Protein discovery using 2-D gel and mass spectrometry proteomic approaches have aided in deciphering the complex biochemical changes following TBI. [6
]. These approaches, although informative, require specialized expertise and equipment. Antibody arrays, however, provide a target-directed methodology that facilitates high-throughput screening of large numbers of known or suspected candidate proteins. The antibody array analysis presented in this study identified 41 candidates whose hippocampal expression levels were significantly altered 24 h after injury, indicating the complexity of the brain’s response to TBI.
Previous studies of individually selected proteins, as well as global gene chip analysis, have indicated the involvement of many different proteins in TBI pathophysiology (for review, see [5
]). Although some candidates may also appear within DNA microarray data sets, literature searches revealed at least 9 of the candidates in have been specifically implicated in injury models or injury-activated pathways. For instance, the increased hypoxia-inducible transcription factor (Hif-1α) protein after TBI is consistent with altered Hif-1α mRNA after TBI [24
], and increased protein expression after ischemic and brain hemorrhage injury [13
]. Gao et al. recently reported a significant decrease in H3 histone acetylation levels after TBI [9
]. This is consistent with increased SMRT expression, a central component of a co-repressor complex that recruits histone deacetylase to the promoter regions of genes [23
]. These data, in conjunction with our confirming altered AF6 and CA150 expression (), indicate a high-throughput characterization of protein expression approach using Ab arrays may successfully identify potential candidates involved in TBI pathophysiology.
In the present study, the selection of COMT for a more detailed analysis was guided by a number of previous studies that have implicated catecholamine signaling in the behavioral sequelae of TBI [4
]. Following their release, catecholamines are either taken up by specific transporters and/or are metabolized by monoamine oxidase or COMT. Transporter-mediated termination of catecholamine signaling plays a prominent role in structures such as the striatum, whereas in structures such as the prefrontal cortex that express low transporter levels, inactivation through metabolism may play a more prominent role [14
]. An increased role for COMT after injury is consistent with the reported decrease in dopamine transporter (DAT) as early as 7 d post-injury, and increases in the levels of TH protein and TH fibers in the frontal cortex [16
]. Western blot data () indicated significant changes in COMT isoform expression occurred in both hemispheres at both 3 and 14 d post-TBI. These data may reflect changes in COMT expression in a wide variety of cell types, including vascular endothelial, microglial and neuronal cell types. Our double immunohistochemical-labeling experiments () indicate that at all time points post-injury, microglia in the ipsilateral injured area exhibit enhanced COMT expression. In addition, increased COMT immunoreactivity in the dentate gyrus, hilus, and fiber tracts was also observed. Interestingly, recent research has shown that microglia express dopamine receptor mRNA, and their migration can be stimulated by the application of dopamine, the D1
receptor agonist dihydrexidine, or the D2
receptor agonist quinpirole [8
]. It is possible that increased dopamine release after injury [20
] might serve as a chemotactic signal to trigger the recruitment of microglia to distressed regions.
Since scavenging is one of the primary microglia roles in the CNS, enhanced microglia COMT expression may aid in removing and metabolizing catecholamines from damaged areas. Alternatively, the increased COMT immunoreactivity detected in the activated microglia () might arise from phagocytic activity as they clean the debris from the dead and dying cells in the injured hippocampus. However, elevated COMT immunoreactivity is still associated with microglia at 14 days post-injury. This would indicate that intrinsically up-regulated COMT expression in microglia is a more likely explaination, since phagocytosed COMT would not be expected to remain stable over long periods. Additional experiments are needed to determine the exact mechanism (transcription, translation, or protein stability) through which microglia regulates COMT expression, and its consequences following TBI.
In conclusion, the present study demonstrates the potential of Ab arrays to explore the complex pathophysiology of CNS injury. In addition to corroborating previous studies, the antibody array identified many novel candidate proteins. For instance, 10 of the 41 candidates possess functions related to actin dynamics, remodeling, or cell adhesion. Since blood-brain barrier integrity is compromised following TBI, defining the changes that occur in tight junction proteins (e.g. AF6, symplekin) or signaling proteins that regulate actin dynamics (e.g. PRK2, ILK, ABR, Rho-GDI 2) may be critical to understanding the mechanisms underlying blood-brain barrier dysfunction. Some of these candidates may also be involved structural remodeling after TBI. The identification of altered COMT expression, in addition to other novel candidates, may be useful for developing therapies to improve TBI outcome.