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Retina  |   November 2010
Effective and Sustained Delivery of Hydrophobic Retinoids to Photoreceptors
Author Affiliations & Notes
  • Peter H. Tang
    From the Departments of Neuroscience and
  • Jie Fan
    Ophthalmology, and
  • Patrice W. Goletz
    Ophthalmology, and
  • Lee Wheless
    the Department of Medicine, Division of Biostatistics and Epidemiology, Medical University of South Carolina, Charleston, South Carolina.
  • Rosalie K. Crouch
    From the Departments of Neuroscience and
    Ophthalmology, and
  • Corresponding author: Rosalie K. Crouch, Department of Ophthalmology, Medical University of South Carolina, 167 Ashley Avenue, Charleston, SC 29425; crouchrk@musc.edu
Investigative Ophthalmology & Visual Science November 2010, Vol.51, 5958-5964. doi:10.1167/iovs.10-5766
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      Peter H. Tang, Jie Fan, Patrice W. Goletz, Lee Wheless, Rosalie K. Crouch; Effective and Sustained Delivery of Hydrophobic Retinoids to Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2010;51(11):5958-5964. doi: 10.1167/iovs.10-5766.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Delivery of hydrophobic compounds to the retina/RPE has been challenging. The purpose of this study was to develop an effective method for the sustained delivery of retinoids to rod and cone photoreceptors of young mice lacking a normal supply of 11-cis retinal.

Methods.: Solubilized basement membrane matrix (Matrigel; BD Biosciences, San Jose, CA) loaded with 9-cis retinal was administered subcutaneously into Rpe65 −/− mouse pups for assessment of delivery to rods and cones and to Rpe65 −/− Rho −/− mouse pups for assessment of delivery to cones. Intraperitoneal injections of 9-cis retinal were used for comparison. Cone density and opsin localization were evaluated with immunohistochemistry. Cone opsin protein levels were assayed with immunoblots, and cone function was analyzed by electroretinography (ERG) recordings. Retinoid content was determined by high-performance liquid chromatography analysis of retinal extracts. Pigment levels were quantified in homogenized retinas by absorption spectroscopy before and after light exposure.

Results.: Single administration of Matrigel loaded with 9-cis retinal to Rpe65 −/− mice increased cone densities in all analyzed regions of the retina compared with mice treated using intraperitoneal delivery. Cone opsin levels increased to near wild-type levels. Similar treatment in Rpe65 −/− Rho −/− mice increased b-wave ERG amplitudes significantly, indicating the maintenance of cone function. Matrigel was shown to continuously release 9-cis retinal for periods up to 1 week.

Conclusions.: As a method for sustained drug delivery, subcutaneous administration using Matrigel proved more efficacious than intraperitoneal injection for in vivo delivery of retinoids to cone photoreceptors. These experiments are the first to show a sustained delivery of retinoids in mice and suggest a strategy for potential clinical therapeutic development.

Sustained therapeutic dosing methods for long-term disease maintenance hold numerous advantages over bolus dosing, including a lower risk of toxicity and an increased duration of therapeutic efficacy. The retina poses unique challenges to sustained therapeutic dosing because of its anatomic isolation. 1 Injection of compounds directly into the vitreal chamber is a common method for treating the retina; however, long-term therapeutic maintenance necessitates multiple injections, which are associated with retinal detachment, hemorrhage, uveitis, endophthalmitis, and infections. 2 The dynamic nature of humoral flow and its subsequent change after vitreal liquefaction in the aged eye further complicate this method of therapeutic delivery. 3 Delivery of hydrophobic molecules such as retinoids presents an additional challenge. 
Polymers provide an alternative route for administration that has the potential to be less invasive than intravitreal injection. This strategy has already been successfully demonstrated using in vitro periocular delivery models. 1,4 Thermosensitive hydrogels are a unique class of polymers that undergo phase change according to temperature. Studies have shown the potential clinical advantages of using gels for ocular drug delivery for glaucoma. 5  
We investigated the efficacy of Matrigel (BD Biosciences, San Jose, CA), a commercially available thermosensitive hydrogel, as a vehicle for sustained drug delivery to the retina. Matrigel is a solubilized basement membrane matrix originally developed to study endothelial cell differentiation in vitro and angiogenic processes in vivo. 6 The components are extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma and contain laminin, collagen, and heparin sulfate proteoglycan, as well as transforming growth factor-β (TGF-β), epidermal growth factor (EGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and other growth factors that occur naturally within the EHS tumor. 6 In our delivery protocol, Matrigel is loaded with therapeutic compounds in its liquid phase at 4°C and placed subcutaneously, where it solidifies into a plug and maintains the therapeutic compounds for sustained delivery. 6  
11-cis Retinal is the chromophore for rod and cone visual pigments and is a highly hydrophobic compound. The protein RPE65 is essential for the production of 11-cis retinal within the RPE cell. 7 Mutations in the Rpe65 gene causes type 2 Leber congenital amaurosis (LCA2), an autosomal recessive form of retinal dystrophy that can cause severe blindness and visual impairment in humans within the first year of life because of decreased endogenous production of 11-cis retinal. 8 The Rpe65 −/− mouse provides an excellent model for studying this disease and a model for testing the uptake of 11-cis retinal. 9,10  
The development of therapeutics such as gene therapy and pharmacologic agents aimed at treating LCA2 patients with RPE65 gene mutations has been greatly aided by the availability of animal models. 11,12 Mouse cones contain two visual pigments that, when bound to 11-cis retinal, are sensitive to either short-wavelength (S) or mid/long-wavelength (M/L) light. In mouse models for LCA2, both forms of cone pigments are mistrafficked throughout the cone, and degeneration follows shortly afterward. 13,14 Previous studies have shown that initiating an early regimen of delivering exogenous 11-cis retinal or its naturally occurring functional analog 9-cis retinal can partially inhibit cone degenerative processes in mouse models for LCA2. 15,16 To maximize the efficacy of retinoid therapy, developing a sustained dosing protocol that is well tolerated at an early age is essential. We report here that Matrigel loaded with 9-cis retinal, applied subcutaneously (SC) to Rpe65 −/− and Rpe65 −/− Rho −/− mice is efficacious for delivering retinoids to photoreceptors with a single administration. 
Materials and Methods
Animals
Rpe65 −/− mice were a generous gift of T. Michael Redmond (National Eye Institute, National Institutes of Health, Bethesda, MD), and Rpe65 −/− Rho −/− mice were a generous gift of Mathias Seeliger (University of Tübingen, Tübingen, Germany). C57BL/6J (Wt) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were reared under cyclic light (12 hours light/12 hours dark with the ambient light intensity at eye level of 85 ± 18 lux) conditions until the initiation of experiments, when they were transferred to a constant dark environment. Mice were killed by CO2 exposure and cervical dislocation. Husbandry was performed under dim red light (GBX-2; Eastman Kodak, Rochester, NY). All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Medical University of South Carolina Animal Care and Use Committee. 
Retinoid Delivery
All retinoids were handled under dim red light. 9-cis Retinal (Sigma-Aldrich, St. Louis, MO; 0.25 mg/animal) was dissolved in 20 μL of 100% ethanol and combined with 180 μL of either Matrigel or Matrigel with reduced growth factors at 4°C for a final volume of 200 μL. The mixture was injected SC into the dorsal torso region. 9-cis Retinal was omitted from the mixture for all control groups. For intraperitoneal (IP) delivery, 9-cis retinal (0.05–0.25 mg/animal) was dissolved in 200 μL vehicle (10% ethanol/10% bovine serum albumin in 0.9% NaCl) and injected into the peritoneal cavity. 
Cone Density Measurements
After the cornea was removed, the retina-lens complex was separated from the RPE-choroid layer and fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS) for 2 hours at room temperature (RT). After washing with PBS (3×, 20 minutes, 4°C), retinas were incubated with 0.02 mg/mL FITC-conjugated peanut agglutinin (PNA; lectin Arachi hypogaea; Sigma-Aldrich) overnight at 4°C. Retinas were washed with PBS (3×, 20 minutes, 4°C), flattened by relaxing cuts, mounted flat on a slide, and coverslipped after application of mounting medium (Fluoromount-G; Southern Biotech, Birmingham, AL). Samples were analyzed by fluorescence microscopy (Axioplan II; Zeiss, Jena, Germany) using a 100-W mercury light source and FITC filters. Cones were counted within six fields of view (Fig. 1A) at 20× magnification that spanned the entire length of the retina along the dorsal ventral axis. Cone densities are expressed as cones/mm2
Figure 1.
 
Total cone density increases in Rpe65 −/− animals with delivery of 9-cis retinal by Matrigel. Data are obtained from P30 dark-reared mice that began treatment at P10. (A) Schematic representation of retinal flat mount, with six regions analyzed for cone density spanning the dorsal ventral axis. FD, far dorsal; MD, mid-dorsal; CD, central dorsal; ONH, optic nerve head; CV, central ventral; MV, mid ventral; FV, far ventral. (B) Quantification of PNA-positive cells by region from Wt (n = 7), Rpe65 −/− mice treated with 9-cis retinal (0.25 mg/animal) using Matrigel (n = 7) or IP injection (0.05 mg/animal ×5) (n = 6) and control Rpe65 −/− mice (n = 6). Matrigel administration of 9-cis retinal significantly increased cone density at all analyzed regions of the retina compared with IP injection; *P < 0.01. Two-way ANOVA was used to test for differences in cone counts by treatment group and retinal region with post hoc pairwise t-tests used to compare individual groups. Differences in maximum trough to peak, between left and right eyes, were averaged for each treatment group and analyzed using ANOVA with Tukey's post hoc tests to compare individual groups. A two-sided P-value of 0.05 was considered significant. Bars indicate ±SD. (C) Images of flat-mounted retina labeled with FITC-conjugated peanut agglutinin lectin.
Figure 1.
 
Total cone density increases in Rpe65 −/− animals with delivery of 9-cis retinal by Matrigel. Data are obtained from P30 dark-reared mice that began treatment at P10. (A) Schematic representation of retinal flat mount, with six regions analyzed for cone density spanning the dorsal ventral axis. FD, far dorsal; MD, mid-dorsal; CD, central dorsal; ONH, optic nerve head; CV, central ventral; MV, mid ventral; FV, far ventral. (B) Quantification of PNA-positive cells by region from Wt (n = 7), Rpe65 −/− mice treated with 9-cis retinal (0.25 mg/animal) using Matrigel (n = 7) or IP injection (0.05 mg/animal ×5) (n = 6) and control Rpe65 −/− mice (n = 6). Matrigel administration of 9-cis retinal significantly increased cone density at all analyzed regions of the retina compared with IP injection; *P < 0.01. Two-way ANOVA was used to test for differences in cone counts by treatment group and retinal region with post hoc pairwise t-tests used to compare individual groups. Differences in maximum trough to peak, between left and right eyes, were averaged for each treatment group and analyzed using ANOVA with Tukey's post hoc tests to compare individual groups. A two-sided P-value of 0.05 was considered significant. Bars indicate ±SD. (C) Images of flat-mounted retina labeled with FITC-conjugated peanut agglutinin lectin.
Histologic Evaluation
To fixate the tissue, a flap was cut into the cornea, and the eye was immediately immersed in 4% PFA in 0.1 M PBS for 2 hours at RT. For morphologic analysis, eyes were dehydrated in ethanol (30%, 50%, and 70%, 15 minutes each) and left overnight in 70% ethanol at 4°C. Eyes were further washed in 90% and 100% ethanol (30 minutes each), and incubated in xylene (2 hours, RT). The eyes were then incubated in paraffin for 2 hours at 60°C under vacuum, embedded, and sectioned at 6-μm thickness. For dewaxing and rehydration, sections were dried under vacuum (20 minutes, 60°C) and washed in xylene followed by ethanol (100%, 90%, 80%, 70%, 50%) and finally deionized water (5 minutes each, RT). Sections were processed for staining with hematoxylin and eosin (H&E) and then dehydrated for coverslip mounting with medium (Permount; Sigma-Aldrich, St. Louis, MO). Slides were analyzed using a light microscope (Axioplan II; Zeiss, Jena, Germany). 
For immunofluorescence analysis, eyes were cryoprotected, embedded, frozen, and sectioned at 10-μm thickness. Sections were incubated as previously described 16 with affinity-purified anti–M/L-opsin antibody (1:500 dilution;, Chemicon/Millipore, Temecula, CA) in a humidified chamber overnight at 4°C. After washing with PBS (1×, 10 minutes, RT), sections were incubated with solution containing Alexa594-conjugated secondary antibody (Molecular Probes, Eugene, OR) and 4′,6-diamidino-2-phenylindole (1:3000 dilution; Invitrogen, Carlsbad, CA) for 2 hours in RT, washed with PBS (2×, 10 minutes, RT), and coverslip-mounted with medium (Fluoromount-G; Southern Biotech) for analysis using a light microscope with appropriate filters. 
Western Blot Analysis
Procedures were modified from methods previously described. 17 Two retinas were homogenized in 1% sodium dodecyl sulfate (Sigma-Aldrich) buffer using sonication (30 seconds). Total proteins (200 μg) from each lysate were loaded onto a 4% to 12% gel (NuPAGE Bis-Tris; Invitrogen) and run at 60 V for 30 minutes, followed by 100 V for 1.5 hours. The proteins were transferred to a nitrocellulose membrane and probed with rabbit anti–M/L-opsin primary antibody (1:2000 dilution; Chemicon/Millipore). The membrane was stripped and reprobed with mouse β-actin antibody (1:1000 dilution; Sigma-Aldrich). Secondary antibodies were goat anti-rabbit IgG (1:10,000 dilution; Vector, Burlingame, CA) and horse anti-mouse IgG (1:10,000 dilution; Vector). Bands were visualized with imaging equipment (VersaDoc; Bio-Rad, Hercules, CA). Densitometry analysis of the bands was performed with imaging and analysis software (Quantity One; Bio-Rad), and the data were expressed as a percentage of the Wt band. 
ERG Recordings
Dark-adapted (12 hours) Rpe65 −/− Rho −/− mice (postnatal day [P] 30) were placed on a DC-powered 37°C warming pad and anesthetized using xylazine (20 mg/kg body weight) and ketamine (80 mg/kg body weight). Pupils were dilated with 1 drop of phenylephrine HCl (2.5%) and atropine sulfate (1%). A reference/ground needle electrode was placed SC at the forehead. ERG responses were measured on a universal testing and electrophysiological system (System 2000; LKC UTAS Visual Diagnostic System with BigShot Ganzfeld) using a contact lens electrode brought into electrical contact with the cornea with 1 drop of methylcellulose (Gonisol; Iolab Pharmaceutical, Claremont, CA). 18 Recordings were averaged in response to five 10-ms flashes using a white spectrum (450–750 λ) from 1.56 to 24.8 cd · s/mm2 intensities with a 5-second-interval between each flash. B-wave amplitudes were measured from the trough to the highest peak and expressed in μV. 
Retinoid Analysis
All procedures were performed under dim red light on dark-adapted animals using methods modified from those previously described. 19 Retinas (4 retinas/sample) were dissected and homogenized in 200 μL PBS buffer using a microtissue grinder (Fisher Scientific, Pittsburgh, PA). Methanol (300 μL) and hydroxylamine (60 μL, 1 M in PBS buffer) were added, and samples were vortexed (30 seconds). After 5 minutes at 22°C, methylene chloride (300 μL) was added, and the samples were vortexed (30 seconds) and centrifuged (14,000g, 1 minute). The lower phase was separated, dried under argon, and dissolved in high-performance liquid chromatography (HPLC) mobile phase (11.2% ethyl acetate/2.0% dioxane/1.4% octanol in hexane, 90 mL). The syn- and anti-9-cis retinal oximes were separated using a 5-mm column (Lichrosphere SI-60; Alltech, Lexington, KY) and quantified by comparison of retention times and absorption properties with pure retinoid isomeric standards. 20  
Pigment Measurements
Retinas were dissected under dim red light from dark-adapted animals and homogenized (2 retinas/sample) with a microtissue grinder in 1 mL of 10 mM Tris buffer (pH 7.5) containing 1 mM EDTA, 1 mM 4-(2-aminoethyl)-benzene sulfonyl fluoride hydrochloride (AEBSF; Roche, Mannheim, Germany), and 10 μg DNase I (Sigma-Aldrich). Samples were shaken at 4°C for 2 hours and were centrifuged (88,000g, 15 minutes). The pellet was resuspended in 100 μL of 1% dodecylmaltoside in 0.1 M PBS, rotated for 2 hours at 4°C, and centrifuged (88,000g, 10 minutes). The supernatant was retrieved for analysis. Measurements were performed on a spectrophotometer (Cary 300; Varian, Walnut Creek, CA). Difference spectra were determined from measurements before and after bleaching with white light in the freshly prepared 20 mM hydroxylamine, pH 7.0. The concentration of isorhodopsin was calculated based on absorption at λmax = 487 nm using the extinction coefficient ε (isorhodopsin) = 43,000 M/cm. 21  
Statistical Analysis
Two-way ANOVA was used to test for differences in cone density evaluation by treatment group and retinal region with post hoc pair-wise t-tests used to compare individual groups. Differences in maximum trough-to-peak b-wave amplitudes of ERG recordings between left and right eyes were averaged for each treatment group and analyzed using ANOVA with Tukey's post hoc tests to compare individual groups. Immunoblotting results were evaluated using two-sample t-tests. Retinoid concentrations and absorbance spectra were compared with their respective reference samples using two-sample t-tests. A two-sided P-value of 0.05 was considered significant. Statistical software (SAS, version 9.1; SAS Institute, Inc., Cary, NC) was used to conduct all analyses. 
Results
Increases in Cone Density after Early 9-cis Retinal Administration in Matrigel
Previous studies have shown that early administration of 9-cis retinal to young (P10) Rpe65 −/− and Lrat −/− mice are effective for preserving rods and cones. 14,22,23 This study was undertaken to evaluate the efficiency of a single administration of Matrigel in delivering 9-cis retinal. Cone density was quantified by counts of PNA positively stained cells in six areas of the retina (Fig. 1A). Mice were dark-reared after the initiation of treatment at P10 and analyzed at age P30. Rpe65 −/− mice were given a single SC injection of Matrigel with and without 9-cis retinal. To determine the optimum dosage of 9-cis retinal for cone preservation in Rpe65 −/− mice with the Matrigel delivery, a range of dosages (0.1–1.0 mg/mouse) were tested, and gains in cone density were observed to stabilize at 0.25 mg/mouse (data not shown). 9-cis Retinal (0.25 mg) with Matrigel in Rpe65 −/− mice produced significant (P < 0.01) increases in cone densities at all six analyzed regions of the retina when compared with mice treated with Matrigel alone, although WT levels were not achieved (Fig. 1B). A multiple IP administration protocol was used for comparison, by which a dose-matched amount of 9-cis retinal was administered by five injections of 0.05 mg at ages P10, P14, P18, P22, and P26. The data show that the Matrigel delivery system produced significantly (P < 0.01) higher cone densities across all six analyzed regions of the retina compared with the IP delivery system (Fig. 1B). 
To rule out the possibility that Matrigel exerts an independent effect on cone densities in the presence of 9-cis retinal, Rpe65 −/− mice were treated by the IP protocol described, and volume-matched Matrigel was also injected SC into the dorsal torso region of the mouse at P10. There was no significant difference in the cone densities between the animals administered 9-cis retinal through IP alone and those coadministered 9-cis retinal through IP and SC Matrigel (data not shown). These data rule out the possibility of a synergistic effect between the two agents. 
Prevention of Cone M/L-Opsin Loss in Rpe65−/− Mice
The immunoreactivity of cone M/L-opsin was evaluated in central dorsal retinal sections of P30 mice. In Wt mice, M/L-opsin was normally abundant within the cone outer segments (Fig. 2A, top). In P30 Rpe65 −/− mice, there is advanced cone outer segment degeneration, highlighted by the decreased presence of M/L-opsin immunoreactivity (Fig. 2A, middle). However, 9-cis retinal administration with Matrigel at P10 resulted in increased M/L-opsin immunoreactivity in the outer segments of Rpe65 −/− retina at P30, with opsin presence also in the pedicles (Fig. 2A, bottom). 
Figure 2.
 
M/L-opsin protein increases with Matrigel delivery of 9-cis retinal in Rpe65 −/− mice. Data obtained from Rpe65 −/− P30 animals. (A) Cross-section images of the central dorsal retina are shown for Wt (top) and Rpe65 −/− mice treated with 9-cis retinal using Matrigel (bottom). For control mice, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only; middle). Paraffin-embedded retinas were sectioned and stained with hematoxylin-eosin (left column); cryoprotected retinas were sectioned and stained with both M/L-opsin-specific primary antibody (red) and DAPI (blue) for nuclei (right column). OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. (B) Western blot images for M/L-opsin (top) and β-actin (bottom) are shown for Wt and Rpe65 −/− Matrigel, IP, and control mice. (C) Densitometry band analyses are displayed as percentages of Wt. Matrigel alone and 9-cis retinal (RAL) intraperitoneally injected Rpe65 −/− mice displayed significantly decreased M/L-opsin protein levels compared with Wt; *P < 0.02. Black bar: Wt, n = 3. White bar: Matrigel alone, n = 3. Red bar: 9-cis retinal IP, n = 3. Blue bar: Matrigel plus 9-cis retinal, n = 3. Data are presented as mean ± SD and were analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Figure 2.
 
M/L-opsin protein increases with Matrigel delivery of 9-cis retinal in Rpe65 −/− mice. Data obtained from Rpe65 −/− P30 animals. (A) Cross-section images of the central dorsal retina are shown for Wt (top) and Rpe65 −/− mice treated with 9-cis retinal using Matrigel (bottom). For control mice, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only; middle). Paraffin-embedded retinas were sectioned and stained with hematoxylin-eosin (left column); cryoprotected retinas were sectioned and stained with both M/L-opsin-specific primary antibody (red) and DAPI (blue) for nuclei (right column). OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. (B) Western blot images for M/L-opsin (top) and β-actin (bottom) are shown for Wt and Rpe65 −/− Matrigel, IP, and control mice. (C) Densitometry band analyses are displayed as percentages of Wt. Matrigel alone and 9-cis retinal (RAL) intraperitoneally injected Rpe65 −/− mice displayed significantly decreased M/L-opsin protein levels compared with Wt; *P < 0.02. Black bar: Wt, n = 3. White bar: Matrigel alone, n = 3. Red bar: 9-cis retinal IP, n = 3. Blue bar: Matrigel plus 9-cis retinal, n = 3. Data are presented as mean ± SD and were analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Cone M/L-opsin quantities in retina homogenates of P30 Rpe65 −/− mice were analyzed by Western blot analysis (Figs. 2B, 2C). Multiple IP administration of 9-cis retinal to mice did not significantly increase overall M/L-opsin levels within the retina compared with mice receiving Matrigel-only injections, with both groups showing <60% of Wt levels (IP, 59% ± 14%; Matrigel-only, 52% ± 12%). Furthermore, no significant differences in M/L-opsin levels were detected between mice receiving Matrigel-only injections and those receiving saline injections after the dosing schedule of the 9-cis retinal/IP group (data not shown). Delivery of 9-cis retinal with Matrigel resulted in (P < 0.05) M/L-opsin levels near Wt levels (Matrigel, 91% ± 15%). 
Maintenance of Cone Function in Rpe65−/−Rho−/− Mice
Rpe65 −/− Rho −/− mice were used for ERG analyses to eliminate interference from rods. 24 Representative ERG traces are provided in Figure 3A, and the data are summarized in Figure 3B. Rpe65 −/− Rho −/− mice were treated at P10 as described with 9-cis retinal (0.25 mg/animal) using either a single Matrigel injection or five IP injections of 0.05 mg each and were maintained in darkness. Controls were animals injected with Matrigel alone. To gain maximum pigment formation and, therefore, maximum function, a single additional dose of 9-cis retinal (0.1 mg/mouse) was administered through the IP route to mice of all treatment groups, including the control group, 24 hours before cone function was evaluated by ERG. Delivery of 9-cis retinal using Matrigel produced significant (P < 0.05) increases in b-wave amplitudes beginning at a stimulus intensity of 1.56 cd · s/mm2, whereas IP delivery of 9-cis retinal did not produce significant increases when compared to animals treated with either a Matrigel-alone injection or with multiple saline injections after the same dosing schedule (data not shown). We did not detect a significant increase in b-wave amplitudes between animals treated with IP delivery of 9-cis retinal compared with those treated with Matrigel-only injections; however, this might have been because our equipment lacked the necessary sensitivity. Earlier studies have shown that treatment by multiple IP injections of 9-cis retinal produced some recovery in cone function; however, these animals were treated with greater doses than those of the present study and were evaluated at a younger age. 16  
Figure 3.
 
Maintenance of cone function in Rpe65 −/− Rho −/− mice with Matrigel delivery of 9-cis retinal. Rpe65 −/− Rho −/− mice were treated with 9-cis retinal using Matrigel (0.25 mg/mouse at P10) or by IP injection (0. 5 mg/animal, beginning at P10, ×5) and were analyzed at P30 with single white flash (intensity range, 1.56–24.8 cd · s/mm2) ERG recordings under dark-adapted conditions. For control mice, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only). (A) Representative ERG traces from Matrigel/9-cis retinal (left), 9-cis retinal IP (middle), and control (right) animals. Dashed line: onset of flash. (B) Quantification of b-wave amplitudes from Matrigel/9-cis retinal (black, n = 5), 9-cis retinal IP (gray, n = 5) and control (dashed, n = 5) animals. Mice administered 9-cis retinal using Matrigel showed significantly increased cone responses from 2.48 to 24.8 cd · s/mm2 flash intensities compared with those that did not receive 9-cis retinal therapy; *P < 0.05. Data are presented as mean ± SD and were analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Figure 3.
 
Maintenance of cone function in Rpe65 −/− Rho −/− mice with Matrigel delivery of 9-cis retinal. Rpe65 −/− Rho −/− mice were treated with 9-cis retinal using Matrigel (0.25 mg/mouse at P10) or by IP injection (0. 5 mg/animal, beginning at P10, ×5) and were analyzed at P30 with single white flash (intensity range, 1.56–24.8 cd · s/mm2) ERG recordings under dark-adapted conditions. For control mice, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only). (A) Representative ERG traces from Matrigel/9-cis retinal (left), 9-cis retinal IP (middle), and control (right) animals. Dashed line: onset of flash. (B) Quantification of b-wave amplitudes from Matrigel/9-cis retinal (black, n = 5), 9-cis retinal IP (gray, n = 5) and control (dashed, n = 5) animals. Mice administered 9-cis retinal using Matrigel showed significantly increased cone responses from 2.48 to 24.8 cd · s/mm2 flash intensities compared with those that did not receive 9-cis retinal therapy; *P < 0.05. Data are presented as mean ± SD and were analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Sustained Delivery of 9-cis Retinal by Matrigel
To evaluate the capability of Matrigel to maintain sustained release of 9-cis retinal to the retina, P45 Rpe65 −/− mice treated with a single dose of 0.25 mg 9-cis retinal either through Matrigel or IP delivery were compared. Because most of the cones had degenerated by P45 in this model, 13,14 it is likely that the rod opsin was responsible for most of the 9-cis retinal uptake to form isorhodopsin, allowing for the quantification of retinoids delivered to the retina. The animals were reared in cyclic light for 2 days immediately after injection and then were transferred to dark-rearing conditions. After either 3 or 7 days of dark rearing, the harvested retinas were homogenized, and the retinoids were extracted for HPLC analysis (Fig. 4A). For controls, Matrigel was used without 9-cis retinal. 
Figure 4.
 
Matrigel maintains a sustained delivery of 9-cis retinal to photoreceptors. P45 Rpe65 −/− animals were administered 9-cis retinal (0.25 mg/animal) with a single subcutaneous Matrigel or IP injection. For control animals, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only). Animals were reared under cyclic light until 2 days after treatment and then were dark reared for either 3 or 7 days, and the retinas were analyzed for extracted retinoids or isorhodopsin levels (4 eyes/sample). (A) Representative HPLC profile of retinoid extract from Rpe65 −/− mice dark reared for 3 days and administered 9-cis retinal using Matrigel. Peaks were identified by comparison with known standards. Detection was at 360 nm. (B) syn-9-cis Retinal oxime levels after 3 and 7 days of dark rearing in Rpe65 −/− mice administered 9-cis retinal with a single injection of Matrigel (black, n = 6), IP administration (gray, n = 6), or control (white, n = 6). Administration of 9-cis retinal with Matrigel significantly increased syn-9-cis retinal oxime levels compared with IP administration; *P < 0.002. Mice administered 9-cis retinal with Matrigel showed significantly increased syn-9-cis retinal oxime levels between 3 and 7 days of dark rearing; †P < 0.05. (C) Isorhodopsin levels from whole retinas of mice administered 9-cis retinal using Matrigel injection after 3 (n = 3) and 7 (n = 3) days of dark rearing. Analysis showed a significant increase in isorhodopsin levels between both time points, indicating sustained delivery of 9-cis retinal; øP < 0.01. Data are presented as mean ± SD and analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Figure 4.
 
Matrigel maintains a sustained delivery of 9-cis retinal to photoreceptors. P45 Rpe65 −/− animals were administered 9-cis retinal (0.25 mg/animal) with a single subcutaneous Matrigel or IP injection. For control animals, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only). Animals were reared under cyclic light until 2 days after treatment and then were dark reared for either 3 or 7 days, and the retinas were analyzed for extracted retinoids or isorhodopsin levels (4 eyes/sample). (A) Representative HPLC profile of retinoid extract from Rpe65 −/− mice dark reared for 3 days and administered 9-cis retinal using Matrigel. Peaks were identified by comparison with known standards. Detection was at 360 nm. (B) syn-9-cis Retinal oxime levels after 3 and 7 days of dark rearing in Rpe65 −/− mice administered 9-cis retinal with a single injection of Matrigel (black, n = 6), IP administration (gray, n = 6), or control (white, n = 6). Administration of 9-cis retinal with Matrigel significantly increased syn-9-cis retinal oxime levels compared with IP administration; *P < 0.002. Mice administered 9-cis retinal with Matrigel showed significantly increased syn-9-cis retinal oxime levels between 3 and 7 days of dark rearing; †P < 0.05. (C) Isorhodopsin levels from whole retinas of mice administered 9-cis retinal using Matrigel injection after 3 (n = 3) and 7 (n = 3) days of dark rearing. Analysis showed a significant increase in isorhodopsin levels between both time points, indicating sustained delivery of 9-cis retinal; øP < 0.01. Data are presented as mean ± SD and analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Untreated mice showed only trace levels of 9-cis retinal after 3 and 7 days of dark rearing (3 days, 2.86 ± 0.13 pmol/retina; 7 days, 2.23 ± 0.13 pmol/retina, n = 6). Administration of 9-cis retinal by IP injection resulted in a modest, but significant, increase of 9-cis retinal in mice after dark rearing for 3 or 7 days compared with untreated mice (3 days, 15.94 ± 3.02 pmol/retina; 7 days, 10.54 ± 1.78 pmol/retina; n = 6; P < 0.05). However, administration of 9-cis retinal in Matrigel showed a significant increase in 9-cis retinal levels at both time points compared with IP injection (3 days, 49.12 ± 5.74 pmol/retina; 7 days, 73.11 ± 14.36 pmol/retina; n = 6, P < 0.05) and with untreated mice (P < 0.01, Fig. 4B). Further, there was a significant increase in 9-cis retinal after 7 days of dark rearing compared with 3 days in the Matrigel-treated animals, showing that the retinoid was still being released. In the IP-injected animals, the levels were decreased at 7 days. This increase in 9-cis retinal levels observed in the Matrigel-treated mice was confirmed by quantification of isorhodopsin pigment levels through absorption difference spectra, where isorhodopsin levels doubled (3 days, 13.83 ± 2.20 pmol/retina; 7 days, 27.44 ± 3.14 pmol/retina; n = 6; P < 0.01; Fig. 4C). 
Role of Growth Factors within Matrigel for Sustained Delivery
Given that Matrigel is derived from the EHS mouse sarcoma, numerous growth factors—such as TGF-β, EGF, IGF, VEGF, and FGF—are present within the compound. 6 To investigate whether these growth factors were involved in the sustained delivery of compounds by Matrigel, P45 Rpe65 −/− mice were treated with a single administration of 9-cis retinal (0.25 mg/animal) using growth factor-reduced (GFR) Matrigel, reared in cyclic light for 2 days immediately after injection, and then transferred to dark-rearing conditions. After either 3 or 7 days of dark rearing, the retinas were homogenized and the retinoids were analyzed by HPLC for comparison with animals treated using normal Matrigel. Results show significantly decreased 9-cis retinal levels per retina at both 3 and 7 days of dark rearing (3 days, 26.80 ± 5.30 pmol/retina; 7 days, 22.84 ± 4.63 pmol/retina) using GFR Matrigel when compared with mice administered 9-cis retinal using normal Matrigel (P < 0.01), and there was no significant difference in 9-cis retinal levels between 3 and 7 days of dark rearing (P = 0.39) in animals treated with GFR Matrigel. 
Growth factors within Matrigel have previously been shown to trigger the infiltration of blood vessels into the solidified compound when injected into the mouse as a plug. 25 To investigate whether the infiltration of blood vessels contributed to the sustained release of 9-cis retinal by Matrigel in our delivery model, plugs from mice treated with normal or GFR Matrigel were retrieved and processed for immunohistochemical analysis. Vascular infiltration of the normal Matrigel plug was observed to be more abundant than was the GFR Matrigel plug (Supplementary Figs. S1A and S1B). The observed structures within the normal Matrigel plug were confirmed to be blood vessels through the histologic identification of endothelial cells (Supplementary Fig. S1C), the presence of erythrocytes in the vessel lumen (Supplementary Fig. S1E), and positive staining of platelet endothelial cell adhesion molecule-1 (PECAM-1, Supplementary Fig. S1D). The presence of 9-cis retinal within the normal Matrigel plug did not produce an observable effect on the amount of vascular infiltration (data not shown). 
Discussion
Our studies have shown that using a collagen gel such as Matrigel increases the efficiency and duration of delivery of retinoids to photoreceptors. We have used the Rpe65 −/−mouse as a model, both to track delivery of a hydrophobic compound and as a model for a clinical disorder. The drawback of this model is that early delivery is critical for cone photoreceptors because cones degenerate rapidly in the absence of the opsin chromophore. 
The retinospatial patterning of cone loss within the Rpe65 −/− retina follows the dorsal ventral axis over time. Previous studies have shown no detectable differences in cone densities within the retinas of 2-week-old Rpe65 −/− and Wt mice. 13 By P30, Rpe65 −/− mice exhibit significant loss of cone densities across all regions of the retina, highlighting the importance of innate 11-cis retinal production for early cone survival. There was no significant variation in the cone densities across the Wt retina, whereas Rpe65 −/− retina exhibited significantly reduced cone densities in central and ventral regions. This pattern of cone loss may be related to the spatial distribution of S- and M/L-opsin expression within cones across the retina. Although mouse cones are capable of expressing both forms of cone opsins, the ratio of M/L- to S-opsin is much higher in cones of the dorsal retina. 26 Our results suggest that a high concentration of M/L-opsin may be favorable for cone survival in the absence, or with limited amounts (<0.1%), of chromophore. 27 This is supported by our finding that cones are present in the far and mid-dorsal retinal regions (rich in M/L opsin cones) of untreated Rpe65 −/− mice that were dark reared for 5 months; however, complete absence of cones was observed in the central and ventral regions (data not shown). Clinical data from LCA2 patients with the RPE65 gene mutation also lend support, whereas loss of cones within the fovea containing high levels of S-opsin occurred earlier than those containing high levels of M/L-opsin. 8 However, variation in the supply of chromophore to the different areas of the retina remains a possibility. 
Opsin mistrafficking may play a key role in the pathophysiology of cone loss in diseases involving a disrupted visual cycle. Previous studies in both Rpe65 −/− and Lrat −/− mice indicate that trafficking of phototransduction proteins, including S- and M/L-opsins, to the cone outer segment is disrupted, with opsins distributed throughout the entire length of the cell at ages younger than P30. 14 This pathologic characteristic may underlie the subsequent onset of rapid cone loss, highlighted by the lack of M/L-opsin immunoreactivity we observed in central dorsal sections of the retina from P30 untreated Rpe65 −/− mice. Cone opsins are speculated to contribute to cone outer segment stability in a manner analogous to rhodopsin, promoting the development and stability of the rod outer segment. 28 Inappropriate trafficking of opsins throughout the cone results in a decreased quantity of opsin reaching the cone outer segment region, causing destabilization and, presumably, triggering degenerative processes. IP administration to mice of either 9- or 11-cis retinal immediately after birth has been shown to partially correct for opsin mistrafficking, resulting in the arrest of cellular degeneration. 13,14,16  
Our data suggested that growth factors present within Matrigel have a role in its ability for the release of hydrophobic compounds. Matrigel is derived from the EHS mouse sarcoma, and it contains numerous growth factors (e.g., TGF-β, EGF, VEGF, and FGF) that have previously been shown to trigger the infiltration of blood vessels into Matrigel when it is injected into the mouse as a plug. 25 Vascular infiltration of the Matrigel plug may contribute to the sustained release of loaded compounds into systemic circulation, as newly formed vessels are characterized by increased permeability and a fenestrated phenotype leading to vascular leakiness, a characteristic implicated in the pathophysiology of neovascularizing diseases such as diabetic retinopathy. 29 When leaky vessels initially infiltrate the periphery of the plug, 9-cis retinal entrapped within the region may be deposited into the vessel lumen through fenestrations, carried into systemic circulation, and ultimately delivered to the retina through the ophthalmic artery. As the growing vasculature continues to infiltrate adjacent regions of the plug, this may possibly result in the release of further amounts of 9-cis retinal into systemic circulation. The vascularized Matrigel plug is completely absorbed by the animal by 4 weeks after injection, with no health detriments observed during this time. 
This demonstration that Matrigel delivers hydrophobic compounds to the retina in a sustained fashion provides an important tool for animal studies that require long-term drug maintenance and suggests approaches to drug delivery for various human ocular diseases. 
Supplementary Materials
Footnotes
 Supported in part by National Institutes of Health Grants R01 EY04939 (RKC) and CO6 RR015455 (Medical University of South Carolina); Foundation Fighting Blindness, Inc. (RKC); an unrestricted grant (Department of Ophthalmology, Medical University of South Carolina) from Research to Prevent Blindness (RPB); RPB Senior Scientific Investigator Award (RKC); and RPB Medical Student Research Fellowship (PHT).
Footnotes
 Disclosure: P.H. Tang, None; J. Fan, None; P.W. Goletz, None; L. Wheless, None; R.K. Crouch, None
The authors thank Masahiro Kono, Ryan Parker, and Bärbel Rohrer (Medical University of South Carolina) for helpful discussions. 
References
Geroski DH Edelhauser HF . Drug delivery for posterior segment eye disease. Invest Ophthalmol Vis Sci. 2000;41:961–964. [PubMed]
Ahmadi MA Lim JI . Pharmacotherapy of age-related macular degeneration. Expert Opin Pharmacother. 2008;9:3045–3052. [CrossRef] [PubMed]
Le-Goff MM Bishop PN . Adult vitreous structure and postnatal changes. Eye. 2008;22:1214–1222. [CrossRef] [PubMed]
Cruysberg LP Nuijts RM Gilbert JA Geroski DH Hendrikse F Edelhauser HF . Delivery of fluorescein-labeled dexamethasone and methotrexate with fibrin sealant. Curr Eye Res. 2005;30:653–660. [CrossRef] [PubMed]
Hsiue GH Chang RW Wang CH Lee SH . Development of in situ thermosensitive drug vehicles for glaucoma therapy. Biomaterials. 2003;24:2423–2430. [CrossRef] [PubMed]
Passaniti A Taylor RM Pili R . A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest. 1992;67:519–528. [PubMed]
Redmond TM Yu S Lee E . RPE65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet. 1998;20:344–351. [CrossRef] [PubMed]
Jacobson SG Aleman TS Cideciyan AV . Human cone photoreceptor dependence on RPE65 isomerase. Proc Natl Acad Sci U S A. 2007;104:15123–15128. [CrossRef] [PubMed]
Van-Hooser JP Aleman TS He YG . Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness. Proc Natl Acad Sci U S A. 2000;97:8623–8628. [CrossRef] [PubMed]
Van-Hooser JP Liang Y Maeda T . Recovery of visual functions in a mouse model of Leber congenital amaurosis. J Biol Chem. 2002;277:19173–19182. [CrossRef] [PubMed]
Maguire A Simonelli F Pierce EA . Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med. 2008;358:2240–2248. [CrossRef] [PubMed]
Bainbridge JW Smith AJ Barker SS . Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med. 2008;358:2231–2239. [CrossRef] [PubMed]
Znoiko SL Rohrer B Lu K Lohr HR Crouch RK Ma JX . Downregulation of cone-specific gene expression and degeneration of cone photoreceptors in the RPE65−/− mouse at early ages. Invest Ophthalmol Vis Sci. 2005;46:1473–1479. [CrossRef] [PubMed]
Fan J Rohrer B Frederick JM Baehr W Crouch RK . RPE65−/− and LRAT−/− mice: comparable models of Leber congenital amaurosis. Invest Ophthalmol Vis Sci. 2008;49:2384–2389. [CrossRef] [PubMed]
Rohrer B Goletz P Znoiko S . Correlation of regenerable opsin with rod ERG signal in RPE65−/− mice during development and aging. Invest Ophthalmol Vis Sci. 2003;44:310–315. [CrossRef] [PubMed]
Rohrer B Lohr HR Humphries P Redmond TM Seeliger MW Crouch RK . Cone opsin mislocalization in RPE65−/− mice: a defect that can be corrected by 11-cis retinal. Invest Ophthalmol Vis Sci. 2005;46:3876–3882. [CrossRef] [PubMed]
Fan J Wu BX Sarna T Rohrer B Redmond TM Crouch RK . 9-Cis retinal increased in retina of rpe65 knockout mice with decrease in coat pigmentation. Photochem Photobiol. 2006;82:1461–1467. [PubMed]
Bayer AU Cook P Brodie SE Maag KP Mittag T . Evaluation of different recording parameters to establish a standard for flash electroretinography in rodents. Vision Res. 2001;41:2173–2185. [CrossRef] [PubMed]
Saari JC Garwin GG Van-Hooser JP Palczewski K . Reduction of all-trans-retinal limits regeneration of visual pigment in mice. Vision Res. 1998;38:1325–1333. [CrossRef] [PubMed]
Groenendijk GW De-Grip WJ Daemen FJ . Quantitative determination of retinals with complete retention of their geometric configuration. Biochim Biophys Acta. 1980;617:430–438. [CrossRef] [PubMed]
Yoshizawa T Wald G . Pre-lumirhodopsin and the bleaching of visual pigments. Nature. 1963;197:1279–1286. [CrossRef] [PubMed]
Zhang H Fan J Li S . Trafficking of membrane-associated proteins to cone photoreceptor outer segments requires the chromophore 11-cis-retinal. J Neurosci. 2008;28:4008–4014. [CrossRef] [PubMed]
Batten ML Imanishi Y Tu DC . Pharmacological and rAAV gene therapy rescue of visual functions in a blind mouse model of Leber congenital amaurosis. PloS Med. 2005;2:e333. [CrossRef] [PubMed]
Seeliger MW Grimm C Stahlberg F . New views on Rpe65 deficiency: the rod system is the source of vision in a mouse model of Leber congenital amaurosis. Nat Genet. 2001;29:70–74. [CrossRef] [PubMed]
Miao RQ Agata J Chao L Chao J . Kallistatin is a new inhibitor of angiogenesis and tumor growth. Blood. 2002;100:3245–3252. [CrossRef] [PubMed]
Applebury ML Antoch MP Baxter LC . The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning. Neuron. 2000;27:513–523. [CrossRef] [PubMed]
Fan J Rohrer B Moiseyev G Ma JX Crouch RK . Isorhodopsin rather than rhodopsin mediates rod function in RPE65 knock-out mice. Proc Natl Acad Sci U S A. 2003;100:13662–13667. [CrossRef] [PubMed]
Lem J Krasnoperova NV Calvert PD . Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc Natl Acad Sci U S A. 1999;96:736–741. [CrossRef] [PubMed]
Roberts WG Palade GE . Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J Cell Sci. 1995;108:2369–2379. [PubMed]
Figure 1.
 
Total cone density increases in Rpe65 −/− animals with delivery of 9-cis retinal by Matrigel. Data are obtained from P30 dark-reared mice that began treatment at P10. (A) Schematic representation of retinal flat mount, with six regions analyzed for cone density spanning the dorsal ventral axis. FD, far dorsal; MD, mid-dorsal; CD, central dorsal; ONH, optic nerve head; CV, central ventral; MV, mid ventral; FV, far ventral. (B) Quantification of PNA-positive cells by region from Wt (n = 7), Rpe65 −/− mice treated with 9-cis retinal (0.25 mg/animal) using Matrigel (n = 7) or IP injection (0.05 mg/animal ×5) (n = 6) and control Rpe65 −/− mice (n = 6). Matrigel administration of 9-cis retinal significantly increased cone density at all analyzed regions of the retina compared with IP injection; *P < 0.01. Two-way ANOVA was used to test for differences in cone counts by treatment group and retinal region with post hoc pairwise t-tests used to compare individual groups. Differences in maximum trough to peak, between left and right eyes, were averaged for each treatment group and analyzed using ANOVA with Tukey's post hoc tests to compare individual groups. A two-sided P-value of 0.05 was considered significant. Bars indicate ±SD. (C) Images of flat-mounted retina labeled with FITC-conjugated peanut agglutinin lectin.
Figure 1.
 
Total cone density increases in Rpe65 −/− animals with delivery of 9-cis retinal by Matrigel. Data are obtained from P30 dark-reared mice that began treatment at P10. (A) Schematic representation of retinal flat mount, with six regions analyzed for cone density spanning the dorsal ventral axis. FD, far dorsal; MD, mid-dorsal; CD, central dorsal; ONH, optic nerve head; CV, central ventral; MV, mid ventral; FV, far ventral. (B) Quantification of PNA-positive cells by region from Wt (n = 7), Rpe65 −/− mice treated with 9-cis retinal (0.25 mg/animal) using Matrigel (n = 7) or IP injection (0.05 mg/animal ×5) (n = 6) and control Rpe65 −/− mice (n = 6). Matrigel administration of 9-cis retinal significantly increased cone density at all analyzed regions of the retina compared with IP injection; *P < 0.01. Two-way ANOVA was used to test for differences in cone counts by treatment group and retinal region with post hoc pairwise t-tests used to compare individual groups. Differences in maximum trough to peak, between left and right eyes, were averaged for each treatment group and analyzed using ANOVA with Tukey's post hoc tests to compare individual groups. A two-sided P-value of 0.05 was considered significant. Bars indicate ±SD. (C) Images of flat-mounted retina labeled with FITC-conjugated peanut agglutinin lectin.
Figure 2.
 
M/L-opsin protein increases with Matrigel delivery of 9-cis retinal in Rpe65 −/− mice. Data obtained from Rpe65 −/− P30 animals. (A) Cross-section images of the central dorsal retina are shown for Wt (top) and Rpe65 −/− mice treated with 9-cis retinal using Matrigel (bottom). For control mice, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only; middle). Paraffin-embedded retinas were sectioned and stained with hematoxylin-eosin (left column); cryoprotected retinas were sectioned and stained with both M/L-opsin-specific primary antibody (red) and DAPI (blue) for nuclei (right column). OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. (B) Western blot images for M/L-opsin (top) and β-actin (bottom) are shown for Wt and Rpe65 −/− Matrigel, IP, and control mice. (C) Densitometry band analyses are displayed as percentages of Wt. Matrigel alone and 9-cis retinal (RAL) intraperitoneally injected Rpe65 −/− mice displayed significantly decreased M/L-opsin protein levels compared with Wt; *P < 0.02. Black bar: Wt, n = 3. White bar: Matrigel alone, n = 3. Red bar: 9-cis retinal IP, n = 3. Blue bar: Matrigel plus 9-cis retinal, n = 3. Data are presented as mean ± SD and were analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Figure 2.
 
M/L-opsin protein increases with Matrigel delivery of 9-cis retinal in Rpe65 −/− mice. Data obtained from Rpe65 −/− P30 animals. (A) Cross-section images of the central dorsal retina are shown for Wt (top) and Rpe65 −/− mice treated with 9-cis retinal using Matrigel (bottom). For control mice, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only; middle). Paraffin-embedded retinas were sectioned and stained with hematoxylin-eosin (left column); cryoprotected retinas were sectioned and stained with both M/L-opsin-specific primary antibody (red) and DAPI (blue) for nuclei (right column). OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. (B) Western blot images for M/L-opsin (top) and β-actin (bottom) are shown for Wt and Rpe65 −/− Matrigel, IP, and control mice. (C) Densitometry band analyses are displayed as percentages of Wt. Matrigel alone and 9-cis retinal (RAL) intraperitoneally injected Rpe65 −/− mice displayed significantly decreased M/L-opsin protein levels compared with Wt; *P < 0.02. Black bar: Wt, n = 3. White bar: Matrigel alone, n = 3. Red bar: 9-cis retinal IP, n = 3. Blue bar: Matrigel plus 9-cis retinal, n = 3. Data are presented as mean ± SD and were analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Figure 3.
 
Maintenance of cone function in Rpe65 −/− Rho −/− mice with Matrigel delivery of 9-cis retinal. Rpe65 −/− Rho −/− mice were treated with 9-cis retinal using Matrigel (0.25 mg/mouse at P10) or by IP injection (0. 5 mg/animal, beginning at P10, ×5) and were analyzed at P30 with single white flash (intensity range, 1.56–24.8 cd · s/mm2) ERG recordings under dark-adapted conditions. For control mice, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only). (A) Representative ERG traces from Matrigel/9-cis retinal (left), 9-cis retinal IP (middle), and control (right) animals. Dashed line: onset of flash. (B) Quantification of b-wave amplitudes from Matrigel/9-cis retinal (black, n = 5), 9-cis retinal IP (gray, n = 5) and control (dashed, n = 5) animals. Mice administered 9-cis retinal using Matrigel showed significantly increased cone responses from 2.48 to 24.8 cd · s/mm2 flash intensities compared with those that did not receive 9-cis retinal therapy; *P < 0.05. Data are presented as mean ± SD and were analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Figure 3.
 
Maintenance of cone function in Rpe65 −/− Rho −/− mice with Matrigel delivery of 9-cis retinal. Rpe65 −/− Rho −/− mice were treated with 9-cis retinal using Matrigel (0.25 mg/mouse at P10) or by IP injection (0. 5 mg/animal, beginning at P10, ×5) and were analyzed at P30 with single white flash (intensity range, 1.56–24.8 cd · s/mm2) ERG recordings under dark-adapted conditions. For control mice, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only). (A) Representative ERG traces from Matrigel/9-cis retinal (left), 9-cis retinal IP (middle), and control (right) animals. Dashed line: onset of flash. (B) Quantification of b-wave amplitudes from Matrigel/9-cis retinal (black, n = 5), 9-cis retinal IP (gray, n = 5) and control (dashed, n = 5) animals. Mice administered 9-cis retinal using Matrigel showed significantly increased cone responses from 2.48 to 24.8 cd · s/mm2 flash intensities compared with those that did not receive 9-cis retinal therapy; *P < 0.05. Data are presented as mean ± SD and were analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Figure 4.
 
Matrigel maintains a sustained delivery of 9-cis retinal to photoreceptors. P45 Rpe65 −/− animals were administered 9-cis retinal (0.25 mg/animal) with a single subcutaneous Matrigel or IP injection. For control animals, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only). Animals were reared under cyclic light until 2 days after treatment and then were dark reared for either 3 or 7 days, and the retinas were analyzed for extracted retinoids or isorhodopsin levels (4 eyes/sample). (A) Representative HPLC profile of retinoid extract from Rpe65 −/− mice dark reared for 3 days and administered 9-cis retinal using Matrigel. Peaks were identified by comparison with known standards. Detection was at 360 nm. (B) syn-9-cis Retinal oxime levels after 3 and 7 days of dark rearing in Rpe65 −/− mice administered 9-cis retinal with a single injection of Matrigel (black, n = 6), IP administration (gray, n = 6), or control (white, n = 6). Administration of 9-cis retinal with Matrigel significantly increased syn-9-cis retinal oxime levels compared with IP administration; *P < 0.002. Mice administered 9-cis retinal with Matrigel showed significantly increased syn-9-cis retinal oxime levels between 3 and 7 days of dark rearing; †P < 0.05. (C) Isorhodopsin levels from whole retinas of mice administered 9-cis retinal using Matrigel injection after 3 (n = 3) and 7 (n = 3) days of dark rearing. Analysis showed a significant increase in isorhodopsin levels between both time points, indicating sustained delivery of 9-cis retinal; øP < 0.01. Data are presented as mean ± SD and analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Figure 4.
 
Matrigel maintains a sustained delivery of 9-cis retinal to photoreceptors. P45 Rpe65 −/− animals were administered 9-cis retinal (0.25 mg/animal) with a single subcutaneous Matrigel or IP injection. For control animals, 9-cis retinal was omitted from the Matrigel preparation (Matrigel-only). Animals were reared under cyclic light until 2 days after treatment and then were dark reared for either 3 or 7 days, and the retinas were analyzed for extracted retinoids or isorhodopsin levels (4 eyes/sample). (A) Representative HPLC profile of retinoid extract from Rpe65 −/− mice dark reared for 3 days and administered 9-cis retinal using Matrigel. Peaks were identified by comparison with known standards. Detection was at 360 nm. (B) syn-9-cis Retinal oxime levels after 3 and 7 days of dark rearing in Rpe65 −/− mice administered 9-cis retinal with a single injection of Matrigel (black, n = 6), IP administration (gray, n = 6), or control (white, n = 6). Administration of 9-cis retinal with Matrigel significantly increased syn-9-cis retinal oxime levels compared with IP administration; *P < 0.002. Mice administered 9-cis retinal with Matrigel showed significantly increased syn-9-cis retinal oxime levels between 3 and 7 days of dark rearing; †P < 0.05. (C) Isorhodopsin levels from whole retinas of mice administered 9-cis retinal using Matrigel injection after 3 (n = 3) and 7 (n = 3) days of dark rearing. Analysis showed a significant increase in isorhodopsin levels between both time points, indicating sustained delivery of 9-cis retinal; øP < 0.01. Data are presented as mean ± SD and analyzed by two-sample t-test, accepting a significance value of P < 0.05.
Supplementary Figure S1
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