Min Zhai

Wound Trauma Alters Ionizing Radiation Dose Assessment

BackgroundWounding following whole-body γ-irradiation (radiation combined injury, RCI) increases mortality. Wounding-induced increases in radiation mortality are triggered by sustained activation of inducible nitric oxide synthase pathways, persistent alteration of cytokine homeostasis, and increased susceptibility to bacterial infection. Among these factors, cytokines along with other biomarkers have been adopted for biodosimetric evaluation and assessment of radiation dose and injury. Therefore, wounding could complicate biodosimetric assessments.ResultsIn this report, such confounding effects were addressed. Mice were given 60Co γ-photon radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, body-weight loss, and wound healing. Analyses of DNA damage in bone-marrow cells and peripheral blood mononuclear cells (PBMCs), changes in hematology and cytokine profiles, and fundamental clinical signs were evaluated. Early biomarkers (1 d after RCI) vs. irradiation alone included significant decreases in survivin expression in bone marrow cells, enhanced increases in γ-H2AX formation in Lin+ bone marrow cells, enhanced increases in IL-1β, IL-6, IL-8, and G-CSF concentrations in blood, and concomitant decreases in γ-H2AX formation in PBMCs and decreases in numbers of splenocytes, lymphocytes, and neutrophils. Intermediate biomarkers (7 – 10 d after RCI) included continuously decreased γ-H2AX formation in PBMC and enhanced increases in IL-1β, IL-6, IL-8, and G-CSF concentrations in blood. The clinical signs evaluated after RCI were increased water consumption, decreased body weight, and decreased wound healing rate and survival rate. Late clinical signs (30 d after RCI) included poor survival and wound healing.ConclusionResults suggest that confounding factors such as wounding alters ionizing radiation dose assessment and agents inhibiting these responses may prove therapeutic for radiation combined injury and reduce related mortality.

Pegylated G-CSF Inhibits Blood Cell Depletion, Increases Platelets, Blocks Splenomegaly, and Improves Survival after Whole-Body Ionizing Irradiation but Not after Irradiation Combined with Burn

Exposure to ionizing radiation alone (radiation injury, RI) or combined with traumatic tissue injury (radiation combined injury, CI) is a crucial life-threatening factor in nuclear and radiological accidents. As demonstrated in animal models, CI results in greater mortality than RI. In our laboratory, we found that B6D2F1/J female mice exposed to 60Co-γ-photon radiation followed by 15% total-body-surface-area skin burns experienced an increment of 18% higher mortality over a 30-day observation period compared to irradiation alone; that was accompanied by severe cytopenia, thrombopenia, erythropenia, and anemia. At the 30th day after injury, neutrophils, lymphocytes, and platelets still remained very low in surviving RI and CI mice. In contrast, their RBC, hemoglobin, and hematocrit were similar to basal levels. Comparing CI and RI mice, only RI induced splenomegaly. Both RI and CI resulted in bone marrow cell depletion. It was observed that only the RI mice treated with pegylated G-CSF after RI resulted in 100% survival over the 30-day period, and pegylated G-CSF mitigated RI-induced body-weight loss and depletion of WBC and platelets. Peg-G-CSF treatment sustained RBC balance, hemoglobin levels, and hematocrits and inhibited splenomegaly after RI. The results suggest that pegylated G-CSF effectively sustained animal survival by mitigating radiation-induced cytopenia, thrombopenia, erythropenia, and anemia.

Ghrelin therapy improves survival after whole-body ionizing irradiation or combined with burn or wound: amelioration of leukocytopenia, thrombocytopenia, splenomegaly, and bone marrow injury.

Exposure to ionizing radiation alone (RI) or combined with traumatic tissue injury (CI) is a crucial life-threatening factor in nuclear and radiological events. In our laboratory, mice exposed to 60Co-γ-photon radiation (9.5 Gy, 0.4 Gy/min, bilateral) followed by 15% total-body-surface-area skin wounds (R-W CI) or burns (R-B CI) experienced an increment of ≥18% higher mortality over a 30-day observation period compared to RI alone. CI was accompanied by severe leukocytopenia, thrombocytopenia, erythropenia, and anemia. At the 30th day after injury, numbers of WBC and platelets still remained very low in surviving RI and CI mice. In contrast, their RBC, hemoglobin, and hematocrit were recovered towards preirradiation levels. Only RI induced splenomegaly. RI and CI resulted in bone-marrow cell depletion. In R-W CI mice, ghrelin (a hunger-stimulating peptide) therapy increased survival, mitigated body-weight loss, accelerated wound healing, and increased hematocrit. In R-B CI mice, ghrelin therapy increased survival and numbers of neutrophils, lymphocytes, and platelets and ameliorated bone-marrow cell depletion. In RI mice, this treatment increased survival, hemoglobin, and hematocrit and inhibited splenomegaly. Our novel results are the first to suggest that ghrelin therapy effectively improved survival by mitigating CI-induced leukocytopenia, thrombocytopenia, and bone-marrow injury or the RI-induced decreased hemoglobin and hematocrit.

Adaptive Redox Response of Mesenchymal Stromal Cells to Stimulation with Lipopolysaccharide Inflammagen: Mechanisms of Remodeling of Tissue Barriers in Sepsis

Acute bacterial inflammation is accompanied by excessive release of bacterial toxins and production of reactive oxygen and nitrogen species (ROS and RNS), which ultimately results in redox stress. These factors can induce damage to components of tissue barriers, including damage to ubiquitous mesenchymal stromal cells (MSCs), and thus can exacerbate the septic multiple organ dysfunctions. The mechanisms employed by MSCs in order to survive these stress conditions are still poorly understood and require clarification. In this report, we demonstrated that in vitro treatment of MSCs with lipopolysaccharide (LPS) induced inflammatory responses, which included, but not limited to, upregulation of iNOS and release of RNS and ROS. These events triggered in MSCs a cascade of responses driving adaptive remodeling and resistance to a “self-inflicted” oxidative stress. Thus, while MSCs displayed high levels of constitutively present adaptogens, for example, HSP70 and mitochondrial Sirt3, treatment with LPS induced a number of adaptive responses that included induction and nuclear translocation of redox response elements such as NFkB, TRX1, Ref1, Nrf2, FoxO3a, HO1, and activation of autophagy and mitochondrial remodeling. We propose that the above prosurvival pathways activated in MSCs in vitro could be a part of adaptive responses employed by stromal cells under septic conditions.

Autophagy-Mediated Defense Response of Mouse Mesenchymal Stromal Cells (MSCs) to Challenge with Escherichia coli

Abstract : Symbiotic microorganisms are spatially separated from their animal host, e.g., in the intestine and skin, in a manner enabling nutrient metabolism as well as evolutionary development of protective physiologic features in the host such as innate and adaptive immunity, immune tolerance, and function of tissue barriers . The major interface barrier between the microbiota and host tissue is constituted by epithelium reticuloendothelial tissue, and mucosa-associated lymphoid tissue (MALT) . Traumatic damage to skin and the internal epithelium in soft tissues can cause infections that account for 7% to 10% of hospitalizations in the United States (4). Moreover, wound infections and sepsis are an increasing cause of death in severely ill patients, especially those with immunosupression due to exposure to cytotoxic agents and chronic inflammation (4). It is well accepted that breakdown of the host-bacterial symbiotic homeostasis and associated infections are the major consequences of impairment of the first line of antimicrobial defense barriers such as the mucosal layers, MALT and reticuloendothelium. Under these impairment conditions of particular interest then is the role of sub-mucosal structures, such as connective tissue stroma, in the innate defense compensatory responses to infections. The mesenchymal connective tissue of different origins is a major source of multipotent mesenchymal stromal cells (i.e., colony-forming-unit fibroblasts). Recent discovery of immunomodulatory function of mesenchymal stromal cells (MSCs) suggests that they are essential constituents that control inflammatory responses.

Captopril Increases Survival after Whole-Body Ionizing Irradiation but Decreases Survival when Combined with Skin-Burn Trauma in Mice

Past and recent radiation events have involved a high incidence of radiation combined injury where victims often succumb to serious infections as a consequence of bacterial translocation and subsequent sepsis. The risk of infection is exacerbated in radiation combined skin-burn injury (RCI), which increase vulnerability. Furthermore, no suitable countermeasures for radiation combined skin-burn injury have been established. In this study, we evaluated captopril as a potential countermeasure to radiation combined skin-burn injury. Captopril is an FDA-approved angiotensin-converting enzyme inhibitor that was previously reported to stimulate hematopoietic recovery after exposure to ionizing radiation. Female B6D2F1/J mice were whole-body bilateral 60Co gamma-photon irradiated (dose rate of 0.4 Gy/min) with 9.5 Gy (LD70/30 for RCI), followed by nonlethal dorsal skin-burn injury under anesthesia (approximately 15% total-body surface-area burn). Mice were provided with acidified drinking water with or without dissolved captopril (0.55 g/l) for 30 days immediately after injury and were administered topical gentamicin (0.1% cream; day 1–10) and oral levofloxacin (90–100 mg/kg; day 3–16). Surviving mice were euthanized on day 30 after analyses of water consumption, body weight and survival. Our data demonstrate that, while treatment with captopril did mitigate mortality induced by radiation injury (RI) alone (55% captopril vs. 80% vehicle; n = 20, P < 0.05), it also resulted in decreased survival after radiation combined skin-burn injury (22% captopril vs. 41% vehicle; n = 22, P < 0.05). Moreover, captopril administration via drinking water produced an uneven dosage pattern among the different injury groups ranging from 74 ± 5.4 to 115 ± 2.2 mg/kg/day. Captopril treatment also did not counteract the negative alterations in hematology, splenocytes or bone marrow cellularity after either radiation injury or radiation combined skin-burn injury. These data suggest that captopril may exert its actions differently between the two injury models (RI vs. RCI) and that captopril dosing, when combined with topical and systemic antibiotic treatments, may not be a suitable countermeasure for RCI.

Autophagy and mitochondrial remodelling in mouse mesenchymal stromal cells challenged with Staphylococcus epidermidis

The bone marrow stroma constitutes the marrow‐blood barrier, which sustains immunochemical homoeostasis and protection of the haematopoietic tissue in sequelae of systemic bacterial infections. Under these conditions, the bone marrow stromal cells affected by circulating bacterial pathogens shall elicit the adaptive stress‐response mechanisms to maintain integrity of the barrier. The objective of this communication was to demonstrate (i) that in vitro challenge of mesenchymal stromal cells, i.e. colony‐forming unit fibroblasts (CFU‐F), with Staphylococcus epidermidis can activate the autophagy pathway to execute antibacterial defence response, and (ii) that homoeostatic shift because of the bacteria‐induced stress includes the mitochondrial remodelling and sequestration of compromised organelles via mitophagy. Implication of Drp1 and PINK1–PARK2‐dependent mechanisms in the mitophagy turnover of the aberrant mitochondria in mesenchymal stromal cells is investigated and discussed.

Up-Regulation of Autophagy Defense Mechanisms in Mouse Mesenchymal Stromal Cells in Response to Ionizing Irradiation Followed by Bacterial Challenge

Mesenchymal stroma along with epithelium, endothelium, reticuloendothelium, and lym‐ phoid components is an essential constituent of tissue barriers that sustain immunochemical homeostatic interactions of tissue with internal and external environments. Thus, mesenchy‐ mal stroma protects the body from infections [1-8]. A breach of immune and structural integrity of tissue barriers under patho-physiological conditions such as complicated injury can lead to translocation of bacteria from different host-associated microbiomes and colonization of vital organs that can ultimately result in multiple organ failure and sepsis [9].

Thrombopoietin Receptor Agonist Mitigates Hematopoietic Radiation Syndrome and Improves Survival after Whole-Body Ionizing Irradiation Followed by Wound Trauma

Ionizing radiation combined with trauma tissue injury (combined injury, CI) results in greater mortality and H-ARS than radiation alone (radiation injury, RI), which includes thrombocytopenia. The aim of this study was to determine whether increases in numbers of thrombocytes would improve survival and mitigate H-ARS after CI. We observed in mice that WBC and platelets remained very low in surviving RI animals that were given 9.5 Gy 60Co-γ-photon radiation, whereas only lymphocytes and basophils remained low in surviving CI mice that were irradiated and then given skin wounds. Numbers of RBC and platelets, hemoglobin concentrations, and hematocrit values remained low in surviving RI and CI mice. CI induced 30-day mortality higher than RI. Radiation delayed wound healing by approximately 14 days. Treatment with a thrombopoietin receptor agonist, Alxn4100TPO, after CI improved survival, mitigated body-weight loss, and reduced water consumption. Though this therapy delayed wound-healing rate more than in vehicle groups, it greatly increased numbers of platelets in sham, wounded, RI, and CI mice; it significantly mitigated decreases in WBC, spleen weights, and splenocytes in CI mice and decreases in RBC, hemoglobin, hematocrit values, and splenocytes and splenomegaly in RI mice. The results suggest that Alxn4100TPO is effective in mitigating CI.

2-0, 3-0 Desulfated Heparin does not Affect Radiation Injury Induced Mortality but Reduces Radiation Combined Skin-burn Injury Induced Survival in Mice

Many radiation events have involved a high incidence of radiation combined injuries. Victims of radiation events succumb to serious infections as a consequence of bacterial translocation and sepsis. Exacerbation of the risk of infection by radiation combined burn injury (RCBI) further heightens vulnerability. There are currently no suitable countermeasures that exist for RCBIs. We evaluated 2-0, 3-0 desulfated heparin (ODSH), an anti-inflammatory and anticoagulant agent as a potential countermeasure to RCBI. Female B6D2F1/J mice (12-week) were subjected to 9.5 Gy (LD70/30 for RCBI) whole-body bilateral 60Co gamma-photon radiation (0.4 Gy/min), followed by dorsal skin burn injury under anesthesia (∼15% total-body-surface area burn). Mice were injected subcutaneously with ODSH (25 mg/kg every 12 h; days 1-2 and 17.5 mg/kg every 12 h; days 3-7) or vehicle (sterile saline of equal volume) for 7 days post-injury and further administered topical gentamicin (0.1% cream; days 1-10) and oral levofloxacin (100 mg/kg; days 3-16). Mice were euthanized on day 30 following water consumption, body mass and survival analysis. Our data showed ODSH had no effect on radiation injury (RI)-induced mortality (45% ODSH vs. 45% VEH; n=20). However interestingly, ODSH treatment significantly reduced survival after RCBI (12% ODSH vs. 41% VEH; n=22, p<0.05). Furthermore, ODSH did not affect water consumption or body mass accrual after RI or RCBI. ODSH was not able to counteract the negative alterations in hematology, splenocytes, or bone marrow cell counts after RI or RCBI. These data illustrate that ODSH in combination with antibiotic treatments, may not be a mitigating countermeasure for RCBI.