Abstract
Purpose :
Neuroinflammation in the retina is a major cause of vision impairment in blinding diseases such as diabetic retinopathy (DR). Previous studies from our laboratory have shown that inhibition of spermine oxidase (SMOX, a member of polyamine oxidase family) using the pharmacological inhibitor MDL 72527 reduced neurodegeneration in models of retinal excitotoxicity and DR. Utilizing the experimental model of retinal excitotoxicity, the present study was undertaken to determine the impact of SMOX blockade in retinal neuroinflammation.
Methods :
Adult mice (8-10 weeks old) were given intravitreal injections (20 nmoles) of NMDA (N-Methyl-D-aspartate) or NMLA (N-Methyl-L-aspartate, control). Intraperitoneal injections of MDL 72527 (40 mg/kg body weight/day) or vehicle (normal saline) were given to NMDA and NMLA treatment groups. Retinal flat mounts or cryostat sections were prepared for immunostaining and fresh frozen retinal samples were used for Western blotting studies. NIH Image J was used for quantitation.
Results :
Immunofluorescence staining of retinal flat mounts using Iba1 (Ionized calcium-binding adaptor molecule 1) antibody was utilized to study activation of microglia. An increase in retinal microglia presenting activated morphology was observed in NMDA retinas (7 days post injury) compared to their NMLA controls. Treatment with MDL 72527 reduced this effect in the NMDA retinas. Quantification studies demonstrated that excitotoxicity–induced upregulation of Iba1 positive cells with activated morphology was significantly reduced in response to SMOX blockade (N=5, p<0.01). The increased protein levels of Iba1 in NMDA retinas were reduced in response to MDL 72527 treatment (p<0.01, N=4). Excitotoxicity-induced upregulation in the number of CD 68 (Cluster of Differentiation 68) positive cells was significantly decreased in response to SMOX inhibition (N=4, p<0.05, 3 days post injury). Analysis of molecular pathways revealed a significant increase in NRF2 expression in MDL 72527 treated excitotoxic retinas compared to respective vehicle group (p<0.05, N=3, 3 days post injury), suggesting the involvement of antioxidant signaling in response to SMOX blockade.
Conclusions :
Our study suggests the critical involvement of SMOX signaling in retinal neuroinflammation thus offering a new therapeutic target for vision disorders.
This is a 2021 ARVO Annual Meeting abstract.