High-intensity corneal collagen crosslinking with riboflavin and UVA in rat cornea

Corneal collagen cross-linking (CXL) halts human corneal ectasias progression by increasing stromal mechanical stiffness. Although some reports describe that this procedure is effective in dealing with some infectious and immunologic corneal thinning diseases, there is a need for more animal models whose corneal thickness more closely resemble those occurring in these patients. To meet this need, we describe here high-intensity protocols that are safe and effective for obtaining CXL in rat corneas. Initially, a range of potentially effective UVA doses were evaluated based on their effectiveness in increasing tissue enzymatic resistance to dissolution. At UVA doses higher than a threshold level of 0.54 J/cm2, resistance to enzymatic digestion increased relative to that in non-irradiated corneas. Based on the theoretical threshold CXL dose, a CXL regimen was established in which the UVA tissue irradiance was 9 mW/cm2, which was delivered at doses of either 2.16, 2.7 or 3.24 J/cm2. Their dose dependent effects were evaluated on ocular surface morphological integrity, keratocyte apoptotic frequency, tissue thickness and endothelial cell layer density. Doses of 2.16 and 2.7 J/cm2 transiently decreased normal corneal transparency and increased thickness. These effects were fully reversed after 14 days. In contrast, 3.24 J/cm2 had more irreversible side effects. Three days after treatment, apoptotic frequency in the CXL-2.16 group was lower than that at higher doses. Endothelial cell losses remained evident only in the CXL-3.24 group at 42 days posttreatment. Stromal fiber thickening was evident in all the CXL-treated groups. We determined both the threshold UVA dose using the high-intensity CXL procedure and identified an effective dose range that provides optimal CXL with minimal transient side effects in the rat cornea. These results may help to provide insight into how to improve the CXL outcome in patients afflicted with a severe corneal thinning disease

Hyperosmolarity-induced AQP5 upregulation promotes inflammation and cell death via JNK1/2 Activation in human corneal epithelial cells

AbstractTear film hyperosmolarity and anterior ocular inflammation are two clinical signs that may be indicative of dry eye disease (DED). This condition can cause pathological and functional changes to the anterior ocular surface tissues. A contributing factor may be dysfunctional aquaporin 5 (AQP5) water channels as they are the AQP subtype that expressed in the corneal epithelium and contribute to fluid efflux needed for corneal function. We determined if described hyperosmolarity-induced increases in proinflammatory cytokine expression and cell death are mediated through AQP5 upregulation and JNK1/2 MAPK signaling activation in both primary human corneal epithelial cells (HCECs), and in a HCEC line. Real time RT-PCR identified rises in IL-1β, IL-6, IL-8, TNF-α, caspase-1, and AQP5 mRNA levels upon step increases in osmolarity up to 550 mOsm. Western blot analysis and the TUNEL assay identified corresponding rises in AQP5 and p-JNK1/2 protein expression and cell death respectively. JNK1/2 inhibition with SP600125, or siRNA AQP5 gene silencing reduced hypertonic-induced rises in proinflammatory cytokine expression and cell death. Taken together, hypertonicity-induced AQP5 upregulation leads to increases in proinflammatory cytokine expression and cell death through JNK1/2 MAPK activation. These results suggest that drug targeting AQP5 upregulation may be a therapeutic option in DED management.</jats:p