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(Investigative Ophthalmology and Visual Science. 2000;41:568-579.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Rapid Upregulation of Fibroblast Growth Factor Receptor 1 (flg) by Rat Photoreceptor Cells after Injury

Shiro Ozaki, Monte J. Radeke and Don H. Anderson

From the Center for the Study of Macular Degeneration, Neuroscience Research Institute, University of California, Santa Barbara.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the mechanism by which basic fibroblast growth factor (bFGF) exerts its neuroprotective effects on degenerating or injured photoreceptors.

METHODS. Confocal immunofluorescence microscopy was used to identify sites of bFGF and FGF receptor 1 (FGFR1) expression after focal injury or experimental retinal detachment in adult rats. FGFR1 expression was analyzed immunohistochemically and at the transcription level in single photoreceptor cells, after reverse transcription (RT), using the polymerase chain reaction (PCR). Real time quantitative RT-PCR was used to measure changes in FGFR1 mRNA levels in the retina in response to injury or detachment.

RESULTS. Confocal immunofluorescence observations showed that FGFR1 immunoreactivity in the rat retina is concentrated primarily in the perinuclear cytoplasm of photoreceptor cell bodies. Reverse transcription of total RNA derived from dissociated rat photoreceptor cells, followed by amplification of FGFR1 cDNA using the PCR, verified the presence of FGFR1 transcripts in normal rat photoreceptor cells; in contrast, no evidence of bFGF transcription was detected. Collectively, these results provide compelling evidence for FGFR1 gene expression by rat photoreceptors in situ. Within hours after experimental retinal detachment or focal injury, there is a twofold increase in FGFR1 immunoreactivity in the outer nuclear layer that persists for at least 7 days; a similar increase in bFGF immunoreactivity in the interphotoreceptor matrix is also apparent. This increase in FGFR1 protein levels after detachment and injury also was confirmed by western blot analysis. Real time quantitative RT-PCR analyses revealed that a rapid upregulation of FGFR1 mRNA occurred within 12 hours after retinal injury/detachment, but then declined to near baseline levels by 24 hours.

CONCLUSIONS. This body of evidence strongly suggests that the photoreceptor rescue effect elicited by retinal injury as well as by injection of exogenous bFGF is mediated, at least in part, by upregulation of the FGFR1 by the photoreceptor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the past few years, a number of growth factors, cytokines, and neurotrophins have been shown to have survival-promoting activity in the neural retina.1 Basic fibroblast growth factor (bFGF) was the first factor demonstrated to have survival-promoting neurotrophic activity in rats with an inherited retinal dystrophy.2 Subsequently, Faktorovich et al.3 speculated that endogenous (injury-induced) bFGF also effects a "rescue" of rat photoreceptors injured by exposure to constant light. Most recently, exogenous bFGF has been shown to retard the photoreceptor cell loss that occurs in a rodent model of retinal aging.4

Collectively, these results indicate that bFGF has the potential to mitigate the degenerative changes in photoreceptor cells that are induced by a broad array of insults to the retina, including genetic defects, focal injury, trauma, ischemia, photooxidative stress, and age. However, the therapeutic potential of bFGF and bFGF-like agents is tempered by in vivo studies demonstrating that exogenous bFGF also can mimic aspects of reactive gliosis induced by retinal injury.5 These contrasting results provide the impetus to identify the retinal cell types involved in, and the molecular events responsible for, eliciting these seemingly divergent effects.

Levels of endogenous bFGF have been shown to increase at sites of injury, thereby suggesting that its release and/or activation from intracellular sources and/or extracellular depots may be an integral part of the "rescue" effect.6 7 8 9 10 11 12 Similarly, the potentially important role of the FGF receptors(FGFRs) in this process also has received increased scrutiny.12 13 Nevertheless, the "rescue" effect in the retina remains poorly characterized in terms of the specific triggering events, the role of the FGFRs, as well as the ensuing cascade of intracellular events and cellular interactions that presumably occurs. In this investigation, we attempt to clarify the response of the photoreceptors to acute retinal injury in vivo. We present several lines of evidence suggesting that the photoreceptor cells play a primary role in their own survival after injury, by rapidly up regulating the high-affinity bFGF receptor (FGFR1) on their respective cell surfaces.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Long–Evans pigmented rats (8–12 weeks of age) were cared for and treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All surgical procedures were carried out under general anesthesia using intraperitoneal injection of ketamine hydrochloride (20 mg/kg; Phoenix Scientific, Inc., St. Joseph, MO) and xylazine hydrochloride (2 mg/kg; Lloyd laboratories, Inc., Shenandoah, IA); supplemental doses were administered as needed.

Retinal Detachment
Under general anesthesia, maximal pupillary dilation was achieved using topical 1% atropine sulfate solution and 1% tropicamide solution (Optipics Laboratories Corp., Fairton, NJ). Local ocular anesthesia was induced with topical 0.5% proparacaine hydrochloride solution (Bausch & Lomb, Tampa, FL). The limbal conjunctiva was cut 90°, and one scleral incision was made approximately 4 mm from the limbus. An experimental retinal detachment was created by subretinal injection of balanced salt solution (BSS; Alcon Laboratories Inc., Ft. Worth, TX) using a glass micropipette. A glass micropipette (tip diameter, 70–100 µm) was inserted through the scleral incision and advanced through the superior retina and vitreous to the opposite inferior retina approximately 5 disc diameters inferior to the optic disc. Using a microinjector, BSS was injected slowly through the micropipette until a retinal detachment was created. At the indicated times the animals were euthanatized using an overdose of sodium pentobarbiturate (Abbott Laboratories, North Chicago, IL) and the eyes were enucleated.

Tissue Preparation
For immunohistochemical analysis, the eyes were fixed overnight in 0.1 M sodium cacodylate buffer, pH 7.4, containing 4% paraformaldehyde. The cornea and lens were removed, and the retina was dissected from the rest of the eye. The retina was then divided into two samples; one included the site of retinal detachment and the other included the penetrating injury site.

Antibodies
A polyclonal rabbit antibody directed against two peptide fragments of bovine pituitary bFGF was obtained from Andrew Baird (anti-bFGF 810, amino acids 30–50). This antibody was purified using protein A-Sepharose column chromatography. Rabbit anti-FGFR1 antibody, directed against the 15 carboxyl-terminal amino acids of FGFR1 gene product was purchased from Santa Cruz Biotechnology [Flg(C-15); Santa Cruz, CA]. The antibody recognizes an intracellular portion of the receptor protein and does not recognize secreted forms of FGFR1.14 15 Anti-rhodopsin monoclonal antibody was generously provided by Robert Molday (rho4D2). This antibody, directed against a highly conserved epitope near the N terminus of rhodopsin, cross-reacts with rhodopsin from a broad range of vertebrate species.16

Dissociated Photoreceptor Immunolabeling
The isolated retina was placed in a tube containing 1 ml of 25 mM HEPES-Dulbecco’s modified Eagle’s medium (DMEM), pH 7.4. (Life Technology, Grand Island, NY). Dissociated photoreceptors were then obtained by vortexing with three 1- to 2-second pulses. After allowing the pieces of retina to settle for ~1 minute, the resulting supernatant was collected, transferred to a new tube containing 1 ml of HEPES-DMEM, and then plated on poly-L-lysine (5 µg/ml)-coated coverslips. After 30 minutes at 4°C, a coverslip with attached cells was rinsed with 0.1 M phosphate-buffered saline (PBS), pH 7.4, and then fixed for 30 minutes with 4% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4. After rinsing with PBS, nonspecific binding sites were blocked with 5% normal donkey serum in PBS, the cells were incubated with both the monoclonal anti-rhodopsin antibody (rho4D2) and the anti-FGFR1 antibody [Flg(C-15)] in PBS containing 0.5% bovine serum albumin, 0.1% Triton X-100, and 0.1% sodium azide (PBTA) for 2 hours. The cells were then rinsed three times with PBTA, incubated for 2 hours with a 1:200 dilution each of Cy3-labeled anti-rabbit IgG and Cy2-labeled anti-mouse IgG (Jackson Immunolaboratories, Inc., West Grove, PA) in PBTA, rinsed three times with PBTA, mounted in 5% n-propyl gallate in glycerol, and then examined by laser scanning confocal microscopy (MRC-1024; Bio-Rad Laboratories, Hercules, CA).

Confocal Microscopy and Immunodensitometry
Tissue specimens were processed according to the method described by Matsumoto and Hale,17 with minor modifications. Briefly, approximately 1-mm2 specimens were cut from the fixed retina samples, rinsed in PBS for at least 2 hours, and then embedded in 5% agarose (Sigma Chemical Co., St. Louis, MO) in PBS. One hundred–micrometer sections were then cut on a Vibratome (Technical Products International, Polysciences, Warrington, PA) and incubated overnight at 4°C in PBS with 5% normal donkey serum. The next day the diluted blocking serum was removed and the sections were then incubated overnight at 4°C with primary antibody diluted in PBTA. The following day, sections were rinsed with PBTA and the Cy3 anti-rabbit IgG (1:200 in PBTA) was added. After an overnight incubation at 4°C, the sections were then washed with PBTA, mounted using 5% n-propyl gallate in glycerol, and examined by laser scanning confocal microscopy (MRC-1024; Bio-Rad Laboratories).

To compare the immunoreactivity levels semi-quantitatively, 10 0.5-µm optical sections of each specimen were captured along the z-axis using identical microscope settings, and a projection series of the images was generated. Labeling intensity in outer nuclear layer (ONL) was then quantified using the Bio-Rad Lasersharp software package.

Protein Extraction and Immunoblotting
Isolated rat retinas were washed in PBS (pH 7.4) and homogenized with a dounce homogenizer in 10 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF, 3 µg/ml leupeptin, and 3 µg/ml pepstatin A, pH 7.4 (homogenization buffer) at 4°C. The homogenate was then centrifuged at 20,000g for 15 minutes at 4°C, and the supernatant was stored at -70°C. The resulting pellet containing the cell membranes was then resuspended in lysis buffer (homogenization buffer containing 1% Triton X-100) and centrifuged again at 20,000g for 15 minutes at 4°C. The resulting supernatant containing the detergent-soluble membrane proteins was collected and stored at -70°C. A volume containing 30 µg of protein was added to an equal volume of 2x sample buffer, boiled for 5 minutes, and electrophoresed on a 6% sodium dodecyl sulfate-polyacrylamide gel.18 Proteins were transferred to nitrocellulose membrane using borate buffer (10 mM sodium tetraborate and 40 mM boric acid, pH 8.5) as the transfer buffer. The protein blot was then blocked with 0.5% nonfat dry milk in immunoblot buffer (50 mM Tris-HCl, 1 mM MgCl2, and 1 mM CaCl2, pH 7.4) at room temperature for 1 hour. After washing with 0.1% Tween-20 in immunoblot buffer, the membrane was incubated overnight at 4°C with a 1:1000 dilution of rabbit anti-FGFR1 antibody (Santa Cruz Biotechnology) in blot buffer containing 0.1% Tween-20. After washing three times in this blot buffer, the membrane was incubated with alkaline phosphate–conjugated anti-rabbit IgG (1:30,000; Sigma Chemical Co.) in blot buffer for 60 minutes at 4°C. The blot was then washed in blot buffer, and the immunoreactive bands were visualized using an alkaline phosphatase detection kit (Bio-Rad Laboratories).

Time Course of FGFR1 Upregulation
A chemiluminescent detection system (Super Signal, West Dura; Pierce, Rockford, IL) was used to quantify levels of FGFR1 protein expression after detachment or injury. Briefly, a volume containing 10 µg retinal protein per sample was loaded into each lane of the gel. The blotted membrane was incubated for 1 hour at room temperature with a 1:5000 stock dilution of rabbit anti-FGFR1 antibody (Santa Cruz Biotechnology) in TBS containing 0.1% Tween-20 (TBS-T). After washing three times in TBS-T, the membrane was incubated with horseradish peroxidase–labeled anti-rabbit IgG (1:10,000; Santa Cruz Biotechnology) for 1 hour at room temperature. After washing the membrane at least six times in TBS-T, the membrane was incubated in detection solution according to the manufacturer’s specifications. The membrane was exposed to X-ray film, and the films were then captured as digital images using an Epson Expression 636 scanner (Epson America, Inc., Torrance, CA). The 145-kDa bands at each time point were then analyzed by densitometry using the NIH Image software version 1.62.

Single Photoreceptor Reverse Transcription–Polymerase Chain Reaction
Cell Harvesting and RNA Preparation.
The single photoreceptor cell reverse transcription (RT)–polymerase chain reaction (PCR) technique used in this study was adapted from single-cell RT-PCR methods described previously by several other investigators.19 20 21 22 A rat retina was dissected on ice and placed in a tube containing 1 ml of 25 mM HEPES-DMEM; pH 7.4 (Life Technology). Photoreceptors were dissociated from the retina by vortexing using three 1- to 2-second pulses. Fifty microliters of the supernatant was collected and diluted with 1 ml HEPES-DMEM. The resulting cell suspension was plated on several poly-L-lysine–coated (5 µg/ml) coverslips. After settling for 30 minutes at 4°C, a coverslip with attached cells was placed in a flow chamber and washed with several hundred milliliters of HEPES-DMEM using a peristaltic pump. Single photoreceptor inner segment–outer segment (IS-OS) fragments were then collected manually by aspiration using a micropipette and a micromanipulator. The tip of the glass micropipette, which contained the single photoreceptor IS-OS, was broken into a 0.2-ml PCR tube filled with 5.0 µl of 0.5% Triton X-100 (Pierce) containing 0.5 unit of RNase inhibitor (5'–3' Prime Inc., Boulder, CO), incubated for 5 minutes on ice, and stored at -70°C for later analysis. Negative ("sham") controls included the same procedure but without the actual harvesting of a photoreceptor.

First-Strand cDNA Synthesis.
After thawing, 0.5 µl of 5.6 µM oligo(dT) (Boehringer Mannheim, Indianapolis, IN) was added to the photoreceptor IS-OSs. This mixture was heated to 70°C for 10 minutes and then chilled on ice for 5 additional minutes. Then 4.5 µl of a stock solution was added to yield 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM each deoxyribonucleotide triphosphate (Boehringer Mannheim), 20 U of recombinant RNase inhibitor (Promega; Madison, WI), and 100 U of Superscript II RNase H- reverse transcriptase (Life Technology). The resulting 10-µl reaction was incubated for 50 minutes at 42°C for the synthesis of single-strand cDNA, after which the reaction was terminated by heat inactivation at 70°C for 15 minutes.

PCR Amplification.
A first round of multiplex PCR amplification was performed using the resultant cDNA and appropriate primer pairs for the following genes of interest: (a) bFGF; (b) FGFR1; (c) phosducin, which modulates phototransduction in retinal photoreceptors,23 was used as a positive control for the presence of photoreceptor-derived cDNA; (d) thy-1 was used to control for the presence of retinal ganglion cell contamination24 ; and (e) glial fibrillary acidic protein (GFAP) was used to control for the presence of retinal glial cell–derived cDNA.25 The sequences of these primers, designated as "outside" primers, are given in Table 1 . To each cDNA reaction, 40 µl of a master mix containing 0.25 µM of each forward and reverse outside primer, 12.5 mM Tris-HCl (pH 8.3), 62.5 mM KCl, 2.5 mM MgCl2, 0.25 mM each of the 4 deoxyribonucleotide triphosphates, and 2.5 U of Taq DNA polymerase (Promega) was added. After a 3-minute incubation at 94°C, PCR amplification was carried out using 40 cycles of the following temperature profile: 30 seconds at 94°C, 30 seconds at 55°C, and 90 seconds at 72°C using a GeneAmp PCR System 9600 thermal cycler (PE Applied Biosystems, Foster City, CA).


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  		Table 1. Primer Pairs for Single-Cell RT-PCR

 
After this first round of PCR, 5 µl of the resulting amplification products were added to 245 µl nanopure water. For each of the five relevant genes, 10 µl of the diluted first round PCR products was placed in a PCR tube containing 10 µl of a PCR reaction mixture, resulting in the following reaction conditions: 0.2 µM of the appropriate forward and reverse inside primers, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM each of the four deoxyribonucleotide triphosphates, and 2.5 U of Taq DNA polymerase. The second round PCR amplifications were performed using the same cycling parameters described above. Ten microliters from each reaction was then loaded on a 1.8% tris-borate-EDTA agarose gel containing 0.5 µg/ml ethidium bromide and electrophoresed at 50 V for 120 minutes. Photographs of the gels were taken under UV light with a Polaroid camera (Polaroid, Cambridge, MA).

Real-Time Quantitative RT-PCR
Quantitative RT-PCR was performed, and data were analyzed using the Taqman PCR fluorescence detection system26 in combination with a Prism 7700 Sequence Detector (PE Applied Biosystem27 ). In brief, a fluorescent oligonucleotide probe that binds to the PCR products was used. These Taqman probes were labeled with a 5' reporter dye, FAM, and a 3' quencher dye, TAMRA. During PCR, the 5'–3' nuclease activity of Taq DNA polymerase releases the reporter dye, which is then detected by the Prism 7700 system. Values corresponding to the PCR cycle number at which the fluorescent emission, monitored in real time, reaches a threshold above the baseline emission were determined Ct (cycle threshold) and relative abundances of the gene(s) of interest were calculated using the standard curve method.

RNA was purified from normal brain and retina at the indicated time points using an RNA extraction kit (Qiagen, Valencia, CA) and was pretreated with RNase-free DNase (Promega). cDNA was generated using DNA-free RNA as described above except that 2.5 µM random hexamers were used in place of the oligo(dT) (First-Strand cDNA Synthesis). The resulting cDNA was then used to set up 25-µl, real-time, quantitative PCR reactions consisting of the following reagents: 1x Taqman PCR buffer containing a reference dye, ROX, 5.5 mM MgCl2, AmpliTaq Gold DNA polymerase (0.025 U/µl), AmpErase uracil N-glycosylase (UNG, 0.01 U/µl), 200 µM each dATP, dGTP, dCTP, and 400 µM dUTP (PE Applied Biosystems). In this study, we used two pairs of primers and probe: A FGFR1 primer (300 nM) and probe (200 nM) as our gene of interest and an 18S ribosomal RNA (18S rRNA) primer (50 nM) and probe (50 nM) as the endogenous control (Table 2) . PCR amplification was carried out using the following temperature profile: 2 minutes at 55°C, 10 minutes at 95°C, and 40 cycles of 15 seconds at 95° and 1 minute at 60°C.


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  		Table 2. Primers and Probes for Real-Time Quantitative RT-PCR

 
Data Analysis
To compare the relative abundance of FGFR1 mRNA, standard curves of both FGFR1 and 18S rRNA were generated using cDNAs synthesized from serial 1:5 dilutions of normal rat brain RNA. For each rat retinal sample, the amount of FGFR1 and 18S rRNA was determined from those standard curves. The resulting FGFR1 amount was divided by the 18S rRNA amount to obtain a normalized value. Finally, each of normalized FGFR1 values was divided by the normalized normal retinal FGFR1 value to generate the relative expression levels of FGFR1 mRNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of FGFR1 Antibody
To assess the specificity of the anti-FGFR1 antibody, rat retinal protein homogenates were separated on 6% polyacrylamide gels under reducing conditions, blotted onto nitrocellulose filters, and then probed with the anti-FGFR1 polyclonal antibody raised against a synthetic peptide corresponding to the carboxyl-terminus of the human FGFR1. This antibody does not recognize the secreted form of FGFR1.14 The antibody detected a single 145-kDa component under these conditions (Fig. 1) in the membrane fraction. The size of this component is consistent with that of the full length, membrane-bound form of the FGFR1.28 29 30



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  		Figure 1. Anti-FGFR1 recognizes a single 145-kDa component on western blot analysis of total retinal homogenates. Retinal homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose membrane, and probed using the anti-FGFR1 antibody, Flg (C-15). A single component with an apparent molecular weight of 145 kDa is detected, which is consistent with the mass predicted for the membrane-bound, tyrosine kinase form of the FGFR1.

 
FGFR1 Immunolabeling in Dissociated Rat Photoreceptor Cells
In dissociated rat photoreceptors, anti-FGFR1 immunoreactivity is concentrated mainly on the cell surface and/or perinuclear region of the photoreceptor cell bodies. Little or no labeling is associated with other attached cell segments. In contrast, the rhodopsin antibody showed diffuse labeling of the presumptive IS-OS region (Fig. 2) .



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  		Figure 2. Immunolocalization of FGFR1 in isolated rat photoreceptors. Dissociated rat retinal photoreceptors were double-immunolabeled using anti-FGFR1 antibody (red) and anti-rhodopsin antibody (green). Anti-FGFR1 immunoreactivity is associated mainly with the photoreceptor cell body (CB), whereas anti-rhodopsin labeling is associated with the attached segments that are presumably remnants of the cylindrically shaped, inner and outer segments (IS-OS). Bar, 5 µm.

 
Expression of FGFR1 mRNA in Dissociated Rat Photoreceptors
As a second method of assessing the expression of FGFR1 by photoreceptors, single-cell PCR was carried out using isolated photoreceptor IS-OSs harvested from freshly dissociated rat retinas. The IS-OS fragments, identified by their characteristic cylindrical morphology, were harvested manually and lysed with detergent in the presence of an RNase inhibitor. This lysate was then used for the synthesis of total cDNA by reverse transcription, and the expression profile of specific genes was assessed using "nested" PCR. A first round of PCR was performed using a multiplex format with five sets of "outside" primer pairs derived from the following rat genes in a single reaction: phosducin, Thy-1, GFAP, bFGF, and FGFR1. The resulting multiplex PCR mixture was then diluted 1000-fold and used as template for detection of each gene using a second round of PCR. Five separate reactions, each containing an "inside" primer pair specific for one of the above listed genes, were carried out and the resulting PCR products were detected by gel electrophoresis and staining with ethidium bromide. Primers were selected such that they spanned at least one intron, to distinguish between PCR products amplified from mRNA or larger genomic DNA fragments. Phosducin23 was used as a marker for photoreceptor cell–derived RNA. Thy-124 and GFAP25 probes were used to determine whether the harvested photoreceptor material contained contaminating fragments derived from ganglion cells, glial cells, or their respective cell lysates. Total RNA derived from whole retina served as a positive control. RNA-free H2O and a "sham" sample, free of cells collected from the chamber containing the photoreceptors, were used to control for the effects of possible contaminating RNAs. Any experiments that showed evidence of RNA or DNA contamination as determined by the detection of specific PCR products in the H2O control samples were excluded from the analysis.

A total of 25 photoreceptor cells and 25 "sham" samples were used for this analysis. Figure 3 shows the results obtained with total retina RNA compared to that obtained from lysates of individual photoreceptor IS-OSs. Using total retina RNA, amplification products of the same length predicted from their mRNA sequence could be detected for all five genes investigated (phosducin, 360 bp; Thy-1, 138 bp; GFAP, 128 bp; bFGF, 311 bp, and FGFR1, 651 bp). In the photoreceptor IS-OSs shown in Figure 3 , phosducin and FGFR1 expression were detected, as determined by the generation of the appropriate length amplification products. A PCR product also was detected using Thy-1 specific primers, but its length of 542 bp was inconsistent with that found using whole retina RNA as well as that predicted from the Thy-1 mRNA sequence. It did, however, correspond to what would be predicted for a product generated from genomic DNA. This was experimentally confirmed by using genomic DNA as template for the nested PCR (data not shown).



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  		Figure 3. Detection of FGFR1 mRNA in single photoreceptors. Amplification products from single-cell RT-PCR visualized by staining with ethidium bromide. Using 1 ng rat retinal total RNA as a positive control, amplification products corresponding to each of the following transcripts are detected: bFGF, 311 bp; FGFR1, 651 bp; phosducin, 360 bp; Thy-1, 138 bp; GFAP, 128 bp. In the single photoreceptor shown, amplification products corresponding to phosducin and FGFR1 transcripts are detected, but no products originating from bFGF, Thy-1, and GFAP mRNAs are observed. (The 542-bp Thy-1 band results from amplification of genomic DNA; see the Results section).

 
Phosducin PCR products were detected in 19 of 25 or 76% of the photoreceptor samples. In addition, 4 of 25 or 16% of the photoreceptor samples were positive for FGFR1 mRNA. No bFGF expression could be detected in any of the photoreceptor samples. In contrast, the mRNAs for phosducin, FGFR1, or bFGF were never detected in any of the sham controls. A {chi}2 test of this data resulted in a P value of less than 0.05 for both phosducin and FGFR1, constituting statistically significant evidence for the expression of these two genes by photoreceptors (Table 3) . However, the results obtained with the two negative controls, Thy-1 and GFAP, were not as clear. Thy-1 and GFAP mRNAs were detected in 3 of 25 and 1 of 25 photoreceptors samples as well as in 2 of 25 and 1 of 25 sham controls, respectively. The {chi}2 test of this data yields a P value greater than 0.5, indicating no significant difference the photoreceptor samples and the sham control, suggesting that photoreceptors do not express significant levels of either Thy-1 or GFAP. In addition to Thy-1 and GFAP mRNA detection, Thy-1 genomic DNA was detected in three photoreceptor samples, and in one of these, GFAP genomic DNA was detected as well (Table 4) . More information about the expression of these genes can be derived from displaying the data from the single-cell PCR in the form of a matrix, which cross-correlates the expression of all five of these genes (Table 4) . From this, one can see that the detection of FGFR1 mRNA always correlates with phosducin mRNA expression (i.e., the photoreceptor marker), but never correlates with the detection of Thy-1 or GFAP mRNA (ganglion cell and glial cell markers). In other words, FGFR1 expression was detected in ~21% of the photoreceptors (as determined by phosducin expression). Also of interest is the positive correlation between detection of genomic DNA [as determined using either the Thy-1 (3 of 4) or GFAP (1 of 4) primer sets] and FGFR1 mRNA. These suggest a possible localization of the FGFR1 mRNA to photoreceptor cell body. Such a subcellular localization also would be consistent with the somewhat low level (21%) detection of the FGFR1 in photoreceptor IS-OSs. Given the anatomy of the photoreceptor, only those photoreceptors whose cell body lies close to the outer limiting membrane would be expected to have a nucleus associated with the IS-OS cell fragment.


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  		Table 3. Frequency of Detection of Each mRNA

 

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  		Table 4. Cross-Correlation between Expression of mRNAs

 
Rapid Upregulation of bFGF and FGFR1 Protein after Retinal Injury
Significant changes in the intensity of both FGFR1 and bFGF immunolabeling were detected within hours after retinal detachment or focal injury. Increased FGFR1 immunoreactivity in the ONL was observed as early as 3 hours after retinal injury (data not shown); it persisted for at least 1 day and then gradually decreased by the 7th postoperative day (Fig. 4A ). To estimate the magnitude of these changes, we quantified the anti-FGFR1 labeling intensity in the ONL. Immunodensitometric analysis of digitized optical sections captured at the same specimen depth showed that the FGFR1 labeling intensities in the ONL at 24 hours after focal injury or detachment was approximately twofold higher than levels measured from normal controls (Fig. 4B) . The upregulation of FGFR1 immunoreactivity also was observed in the inner nuclear layer (INL). This increased INL labeling was detected at 1 day after retinal detachment or retinal injury and then rapidly decreased to normal control levels at the 3- and 7-day time points. This immunolabeling was more diffuse than in the ONL and, therefore, it remains unclear whether this labeling in the INL is membrane-associated, as it appears to be in the ONL. The intensity of bFGF immunoreactivity in the interphotoreceptor matrix also increased after focal injury or detachment and followed approximately the same time course (Fig. 4C) .






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  		Figure 4. (A) Anti-FGFR1 immunoreactivity increases in photoreceptors after retinal detachment or penetrating injury. Tissue sections in the region of the retinal detachment or penetrating injury were labeled using anti-FGFR1 and visualized using laser scanning confocal microscopy. A projection series of 10 0.5-µm optical sections is shown for each condition. Normal retina (a, e); 1, 3, and 7 day(s) detachment (b, c, d); 1, 3, and 7 day(s) after penetrating injury (f, g, h). Ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), photoreceptor inner segments (IS). (B) Semi-quantitative analysis of FGFR1 immunolabeling intensity in the ONL after experimental retinal detachment or injury. The average pixel intensity at the various time points is plotted (n = 2, ±STD). (C) Anti-bFGF immunolabeling in the region of the retinal detachment or penetrating injury visualized using laser scanning confocal microscopy. Anti-bFGF immunoreactivity increases at the level of the photoreceptor inner and outer segments. At higher magnification, this immunoreactivity appears to be associated with the extracellular compartment called the interphotoreceptor matrix (IPM). A projection of 10 0.5-µm optical sections is shown for each condition. Normal retina (a, e); 1, 3, and 7 day(s) detachment (b, c, d); 1, 3, and 7 day(s) after penetrating injury (f, g, h). Ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), photoreceptor inner segments (IS). (D) Quantitative densitometric analysis of FGFR1 protein expression in normal, detached, and wounded rat retina. The results confirm that the levels of putative 145-kDa FGFR1 component increase substantially by 24 hours after injury (WD) or detachment (RD).

 
Elevated levels of bFGFR1 protein, relative to normal controls, were also detected by immunoblot analysis as early as 1 day after retinal injury (Fig. 4D) . The increased levels of bFGFR1 after detachment or focal injury appeared to be sustained for up to 7 days.

Rapid Upregulation of FGFR1 mRNA after Retinal Injury
A concomitant change in the expression of FGFR1 mRNA also was detected after retinal injury or detachment. Quantitative estimates of the relative abundance of FGFR1 mRNA were obtained using real-time RT-PCR at specific time points after focal injury or retinal detachment. A twofold upregulation of FGFR1 mRNA was detected at the earliest time point sampled (6 hours, data not shown). At 12 hours after retinal detachment or injury, FGFR1 mRNA levels in the sample population were 2.5- to 12-fold higher than those measured in the normal control sample (Fig. 5) . Elevated levels of FGFR1 mRNA persisted for at least 7 days after injury or detachment.



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  		Figure 5. FGFR1 mRNA levels rapidly increase after retinal detachment or penetrating injury. Total RNA was purified from normal, detached, or injured regions at the indicated times, and the FGFR1 mRNA level was quantitated using real-time quantitative PCR. The amount of FGFR1 mRNA is expressed relative to that in normal retina (n = 3, ±STD).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Generally, bFGF is expressed as a cell-associated protein that can act as a mitogenic, angiogenic, and/or survival factor under different experimental conditions and in different tissues. The biological activity of bFGF is thought to be mediated by binding to and activating specific high-affinity receptors in responsive cells. To date, at least four such genes have been characterized: FGFR1,31 32 33 FGFR2,34 35 36 37 FGFR3,38 and FGFR4.39

Although the neuroprotective effects of bFGF on photoreceptors have been amply demonstrated in numerous experimental animal models, the mechanism by which bFGF exerts its neuroprotective effects, and the putative role of the FGFRs in that process, are not clearly understood. In this investigation, we sought to clarify the role of FGFR1 in that regard. In theory, the "protective" effects of endogenous or exogenous bFGF could be mediated directly, via binding to FGFRs located on the photoreceptor cell surface. Alternatively, these effects could be secondary to a primary effect associated with another local cell type such as retinal glia40 41 or retinal pigment epithelial (RPE) cells.42 43 44

Indirect evidence of FGF receptor expression by photoreceptor cells has been described previously.45 46 47 Mascarelli et al.46 reported evidence of 125I-bFGF binding to adult bovine rod OSs (ROS); however, the binding activity was very low and could not be identified with a specific FGF receptor subtype. In contrast, Lewis et al.48 did not detect binding of biotinylated bFGF to rabbit or cat photoreceptor OSs, but punctate binding is present in both the ONL and OPL of cat and rat retinas (Anderson et al., unpublished observations). Blanquet and Jonet45 observed FGFR immunoreactivity in the IPL and OPL and in photoreceptor ISs of rat retina using a polyclonal antibody raised against a peptide that includes the acid box region of the chicken FGF receptor.30 Similarly, Raymond et al.47 observed immunoreactivity at the axon terminals of adult goldfish photoreceptors using the same antibody. However, because this extracellular domain of FGFR1 is highly similar in its full-length and truncated forms,30 the retinal labeling pattern obtained with this antibody could include one or both forms of the receptor. Hanneken et al.49 used a pan-specific human FGF receptor monoclonal antibody (Ab6), which is raised against a domain within the third immunoglobulin loop and which recognized both the long, membrane-bound and the truncated forms of human FGF receptors 1, 2, and 3. They also used FGFR antibodies raised against either the juxtamembrane or intracellular domains, which only recognize the membrane-bound form of FGF receptors. Their results showed clearly that these antibodies detect different forms and/or subtypes of the FGF receptor.

In this study, we provide direct evidence of FGFR1 transcription by adult rat photoreceptors in situ, and we show that the expression of FGFR1 by the photoreceptors is rapidly upregulated in response to retinal injury in vivo. An FGFR1 transcript, including the second immunoglobulin-like domain and the transmembrane region, can be amplified from single rat photoreceptors using RT-PCR. This gene product is specific for the full-length, membrane-bound form of FGFR1.28 29 30 These results conform with a recent report obtained from cultured rat photoreceptor cells using RT-PCR methods.50

Secondly, immunolocalization experiments using a peptide antibody specific for FGFR1 show that FGFR1 is appropriately located on photoreceptor cell bodies. FGFR1 immunoreactivity is observed mainly in the perinuclear cytoplasm and/or cell surface of rat photoreceptors in situ and in dissociated cells (Fig. 2) . This labeling pattern is consistent with the pattern of cell surface labeling expected from a tyrosine kinase receptor such as FGFR1. Characterization of the antibody in rat retina confirmed that it recognizes a single 145-kDa component that is consistent with a full-length form of FGFR1.28 29 30 Hanneken et al.,49 using a polyclonal FGFR1-specific antibody raised against a peptide identical with the one used in this study, also noted labeling of rat retinal neurons in the ONL. These results are consistent with our immunohistochemical and western blot data presented here.

Taken together, these immunohistochemical results are consistent with the data obtained from the single-cell RT-PCR experiments. The amplification of FGFR1 mRNA-specific product coincided completely with the detection of phosducin mRNA in the same dissociated photoreceptor preparations (4 of 4). Amplification of the FGFR1 mRNA fragment also strongly correlated with detection of only the genomic DNA fragments derived from Thy-1 (3 of 4) and GFAP (1 of 4), respectively (see Table 4 ). We conclude that rat photoreceptor cells express the full-length, membrane-bound form of FGFR1.

When the retina is perturbed, significant changes occur in the photoreceptors’ expression of FGFR1. FGFR1 immunoreactivity increases rapidly in photoreceptor cell bodies within several hours after either a penetrating retinal injury or experimental retinal detachment (Fig. 4A) . Quantitative estimates indicate that the labeling intensity is approximately 1.8 times higher in the immediate vicinity of the detachment or wound site 24 hours after injury (Fig. 4B) . Immunoblot analysis confirmed that increased levels of FGFR1 follow approximately the same time course (Fig. 4D) .

This same trend is apparent at the transcriptional level. Real-time, quantitative RT-PCR data show that mean FGFR1 mRNA levels in the retina are 2.5- to 12-fold higher at 12 hours after retinal detachment or injury (Fig. 5) , with relative FGFR1 mRNA levels 18 times higher than normal controls in one sample. A similar upregulation of FGFR1 mRNA after focal retinal injury has been detected by densitometric analysis of Northern blots using total rat retinal RNA,13 although the specific cell type(s) involved were not identified in that study.

In contrast to FGFR1, we did not detect any evidence of bFGF expression in normal dissociated photoreceptors using the single-cell RT-PCR technique (Fig. 3) . This suggests that the cytoplasmic bFGF immunolabeling in photoreceptors reported by several investigators10 47 51 may represent internalized ligand rather than a biosynthetic product. Alternatively, bFGF biosynthesis could be upregulated by photoreceptors in response to retinal degeneration, injury, or stress, as has been reported by others.10 52 53 54 Gao and Hollyfield53 reported that upregulation of bFGF mRNA in photoreceptors occurs in the injured or degenerating mouse retina by in situ hybridization.

Detection and distribution of bFGF protein in the retina can be variable and can be influenced by the use of different antibodies, species, tissue handling, fixation, and embeddment procedures.55 In the monkey retina, bFGF appears to be a component of the interphotoreceptor matrix56 : a discrete extracellular structure of aqueous insoluble glycoconjugate that envelops photoreceptor OSs and ISs. Here, using several of the same bFGF antibodies and similar fixation methods, we show that the rat interphotoreceptor matrix also displays bFGF immunoreactivity. In addition, we find that bFGF immunoreactivity in the interphotoreceptor matrix tends to increase in parallel with FGFR1 immunoreactivity in the ONL. If bFGF is not synthesized by the photoreceptors, Müller glial cells and/or the RPE, the two cell types that border the interphotoreceptor space, are the most likely endogenous local source(s) of bFGF in the interphotoreceptor matrix. This conclusion is consistent with studies demonstrating bFGF expression in primary cultures of both cell types.43 57 58

In summary, the results from this study show clearly that the retina’s response to acute injury includes a rapid and sustained upregulation of the high-affinity FGF receptor (FGFR1) by the photoreceptor cells, which appears to be accompanied by a similar increase of bFGF in the interphotoreceptor matrix that could be contributed by neighboring Müller or RPE cells. This satisfactorily describes a paracrine mechanism whereby bFGF, which is released or activated after retinal injury, binds to FGFR1 on photoreceptor target cells, which in turn initiates an intracellular cascade that "protects" the cells from further damage. We propose that intraocular injections of bFGF and, perhaps other neuroprotective agents as well, may amplify this endogenous "injury response" of the photoreceptors to varying degrees and thereby, produce their protective effects on photoreceptors in precisely the same manner.


   Acknowledgements
 
The authors thank Matthew Nealson and Michelle Staples for their laboratory help and assistance and Steve Fisher, Geoffrey Lewis, and Lincoln Johnson for their advice and discussion. The authors are grateful to Andrew Baird for providing the anti-bFGF 810 antibody and Robert Molday for providing the anti-rhodopsin antibody.


   Footnotes
 
Supported by National Institutes of Health, National Eye Institute Grant EY02082, Bethesda, Maryland.

Submitted for publication May 27, 1999; revised September 17, 1999; accepted September 29, 1999.

Commercial relationships policy: N.

Corresponding author: Don H. Anderson, Center for the Study of Macular Degeneration, Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106-5060. d_anders{at}lifesci.ucsb.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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