Wistar rat palpebral conjunctiva contains more slow-cycling stem cells that have larger proliferative capacity: Implication for conjunctival epithelial homeostasis

Purpose: To determine the location of conjunctival epithelial stem cells. Methods: Wistar rats received daily injection of 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks followed by a 1-month BrdU-free period before death. After the rats were sacrificed, the orbital contents and eyelids were exenterated en bloc, fixed in buffer formalin, and embedded in paraffin. To compare the proliferative capacity of ocular epithelial cells, 1.0% phorbol myristate (TPA) in petrolatum was topically applied once daily to both eyes of Wistar rats for 12 days. After 6,12,18, and 24 hours and 2,4, 6, 8, 10, and 12 days of TPA treatment, rats were administered BrdU intraperitoneally 7 hours before they were sacrificed. The ocular epithelium was fixed and processed for immunochemistry, and the labeling index (LI) of every epithelial zone was determined. Results: Slow-cycling cells, detected as label-retaining cells (LRCs), were found in bulbar, fornical, and palpebral epithelia and mucocutaneous junctions, as well as in limbal epithelia. The greatest numbers of LRCs were identified in palpebral epithelium. Under normal situations, in conjunctiva the LI was lowest in palpebral epithelium (2.1+/-0.5)compared with bulbar (2.2+/-0.5), fornical (2.3+/-0.4) epithelia and mucocutaneous junction (3.4+/-0.9), respectively. In cornea, the LI was lowest in limbal epithelium (1.8+/-0.7) compared with central corneal epithelium (3.5+/-0.6). Twenty-four hours after TPA treatment, an 8.2-fold increase in the palpebral epithelial basal cell labeling index was noted compared with 4.7-fold, 5.7-fold, and 3.8-fold increases in bulbar, fornical, and mucocutaneous junction epithelial basal cell labeling indices, and a sevenfold increase in the limbal basal cell labeling indices compared with a 2.1-fold increase in the corneal basal cell labeling index, respectively. Limbal and palpebral epithelia maintained a significantly greater proliferative response (5.5-to 6.3-fold increase, respectively) during chronic stimulation than corneal, bulbar, fornical epithelia, and mucocutaneous junction (0.6- to 2.3-fold increase, respectively). Conclusions: In Wistar rat conjunctiva, slow-cycling cells are primarily located in palpebral epithelium, which has greater proliferative capacity than other conjunctival epithelia. This finding means that, in the Wistar rat, the conjunctival epithelial stem cells are mainly located in palpebral epithelium. These data open new perspectives in ocular epithelial development and are relevant in conjunctival wound repair.