Abstract
Purpose: :
To determine if the response of conjunctival cells to nerves and growth factors is altered in a mouse model of dry eye and if loss of corneal nerves (induced by a corneal wound mimicking refractive surgery) alters this response.
Methods: :
Four week old, female BALB/c mice and 12 week old, female MRL/MPJ Faslpr mice were used as non– dry eye and dry eye models.(n= 9, each strain) Corneal wounds were made through the stromal layer in 6 of 9 mice in each strain by using a trephine, mimicking nerve loss in LASIK surgery. Conjunctival tissue was collected from unwounded eyes and from eyes 2 and 6 days after wounding. Tissue was incubated for 5 minutes with keratinocyte basal media alone, the cholinergic agonist carbachol (Cch) (10–5M), EGF(10–7M), and the beta–adrenergic agonist Isoproterenol (10–5M). The amount of the phospho (activated) proteins AKT (known as protein kinase B), extracellular signal–regulated kinase (ERK), c–Jun N–terminal kinase (JNK) and p38 mitogen–activated protein kinase (MAPK) were measured by multi–plex assay. Results were standardized by measuring the amount of total ERK.
Results: :
Under basal conditions, the amount of phospho JNK in unwounded eyes and phospho ERK 6 days after wounding was higher in the dry eye model than in the non–dry eye model (p= 0.046, 0.033, respectively). Compared to basal, Cch stimulation of JNK phosphorylation in unwounded eyes was lower in the dry eye model than in the non–dry eye model (p=0.048). In the non–dry eye model Cch increased the phosphorylation of p38 MAPK above basal in unwounded eyes (p=0.02), but this stimulation was decreased in tissue 6 days after wounding (p=0.004). Cch did not significantly alter AKT activation. EGF and isoproterenol did not significantly alter activation of any phosphoproteins measured.
Conclusions: :
In a mouse model the response of conjunctival cells to cholinergic agonists appears to be altered by dry eye status and loss of corneal nerves (as could occur in refractive surgery).
Keywords: conjunctiva • phosphorylation • cornea: tears/tear film/dry eye