Abstract
Purpose :
Neuroinflammation is closely associated with neurodegeneration, and is a major cause of vision impairment in blinding diseases such as diabetic retinopathy (DR). Spermine oxidase (SMOX) is a major enzyme in the polyamine oxidation pathway. Previous findings from our laboratory have shown that treatment with MDL 72527, a selective inhibitor for SMOX, reduced microglial activation and upregulated antioxidant signaling in a mouse model of retinal excitotoxicity. In the present study, using the same model, we investigated the impact of SMOX inhibition on the status of inflammatory cells, cytokine levels, and oxidative damage.
Methods :
Mice (8-10 weeks old) received 20 nmoles of NMDA (N-Methyl-D-aspartate) or NMLA (N-Methyl-L-aspartate, control) intravitreally. 40 mg/kg/day of MDL 72527 or vehicle (normal saline) was given to mice through intraperitoneal injections. Retinal cryostat sections were prepared for immunostaining and fresh frozen retinal samples were used for Western blotting or qRT-PCR studies. C8-B4 cells were used for studying the impact of conjugated acrolein in vitro on microglia cells.
Results :
Immunostaining of retinal sections showed a significant increase in the number of inflammatory cells of M1 phenotype, positive for CD (Cluster Differentiation) 68 and CD16/32 in excitotoxicity-induced retinas, while MDL 72527 treatment markedly reduced these changes (N=3-5, P<0.05). SMOX inhibition with MDL 72527 upregulated the number of M2 phenotype cells stained with arginase1 and CD206 (N=4-6, P<0.05). While retinal excitotoxicity upregulated the level of several inflammatory cytokines, MDL 72527 treatment significantly reduced many of these molecules including IL-1β, TNF-α, CCL3, and IL-21 (N = 6-8, P< 0.05). Excitotoxicity-induced upregulation in protein-conjugated acrolein (an indicator of SMOX activation and oxidative damage) was also reduced in response to SMOX inhibition (N=5-6, P<0.05). In-vitro studies using C8-B4 microglia cells showed changes in cellular morphology and an increase in reactive oxygen species formation in response to conjugated acrolein treatment.
Conclusions :
Our findings indicate that the inhibition SMOX pathway reduced neuroinflammation and oxidative damage, and suggests its potential as a therapeutic target to treat neurodegenerative diseases of the eye.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.