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Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. ...

University of Pittsburgh Cancer Institute

Mitochondrial-targeted gramicidin S (GS)-nitroxide, JP4-039, has been demonstrated to be a potent radiation mitigator, and safe over a wide dose range. In addition, JP4-039 has organ-specific effectiveness when locally applied.

Effectiveness of Analogs of the GS-Nitroxide, JP4-039, as Total Body Irradiation Mitigators.

The polycythemia form of Friend leukemia virus (FVP) causes splenomegaly and lethal erythroleukemia in Fv-2-susceptible mouse strains. We sought to determine whether the hematopoietic stem cell (HSC) pool was expanded in Fv-2-resistant mice.

Reduced Competitive Repopulation Capacity of Multipotential Hematopoietic Stem Cells in the Bone Marrow of Friend Virus-infected Fv2-resistant Mice.

The acute lethality of total-body irradiation (TBI) involves damage to multiple organs, including bone marrow and intestine. Ionizing radiation mitigators that are effective when delivered 24 h or later after TBI include the anti-apoptotic drug, JP4-039 and the anti-necroptotic drug, necrostatin-1. In contrast to effective delivery of JP4-039 at 24 h after TBI, necrostatin-1 is most effective when delivery is delayed until 48 h, a time that correlates with the elevation of necroptosis-inducing inflammatory cytokines and necroptosis-induced serine phosphorylation of receptor-interacting serine/threonine-protein kinase-3 (RIP3) in tissues. The goal of this work was to determine whether administration of JP4-039 influenced the optimal delivery time for necrostatin-1. We measured daily levels of 33 proteins in plasma compared to intestine and bone marrow of C57BL/6NTac female mice over a 7-day time period after 9.25 Gy TBI (LD). Protein responses to TBI in plasma were different from those measured in intestine or bone marrow. In mice that were given JP4-039 at 24 h after TBI, we delayed necrostatin-1 delivery for 72 h after TBI based on measured delay in RIP-3 kinase elevation in marrow and intestine. Sequential delivery of these two radiation mitigator drugs significantly increased survival compared to single drug administration.

Improved Total-Body Irradiation Survival by Delivery of Two Radiation Mitigators that Target Distinct Cell Death Pathways.

Squamous cell carcinomas of the head and neck are appearing with increased frequency in both marrow transplanted and non-transplanted Fanconi anemia (FA) patients. FA patients commonly display radiosensitivity of epithelial tissues, complicating effective radiotherapy. Fancd2 mice (C57BL/6J and 129/Sv background) demonstrate epithelial tissue sensitivity to single-fraction or fractionated irradiation to the head and neck and distant marrow suppression (abscopal effect), both ameliorated by intraoral administration of the mitochondrial-targeted antioxidant, GS-nitroxide, JP4-039. We now report that mice of two other FA genotypes, Fancg (B6) and the most prevalent human genotype Fanca (129/Sv), also demonstrate: 1. reduced longevity of hematopoiesis in long-term bone marrow cultures; 2. radiosensitivity of bone marrow stromal cell lines; and 3. head and neck radiation-induced severe mucositis and abscopal suppression of distant marrow hematopoiesis. Intraoral administration of JP4-039/F15, but not non-mitochondrial-targeted 4-amino-Tempo/F15 or F15 alone, prior to each radiation treatment ameliorated both local and abscopal radiation effects. Head and neck irradiated TGF-β-resistant SMAD3 (129/Sv) mice and double-knockout SMAD3 Fancd2 (129/Sv) mice treated daily with TGF-β receptor antagonist, LY364947, still displayed abscopal bone marrow suppression, implicating a non-TGF-β mechanism. Thus, amelioration of both local normal tissue radiosensitivity and distant marrow suppression by intraoral administration of JP4-039 in Fancg and Fanca mice supports a clinical trial of this locally administered normal tissue radioprotector and mitigator during head and neck irradiation in FA patients.

Amelioration of Head and Neck Radiation-Induced Mucositis and Distant Marrow Suppression in Fanca and Fancg Mice by Intraoral Administration of GS-Nitroxide (JP4-039).

In this study, a methodology was evaluated and improved to quickly measure the tensile strength of murine skin in a biomechanical assay for an incisional wound healing model. The aim was to streamline and enhance the wound model, skin specimen preparation, and tensile test so that large numbers of fresh tissue could be tested reliably and rapidly. Linear incisions of 25-mm length were made in the dorsal skin of mice along the spine and metallic staples were used to close the wound. After 20 days, the mice were sacrificed, and a square-shaped section of skin containing the linear incision was excised. Two metallic punches were fabricated and used to punch 15-mm long strips of skin of 2 mm width whose length was orthogonal to the direction of incision. The tensiometer configuration was modified to expedite tensile measurements on fresh skin, and load-to-failure was measured for each strip of skin from the cephalad to the caudal region. We evaluated sources of error in the animal model and the testing protocol and developed procedures to maximize speed and reproducibility in tensile strength measurements. This report provides guidance for efficient and reproducible tensile strength measurement of large numbers of skin specimens from freshly sacrificed animals. •Tattoo placement to identify the two ends of the healing incisional wound assisted in decreasing error in the position and orientation of tensile strips.•Custom-made punches to prepare skin strips for tensile testing helped conduct tensile tests of fresh tissue rapidly.•Alteration of the manual grips of the tensile tester enabled specimens to be gripped rapidly to significantly accelerate testing for each skin strip.

Renee Fisher

Department of Radiation Oncology

University of Pittsburgh Cancer Institute

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis