Exposure to high-dose radiation results in detrimental effects on survival. The effects of combined trauma, such as radiation in combination with hemorrhage, the typical injury of victims exposed to a radiation blast, on survival and hematopoietic effects have yet to be understood. The purpose of this study was to evaluate the effects of radiation injury (RI) combined with hemorrhage (i.e., combined injury, CI) on survival and hematopoietic effects, and to investigate whether hemorrhage (Hemo) enhanced RI-induced mortality and hematopoietic syndrome. Male CD2F1 mice (10 weeks old) were given one single exposure of γ- radiation (60Co) at various doses (0.6 Gy/min). Within 2 hr after RI, animals under anesthesia were bled 0% (Sham) or 20% (Hemo) of total blood volume via the submandibular vein. In these mice, Hemo reduced the LD50/30 for 30-day survival from 9.1 Gy (RI) to 8.75 Gy (CI) with a DMF of 1.046. RI resulted in leukocytopenia, thrombopenia, erythropenia, and bone marrow cell depletion, but decreased the caspase-3 activation response. RI increased IL-1β, IL-6, IL-17A, and TNF-α concentrations in serum, bone marrow, ileum, spleen, and kidney. Some of these adverse alterations were magnified by CI. Erythropoietin production was increased in kidney and blood more after CI than RI. Furthermore, CI altered the global miRNAs expression in kidney and the ingenuity pathway analysis showed that miRNAs viz., let-7e, miR-30e and miR-29b that were associated with hematopoiesis and inflammation. This study provides preliminary evidence that non-lethal Hemo exacerbates RI-induced mortality and cell losses associated with high-dose γ-radiation. We identified some of the initial changes occurring due to CI which may have facilitated in worsening the injury and hampering the recovery of animals ultimately resulting in higher mortality.

Hemorrhage following whole-body γ-irradiation in a combined injury (CI) model increases mortality compared to whole-body γ-irradiation alone (RI). The decreased survival in CI is accompanied by increased bone marrow injury, decreased hematocrit, and alterations of miRNA in the kidney. In this study, our aim was to examine cytokine homeostasis, susceptibility to systemic bacterial infection, and intestinal injury. More specifically, we evaluated the interleukin-6 (IL-6)-induced stress proteins including C-reactive protein (CRP), complement 3 (C3), Flt-3 ligand, and corticosterone. CD2F1 male mice received 8.75 Gy 60Co gamma photons (0.6 Gy/min, bilateral) which was followed by a hemorrhage of 20% of the blood volume. In serum, RI caused an increase of IL-1, IL-2, IL-3, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17A, IL-18, G-CSF, CM-CSF, eotaxin, IFN-γ, MCP-1, MIP, RANTES, and TNF-α, which were all increased by hemorrhage alone, except IL-9, IL-17A, and MCP-1. Nevertheless, CI further elevated RI-induced increases of these cytokines except for G-CSF, IFN- γ and RANTES in serum. In the ileum, hemorrhage in the CI model significantly enhanced RI-induced IL-1β, IL-3, IL-6, IL-10, IL-12p70, IL-13, IL-18, and TNF-α concentrations. In addition, Proteus mirabilis Gram(-) was found in only 1 of 6 surviving RI mice on Day 15, whereas Streptococcus sanguinis Gram(+) and Sphingomonas paucimobilis Gram(-) were detected in 2 of 3 surviving CI mice (with 3 CI mice diseased due to inflammation and infection before day 15) at the same time point. Hemorrhage in the CI model enhanced the RI-induced increases in C3 and decreases in CRP concentrations. However, hemorrhage alone did not alter the basal levels, but hemorrhage in the CI model displayed similar increases in Flt-3 ligand levels as RI did. Hemorrhage alone altered the basal levels of corticosterone early after injury, which then returned to the baseline, but in RI mice and CI mice the increased corticosterone concentration remained elevated throughout the 15 day study. CI increased 8 miRNAs and decreased 10 miRNAs in serum, and increased 16 miRNA and decreased 6 miRNAs in ileum tissue. Among the altered miRNAs, CI increased miR-34 in the serum and ileum which targeted an increased phosphorylation of ERK, p38, and increased NF-κB, thereby leading to increased iNOS expression and activation of caspase-3 in the ileum. Further, let-7g/miR-98 targeted the increased phosphorylation of STAT3 in the ileum, which is known to bind to the iNOS gene. These changes may correlate with cell death in the ileum of CI mice. The histopathology displayed blunted villi and villus edema in RI and CI mice. Based on the in silico analysis, miR-15, miR-99, and miR-100 were predicted to regulate IL-6 and TNF. These results suggest that CI-induced alterations of cytokines/chemokines, CRP, and C3 cause a homeostatic imbalance and may contribute to the pathophysiology of the gastrointestinal injury. Inhibitory intervention in these responses may prove therapeutic for CI and improve recovery of the ileal morp...

Background Compared to radiation injury alone (RI), radiation injury combined wound (CI) further enhances acute radiation syndrome and subsequently mortality. We previously reported that therapy with Ghrelin, the 28-amino-acid-peptide secreted from the stomach, significantly increased 30-day survival and mitigated hematopoietic death by enhancing and sustaining granulocyte-colony stimulating factor (G-CSF) and keratinocyte chemoattractant (KC) in the blood and bone marrow; increasing circulating white blood cell depletion; inhibiting splenocytopenia; and accelerating skin-wound healing on day 30 after CI. Herein, we aimed to study the efficacy of Ghrelin on intestinal injury at early time points after CI. Methods B6D2F1/J female mice were exposed to 60Co-γ-photon radiation (9.5 Gy, 0.4 Gy/min, bilateral), followed by 15% total-body-surface-area skin wounds. Several endpoints were measured: at 4–5 h and on days 1, 3, 7, and 15. Results Ghrelin therapy mitigated CI-induced increases in IL-1β, IL-6, IL-17A, IL-18, KC, and TNF-α in serum but sustained G-CSF, KC and MIP-1α increases in ileum. Histological analysis of ileum on day 15 showed that Ghrelin treatment mitigated ileum injury by increasing villus height, crypt depth and counts, as well as decreasing villus width and mucosal injury score. Ghrelin therapy increased AKT activation and ERK activation; suppressed JNK activation and caspase-3 activation in ileum; and reduced NF-κB, iNOS, BAX and Bcl-2 in ileum. This therapy recovered the tight junction protein and mitigated bacterial translocation and lipopolysaccharides levels. The results suggest that the capacity of Ghrelin therapy to reduce CI-induced ileum injury is mediated by a balanced NF-κB-AKT-MAPK network that leads to homeostasis of pro-inflammatory and anti-inflammatory cytokines. Conclusions Our novel results are the first to suggest that Ghrelin therapy effectively decreases intestinal injury after CI.

Exposure to ionizing radiation alone or combined with traumatic tissue injury is a crucial life-threatening factor in nuclear and radiological incidents. Radiation injuries occur at the molecular, cellular, tissue and systemic levels; their mechanisms, however, remain largely unclear. Exposure to radiation combined with skin wounding, bacterial infection or burns results in greater mortality than radiation exposure alone in dogs, pigs, rats, guinea pigs and mice. In the current study we observed that B6D2F1/J female mice exposed to 60Co gamma-photon radiation followed by 15% total-body-surface-area skin wounds experienced an increment of 25% higher mortality over a 30-day observation period compared to those subjected to radiation alone. Radiation exposure delayed wound healing by approximately 14 days. On day 30 post-injury, bone marrow and ileum in animals from both groups (radiation alone or combined injury) still displayed low cellularity and structural damage. White blood cell counts, e.g., neutrophils, lymphocytes, monocytes, eosinophils, basophils and platelets, still remained very low in surviving irradiated alone animals, whereas only the lymphocyte count was low in surviving combined injury animals. Likewise, in surviving animals from radiation alone and combined injury groups, the RBCs, hemoglobin, hematocrit and platelets remained low. We observed, that animals treated with both pegylated G-CSF (a cytokine for neutrophil maturation and mobilization) and Alxn4100TPO (a thrombopoietin receptor agonist) at 4 h postirradiation, a 95% survival (vehicle: 60%) over the 30-day period, along with mitigated body-weight loss and significantly reduced acute radiation syndrome. In animals that received combined treatment of radiation and injury that received pegylated G-CSF and Alxn4100TPO, survival was increased from 35% to 55%, but did not accelerate wound healing. Hematopoiesis and ileum showed significant improvement in animals from both groups (irradiation alone and combined injury) when treated with pegylated G-CSF and Alxn4100TPO. Treatment with pegylated G-CSF alone increased survival after irradiation alone and combined injury by 33% and 15%, respectively, and further delayed wound healing, but increased WBC, RBC and platelet counts after irradiation alone, and only RBCs and platelets after combined injury. Treatment with Alxn4100TPO alone increased survival after both irradiation alone and combined injury by 4 and 23%, respectively, and delayed wound healing after combined injury, but increased RBCs, hemoglobin concentrations, hematocrit values and platelets after irradiation alone and only platelets after combined injury. Taken together, the results suggest that combined treatment with pegylated G-CSF and Alxn4100TPO is effective for mitigating effects of both radiation alone and in combination with injury.

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BackgroundRadiation injury combined wound (CI) enhances acute radiation syndrome and subsequently mortality as compared to radiation injury alone (RI). We previously reported that ghrelin (a 28-amino-acid-peptide secreted from the stomach) treatment significantly increased a 30-day survival, mitigated hematopoietic death, circulating white blood cell (WBC) depletion and splenocytopenia and accelerated skin-wound healing on day 30 after CI. Herein, we aimed to study the ghrelin efficacy at early time points after CI.MethodsB6D2F1/J female mice were exposed to 60Co-γ-photon radiation at 9.5 Gy (LD50/30) followed by a 15% total-body-surface-area skin wound. Several endpoints were measured at 4–5 h, days 1, 3, 7 and 15.ResultsHistological analysis of sternums on day 15 showed that CI induced more adipocytes and less megakaryocytes than RI. Bone marrow cell counts from femurs also indicated CI resulted in lower bone marrow cell counts on days 1, 7 and 15 than RI. Ghrelin treatment mitigated these CI-induced adverse effects. RI and CI decreased WBCs within 4–5 h and continued to decrease to day 15. Ghrelin treatment mitigated decreases in CI mice, mainly from all types of WBCs, but not RBCs, hemoglobin levels and hematocrit values. Ghrelin mitigated the CI-induced thrombocytopenia and splenocytopenia. CI increased granulocyte-colony stimulating factor (G-CSF) and keratinocyte chemoattractant (KC) in blood and bone marrow. Ghrelin therapy was able to enhance and sustain the increases in serum on day 15, probably contributed by spleen and ileum, suggesting the correlation between G-CSF and KC increases and the neutropenia mitigation. Activated caspase-3 levels in bone marrow cells were significantly mitigated by ghrelin therapy on days 3 and 15.ConclusionsOur novel results are the first to suggest that ghrelin therapy effectively decreases hematopoietic death and splenocytopenia by sustaining circulating G-CSF and KC increases after CI. These results demonstrate efficacy of ghrelin as a radio-mitigator/therapy agent for CI.

Medical treatment becomes challenging when complicated injuries arise from secondary reactive metabolic and inflammatory products induced by initial acute ionizing radiation injury (RI) or when combined with subsequent trauma insult(s) (CI). With such detrimental effects on many organs, CI exacerbates the severity of primary injuries and decreases survival. Previously, in a novel study, we reported that ghrelin therapy significantly improved survival after CI. This study aimed to investigate whether brain hemorrhage induced by RI and CI could be inhibited by ghrelin therapy with pegylated G-CSF (i.e., Neulasta®, an FDA-approved drug). B6D2F1 female mice were exposed to 9.5 Gy 60 Co-γ-radiation followed by 15% total-skin surface wound. Several endpoints were measured at several days. Brain hemorrhage and platelet depletion were observed in RI and CI mice. Brain hemorrhage severity was significantly higher in CI mice than in RI mice. Ghrelin therapy with pegylated G-CSF reduced the severity in brains of both RI and CI mice. RI and CI did not alter PARP and NF-κB but did significantly reduce PGC-1α and ghrelin receptors; the therapy, however, was able to partially recover ghrelin receptors. RI and CI significantly increased IL-6, KC, Eotaxin, G-CSF, MIP-2, MCP-1, MIP-1α, but significantly decreased IL-2, IL-9, IL-10, MIG, IFN-γ, and PDGF-bb; the therapy inhibited these changes. RI and CI significantly reduced platelet numbers, cellular ATP levels, NRF1/2, and AKT phosphorylation. The therapy significantly mitigated these CI-induced changes and reduced p53-mdm2 mediated caspase-3 activation. Our data are the first to support the view that Ghrelin therapy with pegylated G-CSF is potentially a novel therapy for treating brain hemorrhage after RI and CI.

In nuclear and radiological incidents, overexposure to ionizing radiation is life-threatening. It is evident that radiation depletes blood cells and increases circulating cytokine/chemokine concentrations as well as mortality. While microglia cells of female mice have been observed to be less damaged by radiation than in male mice, it is unclear whether sex affects physio-pathological responses in the bone marrow (BM) and gastrointestinal system (GI). We exposed B6D2F1 male and female mice to 0, 1.5, 3, or 6 Gy with mixed-field radiation containing 67% neutron and 33% gamma at a dose rate of 0.6 Gy/min. Blood and tissues were collected on days 1, 4, and 7 postirradiation. Radiation increased cytokines/chemokines in the femurs and ilea of female and male mice in a dose-dependent manner. Cytokines and chemokines reached a peak on day 4 and declined on day 7 with the exception of G-CSF which continued to increase on day 7 in female mice but not in male mice. MiR-34a (a Bcl-2 inhibitor), G-CSF (a miR-34a inhibitor), MAPK activation (pro-cell death), and citrulline (a biomarker of entro-epithelial proliferation), active caspase-3 (a biomarker of apoptosis) and caspase-1activated gasdermin D (a pyroptosis biomarker) were measured in the sternum, femur BM and ileum. Sternum histopathology analysis with H&E staining and femur BM cell counts as well as Flt-3L showed that BM cellularity was not as diminished in females, with males showing a 50% greater decline on day 7 postirradiation, mainly mediated by pyroptosis as indicated by increased gasdermin D in femur BM samples. Ileum injury, such as villus height and crypt depth, was also 43% and 30%, respectively, less damaged in females than in males. The severity of injury in both sexes was consistent with the citrulline and active caspase-3 measurements as well as active caspase-1 and gasdermin D measurements, suggesting apoptosis and pyroptosis occurred. On day 7, G-CSF in the ileum of female mice continued to be elevated by sevenfold, whereas G-CSF in the ileum of male mice returned to baseline. Furthermore, G-CSF is known to inhibit miR-34a expression, which in ileum on day 1 displayed a 3- to 4-fold increase in female mice after mixed-field (67% neutron + 33% gamma) irradiation, as compared to a 5- to 9-fold increase in male mice. Moreover, miR-34a blocked Bcl-2 expression. Mixed-field (60% neutron + 33% gamma) radiation induced more Bcl-2 in females than in males. On day 7, AKT activation was found in the ileums of females and males. However, MAPK activation including ERK, JNK, and p38 showed no changes in the ileum of females (by 0-fold; P > 0.05), whereas the MAPK activation was increased in the ileum of males (by 100-fold; P < 0.05). Taken together, the results suggest that organ injury from mixed-field (67% neutron + 33% gamma) radiation is less severe in females than in males, likely due to increased G-CSF, less MAPK activation, low miR-34a and increased Bcl-2/Bax ratio.