Given the small electrical resistance across the corneal endothelium, we took recourse to the highly sensitive method of ECIS to quantify TER.
26 Previously, we
30 and others
29 have used ECIS to demonstrate changes in the barrier integrity of corneal endothelial monolayers. Specifically, we have shown that exposure to cytochalasin D, an actin-severing agent, induced a dose-dependent decline in TER. This finding is in line with a number of studies that have shown that a destruction of PAMR by cytochalasin D causes a breakdown of the barrier integrity.
48 Based on these observations, the decline in TER that we observed (
Figs. 1,
3,
5) suggest that TNF-α induces a loss in barrier integrity. Further confirmation is given in
Figure 6, in which the cytokine is shown to increase permeability of FITC dextran. These findings are also reflected in our experiments involving immunostaining of ZO-1 and cadherins (
Figs. 7,
8). ZO-1, which is an intracellular adaptor molecule associated with the transmembrane proteins of the TJs, is contiguous at cell-cell borders, and its dislocation is a marked indication of the loss of barrier integrity.
49 Thus, our findings in
Figure 7 are consistent with the loss of TER in
Figures 1,
3, and
5 and denotes a disruption of the integrity of TJs. That the cytokine also breaks down the AJs is demonstrated by dislocation of cadherins at several points along the cell-cell border (
Fig. 8C, arrows). Because the AJs are critical to inducing the cell-cell tethering necessary to bring about the interaction of transmembrane proteins of the TJs, the dispersion of cadherins on exposure to TNF-α is also indicative of the breakdown of the barrier integrity. These modifications at the AJC collectively argue in favor of the loss of barrier integrity via nonapoptotic mechanisms. This is further demonstrated by our TER measurements with ZVAD, a pan-caspase inhibitor. The failure of ZVAD to oppose the TNF-α response (
Fig. 3) indicates that the cytokine, at the doses used, did not cause significant apoptosis. Our additional apoptosis assay involving annexin V-propidium iodide staining showed that TNF-α did not induce significant apoptosis (% apoptotic cells; untreated cells 4.45 vs. TNF-α 5.03;
n = 6 independent trials;
P > 0.05) or necrosis (% necrotic cells; untreated cells 2.22 vs. TNF-α 2.81;
n = 6 independent trials;
P > 0.05) compared with untreated cells Thus, we conclude that the decline in TER was not through a trivial loss of cells from the monolayers but involved molecular mechanisms directed at AJC.