Abstract
Purpose: :
Previously we demonstrated that injury to corneal epithelial cells releases nucleotides leading to purinergic receptor activation and propagation of a localized Ca2+ wave. Our goal was to develop an in vitro co-culture system to test the hypothesis that damage to epithelial cells elicits both localized and regenerative responses.
Methods: :
Trigeminal ganglion were dissected and cultured using a neurobasal medium. Human corneal limbal epithelial cells (HCLEs) were added to cultures and the co-cultures incubated. The expression of purinergic receptors was determined for primary and cultured neurons and glia. The response to agonists or wounding was performed using live cell imaging on cells loaded with Fluo-3AM. FRAP and immunohistochemical analysis were performed.
Results: :
Expression of P2Y 1,2,4,6 and 12 receptors were detected in neuronal enriched and glial cultures and trigeminal explants using RT-PCR and qRT-PCR. There was no difference in the expression pattern of P2Y 1,2,4 and 12, while there was a 6 fold increase in P2Y6 in neuronal cultures. EC50 values for neurons indicated that ADP (ligand for P2Y1) elicited a greater response than ATP (ligand for multiple P2Y and P2X receptors). When injuries were made to the co-cultures, a Ca2+ wave propagated from epithelia to neurons for 500µm; however, the reverse was not detected. To reduce the P2X contribution, experiments were performed in a Ca2+-free buffer and a decreased propagation in HCLEs but not neurons was detected. When cells incubated with connexin inhibitors were injured, there was a reduction in the number of cells responding and a reduction in the distance of propagation to 250µm. FRAP analysis and imunocytochemistry indicated gap junction coupling between HCLEs, but not neurons. When neurons adjacent to HCLEs were injured, a regenerative Ca2+ response was detected in epithelia that lasted longer than 7.5 min. When HCLE wound media was added to neurons a rapid nucleotide response occurred that was ablated with apyrase. When neuronal wound media was added to HCLEs, a regenerative response occurred that was not ablated by apyrase, and was similar to that elicited by glutamate.
Conclusions: :
Injury to co-culture causes damaged epithelial cells to release ATP, activating P2Y receptors in HCLEs and neurons a distance from the wound. Treatment with connexin inhibitors indicate that injury induced wave has both gap junction dependent and independent components. Damaged neuronal processes release a trophic factor, potentially glutamate, that produces a regenerative, long-lasting response in epithelial cells.
Keywords: wound healing • cell-cell communication • neurotransmitters/neurotransmitter systems