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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. Further-more, microgliosis (P < .05), astrogliosis (P < .01), and myelin thinning (P

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.

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.

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.

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.

266 Objectives Salivary gland dysfunction is one of the serious adverse effects of radioactive iodine (RAI) therapy in thyroid cancer patients but there is no established nuclear imaging tool for the quantitative assessment of the salivary gland dysfunction. Here, we attempted to apply the quantitative parameters of single-photon emission computed tomography/computed tomography (SPECT/CT) post injection of Tc-99m to the patients with RAI therapy. Methods Twenty-three patents with well-differentiated thyroid cancer (age, 44.8±11.5y, male:female=9:14) underwent Tc-99m (5~15mCi) quantitative SPECT/CT before (n=18) or 1 yr after (n=5) RAI therapy (dose=116.0±32.1mCi) from August to November 2015. Of the 18 patients who had had the SPECT/CT before the RAI therapy, 8 patients (m:f=5:3, age, 50.6±7.3y) underwent the follow-up SPECT/CT 3 weeks post RAI (dose=118.8±25.9mCi) with the traditional planar salivary gland scintigraphy. The SPECT/CT protocol consisted of the 1st SPECT/CT around 20 min post Tc-99m injection and the second SPECT/CT post sialagogue stimulation around 40 min post Tc-99m injection. %injected dose (%ID) and standardized uptake value (SUV) were obtained for parotid and submandibular glands. %excretion was calculated as 100 × (%ID at the first SPECT/CT - %ID at the second SPECT/CT)/%ID at the first SPECT/CT. The arithmetic mean value of the right and the left salivary glands was used for the statistical analyses. Results The 5 patients with RAI 1yr ago had significantly lower %ID of parotid (0.23±0.12% vs. 0.52±0.17%, p=0.0022), SUVmean of parotid (7.04±3.10 vs. 12.63±5.36, p=0.0253), and SUVmax of parotid (16.24±6.32 vs. 31.28±14.97, p=0.0209) than the 18 patients prior to the RAI therapy. However, submandibular gland function was not significantly different between the 5 patients with RAI 1yr ago and the 18 patients without RAI (0.19±0.20% vs. 0.20±0.10%, p=0.4561 for %ID; 11.60±10.10 vs. 10.41±3.00, p=0.5023 for SUVmean; and 27.33±27.64 vs. 20.29±6.20, p=0.5023 for SUVmax). On the other hand, the 8 patients who had had the SPECT/CT before and 3 weeks post the RAI therapy, showed a significantly increase of %excretion of parotid glands from before (70.74±4.17%) to after (75.09±5.36%, p=0.0078) RAI therapy, whereas %excretion of submandibular glands was not significantly changed (60.18±11.60% vs. 56.15±14.92, p=0.5469). The corresponding planar scintigraphy parameters (52.27±12.14% vs. 55.63±8.78%, p=0.5469 for %excretion of parotid; and 33.36±7.07% vs. 30.15±12.91%, p=0.8438 for %excretion of submandibular) did not show any difference between before and after the RAI therapy. Conclusions The dysfunction of salivary glands post RAI therapy was readily demonstrated in the parotid glands using the quantitative parameters of SPECT/CT. Tc-99m quantitative SPECT/CT may displace the planar salivary scintigraphy because of its high sensitivity for the salivary dysfunction post RAI therapy.

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.

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.

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. 1 H magnetic resonance spectroscopy ( 1 H-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 1 H-MRS showed significant reduction in taurine levels ( P 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).

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. Original submitted 27 September 2011; Revised submitted 12 December 2011; Published online 14 May 2012

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.

Radiotherapy (RT) induced hypoxia recruits bone marrow-derived cells (BMDCs) to tumors through the upregulation of Stromal Derived Factor-1, a chemokine that binds to receptor CXCR4 present on BMDCs. The infiltration of BMDCs, which includes endothelial progenitors, pericyte progenitors, tumor-associated macrophages, immature monocytes, VEGFR+ hemangiocytes, and CD11b+ myeloid cells has been implicated in supporting tumor establishment by contributing to tumor growth, angiogenesis, vasculogenesis, inflammation, and immune suppression. In this work, we have used a rodent glioma in situ allograft model to study the recruitment/migration of BMDCs post RT. Female albino C57 (8-10 weeks old, Jackson Laboratories) animals underwent +DsRed bone marrow transplant (B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J donor mouse). Following engraftment, glioma cells were cranially implanted with 1x105 glioma cells (GL261-Luc, Perkin Elmer), at an average size of 26.4mm3 indicated by a bioluminescence signal of 5.28x108 photons/sec/cm2/sr, animals underwent 6Gy (RadSource 2000, 250kVp, 25mA) of irradiation limited to a 1cm wide slit centered on the tumor. Bioluminescence (tumor size) and fluorescence imaging (+DsRed BMDCs) was performed prior to and at 1- and 2-weeks post-RT to assess tumor growth and measure the influx of +DsRed BMDCs. In addition, flow cytometry was used to measure the number of +DsRed BMDCs in the tumor and brain. To study the effect of BMDCs in tumor regression, tumors were irradiated at a smaller size (9.3mm3) with 10Gy irradiation. At 1-week post-RT, flow cytometry was used to measure the number of BMDCs (CD45+) in the tumor. In our model 6Gy radiation was able to stall the growth of the tumor. Irradiated tumors did not significantly change in tumor size at either 1- or 2-weeks post irradiation compared to pre-RT size. In addition, irradiated tumors were significantly smaller than untreated tumors (p=0.0008) 1-week post-RT. Using flow cytometry 1-week post irradiation, we observed a significant increase in the number of +DsRed BMDCs in the irradiated tumor (23.5% of cells +DsRed BMDCs) compared to the irradiated brain (6.17% cells +DsRed BMDCs, p=0.03) as well as a significant increase in the tumors (16.6% cells +DsRed BMDCs) compared to the brain (11.1% cells +DsRed BMDCs p=0.02). There was no difference in the number of +DsRed BMDCS in tumors ± irradiation or brains ± irradiation. We found similar results in our tumor regression model using flow cytometry 1-week post-RT; we observed an increase in the number of BMDCs CD45+ in the tumor (~20% of cells CD45+) compared to control tumors (~6% of cells CD45+). Indicating that tumor size at the onset of RT did not effect the migration of BMDCs. Fluorescence imaging did not measure any significant change in the DsRed signal at 1- or 2-weeks post-irradiation. We have successfully shown that post-RT there is an increase in BMDCs migration at 1-week post RT regardless of tumor size at RT. Further experimentation is needed to better elucidate the role of BMDCs in tumor inflammation and immune suppression as well as angiogenesis and vasculogenesis. Our next step is to perform immunohistochemistry on our collected tumor tissue to map out the spatial location of migrated BMDCs within the tumor. Citation Format: Janice A. Zawaski, Tien T. Tang, M Waleed Gaber. A rodent in situ glioma allograft model to study bone marrow-derived cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A59.

Background The islets of Langerhans are micro-organs rich in blood vessels. The process of islet isolation and culture disrupts the vasculature of the islets. The reestablishment of an appropriate microvascular supply is an essential prerequisite for long-term survival and function of islet grafts. In this study, we examined the effects on the process of neovascularization of coating the islets with fibrin. Methods Isolated human islets were stained using the dioctadecylindocarbocyanine (DII) dye. An aliquot of the human islets were embedded in 3-dimensional fibrin. Human islets (100 islets-equivalents) were transplanted into a mouse dorsal window model to evaluate angiogenesis over 17 days. Transplanted islets were divided into 2 groups: either free islets or islets coated with fibrin gel. Animals were imaged using intravital microscopy immediately and at 3, 4, 8, 11, and 17 days after surgery. The DII dye caused the islets to be fluorescent and visible using a rhodamine filter. Fluorescein isothiocyanate dextran was used to visualize vasculature structures surrounding the islets. Results Human islets coated with fibrin demonstrated an early appearance of a network of immature blood vessels that produced a significantly higher density/unit area for neovascularization by day 8 after transplantation. Conclusion Our preliminary data showed that fibrin played a role in early neovascularization and support to sustain development of new blood vessels. Fibrin formed a matrix that helped to maintain the 3-dimensional structure of, and therefore reducing the environmental stress on islets.

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.

This study has developed a chitosan-based delivery system to locally administer ellagic acid for brain cancer treatment. We fabricated chitosan/ellagic acid composite films with various concentrations of ellagic acid. In vitro release study was performed by using a UV spectrophotometer, and enzymatic degradation rate was determined by analyzing the increased free amino groups. Viability of brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was measured via direct and indirect cell culture on the films by MTS assay. Caspase-3 activation, Western blot for p53, and anti-angiogenesis assays were also examined. In the in vivo study, GFP-tagged rat C6 glioma cells were implanted subcutaneously at the right flank region of nude mice and treatments were initiated by implanting the films subcutaneously. Tumor growth was evaluated by measuring tumor volume using a caliper, an ultrasound machine, and an optical imaging system. The chitosan/ellagic acid composite films were enzymatically degradable and exhibited a sustained slow release of ellagic acid. These materials could inhibit the cancer cell growth in an ellagic acid concentration-dependent manner by inducing apoptosis of cancer cells as well as suppressing angiogenesis. These materials also significantly suppressed tumor tissue growth in vivo.

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.

Animal models have been used to quantify the genomic and functional changes post-radiotherapy in brain tissue. However, in these models the effects of tumor presence during radiotherapy is not taken into account. We have recently measured significant genomic changes in the rat brain tissue that was occupied by the tumor post-radiotherapy compared to normal irradiated tissue. In our current work we are characterizing the long term functional changes caused by the presence of the tumor during radiotherapy. Sprague Dawley male rats (7wks) were divided between three experimental groups: Sham implant, RT+sham implant, and RT+Tumor (C6-GFP tumor implant glioma). Hypofractionated irradiation (8-6Gy/day for 5 days) was localized to a 1cm strip of the cranium starting 5 days post implant, which resulted in a complete regression of the tumor and prolonged survival. The former tumor area was imaged 65 days post implant using a 9.4T Biospec MRI scanner (Bruker) with a 20cm bore using a quadrature rat brain array. 1H magnetic resonance spectroscopy (1H-MRS) was performed in the former tumor/implant region using a STEAM sequence with the following parameters: TR = 2s/TE = 2.22ms/ # of averages = 512/ voxel size 56mm3. Diffusion tensor imaging (DTI) was performed using a spin echo EPI sequence with a TR = 500ms, TE = 33.2ms, 2 repetitions, slice thickness = 12.8mm, b value = 800 s/mm2, 30 of directions, matrix size 128×256×16 giving a spatial resolution of 150×150×800mm. 1H MRS data was analyzed using LC Model and DTI was analyzed using Medinria. Intravital microscopy was also used to quantify blood brain-barrier (BBB) permeability and leukocyte activity. 1H-MRS revealed that both RT+sham implant and RT+C6-GFP tumor groups had a significant reduction in taurine levels (p In conclusion, using our tumor implant model to study the effect of radiation alone versus radiation combined with the presence of tumor, we have measured several functional changes post-radiotherapy. Our results indicate that radiation might be the most significant influence on neuronal fiber disruption and damage while tumor presence might play an important role in disrupting synaptic transmission as well as exasperating the inflammatory side effects of radiation. Citation Format: Janice A. Zawaski, Omaima M. Sabek, Kelvin Wong, M. Waleed Gaber. Effects of glioma presence during radiotherapy on tissue damage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3321. doi:10.1158/1538-7445.AM2015-3321

We have previously reported that a recombinant protein (M(r) 47 kDa), which contains both active peptide of platelet receptors for types I and III collagen inhibits both types I and III collagen-induced platelet aggregation. In order to eliminate non-reactive portion of the protein, we have constructed a recombinant of rHyB (M r 6 kDa). In addition, we chemically synthesized a hybrid peptide with 30 amino acid residues (cHyB, M r 3 kDa) that contains each of the active peptide derived from platelet receptors for types I and III collagen and a linker of 12 amino acid residues. In the present investigation, we report that both rHyB and cHyB inhibit type I and type III collagen-induced platelet aggregation, and the adhesion of radiolabeled platelets onto rabbit aortic segments in a dose-dependent manner. We have used an animal model, which employs FeCl3 to induce thrombi formation to study the effectiveness of both rHyb and cHyB on preventing thrombi formation. We obtained results that show that both rHyB and cHyB can inhibit thrombi formation in a dose-dependent manner. These results suggest that either rHyB or cHyB may be a possible therapeutic agent in preventing thrombi formation.

The Variable Resolution X-ray (VRX) CT system, developed at the UTHSC, Memphis, has the potential for use in animal imaging. Animal models of tumor progression and pharmacological impact are becoming increasingly important in understanding the molecular and mechanistic basis of tumor development. In general, CT-imaging offers several advantages in animal research: a fast throughput of seconds to minutes reducing the physiological stress animals are exposed to, and it is an inexpensive modality affordable to many animal laboratories. We are developing the VRX CT scanner as a non-invasive imaging modality to measure tumor volume, progression, and metastasis. From the axial images taken by the VRX CT-scanner, tumor area was measured and the tumor volume was calculated. Animals were also imaged using an optical liquid nitrogen-cooled CCD camera to detect tumor fluorescence. A simple image fusion with a planner x-ray image was used to ascertain the position of the tumors, animals were then sacrificed the tumors excised, and the tumor volume calculated by physical measurements. Furthermore, using a specially designed phantom with three spheres of different volumes, we demonstrated that our system allowed us to estimate the volume with up to 10% accuracy; we expect this to increase dramatically in the next few months.

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.