First published online October 27, 2003
The effect of elevated intraocular oxygen on organelle degradation in the embryonic chicken lens
Steven Bassnett1,2,* and
Richard McNulty1
1 Department of Ophthalmology and Visual Sciences, Washington University
School of Medicine, St Louis, MO 63110, USA
2 Department of Cell Biology and Physiology, Washington University School of
Medicine, St Louis, MO 63110, USA
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Fig. 3. Use of acridine orange to delineate the borders of the organelle-free zone
(OFZ) in the developing chicken lens. (A) Midsagittal section of an E16
chicken lens. The lens is shown with its anterior surface facing up. The bulk
of the lens is composed of elongated fiber cells arranged in concentric
layers. Nuclei (arrow) are present in the peripheral cells but are absent from
the oldest, innermost, fiber cells (asterisk). (B) Confocal image of the
section shown in A, following incubation in 1 µg ml–1
acridine orange. Acridine fluorescence is strongest in the peripheral fiber
cells. A clearly demarcated region with very low fluorescence is present in
the center of the lens (arrowhead). This region corresponds to the OFZ. Scale
bar, 250 µm. (C) Tracing of the section shown in B, indicating the spatial
parameters that were measured in this study to analyze the time course of
organelle breakdown in the developing lens. d, distance; D,
diameter; T, thickness.
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Fig. 1. Variation in intraocular oxygen partial pressure (PO)
during embryonic development and following treatment with 50% O2.
An optode was used to measure PO in the anterior vitreous
(open symbols) and mid-vitreous (filled symbols) of normoxic chicken embryos
(circles). Some embryos were incubated from E7 to E13 in an atmosphere of 50%
O2:50% N2. Measurements made on those embryos
(triangles), at E13, indicated that hyperoxia results in a significant
increase in PO in both the anterior and mid-vitreous.
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Fig. 2. Embryonic lens hypoxia visualized using the bioreductive marker
pimonidazole. E17 lenses were incubated in pimonidazole in vitro
(A–C) in solutions equilibrated with 21%, 2% or 0% O2.
Formation of pimonidazole–protein adducts was visualized
immunocytochemically (see text for details). In 21% O2 (A), no
adducts were detected. However, adducts were detected in the cortex of lenses
incubated in 2% O2 (B), and intense cortical staining was present
in lenses incubated in 0% O2 (C). In vivo treatment with
pimonidazole (D–F) resulted in strong cortical immunostaining throughout
development. When pimonidazole was omitted from the intraocular injection
solution, no immunostaining was observed (G), confirming the specificity of
the pimonidazole antibody. Scale bar, 250 µm.
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Fig. 4. Effect of hyperoxia on lens size. Beginning on E7, lenses were incubated in
50% O2:50% N2. Vibratome slices were prepared from
hyperoxic or normoxic lenses and stained with acridine orange. (A) Lens
diameter (D) is consistently larger in hyperoxic lenses (open
circles) compared with normoxic controls (closed circles). (B) Lens axial
thickness (T) is greater in hyperoxic lenses than in normoxic
controls, and this difference reaches statistical significance by E17. (C) The
area of mid-sagittal slices prepared from hyperoxic lenses is greater than
normoxic controls. Asterisks denote significant (P<0.05)
difference between hyperoxic lenses and age-matched controls.
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Fig. 5. Effect of hyperoxia on the distance from the lens surface to the border of
the organelle-free zone (OFZ). (A) At all ages examined, the distance from the
equatorial surface of the lens to the border of the OFZ (d1 and
d2; see Fig. 3B) was
significantly greater in hyperoxic lenses (open circles) compared with
normoxic controls (closed circles). (B) Hyperoxia caused an increase in the
distance from the anterior pole to the OFZ border (d3). (C) The
distance from the posterior pole of the lens to the border of the OFZ
(d4) was significantly greater in hyperoxic lenses.
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Fig. 6. Effect of hyperoxia on the dimensions of the organelle-free zone (OFZ). (A)
The equatorial width of the OFZ is decreased by hyperoxia (open circles). (B)
Hyperoxia caused a consistent decrease in the area of the OFZ compared with
normoxic controls (filled circles). (C) For each lens slice, the fractional
area occupied by the OFZ was calculated. In hyperoxic lenses, a significantly
smaller fraction of the lens slice area was occupied by the OFZ.
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© The Company of Biologists Ltd 2003
