Increased expression of TNF receptor-associated factor 6 after rat traumatic brain injury

Abstract

TRAF6 (TNF receptor-associated factor 6), a member of tumor necrosis factor receptor-associated factors family was identified as a molecule that binds to the cytoplasmic domain of CD40. TRAF6 functions as an adaptor, positively regulating the NF-κB, JNK pathway. Additionally, some studies have reported that TRAF6 is required for apoptosis within the developing CNS and regulates cell fate decisions by inducing caspase 8-dependent apoptosis. However, its distribution and function in the central nervous system (CNS) lesion are not well understood. In this study, we performed an acute traumatic brain injury model in adult rats. And we mainly examined protein expression and cellular localization of TRAF6 during rat traumatic brain injury (TBI). Western blot analysis showed TRAF6 level significantly improved at 7 days after injury, and then declined during the following days. The protein expression of TRAF6 was further analysed by immunohistochemistry. In comparison to contralateral cerebral cortex, we observed a highly significant accumulation of TRAF6 at the ipsilateral brain. Immunofluorescence double-labeling showed that TRAF6 was co-expressed with NeuN and GFAP. Besides, co-localization of TRAF6/active caspase 3 and TRAF6/proliferating cell nuclear antigen (PCNA) were detected in NeuN and GFAP, respectively. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase 3 whose changes were correlated with the expression profiles of TRAF6. In conclusion, this is the first description of TRAF6 expression in traumatic brains. Our data suggested that TRAF6 might play important roles in CNS pathophysiology after TBI.

Fig. 1

Western immunoblot analysis showing the TRAF6 and GAPDH in the cortex surrounding the wound at various survival times after brain injury. Sample immunoblots probed for TRAF6 and GAPDH are shown above. The bar chart below demonstrates the ratio of TRAF6 relative to GAPDH for each time point. a TRAF6 protein level was low in normal cortex, peaked at 7 days, and then gradually rose thereafter. N denotes cortical extracts from normal animals. b Semiquantitative analysis (relative optical density) of the intensity of staining of TRAF6 to GAPDH for each time point. The data are means ± SEM. (n = 3, * P < 0.05, ** P < 0.01, significantly different from the control groups)

Fig. 2

Immunohistochemical staining of TRAF6 in adult rat brain 1 mm distal to the lesion 7 days after TBI. Low power views of cross-sections immunostained with antibody for TRAF6 in contralateral (a) and ipsilateral brain (b). Higher-power views in contralateral (c) and ipsilateral (d) sides of the injured brain. e Negative control. This photomicrograph showed that injured brain in which primary antibody was omitted. f Quantitative analysis of TRAF6 positive cells/mm² between contralateral and ipsilateral brains 7 days after injury. TRAF6 was significantly increased in the ipsilateral brain at 7 days after TBI. Asterisk * indicated significant difference at P < 0.05 compared with contralateral brain. Error bars represent SEM. Scale bars: 200 μm (a, b) and 20 μm (c–e)

Fig. 3

Double immunofluorescence staining for TRAF6 and cells makers in adult rat brain. The tissue from naive and damaged brains were immunostained with TRAF6 (b, e, h, k) and different cell makers, such as NeuN (a, g) and GFAP (d, j), and the co-localization of TRAF6 with different phenotype-specific markers (arrows) were visualized in the merged images (c, f, i, l). a–f Immunostaining for TRAF6 with NeuN and GFAP in naive brain; g–l Immunostaining for TRAF6 with NeuN and GFAP at 7 days after TBI. m Quantitative analysis of different phenotype-specific markers positive cells expressing TRAF6 (%) in naive group and 7 days after injury. The change of TRAF6 was striking in astrocytes and neurons. *^{, #} indicate significant difference at P < 0.05 compared with the naive group. Error bars represent SEM. Scale bars: 20 μm (a–l)

Fig. 4

Association of TRAF6 with proliferation after TBI. a, b Western blot analysis of PCNA in brains after TBI. The expression of PCNA was increased after TBI and peaked at 5 days. c–h Double immunofluorescence staining for PCNA, GFAP, and TRAF6 in brain cortex after TBI. In adult rat brain at 7 days after injury, sections labeled with PCNA (e) and GFAP (c), and the co-localization of PCNA with GFAP (arrows) were shown in brain (g). The majority of reactive astrocytes were PCNA-positive at 7 days after TBI. Moreover, there was co-localization between PCNA and TRAF6. Scale bars: 20 μm (c–h)

Fig. 5

Association of TRAF6 with apoptosis after TBI. a, b Western blot analysis of active caspase 3 in brain after TBI. Active caspase 3 could not be detected in control groups. Its expression was gradually increased after TBI and peaked at 5 days. c–h Double immunofluorescence staining for active caspase 3, NeuN and TRAF6 in brain cortex after TBI. In adult rat brain at 7 days after injury, sections labeled with active caspase 3 (e) and NeuN (c), and the co-localization of active caspase 3 with NeuN (arrows) was shown in brain. The majority of neurons were caspase-3-positive at 7 days after TBI. Moreover, coincidence of TRAF6 (d) reactivity with active caspase 3 (f) by adjacent serial sections. Scale bars: 20 μm (c–h)