Body Irradiated Mice Research Articles

Effects of Bone Marrow Sparing and TGF-β3 Treatment in Total Body Irradiation of C57BL/6J Mice

Introduction: Mortality from acute radiation syndrome is frequently caused by hematopoietic or gastrointestinal radiotoxicity, the latter of which currently has no effective treatment. Transforming growth factor-beta 3 (TGF-β3) may decrease the severity of radiation-induced gastrointestinal damage in mice. In addition, treatment with TGF-β3 may alleviate radiation-induced fibrosis. Objectives: The current study aimed to investigate the effect of TGF-β3 treatment on acute and late radiotoxicity in whole body irradiated mice. Methods: C57BL/6J mice were total body irradiated with 8.5 Gy X-rays with or without shielding of one hind leg to alleviate hematopoietic radiotoxicity. The effects of intravenous TGF-β3 treatment were investigated. Body weight and pain expression were monitored. Intestine, lung, and liver tissues were preserved and analyzed. Alpha smooth muscle actin (α-SMA) expression in MRC-5 cells after 3.5 Gy X-irradiation combined with TGF-β3 treatment was analyzed using flow cytometry. Results: All total body irradiated animals died within ten days after irradiation. Ninety-three percent of femur-shielded mice survived until sampling or termination. No effect of TGF-β3 treatment was observed in either group. No increase in collagen content was detected in the lungs or liver from irradiated mice regardless of TGF-β3 treatment. In vitro, α-SMA expression increased synergistically after irradiation and TGF-β3 treatment. Conclusions: Shielding of the femur during total body irradiation decreased acute gastrointestinal radiation toxicity and increased survival. TGF-β3 treatment did not impact symptoms or survival. TGF-β3 treatment and irradiation increased α-SMA expression in MRC-5 cells synergistically.

After Bone Marrow Transplantation, the Cell-Intrinsic Th2 Pathway Promotes Recipient T Lymphocyte Survival and Regulates Graft-versus-Host Disease.

Recipient T cells can aggravate or regulate lethal and devastating graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In this context, we have shown before that intestinal immune conditioning with helminths is associated with survival of recipient T cells and Th2 pathway-dependent regulation of GVHD. We investigated the mechanism of survival of recipient T cells and their contribution to GVHD pathogenesis in this helminth infection and BMT model after myeloablative preparation with total body irradiation in mice. Our results indicate that the helminth-induced Th2 pathway directly promotes the survival of recipient T cells after total body irradiation. Th2 cells also directly stimulate recipient T cells to produce TGF-β, which is required to regulate donor T cell-mediated immune attack of GVHD and can thereby contribute to recipient T cell survival after BMT. Moreover, we show that recipient T cells, conditioned to produce Th2 cytokines and TGF-β after helminth infection, are fundamentally necessary for GVHD regulation. Taken together, reprogrammed or immune-conditioned recipient T cells after helminth infection are crucial elements of Th2- and TGF-β-dependent regulation of GVHD after BMT, and their survival is dependent on cell-intrinsic Th2 signaling.

TLR2/6 agonist treatment after total body irradiation in mice and nonhuman primates induces proliferation of hematopoietic progenitor cells

Abstract Exposure to high doses of radiation, whether intentional or accidental, may cause injury or death. Therapeutics are urgently needed for mitigating injuries caused by radiation exposure. Previously, we identified a synthetic lipopeptide agonist of TLR2/6, namely fibroblast-stimulating lipopeptide (FSL-1), that prolonged survival of mice exposed to lethal ionizing radiation and mitigated hematopoietic acute radiation syndrome. Here, this agent was tested in several mouse models as well as in nonhuman primates given total body irradiation followed 24 hours later with a single subcutaneous injection of FSL-1. These studies revealed the mitigation of radiation-induced loss of platelets and red blood cells, as well as the promotion of bone marrow progenitor cell recovery. These beneficial hematopoietic outcomes were accompanied with a lack of toxicity, showcasing the safe, efficacious application of a single dose of FSL-1 for mitigating hematopoietic dysfunction resulting from radiation. By investigating the mechanism of action of FSL-1 in response to radiation, we confirmed that FSL-1 radio-mitigation activity was mediated through a TLR2/MyD88-dependent process. In FSL-1 treated irradiated mice, we found enhanced numbers of proliferating Ki67 +bone marrow cells. Finally, examination of cellular pathways in mice and nonhuman primate bone marrow cells indicated FSL-1 induced the activation of NF-kB and MAPK signaling. These data suggest that FSL-1 promotes hematopoietic progenitor cell proliferation through the activation of cell survival and activation pathways downstream of TLR2/6 stimulation, and thus may serve to mitigate the injurious consequences of radiation. Supported by grants from NIH (AI067798) and DOD Congressionally Directed Medical Research Program grant W81XWH-15-1-0574 (JMC).

Cytosolic Release of Mitochondrial DNA and Associated cGAS Signaling Mediates Radiation-Induced Hematopoietic Injury of Mice.

Mitochondrion is an important organelle of eukaryotic cells and a critical target of ionizing radiation (IR) outside the nucleus. The biological significance and mechanism of the non-target effect originating from mitochondria have received much attention in the field of radiation biology and protection. In this study, we investigated the effect, role, and radioprotective significance of cytosolic mitochondrial DNA (mtDNA) and its associated cGAS signaling on hematopoietic injury induced by IR in vitro culture cells and in vivo total body irradiated mice in this study. The results demonstrated that γ-ray exposure increases the release of mtDNA into the cytosol to activate cGAS signaling pathway, and the voltage-dependent anion channel (VDAC) may contribute to IR-induced mtDNA release. VDAC1 inhibitor DIDS and cGAS synthetase inhibitor can alleviate bone marrow injury and ameliorate hematopoietic suppression induced by IR via protecting hematopoietic stem cells and adjusting subtype distribution of bone marrow cells, such as attenuating the increase of the F4/80+ macrophage proportion in bone marrow cells. The present study provides a new mechanistic explanation for the radiation non-target effect and an alternative technical strategy for the prevention and treatment of hematopoietic acute radiation syndrome.

Quercetin 3-O rutinoside prevents gastrointestinal injury through regulation of apoptosis in 7.5 Gy total body irradiated mice

BackgroundDespite the heightened threat to unforeseen nuclear/radiological exposures worldwide, no countermeasures are currently approved to prevent gastrointestinal (GI) toxicity induced by radiation in humans. PurposeIn this study, we aim to establish the gastroprotective role of flavonoid, Quercetin-3-O-rutinoside (Q-3-R) against 7.5 Gy total body gamma radiation dose that contributes to the hematopoietic syndrome. MethodsQ-3-R (10 mg/kg body weight) was administered intramuscularly to C57BL/6 male mice before exposure to 7.5 Gy and monitored for morbidity and mortality. The GI protection against radiation was ascertained by histopathological and xylose absorption studies. Intestinal apoptosis, crypt proliferation and apoptotic signaling were also investigated in different treatment groups. ResultsWe found that Q-3-R prevented the radiation-induced loss of mitochondrial membrane potential, maintained ATP levels, regulated the apoptotic pathway, and activated crypt cell proliferation in the intestine. Radiation-induced villi and crypt damage as well as mal-absorption were significantly minimized in the Q-3-R treated group. We observed 100% survival post Q-3-R administration against 33.3% lethality in 7.5 Gy (LD33.3/30) exposed C57BL/6 mice. The Q-3-R pre-treated mice that survived the 7.5 Gy dose revealed no pathological changes related to the development of fibrosis in the intestine and thickened mucosal wall till 4 months post irradiation. Complete hematopoietic recovery was observed in these surviving mice when compared to age matched control. ConclusionThe findings revealed that Q-3-R regulated the apoptotic process to achieve GI protection against LD33.3/30 dose (7.5 Gy) that primarily caused death due to hematopoietic failure. The recovery observed in mice survivors suggested that this molecule may also have the potential to minimize side effects on normal tissues during radiotherapy.

Microarray analysis identifies coding and non-coding RNA markers of liver injury in whole body irradiated mice

Radiation injury from medical, accidental, or intentional sources can induce acute and long-term hepatic dysregulation, fibrosis, and cancer. This long-term hepatic dysregulation decreases quality of life and may lead to death. Our goal in this study is to determine acute changes in biological pathways and discover potential RNA biomarkers predictive of radiation injury. We performed whole transcriptome microarray analysis of mouse liver tissue (C57BL/6 J) 48 h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray to identify significant expression changes in mRNAs, lncRNAs, and miRNAs, We also validated changes in specific RNAs through qRT-PCR. We used Ingenuity Pathway Analysis (IPA) to identify pathways associated with gene expression changes. We observed significant dysregulation of multiple mRNAs across all doses. In contrast, miRNA dysregulation was observed upwards of 2 Gray. The most significantly upregulated mRNAs function as tumor suppressors: Cdkn1a, Phlda3, and Eda2r. The most significantly downregulated mRNAs were involved in hemoglobin synthesis, inflammation, and mitochondrial function including multiple members of Hbb and Hba. The most significantly upregulated miRNA included: miR-34a-5p, miR-3102-5p, and miR-3960, while miR-342-3p, miR-142a-3p, and miR-223-3p were most significantly downregulated. IPA predicted activation of cell cycle checkpoint control pathways and inhibition of pathways relevant to inflammation and erythropoietin. Clarifying expression of mRNA, miRNA and lncRNA at a short time point (48 h) offers insight into potential biomarkers, including radiation markers shared across organs and animal models. This information, once validated in human models, can aid in development of bio-dosimetry biomarkers, and furthers our understanding of acute pathway dysregulation.

Mesenchymal Stem Cell Derived Extracellular Vesicles Reverse Radiation-Induced Cytokine Storm

Cytokine storm or cytokine release syndrome is a systemic inflammatory response to different triggers which leads to excessive activation of immune cells with the release of a large amount of pro-inflammatory cytokines. These uncontrolled and excessive releases of cytokines accompany multisystem organ failure and death. Acute radiation exposure leads to acute hematopoietic and acute gastrointestinal syndromes with early mortality. Cytokine storm accompanies these syndromes and significantly complicates the clinical outcomes.Recent data has suggested that the inflammasome and associate cytokine storm may play a major role in various injuries and tissue problems after irradiation. Inflammasome activates caspase-1 which give rise to the proinflammatory cytokines such as interleukin-18 (IL-18) and interleukin-1 beta (IL-1β) into their biologically active forms and further induce other cytokines release.Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles, released from cells in the whole body. EVs play an important role in intercellular communication. EVs have been shown to transfer protein, lipid, DNA, mRNAs, and microRNAs to recipient cells, thereby mediating a variety of biological responses. We have been investigating the capacity of marrow-derived mesenchymal stem cell extracellular vesicles (MSC-EV) to reverse radiation damage to murine bone marrow stem/progenitor cells.In this study, we have evaluated the effect of MSC-EVs on reversal of high dose radiation injury to bone marrow cells. C57BL/6 mice received 0, 700, and 950-1000cGy WBI (either single or split dose). After 24 hrs. post-irradiation, mice received an IV infusion of 2X10 9 hMSC EVs daily for three days with a control group receiving vehicle only. Marrow was harvested from 700 cGy exposed mice which were vesicle treated or not. These marrow cells were evaluated for engraftment into 950 cGy exposed mice. The engraftment rates in mice transplanted with marrow from irradiated EV injected animals was 34.66±14.19% at 4 months post transplantation and marrow from the group not treated with EV gave an engraftment rate of 6.92±3.53%. We further evaluated the effect of MSCs-EVs on decreasing the mortality in mice exposed to 950cGy WBI. The MSC-EV untreated mice were dead between 12-18 days post radiation, but MSC-EV treated mice maintained a 60% survival rate at 130 days post radiation, suggesting that MSC-EV treatment could significantly extend the survival rate of mice after exposure to lethal radiation. We evaluated the circulating inflammatory markers in 1000 cGy whole body irradiated mice with/without MSC-EV treatment. Twenty-four hours post-irradiation, mice received an IV infusion of 2x10 9 hMSC EVs every day for three days with a control group receiving vehicle only. The serum was collected after 8 days post radiation for inflammatory response analysis by ProcartaPlex multiplex immunoassays. There were dramatic increases in cytokines secretion in serum including IFN gamma, IL-12p70, IL-13, IL-1 beta, IL-2, IL-5, IL-6, TNF alpha, GM-CSF and IL-18 in irradiated mice compared to non-irradiated mice and a significant reduction in cytokine levels after MSC-EVs treatment. We further found significantly higher levels of IL-1 beta and activated caspase-1 p20 protein expression in the spleen after 1000cGy radiation compared to non-radiated mice. Moreover, we found that with MSC-EV treatment, the expression level of IL-1 beta and active caspase-1 p20 was significantly inhibited in spleen compared to untreated irradiated mice, indicating that the inhibition of cytokine storm post-radiation by MSC-EVs might be mediated by inhibition of inflammasome activation. Our study suggests that EV Inhibition of an inflammasome mediated cytokine storm could be therapeutically important in many disease entities. DisclosuresNo relevant conflicts of interest to declare.

"Longitudinal Fecal Microbiome Study of Total Body Irradiated Mice Treated With Radiation Mitigators Identifies Bacterial Associations With Survival".

Total body irradiation (TBI) has been demonstrated to alter the intestinal microbiome, but the effects of successful small molecule ionizing radiation mitigators on the intestinal microbiome are not well-known. Our survival experiments examined the effects of anti-cell death radiation mitigators on and in conjunction with the host’s microbiota. Mice received 9.25 Gy TBI and then were administered radiation mitigators 24 hours later. Passed stool were collected pre-irradiation, then on days 1, 3, 5, 7, 10, 14, 21, and 30 post-irradiation for 16S rRNA gene (V4 region) sequencing. The Cox proportional hazards (CPH) model was fit with taxonomic composition (time varying covariates) and treatment as predictors. In the first experiment, mice were administered drugs for “granulocyte stimulation and anti-apoptosis” in four protocol combinations: JP4-039 (anti-apoptosis), granulocyte colony-stimulating factor (G-CSF, granulopoietic precursor cell stimulator), both mitigators, and control. Survival improved relative to control (30.0%) for G-CSF (80%, p-value = 0.025), G-CSF/JP4-039 (70%, p-value = 0.084), but not for JP4-039 (50.0%). In the second experiment, mice were administered mitigation drugs “inhibiting programmed cell death” pathways: JP4-039 (anti-apoptosis), necrostatin-1 (anti-necroptosis), and baicalein (anti-ferroptosis), in eight combinations. The survival of JP4-039/baicalein (60.0%, p-value = 0.010) and JP4-039/baicalein/necrostatin-1 (60.0%, p-value = 0.06) treatment combinations were significantly different from the control (26.7%). The JP4-039/necrostatin-1 (46.7%) and baicalein/necrostatin-1 (40.0%) and singlet treatment combinations (26.7%) were not significantly different from the control. Despite differences between the baseline microbiota compositions of the two experiments, consistent changes in composition after irradiation were found: Lactobacillus decreased post-irradiation, relative to baseline. By day 7, microbiota perturbations had incompletely reversed, and no drug-specific differences were identifiable. The CPH model identified Lactobacillus and members of Ruminococcaceae, including Ruminococcus, as protective and Akkermansia as deleterious. By day 30, the microbiota of surviving mice had not returned to baseline, but the differences between experiments suggest the resultant microbiota composition of the survivors are stochastic or batch specific in nature, rather than a requirement for survival. In conclusion, the study determined that key taxa identified in fecal samples, when applied towards the prediction of TBI survival, improves the survival model relative to treatment information alone.

Effects of captopril against radiation injuries in the Göttingen minipig model of hematopoietic-acute radiation syndrome

Our laboratory has demonstrated that captopril, an angiotensin converting enzyme inhibitor, mitigates hematopoietic injury following total body irradiation in mice. Improved survival in mice is correlated with improved recovery of mature blood cells and bone marrow, reduction of radiation-induced inflammation, and suppression of radiation coagulopathy. Here we investigated the effects of captopril treatment against radiation injuries in the Göttingen mini pig model of Hematopoietic-Acute Radiation Syndrome (H-ARS). Minipigs were given captopril orally (0.96 mg/kg) twice daily for 12 days following total body irradiation (60Co 1.79 Gy, 0.42–0.48 Gy/min). Blood was drawn over a time course following irradiation, and tissue samples were collected at euthanasia (32–35 days post-irradiation). We observed improved survival with captopril treatment, with survival rates of 62.5% in vehicle treated and 87.5% in captopril treated group. Additionally, captopril significantly improved recovery of peripheral blood mononuclear cells, and a trend toward improvement in recovery of red blood cells and platelets. Captopril significantly reduced radiation-induced expression of cytokines erythropoietin and granulocyte-macrophage colony-stimulating factor and suppressed radiation-induced acute-phase inflammatory response cytokine serum amyloid protein A. Using quantitative-RT-PCR to monitor bone marrow recovery, we observed significant suppression of radiation-induced expression of redox stress genes and improved hematopoietic cytokine expression. Our findings suggest that captopril activities in the Göttingen minipig model of hematopoietic-acute radiation syndrome reflect findings in the murine model.

Heat Killed Salmonella typhimurium Protects Intestine Against Radiation Injury Through Wnt Signaling Pathway

Gastrointestinal (GI) toxicity caused by ionizing radiation (IR) is a dose limiting factor in radiotherapy and a great threat for individual nuclear-related military missions. However, there are currently no available strategies to effectively prevent the damage on the intestine induced by IR. In the present study, the protective activity of Heat Killed Salmonella typhimurium (HKST) on intestine against IR was investigated. Through mouse intestinal organoids and whole body irradiation of mice, we found that the pretreatment with HKST significantly preserved the structure of small intestine upon IR exposure and promoted the proliferation of intestinal cells post-IR. Further study revealed that the radioprotective effects of HKST were involved in DNA damage response (DDR) signaling. Moreover, the stimulation of DDR signaling by HKST upon radiation damage was mediated by Wnt signaling, in which the inhibition of Wnt signaling diminished the radioprotective effects of HKST. To sum up, our study suggested HKST as a potential radioprotectant used for prevention of IR-induced GI toxicity.

Novel Murine Biomarkers of Radiation Exposure Using An Aptamer-Based Proteomic Technology.

Purpose: There is a need to identify new biomarkers of radiation exposure both for use in the development of biodosimetry blood diagnostics for radiation exposure and for clinical use as markers of radiation injury. In the current study, a novel high-throughput proteomics screening approach was used to identify proteomic markers of radiation exposure in the plasma of total body irradiated mice. A subset panel of significantly altered proteins was selected to build predictive models of radiation exposure and received radiation dose useful for population screening in a future radiological or nuclear event. Methods: Female C57BL6 Mice of 8–14 weeks of age received a single total body irradiation (TBI) dose of 2, 3.5, 8 Gy or sham radiation and plasma was collected by cardiac puncture at days 1, 3, and 7 post-exposure. Plasma was then screened using the aptamer-based SOMAscan proteomic assay technology, for changes in expression of 1,310 protein analytes. A subset panel of protein biomarkers which demonstrated significant changes (p < 0.05) in expression following radiation exposure were used to build predictive models of radiation exposure and radiation dose. Results: Detectable values were obtained for all 1,310 proteins included in the SOMAscan assay. For the Control vs. Radiation model, the top predictive proteins were immunoglobulin heavy constant mu (IGHM), mitogen-activated protein kinase 14 (MAPK14), ectodysplasin A2 receptor (EDA2R) and solute carrier family 25 member 18 (SLC25A18). For the Control vs. Dose model, the top predictive proteins were cyclin dependent kinase 2/cyclin A2 (CDK2. CCNA2), E-selectin (SELE), BCL2 associated agonist of cell death (BAD) and SLC25A18. Following model validation with a training set of samples, both models tested with a new sample cohort had overall predictive accuracies of 85% and 73% for the Control vs. Radiation and Control vs. Dose models respectively. Conclusion: The SOMAscan proteomics platform is a useful screening tool to evaluate changes in biomarker expression. In our study we were able to identify a novel panel of radiation responsive proteins useful for predicting whether an animal had received a radiation exposure and to what dose they had received. Such diagnostic tools are needed for future medical management of radiation exposures.

Differential normal skin transcriptomic response in total body irradiated mice exposed to scattered versus scanned proton beams

Proton therapy allows to avoid excess radiation dose on normal tissues. However, there are some limitations. Indeed, passive delivery of proton beams results in an increase in the lateral dose upstream of the tumor and active scanning leads to strong differences in dose delivery. This study aims to assess possible differences in the transcriptomic response of skin in C57BL/6 mice after TBI irradiation by active or passive proton beams at the dose of 6 Gy compared to unirradiated mice. In that purpose, total RNA was extracted from skin samples 3 months after irradiation and RNA-Seq was performed. Results showed that active and passive delivery lead to completely different transcription profiles. Indeed, 140 and 167 genes were differentially expressed after active and passive scanning compared to unirradiated, respectively, with only one common gene corresponding to RIKEN cDNA 9930021J03. Moreover, protein–protein interactions performed by STRING analysis showed that 31 and 25 genes are functionally related after active and passive delivery, respectively, with no common gene between both types of proton delivery. Analysis showed that active scanning led to the regulation of genes involved in skin development which was not the case with passive delivery. Moreover, 14 ncRNA were differentially regulated after active scanning against none for passive delivery. Active scanning led to 49 potential mRNA-ncRNA pairs with one ncRNA mainly involved, Gm44383 which is a miRNA. The 43 genes potentially regulated by the miRNA Gm44393 confirmed an important role of active scanning on skin keratin pathway. Our results demonstrated that there are differences in skin gene expression still 3 months after proton irradiation versus unirradiated mouse skin. And strong differences do exist in late skin gene expression between scattered or scanned proton beams. Further investigations are strongly needed to understand this discrepancy and to improve treatments by proton therapy.

Hematopoietic protection and mechanisms of ferrostatin-1 on hematopoietic acute radiation syndrome of mice

Purpose Baicalein (an anti-ferroptosis drug) was recently reported to synergistically improve the survival rate of mice following a high dose of total body irradiation with anti-apoptosis and anti-necroptosis drugs. At the same time, our group has demonstrated that ferrostatin-1, a ferroptosis inhibitor, improves the survival rate of a mouse model of hematopoietic acute radiation syndrome to 60% for 150 days (p < .001). These phenomena suggest that ferroptosis inhibition can mitigate radiation damage. In this study, we continued to study the mechanisms by which ferrostatin-1 alleviated radiation-induced ferroptosis and subsequent hematopoietic acute radiation syndrome. Materials and methods Male ICR mice (8–10 weeks old) were exposed to doses of 0, 8, or 10 Gy irradiated from a 137Cs source. Ferrostatin-1 was intraperitoneally injected into mice 72 h post-irradiation. Bone marrow mononuclear cells (BMMCs) and peripheral blood cells were counted. The changes in iron-related parameters, lipid metabolic enzymes, lipid peroxidation repair molecules (glutathione peroxidase 4, glutathione, and coenzyme Q10), and inflammatory factors (TNF-α, IL-6, and IL-1β) were evaluated using biochemical or antibody techniques. Results Ferrostatin-1 increased the number of red and white blood cells, lymphocytes, and monocytes in the peripheral blood after total body irradiation in mice by mitigating the ferroptosis of BMMCs. Total body irradiation induced ferroptosis in BMMCs by increasing the iron and lipid peroxidation levels and depleting the acyl-CoA synthetase long-chain family member 4 (ASCL4), lipoxygenase 15, glutathione peroxidase 4, and glutathione levels. Ferroptotic BMMCs did not release TNF-α, IL-6, or IL-1β at the early stage of radiation exposure. Ferrostatin-1 mitigated the lipid peroxidation of radiation-induced ferroptosis by attenuating increases in levels of hemosiderin and liable iron pool and decreases in levels of ASCL4 and glutathione peroxidase 4. Conclusions The onset of total body irradiation-induced ferroptosis in BMMCs involved changes in iron, lipid metabolic enzymes, and anti-lipid peroxidation molecules. Ferrostatin-1 could be a potential radiation mitigation agent by acting on these targets.

Biological Effects of Scattered Versus Scanned Proton Beams on Normal Tissues in Total Body Irradiated Mice: Survival, Genotoxicity, Oxidative Stress and Inflammation.

Side effects of proton therapy are poorly studied. Moreover, the differences in the method of dose delivery on normal tissues are not taken into account when proton beams are scanned instead of being scattered. We proposed here to study the effects of both modalities of proton beam delivery on blood; skin; lung and heart in a murine model. In that purpose; C57BL/6 mice were total body irradiated by 190.6 MeV proton beams either by Double Scattering (DS) or by Pencil Beam Scanning (PBS) in the plateau phase before the Bragg Peak. Mouse survival was evaluated. Blood and organs were removed three months after irradiation. Biomarkers of genotoxicity; oxidative stress and inflammation were measured. Proton irradiation was shown to increase lymphocyte micronucleus frequency; lung superoxide dismutase activity; erythrocyte and skin glutathione peroxidase activity; erythrocyte catalase activity; lung; heart and skin oxidized glutathione level; erythrocyte and lung lipid peroxidation and erythrocyte protein carbonylation even 3 months post-irradiation. When comparing both methods of proton beam delivery; mouse survival was not different. However, PBS significantly increased lymphocyte micronucleus frequency; erythrocyte glutathione peroxidase activity and heart oxidized glutathione level compared to DS. These results point out the necessity to take into account the way of delivering dose in PT as it could influence late side effects.

Phenotypic and Functional Characteristics of Exosomes Derived from Irradiated Mouse Organs and Their Role in the Mechanisms Driving Non-Targeted Effects.

Molecular communication between irradiated and unirradiated neighbouring cells initiates radiation-induced bystander effects (RIBE) and out-of-field (abscopal) effects which are both an example of the non-targeted effects (NTE) of ionising radiation (IR). Exosomes are small membrane vesicles of endosomal origin and newly identified mediators of NTE. Although exosome-mediated changes are well documented in radiation therapy and oncology, there is a lack of knowledge regarding the role of exosomes derived from inside and outside the radiation field in the early and delayed induction of NTE following IR. Therefore, here we investigated the changes in exosome profile and the role of exosomes as possible molecular signalling mediators of radiation damage. Exosomes derived from organs of whole body irradiated (WBI) or partial body irradiated (PBI) mice after 24 h and 15 days post-irradiation were transferred to recipient mouse embryonic fibroblast (MEF) cells and changes in cellular viability, DNA damage and calcium, reactive oxygen species and nitric oxide signalling were evaluated compared to that of MEF cells treated with exosomes derived from unirradiated mice. Taken together, our results show that whole and partial-body irradiation increases the number of exosomes, instigating changes in exosome-treated MEF cells, depending on the source organ and time after exposure.

Alleviation of acute radiation-induced bone marrow failure in mice with human fetal placental stromal cell therapy

PurposeSelected placental mesenchymal stromal cells isolated from the fetal mesenchymal placental tissues (f-hPSCs) were tested as cell therapy of lethal acute radiation syndrome (ARS) with bone marrow regeneration and induced extramedullary hematopoiesis.Methods and materialsf-hPSCs were isolated from the chorionic plate of human placentae and further expanded in regular culture conditions. 2 × 106 f-hPSCs were injected on days 1 and 4 to 8-Gy total body irradiated (TBI) C3H mice, both intramuscularly and subcutaneously. Pre-splenectomized TBI mice were used to test the involvement of extramedullary spleen hematopoiesis in the f-hPSC-induced hematopoiesis recovery in the TBI mice. Weight and survival of the mice were followed up within the morbid period of up to 23 days following irradiation. The role of hematopoietic progenitors in the recovery of treated mice was evaluated by flow cytometry, blood cell counts, and assay of possibly relevant growth factors.Results and conclusionsThe survival rate of all groups of TBI f-hPSC-treated mice at the end of the follow-up was dramatically elevated from < 10% in untreated to ~ 80%, with a parallel regain of body weight, bone marrow (BM) recovery, and elevated circulating progenitors of blood cell lineages. Blood erythropoietin levels were elevated in all f-hPSC-treated mice. Extramedullary splenic hematopoiesis was recorded in the f-hPSC-treated mice, though splenectomized mice still had similar survival rate. Our findings suggest that the indirect f-hPSC life-saving therapy of ARS may also be applied for treating other conditions with a failure of the hematopoietic system and severe pancytopenia.

Characterization of macrophages in the liver of total body irradiated mice

Abstract Two hepatic macrophage (MΦ) populations have been described in the inflamed liver: F4/80+CD11b+ cells and F4/80+CD68+ cells. In normal conditions, hepatic F4/80+CD11b+ cells are involved in host antibacterial resistance. However, these cells in total body irradiated mice (TBI mice) are unfunctional for the elimination of bacteria translocated from intestinal tracts. In this study, the properties of hepatic MΦ populations in TBI mice were characterized. Normal mice and mice 7 days after exposure to 4 Gy of X-rays were infected i.v. with 107 CFU/mouse of Enterococcus faecalis (E. faecalis). Three day after infection, F4/80+CD11b+ cells and F4/80+CD68+ cells isolated from the liver of these mice were cultured for 24 hrs, and culture fluids were assayed for IL-12, IL-10 and CCL1 by ELISA. NSG mice were inoculated i.v. with these MΦ preparations (2 × 105 cells/mouse), then infected i.v. with 5 × 104 CFU/mouse of E. faecalis. In the results, all TBI mice died within 2 weeks, while all normal mice survived. IL-12 was produced by F4/80+CD11b+ cells from normal mice, but not by both MΦ preparations from TBI mice. IL-10 was produced by F4/80+CD68+ cells from TBI mice (529.9 ± 272.6 pg/ml) and normal mice (445.8 ± 147.7 pg/ml). Large amounts of IL-10 (912.1 ± 103.7 pg/ml) was produced by F4/80+CD11b+ cells from TBI mice, and these cells produced CCL1 (327.5 ± 9.1 pg/ml), while other MΦ preparations did not. NSG mice inoculated with F4/80+CD11b+ cells from normal mice were resistant to infection; however, NSG mice inoculated with F4/80+CD11b+ cells from TBI mice did not. These results indicate hepatic F4/80+CD11b+ cells are polarized to M2bMΦ in response to total body irradiation, and these MΦ are involved in the decreased host antibacterial resistance in TBI mice.

Plasma Proteins as Biomarkers of Mortality After Total Body Irradiation in Mice.

During large-scale acute radiation exposure, rapidly distinguishing exposed individuals from nonexposed individuals is necessary. Identifying those exposed to high and potentially lethal radiation doses, and in need of immediate treatment, is especially important. To address this and find plasma biomarkers to assess ionizing radiation-induced mortality in the early stages, mice were administered a whole-body lethal dose of γ radiation, and radiation-induced damage was evaluated. Multiple blood biomarkers were screened using an antibody array, followed by validation using enzyme-linked immunoassay. The results revealed that irradiation (IR)-induced mortality in mice and caused body weight and blood platelet losses in deceased mice compared to surviving mice. The levels of certain proteins differed after IR between these 2 groups. Specific proteins in preirradiated mice were also found to potentiate radiosensitivity. Plasma levels of interleukin (IL)-22, urokinase, resistin, and IL-6 were associated with radiation-induced mortality in irradiated mice and may be useful as potential mortality predictors. Our results suggest that estimating the levels of certain plasma proteins is a promising alternative to conventional cytogenetic biodosimetry to accurately identify individuals exposed to high radiation doses and those at risk of death due to exposure. This strategy would facilitate the rapid triage of individuals requiring immediate and intensive medical treatment.

Discordant gene responses to radiation in humans and mice and the role of hematopoietically humanized mice in the search for radiation biomarkers

The mouse (Mus musculus) is an extensively used model of human disease and responses to stresses such as ionizing radiation. As part of our work developing gene expression biomarkers of radiation exposure, dose, and injury, we have found many genes are either up-regulated (e.g. CDKN1A, MDM2, BBC3, and CCNG1) or down-regulated (e.g. TCF4 and MYC) in both species after irradiation at ~4 and 8 Gy. However, we have also found genes that are consistently up-regulated in humans and down-regulated in mice (e.g. DDB2, PCNA, GADD45A, SESN1, RRM2B, KCNN4, IFI30, and PTPRO). Here we test a hematopoietically humanized mouse as a potential in vivo model for biodosimetry studies, measuring the response of these 14 genes one day after irradiation at 2 and 4 Gy, and comparing it with that of human blood irradiated ex vivo, and blood from whole body irradiated mice. We found that human blood cells in the hematopoietically humanized mouse in vivo environment recapitulated the gene expression pattern expected from human cells, not the pattern seen from in vivo irradiated normal mice. The results of this study support the use of hematopoietically humanized mice as an in vivo model for screening of radiation response genes relevant to humans.

NLRP6 deficiency aggravates liver injury after allogeneic hematopoietic stem cell transplantation

This study aims to observe the expression and role of NLRP6 in liver injury after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Allo-HSCT model was established through infusion of 5 × 106 bone marrow mononuclear cells into whole body irradiated mice. On days 7, 14, 21 and 28 after transplantation, the peripheral blood was collected to detect liver function. The liver of the mice was obtained to assess the pathological changes of liver tissues after allo-HSCT by H&E staining and Mason staining. Meanwhile, expression of NLRP6, phosphorylated p38-MAPK and IκBα, caspase-1 and NLRP3 in liver were detected by Western blot. ELISA was used for detection of the level of interleukin (IL)-1β, IL-18, tumor necrosis factor (TNF)-α, IL-6, myeloperoxidase (MPO) and tumor growth factor (TGF)-β1. Increased expression of NLRP6, phosphorylated Iκbα, phosphorylated p38-MAPK, pro-caspase-1, and p20, in liver tissue with injury and fibrosis in mice after allo-HSCT were observed. Meanwhile, the level of IL-1β, IL-18, IL-6 and TNF-α was also increased. However, NLRP6−/− mice showed more severe liver damage and liver fibrosis after transplantation together with higher level of phosphorylated Iκbα, phosphorylated p38-MAPK, Pro-caspase-1, p20 expression as well as IL-1β, IL-18, IL-6, and TNF-α secretion compared with wide-type. Interestingly, the expression of NLRP3 in the liver of NLRP6−/− mice was significantly higher than that of wild-type. In conclusion, the expression of NLRP6 in host's liver is associated with liver injury after allo-HSCT. NLRP6 deficiency in host's liver leads to more severe liver damage, indicating a protective role of NLRP6 in host's liver to liver damage after allo-HSCT.