Purpose: To determine whether temperature fluctuation elicits an increase in deimination (conversion of protein bound arginine into citrulline) in retinal astrocytes, and elevated deimination levels can serve as an astrocytic activation marker (along with other activation markers) in the retina.
Methods: Isolated retinal astrocytes obtained from C57BL/6J mice (about 1000 per plate) were cultured and subjected to different incubation temperatures for one hour, followed by a stabilization period of 23 hours at 37°C. The exposed and control astrocytes were evaluated for deimination levels as well as for levels of other established markers of astrocyte activation (for example, glial fibrillary acidic protein: GFAP) using immunohistochemical, Western blot and ELISA analyses.
Results: Optimal growth of retinal astrocytes occurred at 37°C. Increased levels of astrocyte activation markers such as Aquaporin4, and Thrombospondin were found in astrocytes subjected to hypothermia; whereas decreased GFAP, deimination and peptidylarginine deiminase type 2 (PAD2) levels were found in cells subjected to this conditions. The astrocytes subjected to hyperthermia showed an increase in deimination and PAD2 levels. Deimination and PAD2 levels were higher for hyperthermia (approximately 1.5-fold) than for the cells subjected to hypothermia. Hyperthermia was accompanied by an increase in some astrocytic markers such as GFAP and aldehyde dehydrogenase 1 family member L1 (ALDH1L1).
Conclusion: The level of deimination undergoes a shift (change in level compared to baseline) on either side of optimal temperature incubation (37°C) for retinal astrocytes. The level of deimination correlates with astrocyte activation markers in the retina when cells are subjected to temperature treatment. These results agree with our previous work performed on brain cortex astrocytes, also from the C57BL/6J mouse strain, in which a similar pattern is observed.
