The effect of elevated intraocular oxygen on organelle degradation in the embryonic chicken lens
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
* Author for correspondence (e-mail: bassnett{at}vision.wustl.edu)
Accepted 14 August 2003
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Summary |
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Key words: chicken, embryo, confocal microscopy, organelle, oxygen, optode
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Introduction |
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It has been suggested that organelle loss may represent a form of
attenuated apoptosis (Dahm,
1999). In support of this notion, biochemical studies have
demonstrated the proteolysis of classical apoptotic substrates, such as PARP
[poly(ADP-ribose)polymerase], during organelle loss
(Ishizaki et al., 1998
).
Similarly, fiber cell nuclei become TUNEL-positive shortly before they
disappear (Modak and Bollum,
1972
), and an apoptotic-like DNA ladder is generated as the nuclei
are broken down (Appleby and Modak,
1977
). However, despite progress in elucidating the biochemical
mechanism of organelle degradation, the signal that serves to trigger the
process remains obscure.
Experiments with differentiating lentoid cultures suggested that organelle
loss may be triggered through a death receptor pathway involving tumor
necrosis factor (TNF
). TNF
and two TNF
receptors
(TNFR1 and TNFR2; Wride and Sanders,
1998
) are expressed in the lens, but it is unclear how this
pathway might be activated specifically at the border of the OFZ, hundreds of
cell layers beneath the lens surface. In the present study, we examine an
alternative hypothesis.
The lens lacks a blood supply and has a limited and largely inaccessible
(Shestopalov and Bassnett,
2000) extracellular space. Thus, metabolites entering or exiting
the lens volume must do so across its surface. One consequence of this
arrangement is that it leads, inevitably, to the generation of standing
gradients of small, diffusible metabolites within the tissue. The magnitude of
such gradients depends on the rate at which a metabolite is produced or
consumed, the effective diffusion coefficient of the metabolite within the
lens and the size of the lens. An example of this effect is the gradient of
intracellular pH (pHi) observed within the lens. The lens obtains much of its
ATP from anaerobic glycolysis (Winkler and
Riley, 1991
). Lactic acid generated by glycolysis diffuses from
the lens only across its outer surface. Consequently, a standing gradient of
lactic acid is established (Bassnett et
al., 1987
), resulting in values for core lens pHi that are
significantly lower than those in the outer cortex
(Bassnett and Duncan, 1986
;
Mathias et al., 1991
).
Standing gradients may provide spatial cues that act to coordinate the
differentiation process in fiber cells located within a growth shell. Of
particular relevance in this regard may be the intralenticular distribution of
molecular oxygen. Dissolved oxygen enters the lens from the aqueous and
vitreous humors and is expected to diffuse rapidly through the tissue. Oxygen
is consumed within the lens through oxidative phosphorylation within lens
mitochondria and, perhaps, through non-mitochondrial mechanisms such as the
oxidation of ascorbate. As a result, the partial pressure of oxygen
(PO) in the lens core is likely to be significantly lower
than at the surface. In large lenses such as bovine or human lenses, where
invasive measurements are feasible, we have observed such standing gradients
of PO (R. McNulty, R. Truscott and S. Bassnett,
unpublished). Based on these observations, we hypothesize that as
differentiating fiber cells become buried by more recently formed fibers, they
will be subjected to increasingly severe hypoxia. Hypoxia has long been known
to trigger apoptosis in a variety of cells
(Banasiak et al., 2000) and, in
the lens, we postulate that it may serve to trigger the related process of
organelle degradation. To test this directly, in the present study we
incubated chicken eggs under hyperoxic conditions (50% O2). We
reasoned that such treatment should increase intralenticular
PO and thereby inhibit the organelle loss process. The
results indicate that hyperoxic treatment caused an increase in intraocular
PO and a partial inhibition of organelle loss. The data
are consistent with the view that organelle loss may be triggered, at least in
part, by a decline in intralenticular PO2.
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Materials and methods |
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Microscopy
Embryos were decapitated and lenses were removed through an incision in the
posterior globe. Lenses were fixed for two hours at room temperature in 4%
paraformaldehyde/PBS (phosphate-buffered saline). Lenses were then embedded in
4% agar/PBS and sectioned in the sagittal plane at 100 µm intervals using a
vibratome tissue processor (Model 100 plus; TPI, St Louis, MO, USA) as
described (Bassnett, 1995). All
slices were retained for further examination. Slices were stained for 1 h in 1
µg ml1 of acridine orange (Molecular Probes, Eugene, OR,
USA) in PBS. Acridine orange binds to DNA and RNA. The latter is visible in
acridine-stained tissue as diffuse cytoplasmic fluorescence. Because the
levels of cytoplasmic RNA decline precipitously after the dissolution of the
lens fiber nuclei, RNA staining provides a convenient method for delineating
the borders of the organelle-free zone (OFZ). Following incubation in acridine
orange, lens slices were washed for 1 h in PBS, coverslipped and viewed.
Images were collected with an LSM 410 confocal microscope (Carl Zeiss Inc.,
Thornwood, NJ, USA) using the 488 nm laser line for excitation and a 515 nm
long-pass filter for emission. Software provided with the microscope was used
to measure the diameter of lens slices. The largest slice was deemed to be the
midsagittal slice and was subjected to further analysis. The following
parameters were measured from the confocal images: diameter and thickness of
the lens, equatorial diameter and area of the OFZ, and distance from the
border of the OFZ to the lens equator and poles (see
Fig. 3C for clarification).
Measurements were made on hyperoxic lenses and normoxic controls in a masked
fashion.
|
Oxygen measurement
Vitreous PO was measured using a fiber optic detection
system (OxyLab, Oxford Optronix, Oxford, UK). The oxygen-sensing probe
(optode) consisted of a 320 µm-wide optic fiber. An immobilized
ruthenium-based fluorophore at the tip of the fiber served as the
O2 sensor. The sensor was illuminated with oscillating blue light.
Fluorescence emitted by the O2 sensor was transmitted by the fiber
optic to the detector. Fluorescence lifetime was calculated automatically from
the phase delay between the excitation and emission signals. The fluorescence
lifetime of the fluorophore is inversely related to PO, as
described by the SternVolmer equation, and provides an accurate measure
of PO within the physiological range
(Seddon et al., 2001). Optodes
were obtained pre-calibrated by the manufacturer. To measure vitreous
PO, a hole was cut in the egg shell, and the head of the
embryo was exposed. The fiber optic probe was inserted into the eye through
the posterior sclera. A micromanipulator was used to position the optode tip
in the geometric center of the vitreous. Stable measurements were usually
obtained within 20 s. After PO was measured in the central
vitreous, the optode tip was advanced to a position immediately behind the
lens, where a second measurement was made.
Immunocytochemical detection of tissue hypoxia
Eggs were windowed to expose the head of the embryo. An injection of 3
µl of a 10 mg ml1 solution of pimonidazole hydrochloride
(hypoxyprobe-1; NPI, Inc., Belmont, MA, USA) was made into the left eye of the
embryo. The egg was resealed for one hour and then the embryo was decapitated
and the lens was removed and processed for histology. Immunocytochemistry was
performed according to the instructions provided with the hypoxyprobe system.
Briefly, following antigen retrieval with pronase, sections were incubated
with a 1:50 dilution of hypoxyprobe Mab1, an antibody that specifically
recognizes protein adducts formed by pimonidazole under hypoxic conditions.
Antibody distribution was visualized with a horseradish peroxidase-conjugated
secondary antibody, with diaminobenzidine (DAB) as the chromogenic
substrate.
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Results |
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We examined the effect on vitreous PO of incubating eggs under hyperoxic (50% O2) conditions. Eggs were incubated under normoxic conditions until E7 and then switched to hyperoxic conditions until E13. Measurements were made at E13 in the mid-vitreous and anterior vitreous of hyperoxic or normoxic control embryos (Fig. 1). Hyperoxia produced a significant elevation in PO throughout the vitreous. In E13 hyperoxic embryos, the mid-vitreous PO was 3.5±0.7 kPa and the anterior vitreous PO was 1.3±0.2 kPa. These values were both significantly higher than those of normoxic controls (P<0.001, N=6).
The optode oxygen sensor had a diameter of 320 µm and, although this was sufficiently small to allow oxygen measurements in the vitreous humor, it was too large for intralenticular measurements. We therefore evaluated the use of a bioreductive hypoxia marker, pimonidazole, for visualizing the distribution of oxygen within the lens (Fig. 2). Preliminary experiments on E17 lenses in vitro demonstrated that pimonidazoleprotein adducts were only formed in the lens under hypoxic conditions. Following incubation in solutions equilibrated with room air (20.9% O2), no immunostaining was observed (Fig. 2A).However, incubation with solutions equilibrated with 2% O2 resulted in diffuse staining of the lens cortex (Fig. 2B). In general, the most intense staining pattern was observed following incubation in solutions gassed with 100% nitrogen (0% O2; Fig. 2C). In this case, the lens epithelium and cortex were strongly stained for pimonidazoleprotein adducts. Parallel in vivo experiments indicated that the lens existed in a chronically hypoxic state throughout embryonic development. Thus, intraocular injection of pimonidazole at E10 (Fig. 2D), E13 (Fig. 2E) and E17 (Fig. 2F) resulted in adduct formation. In young embryos (<E13), adducts were present throughout the tissue. However, as with the in vitro experiments, at later stages (>E13) immunostaining was restricted to the lens cortex, and pimonidazole adducts were not detected in the lens core (Fig. 2F). Control experiments (Fig. 2G) confirmed that adduct formation was dependent on the presence of the pimonidazole. We examined whether incubation under hyperoxic (50% O2) conditions abolished pimonidazole staining in vivo. Despite the fact that hyperoxia caused an approximate doubling of intraocular PO, no change in pimonidazole-adduct staining within the lens was observed (data not shown).
|
We next determined the effect of hyperoxia on lens organelle loss, using acridine orange staining as a convenient method for visualizing the development of the OFZ (Fig. 3). In acridine orange-stained lens slices, cortical lens fiber cells were strongly fluorescent but, in contrast, the anucleated cells of the lens core were only weakly fluorescent (Fig. 3B). The acridine fluorescence reflected the relative concentrations of cytoplasmic RNA in various regions of the lens. At the ages examined here, the OFZ was approximately spherical, reflecting the organization of the fiber cells in the lens core. Along any given fiber cell the concentration of RNA was constant. Prolonged treatment with RNase A completely abolished the cytoplasmic staining (data not shown). Presumably, most RNAs were relatively short-lived, and because the anucleated fiber cells of the lens core were no longer transcriptionally active, the concentration of RNA within these cells rapidly decreased. The acridine orange fluorescence thus delineated the OFZ and allowed the various dimensions indicated in Fig. 3C to be measured (see Table 1).
|
To examine the effect of hyperoxia on organelle degradation, chicken eggs
were incubated under normoxic (21% O2) conditions until E7, at
which point eggs were switched to 50% O2 for a further 6, 8 or 10
days. In the embryonic chicken lens, organelle degradation commences in the
central fiber cells on E12 (Bassnett and
Beebe, 1992). Thus, embryos were exposed to hyperoxia for five
days before the expected onset of organelle degradation and for various
periods thereafter. Although extended exposure to hyperoxia is toxic in some
settings, we did not observe any deleterious effects of 50% O2 on
the chicken embryos. There was no increased mortality rate and the eyes of the
hyperoxic embryos appeared to be completely normal. The effect of hyperoxia on
the growth of the lens and the formation of the OFZ is shown in Figs
4,
5,
6 and
Table 1.
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|
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Following incubation in elevated oxygen, lens slices were prepared, stained with acridine orange and compared with tissue from normoxic controls. Hyperoxia caused a marked increase in the overall size of the lens (Fig. 4). At all ages examined, the mean diameter (D) of hyperoxic lenses was larger than that of normoxic controls, although the difference did not reach significance (Fig. 4A). The hyperoxic lenses were also consistently thicker (T) than the normoxic lenses, a difference that was more marked in the older embryos (Fig. 4B). Reflecting these differences in linear dimension, measurements of the area of midsagittal slices prepared from normoxic and hyperoxic lenses indicated that the latter were significantly larger at later embryonic ages (Fig. 4C).
A well-defined OFZ was present in the center of normoxic and hyperoxic lenses at the earliest time point examined (E13). Thus, hyperoxic treatment did not completely inhibit organelle breakdown. To examine whether hyperoxia subsequently delayed the organelle breakdown process, we measured the distance from the border of the OFZ to the equatorial or polar surfaces of the lens (Fig. 5). The distance from the lens equator to the OFZ border (mean of d1 and d2) did not vary greatly during development but was consistently and significantly greater in hyperoxic lenses compared with normoxic controls (Fig. 5A). In normoxic lenses, the ratio of this distance to the total lens diameter fell from 0.37 at E13 to 0.32 at E17. In hyperoxic lenses, the ratio fell from 0.39 to 0.33 over the same period.
Similarly, the distance from the anterior or posterior pole to the border of the OFZ (d3 and d4, respectively) was relatively constant over the observation period but was always greater in the hyperoxic lenses (Fig. 5B,C). These data indicate that in hyperoxic lenses, organelle loss is triggered at a greater depth from the lens surface and thus at a later stage of differentiation than would ordinarily occur.
Finally, we examined the absolute and relative size of the OFZ formed in hyperoxic lenses (Fig. 6). Under hyperoxic conditions, the diameter of the OFZ was consistently smaller than under normoxic conditions (Fig. 6A), a difference that reached significance at later embryonic stages. The area of the OFZ in hyperoxic lenses was also consistently smaller than the normoxic controls (Fig. 6B), although the difference was only significant at E15. For each lens, the area of the OFZ was expressed as a percentage of the area of the lens slice. Under both hyperoxic and normoxic conditions, the proportion of the slice area contained within the OFZ increased markedly during the observation period (Fig. 6C). However, in hyperoxic lenses, the OFZ accounted for a consistently smaller fraction of the total slice area than in normoxic controls.
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Discussion |
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Because of the small size of the embryonic lens (in young embryos, <1 mm
in diameter), we used the hypoxia marker pimonidazole to visualize tissue
hypoxia. Under hypoxic conditions, 2-nitroimidazole compounds form long-lived
adducts in cells in vivo and in vitro
(Hale et al., 2002;
Raleigh et al., 1996
;
Varghese et al., 1976
).
Adducts form with thiol groups in proteins, peptides and amino acids and may
be detected immunocytochemically. This approach has been used to visualize
tissue hypoxia in the thymus (Hale et al.,
2002
), liver (Corpechot et
al., 2002
) and various types of tumors
(Airley et al., 2003
;
Bennewith et al., 2002
;
Kaanders et al., 2002
). As
expected, pimonidazole adducts were produced in the lens in vitro
under hypoxic (0% or 2% O2) conditions. In young lenses, adducts
were distributed throughout the lens; however, even in fully deoxygenated
solutions, the cores of lenses from older embryos (>E13) were not stained
by the adduct-specific antibody. The unstained region matched the OFZ in size
and shape. It is likely that terminally differentiated fiber cells in the OFZ
lacked the endogenous bioreductase activity necessary to properly metabolize
the marker. It was not possible to quantify the pimonidazole staining as a
function of PO, in part because of uncertainties regarding
the consumption of oxygen within the lens, and consequently the precise tissue
PO2. However, pimonidazoleprotein
adducts are generally considered to form only at PO values
of <1.3 kPa (Varghese et al.,
1976
). Thus, the present results indicate that the embryonic lens
was relatively hypoxic by E10 and remained so throughout development.
Quantitative in vivo optode measurements of PO in
the vitreous humor immediately behind the lens supported this notion. The
measured PO in this region was <0.4 kPa. Despite an
approximately threefold increase in PO (to 1.3 kPa)
following incubation in 50% O2, PO in the
fluids bathing the posterior surface of the lens remained below the 0.4 kPa
threshold for pimonidazole adduct formation. This probably explains why lenses
from normoxic and hyperoxic embryos were stained similarly with the
pimonidazole adduct-specific antibody.
Incubation in 50% O2 was not associated with increased mortality
or other obvious pathological change in the embryos. Careful measurements,
however, revealed a slight but significant increase in the size of the lens
relative to normoxic controls. This is consistent with earlier observations
that established that the overall growth of chicken embryos was accelerated
under hyperoxic conditions (van Golde et
al., 1998) and that the mass of various organs systems, including
the eye, was significantly greater than those in age-matched normoxic controls
(McCutcheon et al., 1982
;
Stock et al., 1983
).
Furthermore, the degree of growth enhancement was shown to be proportional to
the ambient oxygen concentration. Thus, treatment with 70% O2
caused a greater increase than treatment with 40% or 60% O2
(Stock et al., 1983
). The fact
that embryonic organ systems show a substantial growth response to elevated
ambient O2 suggests that increases in ambient
PO are associated in embryos with an increase in tissue
PO2. Significantly, the vasoconstrictor
response, which in adults serves to limit blood flow to organs under hyperoxic
conditions, is lacking in embryos (van
Golde et al., 1999
). Although the lens was significantly larger in
hyperoxic embryos than age-matched normoxic controls, we did not determine
whether the increase reflected an increase in cell size, cell number or
both.
The OFZ forms on E12 and expands thereafter. Because the expansion of the
OFZ is closely matched by the growth of the tissue
(Bassnett and Beebe, 1992), the
depth of organelle-containing cells remains relatively constant during
development (Fig. 5). Under
normoxic conditions, the border of the OFZ is located 850 µm below the
equatorial surface of the embryonic chicken lens
(Fig. 5A). Under hyperoxic
conditions, the border is situated approximately 100 µm further into the
lens. This increase may be partly explained by the hyperoxia-induced increase
in lens size but it is also associated with a real decrease in the size of the
OFZ, suggesting that hyperoxia inhibits the organelle breakdown process in the
deep cortical cell layers. The PO in this region of the
normoxic or hyperoxic lens was not quantitatively determined due to the very
small size of the tissue. However, we postulate that, due to oxygen
consumption in the outer layers, a standing gradient of PO
must be established within the lens and that organelle loss may be triggered
when local PO falls below a critical threshold value.
Presumably, under hyperoxic conditions, this threshold value is encountered
somewhat deeper into the lens.
Measurements made on other cellular conglomerates indicate that standing
internal gradients of PO (and other small metabolites) are
an inevitable consequence of the avascular state. For example, in
multicellular spheroids (a well-established in vitro model for solid
tumors), growth of a spheroid invariably leads to progressive hypoxia in the
core region (Dubessy et al.,
2000). In vitro, multicellular spheroids show three
phases of development (Dubessy et al.,
2000
). In the first phase, there is cellular proliferation
throughout the spheroid and a concomitant increase in spheroid volume. In the
second phase, two cell layers are distinguished. The outer layer (also known
as the viable rim) is proliferative and the inner layer is quiescent, due to
diffusion-limited access to oxygen and nutrients and a decrease in pH. The
final phase occurs when spheroids reach diameters of 200500 µm. At
this point, the accumulated metabolic deficits in the center of spheroids
result in the formation of a necrotic core region
(Carlsson et al., 1983
). These
phases are reminiscent of embryonic lens development. During lens development,
cell proliferation is restricted to the peripheral layer of tissue. All cells
are initially nucleated. However, when the lens polar diameter reaches
approximately 1 mm, the cells in the lens core abruptly lose their nuclei in
an apoptosis-like process. Thereafter, a thin rind of nucleated cells
(resembling the viable rim of the spheroids) persists at the surface of the
steadily growing tissue.
In summary, incubation of fertilized chicken eggs under hyperoxic
conditions resulted in a marked increase in intraocular PO
and a significant increase in the depth of the organelle-containing layer of
the lens. We hypothesize that an internal gradient of PO,
generated largely by mitochondrial oxygen consumption in the outer layers,
may, in the inner layers, lead to mitochondrial dysfunction. Release of
proapoptotic factors such as cytochrome c from oxygen-starved
mitochondria may trigger an apoptosis-like cascade of events that results in
breakdown of all cytoplasmic organelles. Because the effects of 50%
O2 treatment on the development of the OFZ were relatively modest,
we endeavored to incubate embryos in either normobaric 100% O2 or
hyperbaric 100% O2, reasoning that such treatment might completely
inhibit organelle degradation. Unfortunately, both treatments killed the
developing embryos. Similarly, we reasoned that increased lens hypoxia might
result in accelerated organelle degradation in the superficial layers of the
lens. However, even brief exposure to hypoxic (10% O2) conditions
resulted in high mortality, as reported by others
(Tazawa et al., 1992). The
potential role of intralenticular PO gradients in
triggering organelle loss might, therefore, be better pursued in an organ
culture setting. To our knowledge, however, no organ culture system has been
described in which fiber cell differentiation and, in particular, organelle
loss persist for an extended period in vitro.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Airley, R. E., Loncaster, J., Raleigh, J. A., Harris, A. L., Davidson, S. E., Hunter, R. D., West, C. M. and Stratford, I. J. (2003). GLUT-1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: Relationship to pimonidazole binding. Int. J. Cancer 104,85 -91.[CrossRef][Medline]
Appleby, D. W. and Modak, S. P. (1977). DNA degradation in terminally differentiating lens fiber cells from chick embryos. Proc. Natl. Acad. Sci. USA 74,5579 -5583.[Abstract]
Banasiak, K. J., Xia, Y. and Haddad, G. G. (2000). Mechanisms underlying hypoxia-induced neuronal apoptosis. Prog. Neurobiol. 62,215 -249.[CrossRef][Medline]
Bassnett, S. (1995). The fate of the Golgi apparatus and the endoplasmic reticulum during lens fiber cell differentiation. Invest. Ophthalmol. Vis. Sci. 36,1793 -1803.[Abstract]
Bassnett, S. (1997). Fiber cell denucleation in the primate lens. Invest. Ophthalmol. Vis. Sci. 38,1678 -1687.[Abstract]
Bassnett, S. (2002). Lens organelle degradation. Exp. Eye Res. 74, 1-6.[CrossRef][Medline]
Bassnett, S. and Beebe, D. C. (1992). Coincident loss of mitochondria and nuclei during lens fiber cell differentiation. Dev. Dyn. 194, 85-93.[Medline]
Bassnett, S., Croghan, P. C. and Duncan, G. (1987). Diffusion of lactate and its role in determining intracellular pH in the lens of the eye. Exp. Eye Res. 44,143 -147.[Medline]
Bassnett, S. and Duncan, G. (1986). Variation of pH with depth in the rat lens measured by double-barreled ion sensitive microelectrodes. In The Lens: Transparency and Cataract (ed. G. Duncan), pp. 77-85. Rijswijk, The Netherlands: EURAGE.
Bennewith, K. L., Raleigh, J. A. and Durand, R. E.
(2002). Orally administered pimonidazole to label hypoxic tumor
cells. Cancer Res. 62,6827
-6830.
Carlsson, J., Nilsson, K., Westermark, B., Ponten, J., Sundstrom, C., Larsson, E., Bergh, J., Pahlman, S., Busch, C. and Collins, V. P. (1983). Formation and growth of multicellular spheroids of human origin. Int. J. Cancer 31,523 -533.[Medline]
Corpechot, C., Barbu, V., Wendum, D., Chignard, N., Housset, C.,
Poupon, R. and Rosmorduc, O. (2002). Hepatocyte growth factor
and c-Met inhibition by hepatic cell hypoxia: a potential mechanism for liver
regeneration failure in experimental cirrhosis. Am. J.
Pathol. 160,613
-620.
Dahm, R. (1999). Lens fibre cell differentiation a link with apoptosis? Ophthalmic Res. 31,163 -183.[CrossRef][Medline]
Dubessy, C., Merlin, J. M., Marchal, C. and Guillemin, F. (2000). Spheroids in radiobiology and photodynamic therapy. Crit. Rev. Oncol. Hematol. 36,179 -192.[Medline]
Hale, L. P., Braun, R. D., Gwinn, W. M., Greer, P. K. and
Dewhirst, M. W. (2002). Hypoxia in the thymus: role of oxygen
tension in thymocyte survival. Am. J. Physiol. Heart Circ.
Physiol. 282,H1467
-H1477.
Helbig, H., Hinz, J. P., Kellner, U. and Foerster, M. H. (1993). Oxygen in the anterior chamber of the human eye. Ger. J. Ophthalmol. 2,161 -164.[Medline]
Hoper, J., Funk, R., Zagorski, Z. and Rohen, J. W. (1989). Oxygen delivery to the anterior chamber of the eye a novel function of the anterior iris surface. Curr. Eye Res. 8,649 -659.[Medline]
Ishizaki, Y., Jacobson, M. D. and Raff, M. C.
(1998). A role for caspases in lens fiber differentiation.
J. Cell Biol. 140,153
-158.
Kaanders, J. H., Wijffels, K. I., Marres, H. A., Ljungkvist, A.
S., Pop, L. A., van den Hoogen, F. J., de Wilde, P. C., Bussink, J., Raleigh,
J. A. and van der Kogel, A. J. (2002). Pimonidazole binding
and tumor vascularity predict for treatment outcome in head and neck cancer.
Cancer Res. 62,7066
-7074.
Mathias, R. T., Riquelme, G. and Rae, J. L. (1991). Cell to cell communication and pH in the frog lens. J. Gen. Physiol. 98,1085 -1103.[Abstract]
Maurice, D. M. (1998). The Von Sallmann Lecture 1996: an ophthalmological explanation of REM sleep. Exp. Eye Res. 66,139 -145.[CrossRef][Medline]
McCutcheon, I. E., Metcalfe, J., Metzenberg, A. B. and Ettinger, T. (1982). Organ growth in hyperoxic and hypoxic chick embryos. Respir. Physiol. 50,153 -163.[CrossRef][Medline]
Modak, S. P. and Bollum, F. J. (1972). Detection and measurement of single-strand breaks in nuclear DNA in fixed lens sections. Exp. Cell Res. 75,307 -313.[Medline]
Ormerod, L. D., Edelstein, M. A., Schmidt, G. J., Juarez, R. S., Finegold, S. M. and Smith, R. E. (1987). The intraocular environment and experimental anaerobic bacterial endophthalmitis. Arch. Ophthalmol. 105,1571 -1575.[Abstract]
Raleigh, J. A., Dewhirst, M. W. and Thrall, D. E. (1996). Measuring tumor hypoxia. Semin. Radiat. Oncol. 6,37 -45.[Medline]
Sakaue, H., Tsukahara, Y., Negi, A., Ogino, N. and Honda, Y. (1989). Measurement of vitreous oxygen tension in human eyes. Jpn J. Ophthalmol. 33,199 -203.[Medline]
Seddon, B. M., Honess, D. J., Vojnovic, B., Tozer, G. M. and Workman, P. (2001). Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic electrode in the p22 tumor. Radiat. Res. 155,837 -846.[Medline]
Shestopalov, V. I. and Bassnett, S. (2000).
Expression of autofluorescent proteins reveals a novel protein permeable
pathway between cells in the lens core. J. Cell Sci.
113,1913
-1921.
Stock, M. K., Francisco, D. L. and Metcalfe, J. (1983). Organ growth in chick embryos incubated in 40% or 70% oxygen. Respir. Physiol. 52, 1-11.[CrossRef][Medline]
Tazawa, H., Hashimoto, Y., Nakazawa, S. and Whittow, G. C. (1992). Metabolic responses of chicken embryos and hatchlings to altered O2 environments. Respir. Physiol. 88, 37-50.[CrossRef][Medline]
van Golde, J., Borm, P. J., Wolfs, M., Gerver, W. and Blanco, C. E. (1998). The effect of hyperoxia on embryonic and organ mass in the developing chick embryo. Respir. Physiol. 113, 75-82.[CrossRef][Medline]
van Golde, J. M., Mulder, T. A., Scheve, E., Prinzen, F. W. and
Blanco, C. E. (1999). Hyperoxia and local organ blood flow in
the developing chick embryo. J. Physiol.
515,243
-248.
Varghese, A. J., Gulyas, S. and Mohindra, J. K. (1976). Hypoxia-dependent reduction of 1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol by Chinese hamster ovary cells and KHT tumor cells in vitro and in vivo. Cancer Res. 36,3761 -3765.[Abstract]
Winkler, B. S. and Riley, M. V. (1991). Relative contributions of epithelial cells and fibers to rabbit lens ATP content and glycolysis. Invest. Ophthalmol. Vis. Sci. 32,2593 -2598.[Abstract]
Wride, M. A. and Sanders, E. J. (1998). Nuclear degeneration in the developing lens and its regulation by TNFalpha. Exp. Eye Res. 66,371 -383.[CrossRef][Medline]