Abstract
Purpose :
Hyperglycemia has been shown by us to activate the pro-inflammatory caspase-1 (cas-1)/interleukin-1β (IL-1β) signaling pathway causing retinal pathology in diabetic mice. Under hyperglycemic conditions, Müller cells are a source of active cas-1 and IL-1β. Previously, we have shown that cas-1 is also activated in the retina of galactosemic mice, a mouse model leading to similar retinal pathology as seen in diabetic mice. To date, it has never been tested whether cas-1 activation is crucial for the development of retinal pathology in the galactose model and whether Müller cells are a potential source of active cas-1 in this model as well. Therefore, this study was focused on identifying the effect of galactosemia on cas-1 activation and subsequent retinal pathology in vitro and in vivo.
Methods :
Wild type mice (C57BL/6), cas-1-/-, and IL-1R1-/- mice were fed a normal or 30% galactose enriched diet. Formation of acellular capillaries was determined (number/mm2 retina) to access retinal pathology at 8 months of galactosemia. A rat Müller cell line (rMC-1) was treated with either 5mM glucose or 5mM glucose + 20 mM galactose in the presence or absence of a caspase-1 inhibitor (YVAD-fmk) for up to 72 hours. Cas-1 activity was measured using the fluorescent probe (YVAD-AFC). Cell death was assessed by trypan blue exclusion assay. Oxidative stress was determined using H2DCFDA (dichloro-dihydro-fluorescine diacetate).
Results :
Galactosemia significantly increased cas-1 activity in retinal tissue by 14±3.8% (compared to normal) at 2 months and by 6.2±3.7% at 4 months duration of galactosemia, respectively. Knock down of cas-1 (6.51±1.27 acellular capillaries/mm2) or the IL-1R1 (4.79±3.34 acellular capillaries/mm2) receptor prevented galactosemia-induced retinal pathology (11.72±4.41 acellular capillaries/mm2). Treatment of Müller cells with high galactose significantly induced cas-1 activation by 21.45±9.15% and cell death (47.95±12.13%; normal: 25.35±6.56%). Interestingly, galactosemia did not cause oxidative stress in Müller cells as seen with hyperglycemia treatment. Inhibition of cas-1 activity prevented galactosemia-induced Müller cell death (33.13±6.87%).
Conclusions :
Our data show that galactosemia-induced retinal pathology is dependent on activation of the caspase-1 signaling pathway making the caspase-1/IL-1β pathway a common pathway leading to retinal pathology associated with diabetic retinopathy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.