Is Iba-1 protein expression a sensitive marker for microglia activation in experimental diabetic retinopathy?
Authors:   Department of Ophthalmology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China; Department of Ophthalmology, Changzhou Laser Hospital, Changzhou 213000, Jiangsu Province, China, Shi Fan-Jun , Zhang Chao-Yang ,   Tongji Eye Institute, Department of Regenerative Medicine, and Department of Pharmacology, Tongji University School of Medicine, Shanghai 200092, China; Department of Ophthalmology, Shanghai General Hospital (Shanghai First People&#x2019;s Hospital), Shanghai Jiao Tong University, Shanghai 200080, China; National Clinical Research Center for Eye Diseases, Qin Hai-Feng ,   Department of Ophthalmology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China, Zeng Xin-Wei ,   Department of Ophthalmology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China, Lou Hui ,   Department of Ophthalmology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China, Zhang Lei ,   Department of Ophthalmology, Changzhou Laser Hospital, Changzhou 213000, Jiangsu Province, China, Xu Guo-Tong ,   Tongji Eye Institute, Department of Regenerative Medicine, and Department of Pharmacology, Tongji University School of Medicine, Shanghai 200092, China, Zhang Jing-Fa ,   Department of Ophthalmology, Shanghai General Hospital (Shanghai First People&#x2019;s Hospital), Shanghai Jiao Tong University, Shanghai 200080, China; National Clinical Research Center for Eye Diseases, Xu Guo-Xu ,   Department of Ophthalmology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China
Published: January 2021

AIM: To investigate the changes of Iba-1 and other potential markers for microglia activation in experimental diabetic retinopathy (DR).
METHODS: Male Sprague-Dawley rats were rendered diabetes via intraperitoneal injection of streptozotocin. The retinas were harvested at 1 to 24wk after diabetes onset. Hypoxia-treated mouse microglial cell line (BV2 cells) was employed as the in vitro model to mimic diabetic condition. The expressions of Iba-1, CD11b, ICAM-1 as well as the inflammatory factors were examined with real-time polymerase chain reaction, Western blot and immunofluorescence both in vivo and in vitro.
RESULTS: Compared with age-matched normal control, the number of microglia (Iba-1 positive immunostaining) in diabetic rat retinas was increased from 1 to 24wk of diabetes, which was most obvious at 12wk of diabetes. Iba-1 protein expression detected by Western blot was increased slightly in diabetic rat retinas compared with that in age-matched normal control; however, there was statistically significant between two groups only at 2wk after diabetes onset. The mRNA expression of Iba-1 was decreased significantly at 2 and 4wk of diabetic rat retinas, and remained unchanged at 8 and 12wk of diabetes. In BV2 cells, there was no significant change for the Iba-1 protein expression between normoxia and hypoxia groups; however, its mRNA level was decreased significantly under hypoxia. To further characterize microglial activation, F4/80, CD11b and inflammatory factors were detected both in vivo and in vitro. Compared with normal control, the expressions of F4/80 and CD11b as well as the inflammatory factors, such as ICAM-1, iNOS, COX2, IL-1β and IL-6, were increased significantly both in vivo and in vitro.
CONCLUSION: Iba-1 protein expression might not be a sensitive marker to evaluate the activation of microglia in experimental DR. However, Iba-1 immunostaining, in combination with other markers like CD11b and ICAM-1, could be well reflect the activation of microglia. Thus, it is of great importance to explore other potential marker to evaluate the activation of microglia.