Mechanistic Understanding of Mustard Gas Toxicity in the Retina using a Minipig Model

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ABSTRACT Sulfur mustard gas (SM) has been used repeatedly as a chemical weapon in the last 100 years, most recently in Syria in 2016. Acute ocular injury from SM includes severe ocular pain, corneal abnormalities, photophobia, chemosis, and blindness. Late chronic toxicity also includes mustard gas keratopathy, vascular tortuosity, limbal ischemia, and retinal damage. At present, no comprehensive animal study describing early and late SM toxicity to retinal function exists. A recent human clinical study reported a significant reduction in retinal function in Iranian veterans exposed to SM, exhibiting defects in the inner layers of the retina, bipolar cells, and Müller glial cells. We observed in vivo damage to rabbit retina seen in Iranian veterans exposed to SM while conducting our corneal counteract studies funded through 1U01EY031650 grant. Pilot rabbit in vivo data was verified with a controlled in vitro model of human Müller glial cells (MIO-M1), depictingSM toxicity in retina involves inflammatory milieu, oxidative stress, and cell death via caspase-1/NLRP3 pyroptosis. Collectively, preliminary data collected from rabbit in vivo and Müller glial cell in vitro studies suggested that Müller glial cells exposed to mustard gas caused severe time- and dose-dependent gliosis in rabbit eyes exposed to SM, and Müller glial cell in vitro. A major knowledge gap in the field is the lack of understanding mechanism of SM toxicity in the neural retina. The human clinical, rabbit in vivo and Müller cell in vitro studies led to a central hypothesis that SM exposure to the eye causes biphasic damage to the retina actuating short-term innate immune responses via hyperactivation of glial cells and long-term neurodegenerative functional malady via defective mitophagy. This proposal tests two novel hypotheses: (a) SM disrupts spatial neural framework and causes retinal dysfunction in vivo in a minipig model and (b) SM causes innate immune response via activation of NLRP3 inflammasome and defective mitophagy in retinal microglia and Müller glia cells and leads retinal neurodegeneration in minipig eye. Two specific aims test these hypotheses using in vivo Göttingen minipig SM toxicity model in collaboration with MRI Global, Kansas City, MO and a primary pig microglia (pMicroglia) culture model (published/established in our lab) and commercially available human Müller glial cells (MIO-M1 cells). Aim-1 will characterize in vivo clinical, structural, and functional changes in the retina of a minipig model of mustard gas toxicity using state-of-the-art multi-modal 2D/3D clinical imaging tools and functional assays. Aim- 2 will identify molecular targets involved in retinal degenerative mechanisms initiated by the activation of innate immune responses and defective mitophagy using cellular and biochemical approaches using in vitro models of retinal microglia/Müller glial cells and in vivo Göttingen minipig retina. The successful completion of this project will provide necessary knowledge about retinal neural architecture and neuronal function, retinal neurodegenerative, innate immune responses, and defective mitophagy after SM exposure. Our team has extensive experience, skills, joint publications and grants in the ocular CounterACT field.