Irradiation induces regionally specific alterations in pro-inflammatory environments in rat brain

Abstract

Purpose: Pro-inflammatory environments in the brain have been implicated in the onset and progression of neurological disorders. In the present study, we investigate the hypothesis that brain irradiation induces regionally specific alterations in cytokine gene and protein expression.

Materials and methods: Four month old F344 x BN rats received either whole brain irradiation with a single dose of 10 Gy gamma-rays or sham-irradiation, and were maintained for 4, 8, and 24 h following irradiation. The mRNA and protein expression levels of pro-inflammatory mediators were analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. To elucidate the molecular mechanisms of irradiation-induced brain inflammation, effects of irradiation on the DNA-binding activity of pro-inflammatory transcription factors were also examined.

Results: A significant and marked up-regulation of mRNA and protein expression of pro-inflammatory mediators, including tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and monocyte chemoattractant protein-1 (MCP-1), was observed in hippocampal and cortical regions isolated from irradiated brain. Cytokine expression was regionally specific since TNF-alpha levels were significantly elevated in cortex compared to hippocampus (57% greater) and IL-1beta levels were elevated in hippocampus compared to cortical samples (126% greater). Increases in cytokine levels also were observed after irradiation of mouse BV-2 microglial cells. A series of electrophoretic mobility shift assays (EMSA) demonstrated that irradiation significantly increased activation of activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and cAMP response element-binding protein (CREB).

Conclusion: The present study demonstrated that whole brain irradiation induces regionally specific pro-inflammatory environments through activation of AP-1, NF-kappaB, and CREB and overexpression of TNF-alpha, IL-1beta, and MCP-1 in rat brain and may contribute to unique pathways for the radiation-induced impairments in tissue function.

Figure 1

Irradiation up-regulates mRNA and protein expression of TNF-α in rat brain. F344 × BN rats (n = 4) received either whole brain irradiation with a single dose of 10 Gy or sham-irradiation. The animals were maintained for 4, 8, and 24 h post-irradiation, and the brains were rapidly removed and two different brain regions (hippocampus and cortex) were dissected. The mRNA expression levels of TNF-α in hippocampus and cortex were determined by quantitative real-time RT-PCR (panel A). Using the 2^−ΔΔCT method as described in Materials and methods, the data are presented as fold change in gene expression normalised to a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and relative to the sham-irradiated control. The protein expression levels of TNF-α in hippocampus and cortex were analysed by ELISA (panel B) and fluorescence microscopy (panel C–J). Panel C: sham-irradiation (Control); panel D: 4 h post-irradiation; panel E: 8 h post-irradiation; panel F: 24 h post-irradiation; panel G–J: negative controls. Magnification of the images (panel C–J) is 100×. Data shown are mean ± SEM for each group. *^,**Statistically significant from control (*p < 0.05 and **p < 0.001). ^#Statistically significant from hippocampus (p < 0.05).

Figure 2

Irradiation up-regulates mRNA and protein expression of TNF-α in microglia. BV-2 cells received irradiation with a single dose of 10 Gy (IR 10 Gy) or sham-irradiation (Control). Cells were maintained for 4 and 24 h post-irradiation, and the mRNA and protein expression levels of TNF-α were analysed by quantitative real-time RT-PCR (panel A) and ELISA (panel B), respectively. Data shown are mean ± SEM for each group. *^,**Statistically significant from control (*p < 0.05 and **p < 0.001).

Figure 3

Irradiation up-regulates mRNA and protein expression of IL-1β in rat brain. Experiments were carried out as described in Figure 1. The mRNA expression levels of IL-1β in hippocampus and cortex were determined by quantitative real-time RT-PCR (panel A). The protein expression levels of IL-1β in hippocampus and cortex were determined by ELISA (panel B). Data shown are mean ± SEM for each group. *^,**Statistically significant from control (*p < 0.05 and **p < 0.001). ^#,!Statistically significant from hippocampus (^#p < 0.05 and ^!p < 0.001).

Figure 4

Effects of irradiation on mRNA and protein expression of IL-6 in rat brain. Experiments were carried out as described in Figure 1. The mRNA expression levels of IL-6 in hippocampus and cortex were determined by quantitative real-time RT-PCR (panel A). The protein expression levels of IL-6 in hippocampus and cortex were determined by ELISA (panel B). Data shown are mean ± SEM for each group. *Statistically significant from control (p < 0.05).

Figure 5

Irradiation up-regulates mRNA and protein expression of MCP-1 in rat brain. Experiments were carried out as described in Figure 1. The mRNA expression levels of MCP-1 in hippocampus and cortex were determined by quantitative real-time RT-PCR (panel A). The protein expression levels of MCP-1 in hippocampus and cortex were determined by ELISA (panel B). Data shown are mean ± SEM for each group. *^,**Statistically significant from control (*p < 0.05 and **p < 0.001).

Figure 6

Representative autoradiogram of EMSA of the effects of irradiation on AP-1 DNA-binding activity in rat brain (panel A). F344 × BN rats (n = 4) received either whole brain irradiation with a single dose of 10 Gy or sham-irradiation. The animals were maintained for 4, 8, and 24 h post-irradiation and nuclear extracts were prepared from the brain hippocampus regions and analysed by EMSA. Competition studies were performed by adding excess unlabeled AP-1 probe. Densitometric quantification of the effects of irradiation on AP-1 DNA-binding activity in rat brain (panel B). Experiments were repeated four times, and the intensities of the AP-1-specific bands were measured and statistically analysed. The results are expressed as fold increase over control values. Data shown are the mean ± SEM for each group. *Statistically different from control (p < 0.05).

Figure 7

Representative autoradiogram of EMSA of the effects of irradiation on NF-κB DNA-binding activity in rat brain (panel A). Experiments were carried out as described in Figure 6. Competition studies were performed by adding excess unlabeled NF-κB probe. Densitometric quantification of the effects of irradiation on NF-κB DNA-binding activity in rat brain (panel B). Experiments were repeated four times, and the intensities of the NF-κB-specific bands were measured and statistically analysed. The results are expressed as fold increase over control values. Data shown are the mean ± SEM for each group. *Statistically different from control (p < 0.05).

Figure 8

Representative autoradiogram of EMSA of the effects of irradiation on CREB DNA-binding activity in rat brain (panel A). Experiments were carried out as described in Figure 6. Competition studies were performed by adding excess unlabeled CREB probe. Densitometric quantification of the effects of irradiation on CREB DNA-binding activity in rat brain (panel B). Experiments were repeated four times, and the intensities of the CREB-specific bands were measured and statistically analysed. The results are expressed as fold increase over control values. Data shown are the mean ± SEM for each group. *Statistically different from control (p < 0.05).