Abstract
Purpose::
To test the possibility that LCVS1001, a glycoprotein, protects retinal neurons from cell death induced by AGEs in two in vitro models.
Methods::
Neurosensory retina of SD rat was kept for 9 h in Ame’s medium containing 0, 30, 800 or 2000 uM glyoxal, with or without LCVS1001 (0.2U/mL). Retinal Neuron apoptosis was studied using TUNEL test. Primary culture of retinal neurons from neonatal (one postnatal day) SD rats were established and maintained at least 7 days. Neurons were identified by cell marker RT-PCR and immunocytochemistry (ICC) using antibodies against opsin and Thy1. Cultured cells were treated by glyoxal (0, 30, 150, 800, 2000 uM) with or without LCVS1001(0.2U/mL). The neuronal death was detected by TUNEL and Acridine Orange/Ethidium Bromide (AO/EB) Staining.
Results::
Glyoxal result in significant cell death in the organ culture of neurosensory retina, mainly in GCL and INL. Such apoptosis was prevented by LCVS1001. In the primary cell culture, 90% of the cells showed typical morphology of neurons. The ICC demonstrated opsin-positive (~30%) and Thy1-positive (~21%) cells. Strong expressions of the calbindin 2 and Thy1 were observed by RT-PCR. Glyoxal-mediated AGEs formation (30 to 2000 uM) can effectively induce neuron death, in a dose-dependent manner. The increased cell death can be effectively blocked by 5 uM Aminoguanidine (56.4% reduction), suggesting that AGEs are the cause of glyoxal induced neuron death. After exposure to 30 uM glyoxal, the supplement of LCVS1001 reduced the cell death by 48.6%. In the cell culture system, containing 150, 800 and 2000 uM glyoxal, LCVS1001 treatment reduced the cell death by 62.7%, 35.8% and 45.9%, respectively.
Conclusions::
The cyto-protective functions of LCVS1001 for retinal neurons in two in vitro systems under the stress of AGEs have been demonstrated. Since AGEs-induced apoptosis is associated with diabetes or age-related macular degeneration, the application of LCVS1001 for treatment of these degenerative retinal diseases may be indicated.
Keywords: apoptosis/cell death • diabetic retinopathy • retinal degenerations: cell biology