In this study, we observed diminished numbers of blue-stained
cells in accordance with the subsidence of LPS-induced inflammation.
There are two possibilities to explain this phenomenon: One is that the
transgene may be deleted or transduced cells may undergo programmed
cell death. Under this mechanism, re-exposure of cornea to LPS would
not increase
LacZ-positive cells. Another possibility is
that the transgene remains stable, but gene expression is suppressed.
Under this mechanism, re-exposure of the cornea to LPS would reactivate
gene expression and therefore increase
LacZ-positive
signals. To test these possibilities, another episode of intraocular
inflammation was induced by a second LPS injection 60 days after
rAAV-
LacZ injection. Briefly, both eyes of each rabbit
(
n = 12) were treated with rAAV-
LacZ (10
7 units of infection). Twenty-four hours
later, intraocular inflammation was induced by LPS injection in both
eyes. At 60 days after rAAV-
LacZ injection when the
transient inflammation had already subsided, LPS reinjection was
performed in the right eye, and the contralateral eye was injected with
PBS as a control. Before LPS reinjection, only 3.4% ± 2.1% of
endothelial cells showed a
LacZ-positive signal (
Fig. 4A ). At 1 day after reinjection, most of the endothelial cells (86.1% ±
8.7%) showed a positive
LacZ signal
(Fig. 4C) . At 5 days
after reinjection,
LacZ-positive endothelial cells were
moderately reduced to 43.4% ± 7.6%
(Fig. 4E) . At 15 days after
reinjection, no sign of inflammation was identified in the ocular
anterior segment, and transgene expression decreased to 3.7% ± 2.2%
(Fig. 4G) . In those eyes without a second inflammation, the
LacZ-positive endothelial cells remained low (2%–5%)
before or at 1, 5, and 15 days after PBS injection
(Figs. 4B 4D 4F 4H) . Our results suggest that the diminished number of blue-stained
cells, in accordance with the subsidence of inflammation, was due to
suppression of the gene expression rather than to loss of transduced
cells.