Is Iba-1 protein expression a sensitive marker for microglia activation in experimental diabetic retinopathy?
Fan-Jun Shi1,2, Hai Xie3, Chao-Yang Zhang3,4,5
1Department of Ophthalmology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China.
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.
Insights
Iba-1 protein expression is not a sensitive marker for microglia activation in experimental diabetic retinopathy (DR). However, combining Iba-1 with CD11b and ICAM-1 can reflect microglia activation, highlighting the need for better markers.
Area of Science:
- Ophthalmology
- Neuroscience
- Immunology
Background:
- Diabetic retinopathy (DR) is a leading cause of vision loss.
- Microglia activation plays a critical role in DR pathogenesis.
- Reliable markers for assessing microglia activation in DR are needed.
Purpose of the Study:
- To investigate changes in Iba-1 and other microglia activation markers in experimental DR.
- To evaluate the utility of Iba-1 as a marker for microglia activation in DR.
- To identify potential markers for assessing microglia activation in DR.
Main Methods:
- Experimental diabetes was induced in Sprague-Dawley rats using streptozotocin.
- Retinas were harvested at various time points (1-24 weeks) post-diabetes onset.
- A hypoxia-treated mouse microglial cell line (BV2) served as an in vitro model.
- Expressions of Iba-1, CD11b, F4/80, ICAM-1, and inflammatory factors were analyzed using RT-PCR, Western blot, and immunofluorescence.
Main Results:
- Microglia numbers (Iba-1 positive) increased in diabetic rat retinas, peaking at 12 weeks.
- Iba-1 protein expression showed a slight increase, but mRNA levels decreased significantly in diabetic retinas and BV2 cells under hypoxia.
- Expressions of F4/80, CD11b, ICAM-1, iNOS, COX2, IL-1β, and IL-6 were significantly elevated in both in vivo and in vitro models.
Conclusions:
- Iba-1 protein expression alone may not be a sensitive indicator of microglia activation in experimental DR.
- Combined Iba-1 immunostaining with markers like CD11b and ICAM-1 can effectively reflect microglia activation.
- Further research is warranted to identify more sensitive and specific markers for microglia activation in DR.
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