Preserving Retinal Structure and Function with the Novel Nitroxide Antioxidant, DCTEIO

**Background:** Oxidative stress and neuroinflammation are key contributors to retinal neurodegenerative diseases such as glaucoma, age-related macular degeneration, and diabetic retinopathy. Nitroxide antioxidants, like TEMPOL, have shown promise in modulating oxidative stress and inflammation. The novel nitroxide DCTEIO (5,6-dicarboxy-1,1,3,3-tetraethylisoindolin-2-yloxyl) has previously demonstrated short-term ROS-scavenging ability in vivo. This study investigates the long-term (8-day) neuroprotective effects of DCTEIO in a rat model of retinal ischemia/reperfusion (I/R) injury and establishes a flow cytometry-based method to quantify oxidative stress in cultured photoreceptor cells.

**Methods:** Female Sprague–Dawley rats (8 weeks old, ~250 g) were divided into six groups: (i) IP vehicle, (ii) sham I/R vehicle, (iii) acute I/R vehicle, (iv) IP DCTEIO, (v) sham I/R DCTEIO, and (vi) acute I/R DCTEIO. DCTEIO (20 mg/kg IP) was administered 1 hour before and 1 hour after I/R injury; an intraocular injection (250 µM) was given 30 min into reperfusion. Retinal function was assessed by full-field electroretinography (ERG) on day 0 (pre-treatment) and day 8 (post-treatment). Glial activation was evaluated via GFAP immunohistochemistry, and microglial reactivity via IBA-1 immunohistochemistry with quantification of ramified and activated microglia in the inner retina (IR), outer nuclear layer (ONL), and photoreceptor segments (PRS). Retinal structure was examined by hematoxylin-eosin staining. In vitro, 661W photoreceptor cells were loaded with the redox-sensitive probe ME-TRN (100 nM), treated with DCTEIO or lutein (0.1–500 µM) for 30 min, then exposed to antimycin (AMC, 1 µM) for 15 min. Mean fluorescence intensity (MFI) was measured by flow cytometry.

**Key Results:** ERG showed that DCTEIO did not affect normal retinal function (IP alone: a-wave p=0.4590, b-wave p=0.4892; sham I/R: a-wave p=0.6606, b-wave p=0.9467). In vehicle-treated acute I/R rats, significant reductions in a-wave (647±86 µV vs 275±40 µV, p=0.0028) and b-wave (1534±150 µV vs 580±122 µV, p=0.0006) amplitudes were observed at day 8. DCTEIO preserved a-wave (449±29 µV vs 463±43 µV, p=0.7894) and b-wave (1095±58 µV vs 942±122 µV, p=0.2815) amplitudes, and partially protected cone b-wave (80±12 µV vs 111±18 µV, p=0.2807) and oscillatory potentials (155±21 µV vs 295±64 µV pre-treatment, p=0.06). GFAP expression was significantly upregulated in acute I/R vehicle retinas (p=0.0011) and significantly reduced by DCTEIO (p=0.0004). IBA-1 staining revealed that acute I/R increased total microglial numbers (109.6±1.45 vs 32.26±2.46 in sham, p<0.0001), and DCTEIO significantly reduced both ramified (61.00±1.89 vs 42.22±2.26, p<0.0001) and activated (48.59±1.406 vs 25.95±4.376, p<0.0001) microglia. DCTEIO also reduced microglial numbers in IR, ONL, and PRS to levels not significantly different from sham. Histology confirmed preservation of retinal structure with DCTEIO. In vitro, DCTEIO at 500 µM significantly mitigated AMC-induced reduction in ME-TRN fluorescence (64.56±13.47% mitigation, p=0.0002).

**Clinical Implications:** DCTEIO demonstrates robust long-term neuroprotection in a rat model of retinal I/R injury, preserving both function and structure while suppressing glial activation and microglial reactivity. Its antioxidant and anti-inflammatory properties, combined with a catalytic mechanism of action, make it a promising candidate for treating retinal diseases driven by oxidative stress and neuroinflammation, such as glaucoma, AMD, and diabetic retinopathy. The flow cytometry method using ME-TRN provides a rapid, quantitative tool for screening antioxidant efficacy in retinal cells.