October 2005
Volume 46, Issue 10
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Retinal Cell Biology  |   October 2005
Laser Photocoagulation and, to a Lesser Extent, Photodynamic Therapy Target and Enhance Adenovirus Vector–Mediated Gene Transfer in the Rat Retina
Author Affiliations
  • Kaname Anzai
    From the Department of Ophthalmology, Saitama Medical School, Saitama, Japan; the
  • Shin Yoneya
    From the Department of Ophthalmology, Saitama Medical School, Saitama, Japan; the
  • Peter L. Gehlbach
    Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and
  • Daisuke Imai
    From the Department of Ophthalmology, Saitama Medical School, Saitama, Japan; the
  • Lisa L. Wei
    GenVec Inc., Gaithersburg, Maryland.
  • Keisuke Mori
    From the Department of Ophthalmology, Saitama Medical School, Saitama, Japan; the
Investigative Ophthalmology & Visual Science October 2005, Vol.46, 3883-3891. doi:10.1167/iovs.05-0593
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      Kaname Anzai, Shin Yoneya, Peter L. Gehlbach, Daisuke Imai, Lisa L. Wei, Keisuke Mori; Laser Photocoagulation and, to a Lesser Extent, Photodynamic Therapy Target and Enhance Adenovirus Vector–Mediated Gene Transfer in the Rat Retina. Invest. Ophthalmol. Vis. Sci. 2005;46(10):3883-3891. doi: 10.1167/iovs.05-0593.

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

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Abstract

purpose. To evaluate the transduction efficiency and localization of a reporter gene after intravitreous injection of adenovirus vector in laser photocoagulation (PC)- and photodynamic therapy (PDT)–treated eyes.

methods. Adult Lewis rats received fundus PC, fundus PDT, or no treatment. Intravitreous injection of an adenovirus vector containing the construct expressing β-galactosidase (AdlacZ.11D) was performed in each group. All eyes were then enucleated for histochemistry and processed for quantitative image analysis.

results. In eyes with no treatment, there was moderate to intense staining for lacZ in the anterior segment, but little in the retina. In eyes treated with PC and PDT, there was significantly more LacZ staining in the retina. The increased staining corresponded closely with the sites treated with PC and PDT. Gene transduction in PC-treated eyes was enhanced and extended to at least 135 days after virus delivery, but not extended in PDT-treated eyes. Gene transfer and expression were targeted and enhanced at the site of laser burns, at all doses tested (3 × 105 to 3 × 109 particles per eye).

conclusions. Compared with untreated eyes, eyes treated with PC and to a lesser extent PDT, manifest increased transduction efficiency, in areas of the retina that are targeted by laser treatment. This finding suggests a new and promising strategy for the treatment of retinochoroidal neovascularization. Adenovirus gene therapy in combination with PC or PDT would have the advantage of increased transduction efficiency; increased duration of transgene expression; targeted delivery; and, potentially, a lower effective dose of virus.

Currently adenovirus (AdV), adenoassociated virus, and lentivirus are actively being investigated as vector platforms for ocular gene therapy. 1 2 3 Several factors determine the relative advantages and disadvantages of each platform and include, but are not limited to, the vector tropism, the efficiency of transduction, the size of the transgene, needs related to latency and duration of expression, vector-related toxicity, and the integration necessary for the vector to express. Adenoassociated virus and lentivirus vectors have extended durations of transgene expression compared with AdV. 4 5 There are clinical settings (e.g., chronic disease) in which prolonged transgene expression may be desired. Currently, the risk of prolonged transgene expression is unknown for most proteins and must be evaluated for each protein. In the eye, even increased expression of wild-type rhodopsin or peripherin/rds can result in degeneration of photoreceptors. 6 7  
Replication-deficient AdV vectors are characterized by a large capacity, short latency, transduction in both dividing and nondividing cells, high expression levels, and relative ease of production, allowing concentration to high titers. Local delivery of AdV vector for relatively short-term ocular indications may therefore be desirable. AdV vectors induce a dose-dependent ocular immune response that may contribute to a shorter duration of expression, particularly at higher doses—the major disadvantage of AdV vectors. Enrollment is completed in a phase I clinical trial of AdV vector expressing human pigment epithelium-derived factor (PEDF) 8 for choroidal neovascularization (CNV) in patients with age-related macular degeneration (AMD), and current findings indicate that intravitreous injection of AdV vector expressing PEDF is well-tolerated at all doses tested (Campochiaro PA, et al. IOVS 2004; 45:ARVO E-Abstract 2361). Therefore, local delivery of AdV vector in the eye remains a promising treatment approach. 
It is known that, in normal eyes, intravitreous injection of AdV vector results in transduction of anterior segment tissues and sporadic transduction of retinal cells. 9 10 11 12 13 After intravitreous injection, transgene expression peaks within days and decreases to low levels by 1 month in preclinical mouse models. 13 This transient expression profile has been considered a disadvantage for the treatment of most chronic retinochoroidal disease. Our work and the work of others indicate that AdV transduction and expression profiles in pathologic retina differ from those observed in normal retina. Neovascular and nonneovascular proliferative retinopathy, surgical vitrectomy, and laser induction of CNV appear to increase transduction efficiency. 13 14 15 In this study, laser photocoagulation (PC) and, to a lesser extent, photodynamic therapy (PDT) increased viral transduction efficiency and expression in the area of laser treatment. We designed four experimental protocols to address the following questions: (1) At what time point, relative to PC and PDT, should we deliver AdV vector to maximize transduction efficiency and expression? (2) How do PC and PDT affect the duration of AdV vector expression? (3) What is the dose of AdV vector necessary to achieve enhanced focal expression? (4) By what mechanisms could enhanced transduction efficiency and gene expression occur in laser-treated retina? 
Methods
AdV Vectors Expressing β-galactosidase
A serotype-5 AdV vector, with E1A, E1B, and partial E3 and E4 regions deleted and containing a construct expressing β-galactosidase (LacZ) from a cytomegalovirus (CMV) immediate early promoter expression cassette (AdlacZ.11D), was used in all vector experiments. Similar AdV vectors containing a CMV expression cassette in the E1 region have been described. 13 16 17 The full-length open reading frame for β-galactosidase was cloned into a pAdCMV shuttle plasmid that contains the expression cassette and the left end of the AdV genome. 16 AdV vectors were generated by transfecting complementing cells, with the plasmid carrying the complete genome of the virus. 18 The production, purification and quantification of these vectors has been described in detail. 16  
Animals and Intravitreous Injection of Vector Constructs
Female Lewis rats were obtained at 4 to 8 weeks of age. Animals were anesthetized by intramuscular injections of 80 mg/kg ketamine hydrochloride. Pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride. All animals were treated while under deep general anesthesia in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
At predetermined time points before and after PC or PDT treatment, according to treatment protocol, rats received intravitreous injection of AdlacZ.11D. All injections were performed with a syringe (Hamilton, Reno, NV) fitted with a 33-gauge beveled needle. The rats were anesthetized; the pupils were dilated; and, under an operating microscope, the needle was passed through the sclera at the equator, into the vitreous cavity; and the vector was injected. The needle tip was directly observed to be in the center of the vitreous cavity throughout the procedure. 
Laser PC and PDT
Thermal diode laser PC (532-nm wavelength, 200-μm spot size, 0.5-second duration, 300 mW) was delivered using the slitlamp delivery system (SL130; Carl Zeiss Meditec, Inc., Oberkochen, Germany) and a hand-held cover slip as a contact lens. Fifty PC burns were delivered to each eye, confined to the left half of the posterior fundus. 
PDT was performed with the novel hydrophilic photosensitizer; mono-l-aspartyl chlorin e6 (NPe6, LS11; Light Science, Seattle, WA). 19 20 21 A solution of 10 mg/kg body weight LS11 was administered through the tail vein. Irradiation with a diode laser (664-nm wavelength, 100-μm spot size, 10-second duration, 4.5 mW) was started within 5 minutes after intravenous injection. Five PDT laser spots were placed in the left half of the posterior fundus in each eye equally, using a 664-nm diode laser delivery system (prototype for NPe6; Panasonic, Osaka, Japan). 19  
Experimental Protocols
Animals were enrolled into one of four treatment protocols. 
Protocol 1.
To determine the time point, relative to PC and PDT, when the AdV vector should be delivered, to maximize transduction efficiency and expression, we administered intravitreous injections to the rats of AdlacZ.11D at a concentration of 3 × 109 particles per eye on days 1, 3, 7, and 28 after laser treatment. Five days after viral delivery, all eyes were enucleated and examined histochemically for LacZ staining. 
Protocol 2.
To evaluate how PC and PDT affects the duration of AdV vector expression in the retina, we injected rats intravitreously with AdlacZ.11D, 3 × 109 particles per eye, 3 days after laser treatment. The eyes were enucleated and examined histochemically on days 5, 14, 28, 90, 135, and 180 after viral delivery. 
Protocol 3.
To investigate the dose–response of transgene expression in laser treated retina, we injected rats intravitreously with AdlacZ.11D at a concentration of 3 × 105, 3 × 106, 3 × 107, 3 × 108, or 3 × 109 particles per eye, 3 days after laser photocoagulation. Eyes were enucleated and examined histochemically 5 days after viral delivery. 
Protocol 4.
To explore potential mechanisms of enhanced transduction and expression by AdV vector in laser treated retina, we treated five C57/BL6 mice with fundus PC with a 532-nm diode laser and left five mice untreated. Twenty-four hours after treatment, the eyes were enucleated, and total RNA was extracted from laser photocoagulated retina and choroid. The samples were processed for quantitative real-time PCR (qRT-PCR) with a sequence-detection system (Prism 7700; Applied Biosystems, Inc. [ABI], Foster City, CA), to quantify mRNA expression of coxsackie AdV receptor (CAR) and the integrins αV, β3, and β5, which have been shown to play a role in cell surface interaction and internalization of AdV vector during the transduction process. 22 23  
Histochemical Examination and Image Analysis
Histochemical analysis consisted of fixing eyes in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) for 60 minutes and rinsing five times for 10 minutes in PBS. The anterior segments were removed, and the remaining posterior segments were incubated overnight in 1 mg/mL 5-bromo-4-chloro-3-indolyl galactopyranoside (X-gal; Sigma-Aldrich, St. Louis, MO) in a solution containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6-3H2O, and 1 mM MgCl2 in PBS. Eyes were postfixed for 15 minutes and then rinsed with PBS. 
For quantitative analysis of X-gal staining in the posterior segment of eyes, a dissecting microscope and its camera system (MZ 8 and MPS 30; Leica, Wetzlar, Germany) were used. Images were captured, digitized, and analyzed by image-analysis software (Win ROOF; Mitani Corp., Fukui, Japan) to delineate X-gal-stained areas. Area ratios (X-gal-stained area to total retinal area) were calculated for each eye. Statistical analysis comparing the area ratio between the treated left hemisphere and the untreated right hemisphere was performed using the Wilcoxon signed-rank test. P < 0.05 was prospectively assigned as the level at which a finding would be considered statistically significant. 
Quantitative Real-Time PCR
Total RNA for qRT-PCR was isolated from samples composed of the retina and choroid (including retinal pigment epithelium). The samples were homogenized (TRIzol reagent; Invitrogen-Gibco, Grand Island, NY) and treated with RNase-free DNase (DNase I; Invitrogen-Gibco) to remove genomic DNA contamination. First-strand cDNA was synthesized by reverse transcription of total RNA using reverse transcriptase (Superscript II; Invitrogen, Carlsbad, CA) with random hexamers as primers in total reaction volume of 20 μL. Amplification of the control gene ARP (acidic ribosomal phosphoprotein P0) was used for normalization. 24 25 CAR, integrin αV, β3, β5, and ARP were amplified using commercially available primers and probe sets (assay IDs: Mm00438361_m1 for CAR, Mm00434506_m1 for αV, Mm00443980_m1 for β3, Mm00439825_m1 for β5, Mm00725448_s1 for ARP; ABI). The expression levels of CAR and integrin were assigned arbitrary units (relative to baseline samples) using the comparative Ct method. 25 26 All qRT-PCR experiments were performed according to the guidelines supplied by ABI. Statistical analysis was performed with Student’s unpaired t-test. P < 0.05 was prospectively assigned as the level at which a finding would be considered statistically significant. 
Results
AdV Vector–Mediated Expression of Reporter Genes in Untreated Rats
The localization of expression from reporter genes was investigated with AdlacZ.11D. Six naïve eyes without laser treatments were given an intravitreous injection of 3 × 109 particles. Five days after injection cornea, iris, and retina flatmounts and cryosections were histochemically stained for LacZ. There was strong staining for LacZ in the anterior segment, including the corneal endothelium, the trabecular meshwork, and the pigment epithelium of the iris (Figs. 1A 1B 1D) . In contrast, AdlacZ.11D resulted in only limited focal and sporadic expression in the posterior segment of these eyes (Figs. 1C 1E)
Enhanced Focal Gene Transfer in Rat Retina at Various Time Points after PC and PDT
To determine the optimum time for AdV vector delivery after PC and PDT, we injected rats intravitreously with AdlacZ.11D at a concentration of 3 × 109 particles on days 1, 3, 7, and 28 after laser therapy. Only the left hemisphere of the fundus was laser treated. The untreated right hemisphere served as an internal control. Retinal flatmounts from these eyes revealed many heavily blue-stained lesions in the left hemisphere corresponding to the position of the laser burns (Fig. 2A 2B 2C 2D) . The right hemisphere demonstrated very limited and sporadic staining, similar to that in naïve controls eyes (Fig. 1) . Cryosection through photocoagulated lesions revealed numerous dark-stained linear structures that spanned the retina, consistent with Müller cell morphology (Fig. 2E) . PDT-treated eyes showed findings similar to those observed in PC-treated eyes, but to a lesser extent (Fig. 3) . There was a significant difference in the ratio of area stained between the PC-treated left and untreated right fundus hemispheres on days 1, 3, and 7 and between PDT-treated left and untreated right fundus hemispheres on day 3 (Fig. 4)
The Time Course of LacZ Expression in Laser-Treated Rat Retina after Intravitreous Injection
To evaluate how long enhanced gene expression persists after viral delivery to laser-treated eyes, we administered intravitreous injection of AdlacZ.11D and then examined the results histochemically at selected time points over a 180-day period. Gene transduction of all PC-treated eyes was significantly enhanced in the PC-treated left fundus hemisphere on days 5, 14, 90, and 135 after injection (P < 0.05; Fig. 5A 5B 5C 5D 5E 5F 5G ). In contrast, PDT-treated eyes demonstrated significantly enhanced X-gal expression only 5 days after injection (P < 0.05; Fig. 5H ). PDT-treated eyes after 5 and before 14 days after vector treatment have not been examined. 
Dose Effect of Transgene Expression in Laser-Treated Retina
To evaluate the dose–response of transgene expression in laser-treated retina, we injected rats intravitreously withAdlacZ.11D at concentrations of 3 × 105, 3 × 106, 3 × 107, 3 × 108, or 3 × 109 particles per eye, 3 days after PC. They were then examined histochemically. Intense enhancement of expression at laser burns was present after intravitreous injection of vector at all the tested doses (P < 0.05, for all doses evaluated; Fig. 6 ). 
Quantitative Real-Time PCR
qRT-PCR was used to quantify the levels of CAR and integrin αV, β3, and β5 mRNA in the photocoagulated retina and choroid. No significant difference in mRNA expression was measurable for any of these genes when photocoagulated retina was compared with untreated retina. The mean mRNA expression for CAR and integrin β5 in photocoagulated choroid was approximately two times higher than untreated choroid, but this was not statistically significant (P > 0.05; Fig. 7 ). 
Discussion
In this study laser treatment with PC and to a lesser extent PDT resulted in observable targeting of AdV to the laser-treated retina. Targeting was evident by enhanced transduction and expression of the reporter gene β-galactosidase (LacZ). Enhanced staining corresponded closely to points of laser treatment and was sustained for at least 135 days, but <180 days in the case of PC-treated eyes. Intense enhancement of expression after PC treatment was present after intravitreous injection of viral vector at all tested doses. PC and PDT are established clinical treatments for retinal and choroidal neovascularization as well as other vitreoretinal conditions. AdV-vectored PEDF is now in clinical trial as a treatment for advanced exudative AMD. This study provides evidence supportive of treatment strategies that combine intravitreous injection of AdV vectors containing potentially therapeutic transgenes and laser PC or PDT treatments. 
In current clinical practice the most limiting factor for laser treatment (PC or PDT) of choroidal neovascularization is the high incidence of either persistence or recurrence of treated CNV; approximately 50% in patients treated by PC and 90% by PDT. 27 28 29 Mechanistically, both PC and PDT attempt to occlude the microvascular networks of the CNV, but neither inhibits or blocks specific angiogenic stimuli. Treatments that are potentially angiostatic, thereby inhibiting regrowth of CNV or alternatively treatments that induce further regression of CNV are logical next steps to add on to traditional monotherapy. 
Laser photocoagulation is currently a principle treatment for retinal neovascularization and macular edema. Despite widespread use and clinical trial evidence of efficacy of laser PC in the setting of diabetic retinopathy, diabetes remains the leading cause of severe vision loss in the working aged population in the developed world. PC in the setting of ischemic central retinal vein occlusion associated with persistent macular edema does not significantly improve vision. Extensive scattered PC ablates normal retina and choroid leading to a significant loss of peripheral and night vision. 30 31 32 Diabetic macular edema is the most common cause of moderate vision loss in middle-aged patients in developed countries. 33 Focal PC in the macula is beneficial to the diabetic population meeting studied clinical criteria, but its usefulness is limited by side effects, inability to restore vision, and lack of efficacy in some individuals. 34 Therefore, there remains an urgency for development and deployment of novel treatments that may supplement current laser strategies. 
Until recently, there has been little in the way of treatment directed at controlling the specific molecular signals that lead to increased vascular permeability and the development of ocular neovascularization. Currently, several new approaches hold promise in this regard. 8 35 36 37 38 39 40 41 42 Drug delivery for antiangiogenesis and antivasopermeability in the retina is complicated by relative isolation of the retina by the blood–retinal barrier and other anatomic structures. The delivery of proteins to the retina is particularly problematic and may require repeated intraocular injections, which are attended by risk and are poorly tolerated. Gene transfer of an expression construct having antiangiogenic and antivasopermeability properties is an appealing alternative. Intraocular gene therapy approaches allow for sustained levels of a transgene product in the eye, with little or no systemic exposure. In prior work, we have shown that intraocular injection of AdV vectors expressing PEDF results in both inhibition and regression of ocular neovascularization, indicating that PEDF not only stops new abnormal blood vessel growth but also induces regression of existing abnormal vessels in the eye. 17 43 An antivasopermeability effect of PEDF and the active site for this activity has recently been reported. 44  
Results of a phase I clinical trial of AdPEDF.11 indicate that intravitreous injection of up to 109.5 viral particles of AdPEDF.11 is well-tolerated in the human eye. 8 In the current study, we evaluated an AdV vector similar to that evaluated in the clinical trial but with a reporter gene occupying the transgene position. At viral particle doses of 105 to 109 particles per eye, enhanced expression of transgene at the site of PC treatment was present. With conservative adjustment for eye volume (rat, 30–50 μL: human, 1.0 mL) the 105 viral particle dose used in the rat eye corresponded to a dose that was considerably lower than that shown to be well tolerated in the human eye (109.5 viral particles). This estimation suggests that targeted enhancement of expression in laser lesions may allow for even lower viral vector doses when used in combination with laser therapy. 
Vector delivery after PC increases transduction efficiency, prolongs duration of transgene expression, and targets delivery to the site of PC. Vector delivery after PDT, however, enhances the duration of transgene expression to a lesser extent. Although we do not fully understand the reasons for this difference at this time, we may speculate that it relates to differences inherent in the two laser therapies. PC results in greater retinal destruction at the time of delivery than PDT, resulting in significant morphologic alteration, which may allow better penetration of viral vectors into the retina. Alternatively, there may be differences in induction of endogenous gene expression in the retinochoroidal tissues. CAR and the integrins αV, β3, and β5 have been shown to play a role in cell surface interaction and internalization of adenoviral vector during the transduction process. 22 23 The qRT-PCR analysis of retina and choroid in laser and nonlasered eyes shows that the mean mRNA expression of CAR and integrin β5 in photocoagulated choroid is approximately two times higher than that in untreated choroid, but this finding did not reach statistical significance. Another possibility is that PC induces greater local cellular proliferative response than does PDT. 
Lai et al. 15 have shown that proliferating retinal pigment epithelial cells in laser-induced choroidal neovascularization are preferentially transduced by subretinal injection of recombinant adenoviral vector. We have shown that, in transgenic mice that model proliferative vitreoretinopathy, there is preferential transduction of proliferating cells. 13 Murata et al. 45 have reported that subretinal injection of retrovirus vector in PC-treated rabbit and monkey eyes results in gene transfer into photocoagulated retinal pigment epithelial cells and macrophages. This finding is consistent with the ability of retrovirus to transduce dividing and proliferating cells, but not terminally differentiated postmitotic cells. In contrast, although AdV transduces nondividing cells, the efficiency of AdV transduction correlates with the growth state in the retina. 46 Further studies directed at characterizing the mechanism of preferential transduction of proliferating cells by AdV will have implications with regard to advances in ocular gene therapy and its clinical applications. 
It is well recognized that altering the tropism of a viral vector for the purpose of increasing specificity or for redirecting the vector to alternative targets is a potentially useful strategy for increasing vector utility. 47 The logic of this approach is made especially clear when systemic delivery of a potentially therapeutic vector is contemplated. Herein we have described, characterized, and quantified a naturally occurring property of AdV vectors that targets selective tissue previously lasered either by PC or PDT. As the eye is relatively isolated from the systemic circulation by the blood–retinal barrier and systemic exposure to treat the small ocular volume is not desirable, local gene therapy approaches now predominate in ocular gene therapy. Intraocular gene therapy requires injection of virus and bears the immediate risks associated with injection. Targeting gene therapy to the region of the disease, enhancing the local expression of therapeutic transgenes with lower doses of vector, and increasing the duration of therapeutic response as is reported herein has the potential to increase the efficacy and safety of ocular gene therapy and to broaden its scope of application to include combination therapy with laser modalities. 
 
Figure 1.
 
Naïve eyes without laser treatment with histochemical X-gal staining. Corneal (A), iridal (B), and retinal (C) flatmounts. ( Image not available ) Optic disc. Arrow: sporadic X-gal staining. Cryosections of anterior segment (D) and the retina (E). Intravitreous injection of AdlacZ.11D resulted in strong expression in cornea and iris, but only focal and sporadic expression in the retina. Bar: (A, B) 70 μm; (C) 180 μm; (D, E) 100 μm.
Figure 1.
 
Naïve eyes without laser treatment with histochemical X-gal staining. Corneal (A), iridal (B), and retinal (C) flatmounts. ( Image not available ) Optic disc. Arrow: sporadic X-gal staining. Cryosections of anterior segment (D) and the retina (E). Intravitreous injection of AdlacZ.11D resulted in strong expression in cornea and iris, but only focal and sporadic expression in the retina. Bar: (A, B) 70 μm; (C) 180 μm; (D, E) 100 μm.
Figure 2.
 
Photocoagulated retina with histochemical X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles/eye on days 1 (A), 3 (B, E), 7 (C), and 28 (D) after laser photocoagulation. Half of the posterior fundus (left hemisphere) was photocoagulated and the remaining right hemisphere was untreated for internal controls (line: the border between PC-treated and untreated hemispheres). In the retinal flatmounts (A-D) each blue X-gal-stained lesion corresponds to laser burns (arrowheads). The cryosection (E) through photocoagulated lesion (between white arrows) demonstrated darker stained linear structures (black arrows) probably representing Müller cells. Bar: (AD) 500 μm; (E) 120 μm.
Figure 2.
 
Photocoagulated retina with histochemical X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles/eye on days 1 (A), 3 (B, E), 7 (C), and 28 (D) after laser photocoagulation. Half of the posterior fundus (left hemisphere) was photocoagulated and the remaining right hemisphere was untreated for internal controls (line: the border between PC-treated and untreated hemispheres). In the retinal flatmounts (A-D) each blue X-gal-stained lesion corresponds to laser burns (arrowheads). The cryosection (E) through photocoagulated lesion (between white arrows) demonstrated darker stained linear structures (black arrows) probably representing Müller cells. Bar: (AD) 500 μm; (E) 120 μm.
Figure 3.
 
PDT and vector-treated eyes were examined after X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles per eye, on days 1 (A), 3 (B, E), 7 (C), and 28 (D) after PDT. Half of the posterior fundus (left hemisphere) was treated with PDT, the remaining right hemisphere was untreated and served as an internal control (white line: the border between PDT-treated and untreated hemispheres). In the retinal flatmounts (AD) each blue X-gal stained lesion corresponds to a PDT-treated area. LacZ expression peaked at day 3 in PDT-treated eyes. Cryosection (E) through PDT-treated retina (between white arrows) demonstrates enhanced staining and linear structures (arrows) consistent with Müller cell morphology. Bar: (AD) 500 μm; (E) 120 μm.
Figure 3.
 
PDT and vector-treated eyes were examined after X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles per eye, on days 1 (A), 3 (B, E), 7 (C), and 28 (D) after PDT. Half of the posterior fundus (left hemisphere) was treated with PDT, the remaining right hemisphere was untreated and served as an internal control (white line: the border between PDT-treated and untreated hemispheres). In the retinal flatmounts (AD) each blue X-gal stained lesion corresponds to a PDT-treated area. LacZ expression peaked at day 3 in PDT-treated eyes. Cryosection (E) through PDT-treated retina (between white arrows) demonstrates enhanced staining and linear structures (arrows) consistent with Müller cell morphology. Bar: (AD) 500 μm; (E) 120 μm.
Figure 4.
 
For determining the best time to deliver AdV vectors after laser treatment, rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles on days 1, 3, 7, and 28 after PC (A) and PDT (B). There was a significant difference in the area ratio of lacZ-stained/total area between photocoagulated and untreated hemispheres on days 1, 3, and 7 after PC and on day 3 after PDT. (•) PC- or PDT-treated left hemispheres; (○) untreated right hemispheres. Bar: SE. *P < 0.05.
Figure 4.
 
For determining the best time to deliver AdV vectors after laser treatment, rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles on days 1, 3, 7, and 28 after PC (A) and PDT (B). There was a significant difference in the area ratio of lacZ-stained/total area between photocoagulated and untreated hemispheres on days 1, 3, and 7 after PC and on day 3 after PDT. (•) PC- or PDT-treated left hemispheres; (○) untreated right hemispheres. Bar: SE. *P < 0.05.
Figure 5.
 
Flatmounts of PC-treated retina after X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles per eye, 3 days after laser treatment. PC-treated eyes were enucleated and examined after X-gal staining on days 5 (A), 14 (B), 28 (C), 90 (D), 135 (E), and 180 (F) after vector delivery (G). PDT-treated eyes were similarly examined on days 5, 14, 28, and 90 (H). Enhanced staining at laser burn sites continued to be significant 135 days after PC (G) and 5 days after PDT (H). (•) PC- or PDT-treated left hemispheres; (○) untreated right hemispheres. Bar: 300 μm. (G, H) Data are the mean ± SE. *P < 0.05.
Figure 5.
 
Flatmounts of PC-treated retina after X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles per eye, 3 days after laser treatment. PC-treated eyes were enucleated and examined after X-gal staining on days 5 (A), 14 (B), 28 (C), 90 (D), 135 (E), and 180 (F) after vector delivery (G). PDT-treated eyes were similarly examined on days 5, 14, 28, and 90 (H). Enhanced staining at laser burn sites continued to be significant 135 days after PC (G) and 5 days after PDT (H). (•) PC- or PDT-treated left hemispheres; (○) untreated right hemispheres. Bar: 300 μm. (G, H) Data are the mean ± SE. *P < 0.05.
Figure 6.
 
Flatmounts of photocoagulated retina with X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 105 (A), 3 × 106 (B), 3 × 107 (C), 3 × 108 (D), and 3 × 109 (E) particles/eye, 3 days after laser photocoagulation. Only the left hemisphere was exposed to laser and intense enhancement of expression at the site of laser burns is evident at all the tested doses (3 × 105 to 3 × 109 particles/eye (F). (•) PC-treated left hemispheres; (○) untreated right hemispheres. Bar, 300 μm. Data are the mean ± SE. *P < 0.05.
Figure 6.
 
Flatmounts of photocoagulated retina with X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 105 (A), 3 × 106 (B), 3 × 107 (C), 3 × 108 (D), and 3 × 109 (E) particles/eye, 3 days after laser photocoagulation. Only the left hemisphere was exposed to laser and intense enhancement of expression at the site of laser burns is evident at all the tested doses (3 × 105 to 3 × 109 particles/eye (F). (•) PC-treated left hemispheres; (○) untreated right hemispheres. Bar, 300 μm. Data are the mean ± SE. *P < 0.05.
Figure 7.
 
CAR and integrin-αV, -β3, and -β5 mRNA levels in photocoagulated and untreated retina and choroid quantified by qRT-PCR. There was no significant difference in mRNA expression for any of the genes evaluated in laser-treated ( Image not available ) vs. (▪) untreated eyes. Bar: SE (P > 0.05 in all comparisons).
Figure 7.
 
CAR and integrin-αV, -β3, and -β5 mRNA levels in photocoagulated and untreated retina and choroid quantified by qRT-PCR. There was no significant difference in mRNA expression for any of the genes evaluated in laser-treated ( Image not available ) vs. (▪) untreated eyes. Bar: SE (P > 0.05 in all comparisons).
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Figure 1.
 
Naïve eyes without laser treatment with histochemical X-gal staining. Corneal (A), iridal (B), and retinal (C) flatmounts. ( Image not available ) Optic disc. Arrow: sporadic X-gal staining. Cryosections of anterior segment (D) and the retina (E). Intravitreous injection of AdlacZ.11D resulted in strong expression in cornea and iris, but only focal and sporadic expression in the retina. Bar: (A, B) 70 μm; (C) 180 μm; (D, E) 100 μm.
Figure 1.
 
Naïve eyes without laser treatment with histochemical X-gal staining. Corneal (A), iridal (B), and retinal (C) flatmounts. ( Image not available ) Optic disc. Arrow: sporadic X-gal staining. Cryosections of anterior segment (D) and the retina (E). Intravitreous injection of AdlacZ.11D resulted in strong expression in cornea and iris, but only focal and sporadic expression in the retina. Bar: (A, B) 70 μm; (C) 180 μm; (D, E) 100 μm.
Figure 2.
 
Photocoagulated retina with histochemical X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles/eye on days 1 (A), 3 (B, E), 7 (C), and 28 (D) after laser photocoagulation. Half of the posterior fundus (left hemisphere) was photocoagulated and the remaining right hemisphere was untreated for internal controls (line: the border between PC-treated and untreated hemispheres). In the retinal flatmounts (A-D) each blue X-gal-stained lesion corresponds to laser burns (arrowheads). The cryosection (E) through photocoagulated lesion (between white arrows) demonstrated darker stained linear structures (black arrows) probably representing Müller cells. Bar: (AD) 500 μm; (E) 120 μm.
Figure 2.
 
Photocoagulated retina with histochemical X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles/eye on days 1 (A), 3 (B, E), 7 (C), and 28 (D) after laser photocoagulation. Half of the posterior fundus (left hemisphere) was photocoagulated and the remaining right hemisphere was untreated for internal controls (line: the border between PC-treated and untreated hemispheres). In the retinal flatmounts (A-D) each blue X-gal-stained lesion corresponds to laser burns (arrowheads). The cryosection (E) through photocoagulated lesion (between white arrows) demonstrated darker stained linear structures (black arrows) probably representing Müller cells. Bar: (AD) 500 μm; (E) 120 μm.
Figure 3.
 
PDT and vector-treated eyes were examined after X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles per eye, on days 1 (A), 3 (B, E), 7 (C), and 28 (D) after PDT. Half of the posterior fundus (left hemisphere) was treated with PDT, the remaining right hemisphere was untreated and served as an internal control (white line: the border between PDT-treated and untreated hemispheres). In the retinal flatmounts (AD) each blue X-gal stained lesion corresponds to a PDT-treated area. LacZ expression peaked at day 3 in PDT-treated eyes. Cryosection (E) through PDT-treated retina (between white arrows) demonstrates enhanced staining and linear structures (arrows) consistent with Müller cell morphology. Bar: (AD) 500 μm; (E) 120 μm.
Figure 3.
 
PDT and vector-treated eyes were examined after X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles per eye, on days 1 (A), 3 (B, E), 7 (C), and 28 (D) after PDT. Half of the posterior fundus (left hemisphere) was treated with PDT, the remaining right hemisphere was untreated and served as an internal control (white line: the border between PDT-treated and untreated hemispheres). In the retinal flatmounts (AD) each blue X-gal stained lesion corresponds to a PDT-treated area. LacZ expression peaked at day 3 in PDT-treated eyes. Cryosection (E) through PDT-treated retina (between white arrows) demonstrates enhanced staining and linear structures (arrows) consistent with Müller cell morphology. Bar: (AD) 500 μm; (E) 120 μm.
Figure 4.
 
For determining the best time to deliver AdV vectors after laser treatment, rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles on days 1, 3, 7, and 28 after PC (A) and PDT (B). There was a significant difference in the area ratio of lacZ-stained/total area between photocoagulated and untreated hemispheres on days 1, 3, and 7 after PC and on day 3 after PDT. (•) PC- or PDT-treated left hemispheres; (○) untreated right hemispheres. Bar: SE. *P < 0.05.
Figure 4.
 
For determining the best time to deliver AdV vectors after laser treatment, rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles on days 1, 3, 7, and 28 after PC (A) and PDT (B). There was a significant difference in the area ratio of lacZ-stained/total area between photocoagulated and untreated hemispheres on days 1, 3, and 7 after PC and on day 3 after PDT. (•) PC- or PDT-treated left hemispheres; (○) untreated right hemispheres. Bar: SE. *P < 0.05.
Figure 5.
 
Flatmounts of PC-treated retina after X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles per eye, 3 days after laser treatment. PC-treated eyes were enucleated and examined after X-gal staining on days 5 (A), 14 (B), 28 (C), 90 (D), 135 (E), and 180 (F) after vector delivery (G). PDT-treated eyes were similarly examined on days 5, 14, 28, and 90 (H). Enhanced staining at laser burn sites continued to be significant 135 days after PC (G) and 5 days after PDT (H). (•) PC- or PDT-treated left hemispheres; (○) untreated right hemispheres. Bar: 300 μm. (G, H) Data are the mean ± SE. *P < 0.05.
Figure 5.
 
Flatmounts of PC-treated retina after X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 109 particles per eye, 3 days after laser treatment. PC-treated eyes were enucleated and examined after X-gal staining on days 5 (A), 14 (B), 28 (C), 90 (D), 135 (E), and 180 (F) after vector delivery (G). PDT-treated eyes were similarly examined on days 5, 14, 28, and 90 (H). Enhanced staining at laser burn sites continued to be significant 135 days after PC (G) and 5 days after PDT (H). (•) PC- or PDT-treated left hemispheres; (○) untreated right hemispheres. Bar: 300 μm. (G, H) Data are the mean ± SE. *P < 0.05.
Figure 6.
 
Flatmounts of photocoagulated retina with X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 105 (A), 3 × 106 (B), 3 × 107 (C), 3 × 108 (D), and 3 × 109 (E) particles/eye, 3 days after laser photocoagulation. Only the left hemisphere was exposed to laser and intense enhancement of expression at the site of laser burns is evident at all the tested doses (3 × 105 to 3 × 109 particles/eye (F). (•) PC-treated left hemispheres; (○) untreated right hemispheres. Bar, 300 μm. Data are the mean ± SE. *P < 0.05.
Figure 6.
 
Flatmounts of photocoagulated retina with X-gal staining. Rats received intravitreous injection of AdlacZ.11D at a concentration of 3 × 105 (A), 3 × 106 (B), 3 × 107 (C), 3 × 108 (D), and 3 × 109 (E) particles/eye, 3 days after laser photocoagulation. Only the left hemisphere was exposed to laser and intense enhancement of expression at the site of laser burns is evident at all the tested doses (3 × 105 to 3 × 109 particles/eye (F). (•) PC-treated left hemispheres; (○) untreated right hemispheres. Bar, 300 μm. Data are the mean ± SE. *P < 0.05.
Figure 7.
 
CAR and integrin-αV, -β3, and -β5 mRNA levels in photocoagulated and untreated retina and choroid quantified by qRT-PCR. There was no significant difference in mRNA expression for any of the genes evaluated in laser-treated ( Image not available ) vs. (▪) untreated eyes. Bar: SE (P > 0.05 in all comparisons).
Figure 7.
 
CAR and integrin-αV, -β3, and -β5 mRNA levels in photocoagulated and untreated retina and choroid quantified by qRT-PCR. There was no significant difference in mRNA expression for any of the genes evaluated in laser-treated ( Image not available ) vs. (▪) untreated eyes. Bar: SE (P > 0.05 in all comparisons).
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