Wound trauma increases radiation-induced mortality by activation of iNOS pathway and elevation of cytokine concentrations and bacterial infection

Abstract

Abstract Although it is documented that concurrent wounding increases mortality from radiation injury, the molecular mechanism of combined injury is unknown. In this study, mice were exposed to gamma radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, reducing the LD(50/30) from 9.65 Gy to 8.95 Gy. Analyses of histopathology, inducible nitric oxide synthase (iNOS), and serum cytokines were performed on mouse ileum and skin at various times after 9.75 Gy and/or wounding. In the ileum, the villi were significantly shortened 3 days postirradiation but not after wounding; combined injury resulted in decreased villus width and tunica muscularis thickness. The skin of mice subjected to combined injury was less cellular and had a smaller healing bud than the skin of mice subjected to wounding alone. Combined injury significantly delayed wound closure times; it also prolonged the increased levels of iNOS protein in the skin and ileum. iNOS up-regulation was correlated with increases in transcription factors, including NF-kappaB and NF-IL6. The increase in NF-IL6 may be due to increases in cytokines, including IL-1beta, -6, -8, -9, -10 and -13, G-CSF, eotaxin, INF-gamma, MCP-1, MIP-1alpha and MIP-1beta. Combined injury resulted in early detection of bacteria in the blood of the heart and liver, whereas radiation alone resulted in later detection of bacteria; only a transient bacteremia occurred after wounding alone. Results suggest that enhancement of iNOS, cytokines and bacterial infection triggered by combined injury may contribute to mortality. Agents that inhibit these responses may prove to be therapeutic for combined injury and may reduce related mortality.

FIG. 1.

Panel A: Wound trauma increases radiation-induced mortality. Thirty-day time course of survival (%) after sham treatment, wounding, irradiation or combined injury. SHAM: no irradiation and no wounding. Data are shown only for 8.5 and 9.75 Gy, which were representative of all doses tested. Panel B: Combined injury has a lower LD_50/30 than radiation. Mice (n = 20 per group) received γ radiation followed by sham treatment or skin wounding. The LD_50/30 was calculated by probit analysis. RI: radiation; CI: radiation + wounding.

FIG. 2.

Wound trauma enhances radiation-induced body weight reduction (panel A), increases radiation-induced water intake (panel B), and delays wound closure time (panel C). Mice received sham treatments, wounding, 9.75 Gy, or 9.75 Gy + wounding (n = 20 per group). For panel A, *P < 0.05 compared to sham, wounding and radiation injury; **P < 0.05 compared to sham, wounding and combined. For panel B, *P < 0.05 compared to sham, wounding and combined injury; **P < 0.05 compared to sham, radiation and wounding; ***P < 0.05 compared to sham, radiation and combined injury; ****P < 0.05 compared to sham and radiation. For panel C, *P < 0.05 compared to wounding, determined by two-way ANOVA. SHAM: no radiation, no wounding; RI: 9.75 Gy; CI: 9.75 Gy + wounding.

FIG. 3.

Radiation injury delays wound healing. Panels A–D: Representative histology of skin from sham-treated, irradiated, wounded or irradiated + wounded mice at day 3 (n = 6 per group). Arrows indicate healing bud. Panel D: Skin (epidermis + dermis) thickness measured at the edge of the wound was further decreased by combined injury. Sham: no radiation, no wounding; RI: 9.75 Gy; CI: 9.75 Gy + wounding.

FIG. 4.

Wound trauma, radiation and a combination or both alter ileal structure. Panel A–D: Representative histology of ileum from sham-treated, irradiated, wounded or irradiated + wounded mice at day 3 (n = 6 per group). Arrows indicate tunica muscularis layer. Magnified images of crypt and tunica muscularis are presented in panels I and J. Villus height (panel E), villus width (panel F), crypt depth (panel G), and tunica muscularis thickness (panel H) were measured. *P < 0.05 compared to other three groups. **P < 0.05 compared to sham and radiation injury, determined by χ² test. Sham: no radiation and no wounding; W: wounding; RI: 9.75 Gy; CI: 9.75 Gy + wounding.

FIG. 5.

Combined injury induces more iNOS in ileum than radiation and wounding. Panel A: Representative gels of iNOS protein from ileal lysates prepared from sham-treated, wounded, irradiated or combined injury mice (n = 6 per group). Panel B: iNOS bands were quantified densitometrically and normalized to IgG. *P < 0.05 compared to sham; **P < 0.05 compared to sham, radiation and wounding, determined by two-way ANOVA and studentized-range test. Sham: no radiation, no wounding; W: wounding; RI: 9.75 Gy; CI: 9.75 Gy + wounding.

FIG. 6.

Combined injury and wounding induce more iNOS in skin than radiation. Panel A: Representative gels of iNOS protein from skin lysates prepared from sham-treated, wounded, irradiated or irradiated + wounded mice (n = 6 per group). iNOS bands were quantified densitometrically and normalized to IgG. *P < 0.05 compared to sham; **P < 0.05 compared to sham, wounding and radiation; ***P < 0.05 compared to sham and radiation, determined by two-way ANOVA and studentized-range test. Sham: no radiation, no wounding; W: wounding; RI: 9.75 Gy; CI: 9.75 Gy + wounding.

FIG. 7.

Increases in iNOS induced by radiation or combined injury correlate with increases in transcription. Representative gels of iNOS mRNA (panel A), NF-κB-65 protein (panel B), and NF-IL6 protein (panel C) from ileal lysates prepared from sham-treated, wounded, irradiated or combined injury mice (n = per group). *P < 0.05 compared to sham; **P < 0.05 compared to sham and combined injury; ***P < 0.05 compared to sham, radiation and wounding, determined by two-way ANOVA and studentized-range test. Sham: no radiation, no wounding; W: wounding; RI: 9.75 Gy; CI: 9.75 Gy + wounding.

FIG. 8.

Radiation exposure increases cytokine concentrations in serum. Mice received sham treatments or 9.75 Gy (n = 6 per group). *P < 0.05 compared to sham, determined by Student’s t test. Sham: no radiation, no wounding; RI: 9.75 Gy.

FIG. 9.

Wound trauma enhances radiation-induced increases in cytokine concentrations and types in serum. Mice received sham treatments, wounding, 9.75 Gy or 9.75 Gy + wounding (n = 6 per group). *P < 0.05 compared to sham; **P < 0.05 compared to sham, radiation and combined injury, determined by two-way ANOVA and studentized-range test. Sham: no radiation, no wounding; W: wounding; RI: 9.75 Gy; CI: 9.75 Gy + wounding.

FIG. 10.

Wound trauma enhances radiation-induced bacterial infection. Panel A: Bacteria isolated and identified in heart and/or liver blood (n = 12–17). Panel B: Combined injury resulted in more mice with early bacterial infection. Sham: no radiation, no wounding; W: wounding; RI: 9.75 Gy; CI: 9.75 Gy + wounding.