A major criticism of the lactate flux model
14 of the endothelial pump is that de-epithelialized corneas, which retain all the pump functions, produce significantly less lactate and could therefore be incompatible with the model. The lactate flux model, however, does not rely on an absolute amount of lactate production; rather, it depends on the endothelium generating a small apical to basolateral [lactate] gradient with the lactate that is available. Whereas methods to measure small concentration gradients across cell layers are unavailable, we can ask if the relationship between water and lactate efflux observed in intact corneas
14 is similar in de-epithelialized corneas producing less lactate. De-epithelialized corneas were mounted and perfused on the endothelial side with GBR. The perfusate was collected over 30-minute intervals and later analyzed for lactate. After 90 minutes, either GBR was continued or the perfusing solution was switched to one containing 100 µM Ouabain, 100 µM acetazolamide (ACTZ) in GBR, or BF Ringers.
Figure 1A shows that ouabain produced the most corneal swelling relative to GBR alone, then BF, and a modest amount of swelling was produced with ACTZ, the same rank order as found in intact corneas.
14 Figure 1B shows the lactate efflux over the course of the experiment, indicating that flux was greatest in GBR, slightly less in ACTZ, much less in BF, and least with ouabain. This finding is summarized in
Figure 1C showing the total lactate efflux from 120 to 300 minutes. Last,
Figure 1D shows that corneas that swelled the least and had the greatest lactate efflux had the lowest amount of lactate retained in the cornea at the end of the experiment, similar to what was observed in intact corneas.
14 However, the absolute lactate efflux and the amount retained in the cornea of de-epithelialized corneas was approximately 50% of that in intact corneas, for example, the lactate efflux was 1300 nanomoles in intact corneas
14 and 700 nanomoles in de-epithelialized corneas (
Fig. 1C) during GBR perfusion.