Lipopolysaccharide sensitized male and female juvenile brains to ionizing radiation

Abstract

Radiotherapy is an effective tool in the treatment of pediatric malignancies but it is associated with adverse side effects, both short- and long-term. One common long-term side effect after cranial radiotherapy is cognitive impairment and this is, at least partly, thought to be caused by reduced hippocampal neurogenesis. Neuroinflammation and a perturbed microenvironment are thought to be important in the dysregulation of neurogenesis seen after irradiation (IR). We investigated the effects of a pre-existing, lipopolysaccharide (LPS)-induced systemic inflammation at the time of IR in both males and females. A single dose of 8 Gy to the brain of postnatal day 14 mice caused an upregulation of cytokines/chemokines (IL-1β, MIP-1β, IL-12, GM-CSF, MIP-1α, IL-17, CCL2 and KC) 6 h after IR, more so in females. Caspase-3 activity, reflecting apoptosis and possibly microglia activation, was elevated 6 h after IR. Females treated with LPS before IR showed a higher caspase-3 activity compared with males. During the chronic phase (3 months post IR), we found that LPS-induced inflammation at the time of IR aggravated the IR-induced injury in both male and female mice, as judged by reduced bromodeoxyuridine incorporation and neurogenesis (doublecortin-positive cells) in the hippocampus. At this late time point, the microglia density was increased by IR, more so in females, indicating long-term effects on the microenvironment. IR increased anxiety-related behavior in vehicle-, but not LPS-, treated animals. However, exploratory behavior was affected by IR in both vehicle- and LPS-treated mice. In conclusion, we found that LPS administration before IR of the young mouse brain aggravated the injury, as judged by reduced hippocampal neurogenesis. This supports the clinical practice to postpone radiotherapy if the patient shows signs of infection. Systemic inflammation is not always obvious, though, for example because of concurrent corticosteroid treatment, so careful monitoring of inflammation is warranted.

Figure 1

An overview of the timeline of the study. On P13, animals received either an i.p. injection of LPS or vehicle, 24 h before IR. On P14, the animals were subjected to IR (8 Gy). One group of animals were euthanized 6 h post IR and used for caspase activity and Luminex assay. The second group was subjected to elevated plus maze, open field and place recognition tests at an age of 10 weeks, subsequently given four consecutive doses of BrdU at the age of 12 weeks and euthanized 4 weeks later (16 weeks of age). The brains collected in the second group were used for immunohistochemistry

Figure 2

Caspase-3-like activity (DEVDase) 6 h post IR in brain homogenate (one hemisphere). All data shown as mean±S.E.M., n=6–7 per group. # for irradiation, ¤ for sex. ^###P<0.001, ^¤P<0.05

Figure 3

(a) A schematic illustration of the DG including the GCL, the hilus and the ML. (b) The volume of the DG, (c) the GCL, (d) the ML and (e) the hilus in mm³ in vehicle- and LPS-treated animals post IR. All data shown as mean±S.E.M., n=11–14 per group. ^##P<0.01, ^###P<0.001

Figure 4

(a) A representative microphotograph of Iba-1⁺ cells in the DG from a control animal. Quantification of Iba-1⁺ cells in the DG post IR in vehicle- and LPS-treated animals. (b) Total cells and (c) density. All data shown as mean±S.E.M., n=11–14 per group. * for interaction between sex and irradiation, # for irradiation, ^¤ for sex. *P<0.05, ^##P<0.01, P<0.05 for sex

Figure 5

(a) A representative microphotograph of BrdU⁺ cells in the GCL. (b) Quantification of BrdU⁺ cells in the DG and (c) in the GCL in vehicle- and LPS-treated animals post IR. (d and e) Quantification of BrdU⁺ cells in the DG and the GCL, respectively, in only vehicle- and LPS-treated animals subjected to IR. All data shown as mean±S.E.M., n=12–14 per group. *Interaction between sex and irradiation, # for irradiation. **P<0.01, ***P<0.001, ^#P<0.025, ^###P<0.001

Figure 6

(a) A representative microphotograph of DCX⁺ cells in the GCL. (b) Quantification of DCX⁺ cells in the GCL in vehicle- and LPS-treated animals post IR. (c) Quantification of DCX⁺ cells in the GCL in only vehicle- and LPS-treated animals subjected to IR. All data shown as mean±S.E.M., n=12–15 per group. # for irradiation. ^###P<0.001

Figure 7

(a) Time per visit in the elevated plus maze and (b) distance to the walls in the open-field arena in vehicle- and LPS-treated animals. (c) The total latency, that is, the time it took for the animals to explore both objects on the first day of the place recognition test in vehicle- and LPS-treated animals. All data shown as mean±S.E.M., n=12–15 per group. # for irradiation, ^#P<0.05, ^##P<0.01, ^###P<0.001