Biomarkers for Radiation Biodosimetry and Injury Assessment after Mixed-field (Neutron and Gamma) Radiation in the Mouse Total-body Irradiation Model.

The risk of potential radiation exposure scenarios that include detonation of nuclear weapons, terrorist attacks on nuclear reactors, and the use of conventional explosives to disperse radioactive substances has increased in recent years. The majority of radiation biodosimetry and countermeasure studies have been performed using photon radiation even though many exposure scenarios predict mixed-field (neutron and photon) radiation. Hence, there is a need to evaluate biomarkers and accurately determine exposure levels of mixed-field combinations of neutrons and photons for an individual. These biomarkers will be critical for biodosimetry triage, treatment, and follow-up visits with such individuals. We evaluated the utility of multiple blood biomarkers for early response assessment of radiation exposure using a mouse (B6D2F1, males and females) total-body irradiation model exposed to a mixed-field (neutrons and gamma rays) using the Armed Forces Radiobiology Research Institute's Mark F nuclear research reactor. Total-body irradiation was given as a single exposure over a dose range from 1.5 to 6 Gy, dose rates of 0.6 and 1.9 Gy min, and different proportions of neutrons and gammas: either (67% neutrons + 33% gammas) or (30% neutrons + 70% gammas). Blood was collected 1, 2, 4, and 7 d after total-body irradiation. Radiation-responsive protein biomarkers were measured using the Meso Scale Diagnostics' high-throughput MULTI-ARRAY plate-format platform (QuickPlex 120 Imager) and enzyme-linked immunosorbent assay kits. Results demonstrate (1) dose- and time-dependent changes in fms-related tyrosine kinase 3 ligand, interleukins IL-5, IL-10, IL-12, and IL-18, granulocyte and granulocyte-macrophage colony-stimulating factors, thrombopoietin, erythropoietin, acute-phase proteins (serum amyloid A and lipopolysaccharide binding protein), surface plasma neutrophil (CD45) and lymphocyte (CD27) markers, ratio of CD45 to CD27, and procalcitonin; (2) dose- and time-dependent changes in blood cell counts (lymphocytes, neutrophils, platelets, red blood cells, and ratio of neutrophils to lymphocytes); (3) levels of IL-18, granulocyte and granulocyte-macrophage colony-stimulating factors, serum amyloid A, and procalcitonin were significantly higher in animals irradiated with 67% neutrons + 33% gammas compared to those irradiated with 30% neutrons + 70% gammas (p < 0.015), while no significant differences (p > 0.114) were observed in hematological biomarker counts; (4) exposure with 3-fold difference in dose rate (0.6 or 1.9 Gy min) revealed no significant differences in hematological and protein biomarker levels (p > 0.154); and (5) no significant differences in hematological and protein biomarker levels were observed in the sex-comparison study for any radiation dose at any time after exposure (p > 0.088). Results show that the dynamic changes in the levels of selected hematopoietic cytokines, organ-specific biomarkers, and acute-phase protein biomarkers reflect the time course and severity of acute radiation syndrome and may function as prognostic indicators of acute radiation syndrome outcome. These studies supplement an ongoing effort to deliver U.S. Federal Drug Administration-approved biodosimetry capabilities, which assess mixed-field radiation exposure.

Comparison of Biodosimetry Biomarkers for Radiation Dose and Injury Assessment After Mixed-Field (Neutron and Gamma) and Pure Gamma Radiation in the Mouse Total-Body Irradiation Model.

The detonation of a nuclear weapon and the occurrence of a nuclear accident represent possible mass-casualty events with significant exposure to mixed neutron and gamma radiation fields in the first few minutes after the event with the ensuing fallout, extending for miles from the epicenter, that would result primarily in photon (gamma- and/or x-ray) exposure. Circulating biomarkers represent a crucial source of information in a mass-casualty radiation exposure triage scenario. We evaluated multiple blood biodosimetry and organ-specific biomarkers for early-response assessment of radiation exposure using a mouse (B6D2F1, males and females) total-body irradiation model exposed to Co gamma rays over a broad dose range (3-12 Gy) and dose rates of either 0.6 or 1.9 Gy min and compared the results with those obtained after exposure of mice to a mixed field (neutrons and gamma rays) using the Armed Forces Radiobiology Research Institute Co gamma-ray source and TRIGA Mark F nuclear research reactor. The mixed-field studies were performed previously over a broad dose range (1.5-6 Gy), with dose rates of either 0.6 or 1.9 Gy min, and using different proportions of neutrons and gammas: either (67% neutrons + 33% gammas) or (30% neutrons + 70% gammas). Blood was collected 1, 2, 4, and 7 d after total-body irradiation. Results from Co gamma-ray studies demonstrate: (1) significant dose- and time-dependent reductions in circulating mature hematopoietic cells; (2) dose- and time-dependent changes in fms-related tyrosine kinase 3 ligand, interleukins IL-5, IL-10, IL-12, and IL-18, granulocyte colony-stimulating factors, thrombopoietin, erythropoietin, acute-phase proteins (serum amyloid A and lipopolysaccharide binding protein), surface plasma neutrophil (CD45) and lymphocyte (CD27) markers, ratio of CD45 to CD27, procalcitonin but not in intestinal fatty acid binding protein; (3) no significant differences were observed between dose-rate groups in hematological and protein profiles (fms-related tyrosine kinase 3 ligand, IL-5, IL-12, IL-18, erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, CD27, CD45, and ratio of CD45 to CD27) for any radiation dose at any time after exposure (p > 0.148); (4) no significant differences were observed between sex groups in hematological and protein profiles (fms-related tyrosine kinase 3 ligand, IL-18, erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, serum amyloid A, CD45) for any radiation dose at any time after exposure (p > 0.114); and (5) PCT level significantly increased (p < 0.008) in mice irradiated with 12 Gy on day 7 post-total-body irradiation without significant differences between groups irradiated at dose rates of either 0.6 or 1.9 Gy min (p > 0.287). Radiation-quality comparison results demonstrate that: (1) equivalent doses of pure gamma rays and mixed-field radiation do not produce equivalent biological effects, and hematopoietic syndrome occurs at lower doses of mixed-field radiation; (2) ratios of hematological and protein biomarker means in the Co study compared to mixed-field studies using 2× Co doses vs. 1× TRIGA radiation doses (i.e., 3 Gy Co vs. 1.5 Gy TRIGA) ranged from roughly 0.2 to as high as 26.5 but 57% of all ratios fell within 0.7 and 1.3; and (3) in general, biomarker results are in agreement with the relative biological effectiveness = 1.95 (Dn/Dt = 0.67) reported earlier by Armed Forces Radiobiology Research Institute scientists in mouse survival countermeasure studies.

Connexin 43 in astrocytes contributes to motor neuron toxicity in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons in the CNS. Astrocytes play a critical role in disease progression of ALS. Astrocytes are interconnected through a family of gap junction proteins known as connexins (Cx). Cx43 is a major astrocyte connexin conducting crucial homeostatic functions in the CNS. Under pathological conditions, connexin expression and functions are altered. Here we report that an abnormal increase in Cx43 expression serves as one of the mechanisms for astrocyte-mediated toxicity in ALS. We observed a progressive increase in Cx43 expression in the SOD1(G93A) mouse model of ALS during the disease course. Notably, this increase in Cx43 was also detected in the motor cortex and spinal cord of ALS patients. Astrocytes isolated from SOD1(G93A) mice as well as human induced pluripotent stem cell (iPSC)-derived astrocytes showed an increase in Cx43 protein, which was found to be an endogenous phenomenon independent of neuronal co-culture. Increased Cx43 expression led to important functional consequences when tested in SOD1(G93A) astrocytes when compared to control astrocytes over-expressing wild-type SOD1 (SOD1(WT) ). We observed SOD1(G93A) astrocytes exhibited enhanced gap junction coupling, increased hemichannel-mediated activity, and elevated intracellular calcium levels. Finally, we tested the impact of increased expression of Cx43 on MN survival and observed that use of both a pan Cx43 blocker and Cx43 hemichannel blocker conferred neuroprotection to MNs cultured with SOD1(G93A) astrocytes. These novel findings show a previously unrecognized role of Cx43 in ALS-related motor neuron loss. GLIA 2016;64:1154-1169.

Human glial progenitor engraftment and gene expression is independent of the ALS environment.

Although Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease, basic research studies have highlighted that astrocytes contribute to the disease process. Therefore, strategies which replace the diseased astrocyte population with healthy astrocytes may protect against motor neuron degeneration. Our studies have sought to evaluate astrocyte replacement using glial-restricted progenitors (GRPs), which are lineage-restricted precursors capable of differentiating into astrocytes after transplantation. The goal of our current study was to evaluate how transplantation to the diseased ALS spinal cord versus a healthy, wild-type spinal cord may affect human GRP engraftment and selected gene expression. Human GRPs were transplanted into the spinal cord of either an ALS mouse model or wild-type littermate mice. Mice were sacrificed for analysis at either the onset of disease course or at the endstage of disease. The transplanted GRPs were analyzed by immunohistochemistry and NanoString gene profiling which showed no gross differences in the engraftment or gene expression of the cells. Our data indicate that human glial progenitor engraftment and gene expression is independent of the neurodegenerative ALS spinal cord environment. These findings are of interest given that human GRPs are currently in clinical development for spinal cord transplantation into ALS patients.