Supporting data for "Diabetic corneal nerve injury: its diagnostic value in diabetic peripheral neuropathy and underlying mechanisms associated with deleted in liver cancer 2 (DLC2)"

Impairment of small nerve fibers is considered the earliest alteration in diabetic peripheral neuropathy, which needs an objective and non-invasive biomarker for its detection. The cornea contains small nerve fibers and their alterations can be monitored with non-invasive in vivo microscopy, which may help to identify the early peripheral nerve status of diabetic patients. Hyperglycemia can activate RhoA and spinal Deleted in liver cancer 2 (DLC2)/RhoA pathway was involved in inflammatory and neuropathic pain; but few studies focused on cornea. We aimed to non-invasively investigate corneal nerve injury and its association with intraepidermal nerve changes upon hyperglycemia and explore underlying pathogenesis.

Hyperglycemia was induced in DLC2-/-YFP mice and DLC2+/+ YFP mice expressing neural YFP by streptozotocin. At different times, body weight and blood glucose levels were measured and central corneal sensitivity was measured with Cochet-Bonnet esthesiometer, while corneal nerves were visualized with in vivo confocal microscopy. Corneal and intraepidermal nerve analysis were performed on corneal flat mount and leg skin, using IMARIS and Image J software.

After STZ injection, hyperglycemia resulted in the reduction of corneal nerve fiber density(CNFD) and corneal nerve fiber length(CNFL). The central cornea of hyperglycemic DLC2+/+ YFP mice showed significant loss in CNFD at 3 months of hyperglycemia, which commenced at 6 months of hyperglycemia in the peripheral cornea. Decrease of CNFD was most significant in the central cornea and inferior quadrants of the paracentral cornea. CNFL also significantly decreased at 6 months of hyperglycemia. More importantly, these pathological structural changes occurred earlier than corneal functional loss and intraepidermal nerve fiber changes. Compared with age-matched hyperglycemic DLC2+/+ YFP mice, hyperglycemic DLC2-/-YFP mice displayed significantly higher blood glucose levels and exhibited more severe corneal nerve damage, indicating that deficiency of DLC2 exacerbated hyperglycemia-induced corneal nerve damage. Western blot results showed increase in activated RhoA expression in the cornea of hyperglycemic DLC2-/- YFP mice.

Corneal nerve damage occurred earlier than intraepidermal nerves upon hyperglycemia, suggesting that monitoring of corneal nerve fiber may help in early diagnosis of diabetic peripheral neuropathy. DLC2/RhoA may play a role in diabetic corneal nerve injury and may serve as a potential therapeutic target in the treatment of diabetic corneal neuropathy.