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Regular Articles |
From the Departments of Ophthalmology and
Neuroscience*
and Molecular Biology and
Genetics,
The Johns Hopkins University School
of Medicine, Baltimore, Maryland; and the Departments of Ophthalmology
and Cell Biology and Anatomy,
Weill Medical
College of Cornell University, New York, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early in the course of retinal degenerations, light exposure, or other types of insults such as trauma to the retina or cutting of the optic nerve, there is increased expression of FGF2 in the retina.5-8 The increased expression of FGF2 may represent an attempt to limit cell death by increasing production of a survival factor. This hypothesis is supported by demonstrations in rats that several of these same insults that increase expression of FGF2, also decrease photoreceptor cell death from subsequent insults.9-11
Thus, there is substantial evidence indicating that FGF2 is likely to be a trophic factor for photoreceptors, but there is one piece of evidence that is not supportive of this hypothesis; the survival-promoting effects of FGF2 in the retina seem to be quite different among species. Although intravitreous injections of FGF2 decrease photoreceptor cell death in the RCS rat model of inherited retinal degeneration or rats exposed to constant light,2,4,10 they have little or no effect in mouse models.12 This raises questions as to whether the findings in rats are predictive of the situation in humans, and mechanistic questions about how rats and mice might differ. However, because of the smaller size of mouse eyes compared to rat eyes, the reported lack of survival-promoting effects of FGF2 in mice may be because of the technical difficulty of intraocular injections in mice rather than a true biological difference. Furthermore, it is difficult to rule out a survival-promoting effect of Fgf2 in mice based on lack of efficacy of a single injection, because more sustained delivery may be needed to achieve a recognizable effect. Perhaps Fgf2 provides protection for both rat and mouse photoreceptors, but Fgf receptor density is somewhat greater in rat photoreceptors, so that a single intravitreous injection is sufficient to see an effect in rats, but not mice. In fact, this explanation has been suggested, because injury and light exposure cause a much smaller increase in Fgf receptor 1 mRNA expression in mice than rats.6,12-14
To ensure sustained delivery of FGF2 to photoreceptors in mice, we used a genetic approach and made transgenic mice with a rhodopsin promoter/(human)FGF2 fusion gene. We have previously used this approach to explore the role of FGF2 in ocular neovascularization15 and in this study we used it to investigate whether or not FGF2 promotes photoreceptor survival in various types of retinal degeneration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation and characterization of rhodopsin promoter/Fgf2 transgenic mice (Rho/FGF2 mice) have been described.15 These mice have been mated into a C57BL/6 background. Both homozygous and heterozygous Rho/Fgf2 mice have morphologically normal retinas.
Breeding Rho/Fgf2 Mice with rd Mice
FVB/NJ rd mice (Jackson Laboratories, Bar Harbor, ME), which are homozygous for a mutation in the ß subunit of phosphodiesterase, were mated with homozygous Rho/Fgf2 mice. Offspring were genotyped by polymerase chain reaction on tail DNA with transgene-specific primers as previously described.15 Mice that inherited a Rho/Fgf2 transgene and were heterozygous for the rd allele were mated with rd mice, and offspring were genotyped by polymerase chain reaction to identify mice carrying a Rho/Fgf2 transgene. The method of Pittler and Baehr16 was used to identify mice with two mutant rd alleles. At P21 mice were sacrificed and eyes were removed and frozen in optimal cutting temperature embedding compound (OCT; Miles Diagnostics, Elkhart, IN). Frozen sections were stained with methyl green and outer nuclear layer thickness was examined by light microscopy.
Breeding Rho/Fgf2 Mice with Q344ter Mice
Transgenic mice that express a truncated rhodopsin because of a mutation resulting in a stop codon at position 344 develop retinal degeneration and are referred to as Q344ter mice.17 Mice with a C57BL/6 background heterozygous for the Q344ter transgene were mated with mice heterozygous for the Rho/Fgf2 transgene. The offspring were genotyped and at P21 they were sacrificed, followed by the same procedures described above to examine outer nuclear layer thickness.
Hyperoxia-Induced Retinal Degeneration
We had previously noted that exposure of adult mice to 75% oxygen for longer than 1 week results in photoreceptor degeneration.18 Mice heterozygous for the Rho/Fgf2 transgene were mated with wild-type C57BL/6 mice. Offspring were genotyped and after 6 weeks of age they were exposed to 75% oxygen for 0, 1, or 2 weeks and then sacrificed. Eyes were removed, frozen in OCT, and sections were stained with hematoxylin. Outer nuclear layer thickness was examined by light microscopy.
For quantitative analysis of outer nuclear layer thickness, eyes were marked for orientation and embedded in OCT. Ten-µm sections were cut serially along the vertical meridian, and the section through the center of the optic nerve was stained with methyl green. Sections were examined with an Axioskop microscope (Zeiss, Thornwood, NY) and images were digitized using a three-charge-coupled device color video camera (IK-TU40A; Toshiba, Tokyo, Japan) and a frame grabber. Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) was used to measure the thickness of the outer nuclear layer measured 100 µm from each edge of the optic nerve. The mean of the two measurements from each eye was used as a single experimental value.
Northern Analysis
RNA blot hybridization analysis was done as previously described19,20 using 10 µg of total retinal RNA. The probe was a 0.8-kb cDNA fragment including most of exon 2 and all of exon 3 of the human FGF2 gene. The membrane was hybridized at 42°C and washed twice for 30 minutes at 60°C in 1x SSC, 0.2% sodium dodecyl sulfate, followed by two washes for 30 minutes each at 65°C in 0.2x SSC, 0.2% sodium dodecyl sulfate. Washed blots were exposed to XAR film (Kodak, Rochester, NY) for various times to obtain exposures within the linear range for autoradiographic signals. Blots were stripped and rehybridized with a probe for 18S ribosomal RNA to control for potential differences in RNA loading.
Immunohistochemistry
FGF2 transgenic mice and littermate controls were exposed to 75% oxygen for 1, 2, or 3 weeks and then sacrificed; their eyes were removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Ten-µm ocular sections were stained immunohistochemically as previously described19 with a 1:200 dilution of a rabbit anti-FGF2 peptide antibody generated against the 15 amino acids at the amino terminus of FGF2 (a gift from Dr. Leonard Hjelmeland, Davis, CA). This antibody has been extensively characterized and does not recognize FGF1.21 Specificity of staining was assessed by comparison with staining using nonimmune serum for primary antibody.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The retinal degeneration (rd) mouse is an animal
model of autosomal-recessive retinitis pigmentosa in which there is a
naturally occurring mutation in the ß subunit of phosphodiesterase,
which also occurs in some patients with retinitis
pigmentosa.22
Mice homozygous for the rd
mutation undergo degeneration of photoreceptors that begins at P10 and
is nearly complete by P21, whereas heterozygotes have normal retinas.
As expected, mice that carried only one copy of the rd
mutation, whether or not they carried the Rho/Fgf2
transgene, showed normal retinas with no evidence of retinal
degeneration (Figure 1, A and B)
. Mice
that inherited two mutant rd alleles with or without a
Rho/Fgf2 transgene showed extensive degeneration of
photoreceptors with only one row remaining at P21 (Figure 1, C and D)
.
Extensive degeneration was a consistent finding in 10 rd
mice that carried the Rho/Fgf2 transgene, indicating that
expression of high levels of FGF2 in photoreceptors does not prevent
photoreceptor cell death in rd mice.
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A mutation that generates a stop codon at position 344 of
rhodopsin results in a truncated protein and is found in some patients
with autosomal-dominant retinitis pigmentosa.23
Transgenic
mice that carry one copy of the Q344ter transgene, develop retinal
degeneration starting at P10, which is complete by P21.17
Mice that inherited the Q344ter transgene, but not the
Rho/Fgf2 transgene, showed severe retinal degeneration with
only one row of photoreceptors remaining at P21 (Figure 1E)
. Mice that inherited both transgenes showed equally severe
degeneration and this was a consistent finding among a total of 10 mice
(Figure 1F)
. At P11, Rho/FGF2 transgenics showed high-level transgene
expression whether or not they carried a Q344ter transgene
(Figure 2)
. By P14, there is substantial
photoreceptor degeneration, and as expected Rho/FGF2-Q344ter-double
transgenics showed less Fgf2 transgene mRNA compared to
Rho/FGF2 mice. These data indicate that expression of high levels of
FGF2 in photoreceptors before onset of degeneration does not prevent
retinal degeneration because of expression of truncated rhodopsin.
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In a previously published study, we observed that exposure of
adult mice to 75% oxygen for 2 or 3 weeks resulted in thinning of the
outer nuclear layer.18
This is consistent with a previous
report of damaging effects on photoreceptors of hyperoxia, presumably
because of oxidative damage.24
After exposure to hyperoxia
for 2 weeks, wild-type mice that did not carry the
Rho/Fgf2 transgene, developed progressive thinning of the
retina that was most marked in the posterior retina, adjacent to the
optic nerve (Figure 3A)
. In contrast,
exposure to 75% oxygen for 2 weeks did not result in thinning of the
outer nuclear layer in mice that carried the Rho/Fgf2
transgene (Figure 3C)
. Figure 3B
shows an ocular section from a
transgene-negative mouse extending from the aura serrata on the left to
the posterior retina on the right. It demonstrates that the outer
nuclear layer has relatively normal thickness in the periphery, but
becomes thinner posteriorly. In contrast, Figure 3D
shows an ocular
section from a Rho/Fgf2 transgene-positive mouse extending
from the aura serrata on the left to the posterior retina on the right,
and the outer nuclear layer becomes thicker scanning from the
peripheral to posterior retina. Sections at 100 µm from the optic
nerve were compared in Rho/Fgf2 transgene-positive and
-negative mice after 0, 1, or 2 weeks of oxygen exposure. There was no
significant difference in outer nuclear layer thickness in unexposed
Rho/Fgf2 transgene-negative or transgene-positive
littermates (Figure 3, E
versus F) or those exposed to
hyperoxia for 1 week (Figure 3, G
versus H). However, after
2 weeks of oxygen exposure, transgene-negative mice showed significant
thinning of the outer and inner nuclear layers, which in some mice was
dramatic (Figure 3I)
. The situation was strikingly different in
Rho/FGF2 transgene-positive mice exposed to oxygen for 2
weeks in which the outer nuclear layer was consistently well-preserved
(Figure 3J)
. Measurement of outer nuclear layer thickness by image
analysis with the examiner masked with respect to genotype
(n = 5 in each group), confirmed that sustained
expression of FGF2 in photoreceptors significantly protects them from
hyperoxia-induced death (P = 0.0001; Figure 4
).
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In rats, exposure to light or mechanical trauma causes increased
expression of Fgf2 in the retina and preconditioning with such insults
can decrease photoreceptor cell death from subsequent prolonged
exposure to light.6,14
Because ectopic FGF2 decreases
hyperoxia-induced photoreceptor cell death in mice, we sought to
determine whether hyperoxia caused increased expression of endogenous
Fgf2, which could suggest normal control mechanisms meant to protect
against oxidative damage in the retina. Northern analysis of retinas
from wild-type mice exposed to hyperoxia showed increased expression of
Fgf2 mRNA at 1 and 2 weeks, which decreased at 3 weeks
(Figure 5)
. Immunohistochemical staining
for Fgf2 in the retinas of unexposed transgene-negative mice,
showed faint staining throughout the retina (Figure 6A)
, whereas unexposed transgene-positive
mice showed dark staining in the outer nuclear layer and around retinal
blood vessels (Figure 6B)
. After 1 week of hyperoxia, staining for FGF2
was slightly decreased in both transgene-negative and
transgene-positive mice (Figure 6, C and D)
. After 2 weeks of
hyperoxia, transgene-negative mice showed only one remaining row of
photoreceptors, many of which were stained for Fgf2, as were cells in
the inner nuclear layer (Figure 6E)
. Transgene-positive mice exposed to
hyperoxia for 2 weeks (Figure 6F)
, showed no photoreceptor degeneration
and retinal staining for Fgf2 was similar to that in the retinas of
transgenics exposed to hyperoxia for 1 week, but less than that seen in
unexposed transgenics. One possible interpretation as to why FGF2
staining is reduced after 2 weeks of hyperoxia, is that hyperoxia may
result in increased Fgf2 turnover in an attempt to protect against
greater oxidative stress. In transgenics, the high levels of FGF2
produced by the Rho/Fgf2 transgene may be sufficient to keep
pace with the increased turnover so that although the steady-state
level is lower than baseline, the dynamic FGF2 may be sufficient to
prevent photoreceptor degeneration. In contrast, despite increased
Fgf2 mRNA levels in wild-type mice, the increased endogenous
Fgf2 expression may not be sufficient to keep pace with the higher
turnover and therefore photoreceptors degenerate.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that FGF2 promotes photoreceptor survival in RCS rats,2 which have a defect in phagocytosis of rod outer segments by RPE cells because of a mutation in the receptor tyrosine kinase gene Mertk.3 In albino rats, FGF2 protects photoreceptors from the damaging effects of constant light.4,10 However, intravitreous injections of FGF2 do not decrease photoreceptor degeneration from constant light or several different mutations in mice.12 In this study, we have demonstrated that sustained increased expression of FGF2 also fails to rescue photoreceptors from a recessive inherited degeneration or a dominant inherited degeneration in mice. But, increased expression of FGF2 in photoreceptors protects them from cell death because of sustained increased inspired oxygen. This suggests that the mechanisms involved in cell death because of hyperoxia, which are likely to involve oxidative damage, and cell death because of mutations in photoreceptor-specific genes are different and that FGF2 provides protection against the former, but not the latter.
Why does FGF2 decrease photoreceptor cell death in RCS rats that have an inherited degeneration and FGF2 has no effect in rd or Q344ter mice? The mutation in RCS rats is in a gene coding for a receptor in RPE cells involved in phagocytosis and results in a defect in phagocytosis of rod outer segments and accumulation of debris between photoreceptors and RPE cells.3,29 It is possible that photoreceptors die in RCS rats because the debris prevents trophic interaction between the RPE and photoreceptors and injection of FGF2 provides the missing trophic support thereby preventing photoreceptors from dying. In contrast, rd mice and Q344ter mice express mutant proteins in photoreceptor cells and whereas the mechanism by which the mutant proteins cause photoreceptors to die is unknown, it is likely to be something different from withdrawal of a trophic factor. Our data suggest that whatever the mechanism, increased expression of FGF2 in photoreceptors is unable to prevent the damage.
What is the evidence that FGF2 decreases photoreceptor cell death in rats expressing mutant proteins in photoreceptors? Intravitreous injection of FGF2 provides essentially no rescue of photoreceptors in several lines of transgenic rats expressing mutated rhodopsin (Matthew LaVail, PhD, personal communication, August, 2000); however, increased expression of FGF2 by intraocular injection of AAV.CMV-FGF2 decreases photoreceptor death in the same transgenic lines.30 Therefore, sustained increased expression of FGF2 by gene transfer can prevent photoreceptor cell death from mutated rhodopsin in rats.
Why does increased expression of FGF2 by intraocular injection of AAV.CMV-FGF2 rescue photoreceptors from mutated rhodopsin-induced cell death in rats, whereas increased expression of FGF2 in photoreceptors of Rho/FGF2 transgenic mice does not? One possible explanation is that there is some type of species difference between rats and mice, either in the mechanism of photoreceptor cell death after expression of mutated proteins, or in the effectiveness of FGF2 as a survival factor. There are some data that suggest that the latter might be true. In rats, primary limited photoreceptor cell injury results in rescue of photoreceptors from several types of secondary insults, a phenomenon that has been referred to as an injury response.6 One report has suggested that mice have little or no injury response.13 However, another study using retinal whole mounts to quantitate photoreceptor cell number, found evidence of an injury response in mice,31 and it may be that there is an injury response in both species, but it is less prominent in mice. One possible explanation is that although both rats and mice show increased expression of FGF2 in photoreceptors after trauma or other insults to the retina, rats also show increased expression of FGF receptors, whereas mice do not.13 Also, although intravitreous injections of FGF2 increase survival of photoreceptors exposed to constant light in rats,4 a similar effect is not seen in mice.12
However, species differences may not be the only explanation. Gene transfer experiments in rats have suggested that the level of expression of FGF2 is critical for the rescue effect, with levels above or below an optimal range leading to little or no rescue (John Flannery, PhD, personal communication, August, 2000). Perhaps the level of expression of FGF2 in our transgenic mice is less than that achieved in some rats with FGF2 gene transfer and is insufficient to protect photoreceptors from mutation-induced cell death. Additional experiments are needed to investigate this possibility.
A third possibility that is more intriguing is that the location of increased expression may be critical. Intraocular injections of constructs in AAV.CMV vectors result in expression in other cells in addition to photoreceptors, particularly RPE cells. Perhaps expression of FGF2 in RPE cells results in production of a more effective neurotrophic factor that rescues photoreceptors.
Although it will be important to address the above issues in future studies and determine whether other experimental approaches to increase expression of FGF2 can prevent mutation-induced photoreceptor cell death in mice, our data clearly demonstrate that FGF2 can protect photoreceptors from oxidative damage in mice. This exciting finding is consistent with other recent observations. For instance, FGF2 protects cultured human RPE cells or vascular endothelial cells from oxidative damage.20,32
Oxidative damage has been implicated in the pathogenesis of age-related macular degeneration, the most common cause of severe visual loss in patients older than the age of 60.33 In this study, we have described a new model of hyperoxia-induced photoreceptor degeneration that mimics an important aspect of macular degeneration, predilection for involvement of the posterior (central) retina compared to the anterior (peripheral) retina. Perhaps the greater blood flow in the posterior portion of the choroid compared to the anterior portion results in more severe oxidative damage to the posterior retina. Elucidation of the exact mechanism of the topographical pattern of the degeneration in our model may provide new clues to help unlock the mystery of macular degeneration. However, our study also suggests that important species differences may exist with regard to the effect of growth factors on photoreceptors. Despite this note of caution, if the ability of FGF2 to inhibit oxidative damage occurs in human photoreceptors as well as those of mice, increased expression of FGF2 in photoreceptors and/or RPE cells may provide a viable therapeutic approach in diseases such as age-related macular degeneration.
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Footnotes |
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Supported by a grant from Foundation Fighting Blindness, grant EY05951 and P30EY1765 from the National Eye Institute, a NRSA training grant (to N. B.), a Lew R. Wasserman Merit Award (to P. A. C.), a career development award (to D. J. Z.), and unrestricted funds from Research to Prevent Blindness, a grant from the Charles M. Moon, Jr. and Dr. P. Thomas Manchester Research Fund, a grant from Mrs. Harry J. Duffey, a grant from Dr. and Mrs. William Lake, a grant from the Steinbach Fund (to P. A. C. and D. J. Z.). P. A. C. is the George S. and Dolores Dore Eccles Professor of Ophthalmology.
Present address of Haruhiko Yamada and Eri Yamada: Kansai Medical University, Moriguchi, Osaka, Japan.
Accepted for publication May 17, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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