampal fimbria (arrows). See Figure 1 legend for methodologic details.