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

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.

Grant Number: 1R56EY035223-01
NIH Institute/Center: NIH
Principal Investigator: Shyam Chaurasia