High variability in short and long-term recovery kinetic of blood cell count and blood chemistry in a partial body irradiation mouse model.

PURPOSE: In the aftermath of a nuclear disaster or accident, survivors will suffer from radiation-induced normal tissue damage. Recovery after radiation exposure is dictated by several factors, one of which is degree of shielding at time of exposure. This study aims to characterize the short and late term changes in kinetics and magnitude of pancytopenia and blood chemistry in a model of heterogeneous radiation exposure, or partial body irradiation (PBI), compared to whole body irradiation (WBI). MATERIALS AND METHODS: Male C57BL/6 mice, 8-10 weeks of age, were WBI at 6 different doses (6, 6.1. 6.15, 6.2, 6.5, and 7.5 Gy) to establish the LD50. To determine the effect of shielding on blood cell counts and chemistry, animals were either WBI at 6 Gy (LD2230) or 6 Gy PBI with one leg shielding (LD030). Complete blood counts and chemistry were measured at 1, 5-, 10-, 20-, 30- and 120-days post-irradiation. RESULTS AND CONCLUSIONS: Irradiated animals had significant depletion of white blood cells, red blood cells and platelets up to 10 days post-irradiation. Separation between PBI and WBI were observed at 10- and 20-days post-irradiation at which point PBI animals showed sign of recovery while overall cell count remains depleted in WBI animals up to 30 days post-irradiation. In addition, significant changes were found in parameters indicative of hematopoietic injury including hemoglobin count, hematocrit count and white blood cell population. Significant changes were observed in kidney function with changes to blood urea nitrogen and calcium concentration at 5-days post-irradiation. At 10-days post-irradiation. liver function changes differentiated WBI from PBI animals. Long-term, irradiated animal's chemistry values and many blood counts were not significantly different from Sham. In conclusion, partial shielding ensured complete survival and demonstrated a different recovery kinetics of blood and chemistry parameters after irradiation compared to survivors of whole body irradiation and no single hemopoietic parameter was able to consistently differentiate irradiated from Sham animals. This seems to indicate that there is no single robust hemopoietic parameter to differentiate those exposed from those who were not due to the inherent variability in individual responses. Furthermore, there were no significant long-term effects on these blood parameters between survivors of WBI and PBI except that shielding accelerated recovery.

The Need for Biological Countermeasures to Mitigate the Risk of Space Radiation-Induced Carcinogenesis, Cardiovascular Disease, and Central Nervous System Deficiencies.

NASA's currently planned long-duration, deep space exploration missions outside of low Earth orbit (LEO) will result in the exposure of astronauts to relatively high lifetime doses of ionizing radiation (IR), exceeding what humans have previously encountered in space. Of concern to this exposure are the long-term health consequences of radiation carcinogenesis, cardiovascular and degenerative disease, and central nervous system decrements. Existing engineering solutions are insufficient to decrease the lifetime accumulated IR exposure to levels currently allowable by agency standards, therefore appropriate countermeasure and mitigation strategies must be developed to enable long duration missions. Emerging discoveries in the fields of radiation oncology and the mitigation of Acute Radiation Syndrome (ARS) have demonstrated the potential for compound-based/biological radiomodifiers to drastically improve clinical outcomes and represent a promising strategy for space radiation countermeasure development. This review outlines the unique challenges posed by the space radiation environment, defines the limits of terrestrial radiation protection strategies in space, describes a brief overview of current space radiation countermeasure development strategies, highlights potential new approaches for countermeasure identification and development, and speculates on the potential benefits beyond space exploration.

Cognitive and Imaging Differences After Proton and Photon Whole Brain Irradiation in a Preclinical Model.

PURPOSE: Compared with photon cranial radiation therapy (X-CRT), proton cranial radiation therapy (P-CRT) offers potential advantages in limiting radiation-induced sequalae in the treatment of pediatric brain tumors. This study aims to identify cognitive, functional magnetic resonance and positron emission tomography imaging markers and molecular differences between the radiation modalities. METHODS AND MATERIALS: Juvenile rats received a single faction of 10 Gy (relative biological effectiveness-weighted dose) delivered with 6 MV X-CRT or at the midspread out Bragg peak of a 100 MeV P-CRT beam. At 3, 6, and 12 months post-CRT, executive function was measured using 5-choice serial reaction time task. At ∼12 months post-CRT, animals were imaged with 18F-Flurodeoxy-glucose positron emission tomography imaging followed by functional ex vivo magnetic resonance imaging and stained for markers of neuroinflammation. RESULTS: Irradiated animals had cognitive impairment with a higher number of omissions and lower incorrect and premature responses compared with sham (P ≤ .05). The accuracy of the animals' X-CRT was less than that of sham (P ≤ .001). No significant difference in rates of cognitive change were found between the radiation modalities. At 12 months post-CRT, glucose metabolism was significantly higher than sham in X-CRT (P = .04) but not P-CRT. Using diffusion tensor imaging, P-CRT brains were found to have higher white matter volume and fiber lengths compared with sham (P < .03). Only X-CRT animals had higher apparent diffusion coefficient values compared with sham (P = .04). P-CRT animals had more connectomic changes compared with X-CRT. Correlative analysis identified several imaging features with cognitive performance. Furthermore, microgliosis (P < .05), astrogliosis (P < .01), and myelin thinning (P <.05) were observed in both radiation modalities, with X-CRT showing slightly more inflammation. CONCLUSIONS: Both P-CRT and X-CRT lead to neurocognitive changes compared with sham. Although no significant difference was observed in neuroinflammation between the irradiated groups, differences were found in late-term glucose metabolism and brain connectome. Our results indicate that despite relative biological effectiveness weighting of the proton dose there are still differential effects which warrants further investigation.

NF-κB Blockade by NEMO Binding Domain Peptide Ameliorates Inflammation and Neurobehavioral Sequelae After Cranial Radiation Therapy in Juvenile Mice.

PURPOSE: Cranial radiation therapy (CRT) is a common treatment for pediatric brain tumor patients. However, side effects include significant neurobehavioral dysfunction in survivors. This dysfunction may in part be caused by inflammation, including increased production of tumor necrosis factor alpha (TNFα) and its receptor TNFR1, which can activate the nuclear factor kappa light-chain enhancer of activated B cells (NF-κB). The TNFα blockade abrogates this inflammatory response, although it presents immunologic risks. Thus, modulation of pathway subsets may be preferable. Here, we test whether inhibition of NF-κB activation using an NF-κB essential modulator binding domain (NBD) peptide mitigates CRT-induced neuroinflammation and improves behavioral outcomes. METHODS AND MATERIALS: Male C57BL/6J 28-day old mice were randomized to saline (sham), 5 Gy whole-brain CRT, or CRT + NBD-peptide. Brain tissue was collected after 4 hours or 3 months for Western blot or immunohistochemistry. The cortex, corpus callosum (CC), and dentate gyrus were variably imaged for NF-κB-p65, IκBα, proliferation, apoptosis, necroptosis, TNFα, TNFR1, IBA-1, doublecortin, CD11c, and GFAP. Neurobehavioral changes were assessed by open field and elevated plus maze tests 3 months post-CRT. RESULTS: NF-κB expression increased in whole and nuclear fractions 4 hours after CRT and was abrogated by NBD treatment. Cell death increased and proliferation decreased after CRT, including within neuronal progenitors, with some loss mitigated by NBD. Increased levels of TNFα, IBA-1, and GFAP were found in the CC and cortex months after CRT and were limited by NBD. The anti-NF-κB peptide also improved neurobehavioral assessments, yielding improvements in anxiety and exploration. CONCLUSIONS: Results suggest a role for NF-κB modulation by NBD peptide in the reduction of neuroinflammation and mitigation of behavioral complications after pediatric radiation therapy.

Differentiation of Heterogeneous Radiation Exposure Using Hematology and Blood Chemistry.

In the aftermath of a nuclear incident, survivors will suffer the deleterious effects from acute radiation exposure. The majority of those affected would have received heterogeneous radiation exposure, reflected in hematological metrics and blood chemistry. Here, we investigated the acute and long-term changes in kinetics and magnitude of pancytopenia and blood chemistry in rats irradiated using varying degrees of body shielding. We hypothesized that, although a single blood count may not be able to differentiate the degree of radiation exposure, a combination of measurements from complete blood cell counts (CBCs) and blood chemistry tests is able to do so. Male Sprague Dawley rats, 8-10 weeks of age, received single-dose 7.5 Gy (160 kVp, 25 mA, 1.16 Gy/min) whole-body irradiation (WBI, LD100/30) or partial-body irradiation (PBI), as follows: one leg shielded (1LS, LD0/30), two legs shielded (2LS, LD0/30) or the upper half of the body shielded (UHS, LD0/30). Animal morbidity and weights were measured. Blood was drawn at 1, 5, 10, 20 and 30 days postirradiation (n = 4-11). For kidney and liver function measurements, CBC and blood chemistry analyses were performed. WBI animals on average survived 9 ± 0.4 days postirradiation. In contrast, all PBI animals survived the 30-day study period. CBC analysis revealed that both white blood cell (WBC) and platelet counts were most affected after irradiation. While WBC counts were significantly lower in all irradiated groups on days 1, 5 and 10, platelets were only significantly lower on days 5 and 10 postirradiation. In addition, on day 5 postirradiation both WBC and platelet counts were able to differentiate WBI (non-survivors) from PBI 2LS and UHS animals (survivors). Using four blood parameters (platelets, percentage lymphocytes, percentage neutrophils and percentage monocytes) on day 5 after 7.5 Gy irradiation and a linear discrimination analysis (LDA), we were able to predict the degree of body exposure in animals with a 95.8% accuracy. Alkaline phosphatase (ALP) was significantly lower in all groups on days 5 and 10 postirradiation compared to baseline. Furthermore, ALP was significantly higher in the UHS than WBI animals. The AST:ALT ratio was significantly higher than baseline in all irradiated groups on day 1 postirradiation. In conclusion, four CBC parameters, on day 5 after receiving a 7.5 Gy dose of radiation, can be employed in a LDA to differentiate various degrees of exposure (shielding). The characterization presented in this work paves the way for further studies in differences caused by heterogeneous body exposure to radiation and a new metric for biodosimetry.

A comprehensive preclinical assessment of late-term imaging markers of radiation-induced brain injury.

BACKGROUND: Cranial radiotherapy (CRT) is an important part of brain tumor treatment, and although highly effective, survivors suffer from long-term cognitive side effects. In this study we aim to establish late-term imaging markers of CRT-induced brain injury and identify functional markers indicative of cognitive performance. Specifically, we aim to identify changes in executive function, brain metabolism, and neuronal organization. METHODS: Male Sprague Dawley rats were fractionally irradiated at 28 days of age to a total dose of 30 Gy to establish a radiation-induced brain injury model. Animals were trained at 3 months after CRT using the 5-choice serial reaction time task. At 12 months after CRT, animals were evaluated for cognitive and imaging changes, which included positron emission tomography (PET) and magnetic resonance imaging (MRI). RESULTS: Cognitive deficit with signs of neuroinflammation were found at 12 months after CRT in irradiated animals. CRT resulted in significant volumetric changes in 38% of brain regions as well as overall decrease in brain volume and reduced gray matter volume. PET imaging showed higher brain glucose uptake in CRT animals. Using MRI, irradiated brains had an overall decrease in fractional anisotropy, lower global efficiency, increased transitivity, and altered regional connectivity. Cognitive measurements were found to be significantly correlated with six image features that included myelin integrity and local organization of the neural network. CONCLUSIONS: These results demonstrate that CRT leads to late-term morphological changes, reorganization of neural connections, and metabolic dysfunction. The correlation between imaging markers and cognitive deficits can be used to assess late-term side effects of brain tumor treatment and evaluate efficacy of new interventions.

Image-based Classification of Tumor Type and Growth Rate using Machine Learning: a preclinical study.

Medical images such as magnetic resonance (MR) imaging provide valuable information for cancer detection, diagnosis, and prognosis. In addition to the anatomical information these images provide, machine learning can identify texture features from these images to further personalize treatment. This study aims to evaluate the use of texture features derived from T1-weighted post contrast scans to classify different types of brain tumors and predict tumor growth rate in a preclinical mouse model. To optimize prediction models this study uses varying gray-level co-occurrence matrix (GLCM) sizes, tumor region selection and different machine learning models. Using a random forest classification model with a GLCM of size 512 resulted in 92%, 91%, and 92% specificity, and 89%, 85%, and 73% sensitivity for GL261 (mouse glioma), U87 (human glioma) and Daoy (human medulloblastoma), respectively. A tenfold cross-validation of the classifier resulted in 84% accuracy when using the entire tumor volume for feature extraction and 74% accuracy for the central tumor region. A two-layer feedforward neural network using the same features is able to predict tumor growth with 16% mean squared error. Broadly applicable, these predictive models can use standard medical images to classify tumor type and predict tumor growth, with model performance, varying as a function of GLCM size, tumor region, and tumor type.

Echocardiography Differentiates Lethally Irradiated Whole-Body From Partial-Body Exposed Rats.

Background: Acute radiation syndrome (ARS) affects morbidity and mortality dependent on the amount of body exposed. We propose the use of echocardiography (EC) to differentiate between survivors and non-survivors by measuring changes in cardiac function (CF) and pulmonary arterial function (PAF). We also investigate the role of rheology in our observed changes. Methods and Results: Rats were irradiated to the whole body (WB) or partial body with two-legs shielded (2LS) at a lethal dose of 7.5Gy. EC and magnetic resonance imaging were performed, and rheological measurements conducted. Only 2LS survived past 12-days post-exposure and their CF and PAR were not significantly different from baseline. WB was significantly different from both baseline and 2LS in stroke volume (P < 0.05), velocity time integral (VTI; P < 0.05) and pulmonary artery acceleration time (PAAT; P < 0.05). Differences were identified as early as six-days post-exposure, where VTI and PAAT were significantly (P < 0.05) decreased in WB versus baseline but only PAAT was different from 2LS. Blood viscosity was significantly lower in the WB versus baseline and 2LS (P < 0.0001). WB exhibited a significant rise in dense red blood cells versus baseline (P < 0.01) and 2LS (P < 0.01). Cell-free hemoglobin, a contributor to pulmonary artery hypertension and vasculopathy, was significantly elevated in WB vs. sham. Conclusions: Non-invasive and readily available imaging can be used to identify critically affected victims. Our findings point to heart failure as one possible cause of death in WB exposed animals, potentially exacerbated by rheological, hemolytic, and pulmonary factors, and the importance of developing radiomitigators against cardiac ARS mortality.

Exercise ameliorates neurocognitive impairments in a translational model of pediatric radiotherapy.

Background: While cranial radiation therapy (CRT) is an effective treatment, healthy areas surrounding irradiation sites are negatively affected. Frontal lobe functions involving attention, processing speed, and inhibition control are impaired. These deficits appear months to years after CRT and impair quality of life. Exercise has been shown to rejuvenate the brain and aid in recovery post-injury through its effects on neurogenesis and cognition. Methods: We developed a juvenile rodent CRT model that reproduces neurocognitive deficits. Next, we utilized the model to test whether exercise ameliorates these deficits. Fischer rats (31 days old) were irradiated with a fractionated dose of 4 Gy × 5 days, trained and tested at 6, 9, and 12 months post-CRT using 5-choice serial reaction time task. After testing, fixed rat brains were imaged using diffusion tensor imaging and immunohistochemistry. Results: CRT caused early and lasting impairments in task acquisition, accuracy, and latency to correct response, as well as causing stunting of growth and changes in brain volume and diffusion. Exercising after irradiation improved acquisition, behavioral control, and processing speed, mitigated the stunting of brain size, and increased brain fiber numbers compared with sedentary CRT values. Further, exercise partially restored global connectome organization, including assortativity and characteristic path length, and while it did not improve the specific regional connections that were lowered by CRT, it appeared to remodel these connections by increasing connectivity between alternate regional pairs. Conclusions: Our data strongly suggest that exercise may be useful in combination with interventions aimed at improving cognitive outcome following pediatric CRT.

Effect of Brain Tumor Presence During Radiation on Tissue Toxicity: Transcriptomic and Metabolic Changes.

PURPOSE: Radiation therapy (RT) causes functional and transcriptomic changes in the brain; however, most studies have been carried out in normal rodent brains. Here, the long-term effect of irradiation and tumor presence during radiation was investigated. METHODS AND MATERIALS: Male Wistar rats ∼7 weeks old were divided into 3 groups: sham implant, RT+sham implant, and RT+tumor implant (C6 glioma). Hypofractionated irradiation (8 or 6 Gy/day for 5 days) was localized to a 1-cm strip of cranium starting 5 days after implantation, resulting in complete tumor regression and prolonged survival. Biopsy of tissue was performed in the implant area 65 days after implantation. RNA was hybridized to GeneChip Rat Exon 1.0 ST array. Data were analyzed using significant analysis of microarrays and ingenuity pathway analysis. 1H magnetic resonance spectroscopy (1H-MRS) imaging was performed in the implantation site 65 to 70 days after implantation using a 9.4 T Biospec magnetic resonance imaging scanner with a quadrature rat brain array. Immunohistochemical staining for astrogliosis, HMG-CoA synthase 2, γ-aminobutyric acid (GABA) and taurine was performed at ∼65 days after implantation. RESULTS: Eighty-four genes had a false discovery rate <3.5%. We compared RT+tumor implant with RT+sham implant animals. The tumor presence affected networks associated with cancer/cell morphology/tissue morphology. 1H-MRS showed significant reduction in taurine levels (P<.04) at the implantation site in both groups. However, the RT+tumor group also showed significant increase in levels of neurotransmitter GABA (P=.02). Hippocampal taurine levels were only significantly reduced in the RT+tumor group (P=.03). HMG-CoA synthase 2, GABA and taurine levels were confirmed using staining. Glial fibrillary acidic protein staining demonstrated a significant increase in inflammation that was heightened in the RT+tumor group. CONCLUSIONS: Our data indicate that tumor presence during radiation significantly affects long-term functional transcriptomics landscape and neurotransmitter levels at the tumor implantation site/normal tissue, accompanied by increased inflammation (astrogliosis).

Investigating the Abscopal Effects of Radioablation on Shielded Bone Marrow in Rodent Models Using Multimodality Imaging.

The abscopal effect is the response to radiation at sites that are distant from the irradiated site of an organism, and it is thought to play a role in bone marrow (BM) recovery by initiating responses in the unirradiated bone marrow. Understanding the mechanism of this effect has applications in treating BM failure (BMF) and BM transplantation (BMT), and improving survival of nuclear disaster victims. Here, we investigated the use of multimodality imaging as a translational tool to longitudinally assess bone marrow recovery. We used positron emission tomography/computed tomography (PET/CT), magnetic resonance imaging (MRI) and optical imaging to quantify bone marrow activity, vascular response and marrow repopulation in fully and partially irradiated rodent models. We further measured the effects of radiation on serum cytokine levels, hematopoietic cell counts and histology. PET/CT imaging revealed a radiation-induced increase in proliferation in the shielded bone marrow (SBM) compared to exposed bone marrow (EBM) and sham controls. T2-weighted MRI showed radiation-induced hemorrhaging in the EBM and unirradiated SBM. In the EBM and SBM groups, we found alterations in serum cytokine and hormone levels and in hematopoietic cell population proportions, and histological evidence of osteoblast activation at the bone marrow interface. Importantly, we generated a BMT mouse model using fluorescent-labeled bone marrow donor cells and performed fluorescent imaging to reveal the migration of bone marrow cells from shielded to radioablated sites. Our study validates the use of multimodality imaging to monitor bone marrow recovery and provides evidence for the abscopal response in promoting bone marrow recovery after irradiation.

Imaging Radiation-Induced Gastrointestinal, Bone Marrow Injury and Recovery Kinetics Using 18F-FDG PET.

Positron emission tomography using 18F-Fluro-deoxy-glucose (18F-FDG) is a useful tool to detect regions of inflammation in patients. We utilized this imaging technique to investigate the kinetics of gastrointestinal recovery after radiation exposure and the role of bone marrow in the recovery process. Male Sprague-Dawley rats were either sham irradiated, irradiated with their upper half body shielded (UHBS) at a dose of 7.5 Gy, or whole body irradiated (WBI) with 4 or 7.5 Gy. Animals were imaged using 18F-FDG PET/CT at 5, 10 and 35 days post-radiation exposure. The gastrointestinal tract and bone marrow were analyzed for 18F-FDG uptake. Tissue was collected at all-time points for histological analysis. Following 7.5 Gy irradiation, there was a significant increase in inflammation in the gastrointestinal tract as indicated by the significantly higher 18F-FDG uptake compared to sham. UHBS animals had a significantly higher activity compared to 7.5 Gy WBI at 5 days post-exposure. Animals that received 4 Gy WBI did not show any significant increase in uptake compared to sham. Analysis of the bone marrow showed a significant decrease of uptake in the 7.5 Gy animals 5 days post-irradiation, albeit not observed in the 4 Gy group. Interestingly, as the metabolic activity of the gastrointestinal tract returned to sham levels in UHBS animals it was accompanied by an increase in metabolic activity in the bone marrow. At 35 days post-exposure both gastrointestinal tract and bone marrow 18F-FDG uptake returned to sham levels. 18F-FDG imaging is a tool that can be used to study the inflammatory response of the gastrointestinal tract and changes in bone marrow metabolism caused by radiation exposure. The recovery of the gastrointestinal tract coincides with an increase in bone marrow metabolism in partially shielded animals. These findings further demonstrate the relationship between the gastrointestinal syndrome and bone marrow recovery, and that this interaction can be studied using non-invasive imaging modalities.

Radiation-Induced Growth Retardation and Microstructural and Metabolite Abnormalities in the Hippocampus.

Cranial radiotherapy (CRT) increases survival in pediatric brain-tumor patients but can cause deleterious effects. This study evaluates the acute and long-term impact of CRT delivered during childhood/adolescence on the brain and body using a rodent model. Rats received CRT, either 4 Gy fractions × 5 d (fractionated) or a cumulative dose of 20 Gy (single dose) at 28 d of age. Animals were euthanized 1 d, 5 d, or 3.5 mo after CRT. The 3.5 mo group was imaged prior to euthanasia. At 3.5 mo, we observed significant growth retardation in irradiated animals, versus controls, and the effects of single dose on brain and body weights were more severe than fractionated. Acutely single dose significantly reduced body weight but increased brain weight, whereas fractionation significantly reduced brain but not body weights, versus controls. CRT suppressed cell proliferation in the hippocampal subgranular zone acutely. Fractional anisotropy (FA) in the fimbria was significantly lower in the single dose versus controls. Hippocampal metabolite levels were significantly altered in the single dose animals, reflecting a heightened state of inflammation that was absent in the fractionated. Our findings indicate that despite the differences in severity between the doses they both demonstrated an effect on cell proliferation and growth retardation, important factors in pediatric CRT.

Mapping Radiation Injury and Recovery in Bone Marrow Using 18F-FLT PET/CT and USPIO MRI in a Rat Model.

UNLABELLED: We present and test the use of multimodality imaging as a topological tool to map the amount of the body exposed to ionizing radiation and the location of exposure, which are important indicators of survival and recovery. To achieve our goal, PET/CT imaging with 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) was used to measure cellular proliferation in bone marrow (BM), whereas MRI using ultra-small superparamagnetic iron oxide (USPIO) particles provided noninvasive information on radiation-induced vascular damage. METHODS: Animals were x-ray-irradiated at a dose of 7.5 Gy with 1 of 3 radiation schemes-whole-body irradiation, half-body shielding (HBS), or 1-leg shielding (1LS)-and imaged repeatedly. The spatial information from the CT scan was used to segment the region corresponding to BM from the PET scan using algorithms developed in-house, allowing for quantification of proliferating cells, and BM blood volume was estimated by measuring the changes in the T2 relaxation rates (ΔR2) collected from MR scans. RESULTS: (18)F-FLT PET/CT imaging differentiated irradiated from unirradiated BM regions. Two days after irradiation, proliferation of 1LS animals was significantly lower than sham (P = 0.0001, femurs; P < 0.0001, tibias) and returned to sham levels by day 10 (P = 0.6344, femurs; P = 0.3962, tibias). The degree of shielding affected proliferation recovery, showing an increase in the irradiated BM of the femurs, but not the tibias, of HBS animals when compared with 1LS (P = 0.0310, femurs; P = 0.5832, tibias). MRI of irradiated spines detected radiation-induced BM vascular damage, measured by the significant increase in ΔR2 2 d after whole-body irradiation (P = 0.0022) and HBS (P = 0.0003) with a decreasing trend of values, returning to levels close to baseline over 10 d. Our data were corroborated using γ-counting and histopathology. CONCLUSION: We demonstrated that (18)F-FLT PET/CT and USPIO MRI are valuable tools in mapping regional radiation exposure and the effects of radiation on BM. Analysis of the (18)F-FLT signal allowed for a clear demarcation of exposed BM regions and elucidated the kinetics of BM recovery, whereas USPIO MRI was used to assess vascular damage and recovery.

Radiation combined injury models to study the effects of interventions and wound biomechanics.

In the event of a nuclear detonation, a considerable number of projected casualties will suffer from combined radiation exposure and burn and/or wound injury. Countermeasure assessment in the setting of radiation exposure combined with dermal injury is hampered by a lack of animal models in which the effects of interventions have been characterized. To address this need, we used two separate models to characterize wound closure. The first was an open wound model in mice to study the effect of wound size in combination with whole-body 6 Gy irradiation on the rate of wound closure, animal weight and survival (morbidity). In this model the addition of interventions, wound closure, subcutaneous vehicle injection, topical antiseptic and topical antibiotics were studied to measure their effect on healing and survival. The second was a rat closed wound model to study the biomechanical properties of a healed wound at 10 days postirradiation (irradiated with 6 or 7.5 Gy). In addition, complete blood counts were performed and wound pathology by staining with hematoxylin and eosin, trichrome, CD68 and Ki67. In the mouse open wound model, we found that wound size and morbidity were positively correlated, while wound size and survival were negatively correlated. Regardless of the wound size, the addition of radiation exposure delayed the healing of the wound by approximately 5-6 days. The addition of interventions caused, at a minimum, a 30% increase in survival and improved mean survival by ∼9 days. In the rat closed wound model we found that radiation exposure significantly decreased all wound biomechanical measurements as well as white blood cell, platelet and red blood cell counts at 10 days post wounding. Also, pathological changes showed a loss of dermal structure, thickening of dermis, loss of collagen/epithelial hyperplasia and an increased density of macrophages. In conclusion, we have characterized the effect of a changing wound size in combination with radiation exposure. We also demonstrated that the most effective interventions mitigated insensible fluid loss, which could help to define the most appropriate requirements of a successful countermeasure.

Neurogenesis, exercise, and cognitive late effects of pediatric radiotherapy.

Brain cancer is a common type of childhood malignancy, and radiotherapy (RT) is a mainstay of treatment. RT is effective for tumor eradication, and survival rates are high. However, RT damages the brain and disrupts ongoing developmental processes, resulting in debilitating cognitive "late" effects that may take years to fully manifest. These late effects likely derive from a long-term decrement in cell proliferation, combined with a neural environment that is hostile to plasticity, both of which are induced by RT. Long-term suppression of cell proliferation deprives the brain of the raw materials needed for optimum cognitive performance (such as new neurons in the hippocampus and new glia in frontal cortex), while chronic inflammation and dearth of trophic substances (such as growth hormone) limit neuroplastic potential in existing circuitry. Potential treatments for cognitive late effects should address both of these conditions. Exercise represents one such potential treatment, since it has the capacity to enhance cell proliferation, as well as to promote a neural milieu permissive for plasticity. Here, we review the evidence that cognitive late effects can be traced to RT-induced suppression of cell proliferation and hostile environmental conditions, as well as emerging evidence that exercise may be effective as an independent or adjuvant therapy.

Vascular-targeted photothermal therapy of an orthotopic murine glioma model.

AIM: To develop nanoshells for vascular-targeted photothermal therapy of glioma. MATERIALS & METHODS: The ability of nanoshells conjugated to VEGF and/or poly(ethylene glycol) (PEG) to thermally ablate VEGF receptor-2-positive endothelial cells upon near-infrared laser irradiation was evaluated in vitro. Subsequent in vivo studies evaluated therapy in mice bearing intracerebral glioma tumors by exposing tumors to near-infrared light after systemically delivering saline, PEG-coated nanoshells, or VEGF-coated nanoshells. The treatment effect was monitored with intravital microscopy and histology. RESULTS: VEGF-coated but not PEG-coated nanoshells bound VEGF receptor-2-positive cells in vitro to enable targeted photothermal ablation. In vivo, VEGF targeting doubled the proportion of nanoshells bound to tumor vessels and vasculature was disrupted following laser exposure. Vessels were not disrupted in mice that received saline. The normal brain was unharmed in all treatment and control mice. CONCLUSION: Nanoshell therapy can induce vascular disruption in glioma.

Effects of irradiation on brain vasculature using an in situ tumor model.

PURPOSE: Damage to normal tissue is a limiting factor in clinical radiotherapy (RT). We tested the hypothesis that the presence of tumor alters the response of normal tissues to irradiation using a rat in situ brain tumor model. METHODS AND MATERIALS: Intravital microscopy was used with a rat cranial window to assess the in situ effect of rat C6 glioma on peritumoral tissue with and without RT. The RT regimen included 40 Gy at 8 Gy/day starting Day 5 after tumor implant. Endpoints included blood-brain barrier permeability, clearance index, leukocyte-endothelial interactions and staining for vascular endothelial growth factor (VEGF) glial fibrillary acidic protein, and apoptosis. To characterize the system response to RT, animal survival and tumor surface area and volume were measured. Sham experiments were performed on similar animals implanted with basement membrane matrix absent of tumor cells. RESULTS: The presence of tumor alone increases permeability but has little effect on leukocyte-endothelial interactions and astrogliosis. Radiation alone increases tissue permeability, leukocyte-endothelial interactions, and astrogliosis. The highest levels of permeability and cell adhesion were seen in the model that combined tumor and irradiation; however, the presence of tumor appeared to reduce the volume of rolling leukocytes. Unirradiated tumor and peritumoral tissue had poor clearance. Irradiated tumor and peritumoral tissue had a similar clearance index to irradiated and unirradiated sham-implanted animals. Radiation reduces the presence of VEGF in peritumoral normal tissues but did not affect the amount of apoptosis in the normal tissue. Apoptosis was identified in the tumor tissue with and without radiation. CONCLUSIONS: We developed a novel approach to demonstrate that the presence of the tumor in a rat intracranial model alters the response of normal tissues to irradiation.

Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II.

INTRODUCTION: Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA). METHODS: We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence. RESULTS: We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint. CONCLUSIONS: Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.

Radiation-induced astrogliosis and blood-brain barrier damage can be abrogated using anti-TNF treatment.

PURPOSE: In this article, we investigate the role of tumor necrosis factor-alpha (TNF) in the initiation of acute damage to the blood-brain barrier (BBB) and brain tissue following radiotherapy (RT) for CNS tumors. METHODS AND MATERIALS: Intravital microscopy and a closed cranial window technique were used to measure quantitatively BBB permeability to FITC-dextran 4.4-kDa molecules, leukocyte adhesion (Rhodamine-6G) and vessel diameters before and after 20-Gy cranial radiation with and without treatment with anti-TNF. Immunohistochemistry was used to quantify astrogliosis post-RT and immunofluorescence was used to visualize protein expression of TNF and ICAM-1 post-RT. Recombinant TNF (rTNF) was used to elucidate the role of TNF in leukocyte adhesion and vessel diameter. RESULTS: Mice treated with anti-TNF showed significantly lower permeability and leukocyte adhesion at 24 and 48 h post-RT vs. RT-only animals. We observed a significant decrease in arteriole diameters at 48 h post-RT that was inhibited in TNF-treated animals. We also saw a significant increase in activated astrocytes following RT that was significantly lower in the anti-TNF-treated group. In addition, immunofluorescence showed protein expression of TNF and ICAM-1 in the cerebral cortex that was inhibited with anti-TNF treatment. Finally, administration of rTNF induced a decrease in arteriole diameter and a significant increase in leukocyte adhesion in venules and arterioles. CONCLUSIONS: TNF plays a significant role in acute changes in BBB permeability, leukocyte adhesion, arteriole diameter, and astrocyte activation following cranial radiation. Treatment with anti-TNF protects the brain's microvascular network from the acute damage following RT.