FIGURE 1

Reporter gene or PEDF expression after periocular injection of adenoviral vectors. In all, 13 adult C57BL/6 mice were given a 3 l injection containing 5 10⁹ particles of AdLacZ.10 in one eye and killed 1 (n=4), 4 (n=4), or 14 days (n=5) after injection. Periocular and ocular expression of LacZ was assessed on day 4 and expression was evaluated in the liver on days 1, 4, and 14. (a) Orbital sections through regions adjacent to the site of injection showed strong staining for LacZ in the rectus muscle (asterisk), connective tissue, and fat. The episclera was often stained all the way around the globe (arrow). (b) High magnification view of the left side of the section from (a) shows faint LacZ staining in the retina (arrow head), indicating that a small amount of virus was able to pass through the sclera and transfect some retinal cells. There is strong staining in the episclera (arrow) and rectus muscle (above the asterisk). (c) High magnification view of the right side of the section from (a) shows strong staining in the episclera (arrow). (d) A section through the optic nerve (ON) shows strong staining of adjacent sclera and connective tissue, but no staining of the nerve. (e) Lacrimal gland from injected orbits (panel 1), contralateral orbits (panel 3), and orbits of uninjected mice (panel 2) all had a faint blue coloration suggesting endogenous -galactosidase-like activity rather than transduction of lacrimal gland. (f) High magnification shows staining of muscle fibers (asterisk) and episclera (arrow). (g) A section through a contralateral orbit and adjacent region of brain (B) and sinus (S) separated from the orbit by bone (red arrowheads) shows no staining for LacZ. (h and i) High magnification views of the contralateral orbit shown in (g) demonstrate the absence of LacZ staining in connective tissue (asterisk) and sclera (arrows). (j) Liver sections showed no evidence of LacZ staining 1 day after periorbital injection of AdLacZ.10. (k) Liver sections typically showed a few LacZ-positive cells (arrowheads) 4 days after injection. (l) LacZ-positive cells were no longer seen in liver sections 2 weeks after injection. Immunohistochemistry for PEDF was carried out on ocular sections 4 days after periocular injection of 5 10⁹ particles of AdPEDF.11 or control vector encoding soluble Flt-1 (AdFlt-1.10). (m) Red reaction product indicates PEDF in the retina and orbital tissue (OT). The arrowhead indicates the surface of the retina, INL indicates the inner nuclear layer, and ONL indicates the outer nuclear layer. At high magnification (p) staining is seen in the sclera (arrow). (n) Incubation of primary antibody with PEDF eliminates staining in the retina (arrowhead shows surface) and orbital tissue (OT). At high magnification, there is no staining in the sclera (q). (o) Four days after injection of AdFlt-1.10, there is no staining for PEDF in the retina (arrowhead) or orbital tissue (OT). At high magnification, no staining in the sclera is detected (r).