|Molecular Vision 2004;
Received 3 June 2003 | Accepted 20 January 2004 | Published 10 February 2004
GDNF gene therapy attenuates retinal ischemic injuries in rats
Wei-Chi Wu,1,2 Chi-Chun Lai,1 Show-Li Chen,3
Ming-Hui Sun,1,2 Xiao Xiao,4 Tun-Lu Chen,1 Ray
Jui-Fang Tsai,1 Shu-Wen Kuo,5
1Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 2Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan; 3Department of Microbiololgy and Immunology, National Defense Medical Center, Taipei, Taiwan; 4Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania; 5Department of Medical Research, Veteran's General Hospital, Taipei, Taiwan; 6Department of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan
Correspondence to: Yeou-Ping Tsao, Department of Ophthalmology, Mackay Memorial Hospital, Number 92, Sec. 2, Chung Shan North Road, Taipei, Taiwan; Phone: 886-2-25433535; FAX: 886-2-25433642; email: email@example.com
Purpose: To examine the protective effects of glial cell line-derived neurotrophic factor (GDNF) on retinal ischemia-reperfusion injury by using gene delivery.
Methods: Gene delivery to retinal cells was achieved through intravitreal injections of recombinant adeno-associated virus expressing GDNF (rAAV-GDNF) in the right eyes and AAV expressing Escherichia coli LacZ (rAAV-LacZ) in the left eyes of Sprague-Dawley rats. Ischemic injury was introduced three weeks after gene delivery. The synthesis and accumulation of GDNF within the retina were determined using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) three weeks after gene delivery. The neuroprotective effects of GDNF were evaluated by determining the preservation of the inner retina thickness and the cell counts in the retinal ganglion cell (RGC) layer one week after reperfusion. In addition, eletroretinograms (ERGs) were performed to determine the functionality of the retinas. Finally, the levels of RGC apoptosis were measured using the TdT-dUTP terminal nick-end labeling (TUNEL) method 6 h after reperfusion.
Results: Gene expression of GDNF was demonstrated through immunohistochemistry and ELISA. Thinning of the inner retina and decreased numbers of cells in RGC layer were noted after ischemia in all of the eyes. However, the thickness of the inner retina and the numbers of cells in RGC layer were better preserved in rAAV-GDNF treated eyes than in rAAV-LacZ treated eyes seven days after reperfusion (p=0.028 and p<0.001, respectively). Also, seven days after reperfusion, the rAAV-GDNF treated eyes had retained larger b-wave amplitudes than rAAV-LacZ treated eyes (p=0.003). Finally, rAAV-GDNF treated eyes had statistically fewer apoptotic cells in the RGC layer than the control eyes (p=0.011).
Conclusions: In these experiments, GDNF moderately protected rat retina from ischemia-reperfusion injury, possibly by preventing apoptosis in retinal cells.
Retinal ischemia is a serious and common problem that occurs as a result of acute vascular occlusion and leads to loss of vision in a number of ocular diseases such as acute glaucoma , diabetic retinopathy , hypertensive retinopathy , and retinal vascular occlusion . Recent studies have shown that most of the neuronal cell death associated with retinal ischemia-reperfusion injuries is due to apoptosis . Although not completely understood, glutamate toxicity  and the production of free radicals [7,8] play an important role in the pathogenesis of retinal neuronal cell death. Possible therapeutic agents, such as MK-801 (a N-methyl-D-asparate [NMDA] receptor inhibitor) [9,10], catalase and thioredoxin (free radical scavengers) [11,12], calcium channel blockers [13,14], nitric oxide synthase inhibitors [15,16], and erythropoietin  have been shown to have neuroprotective effects against retinal ischemia-reperfusion injury by inhibiting the production of glutamate and reactive oxygen intermediates. In addition, neurotrophic and growth factors, such as ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), pigment epithelium derived factor (PEDF), and hepatocyte growth factor (HGF) have demonstrated neuroprotective effects against retinal ischemia-reperfusion injury [18-21]. Due to the chronic status of these retinal ischemia-associated diseases, therapeutic peptides would need to be introduced or made at high concentrations for extended time periods in order to achieve lasting protection of the retinal cells. Because of the short half-lives of these therapeutic agents, repeated intravitreal injections would be necessary. This would likely cause many undesirable side effects. To overcome the damage induced by repeated injections, neurotrophic factors could be delivered to the retina using gene therapy. In fact, viral vectors have been proven to be useful in transfecting ocular cells [22-26]. In this study, recombinant adeno-associated virus (rAAV) vector was used to transfer genetic material to the target cells.
Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor that has been shown to reduce ischemic injury in both the brain [27,28] and the spinal cord . In studies involving the retina, GDNF protects photoreceptors in several different models of retinal degeneration [30,31]. GDNF also promotes the survival of axotomized retinal ganglion cells (RGC) in adult rats [32-35]. In a study of neuronal cultures from the retina, GDNF was found to stimulate cell cycle progression, promote neuroblast proliferation, and delay the onset of apoptosis, thus improving differentiation and acting as a trophic factor for photoreceptors . In addition, the combination of GDNF and docosahexaenoic acid (DHA) had an additive effect both on photoreceptor survival and on opsin expression . From these observations, we hypothesize that GDNF may also protect the retina from ischemia-reperfusion injury.
In this study, we used immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) to investigate the retinal expression of GDNF after intravitreal injection of rAAV-GDNF. To establish that GDNF can act as a protective agent against ischemia-reperfusion injury, we measured inner retinal thickness, cell counts in RGC layer, and electroretinograms (ERG). The number of apoptotic cells was determined using the TdT-dUTP terminal nick-end labeling (TUNEL) method.
Generation of rAAV-GDNF
Recombinant AAV encoding rat GDNF cDNA was constructed in previous work  using a three-plasmid cotransfection system, as previously described in the literature . The recombinant AAV was purified twice by cesium chloride ultracentrifugation and the titers for rAAV-GDNF and rAAV-LacZ were determined by dot blot hybridization with GDNF and LacZ DNA probes, respectively .
Animals and transduction of virus
Sprague-Dawley rats weighing 150-200 g were used in all subsequent experiments. The animals were handled in accordance with the ARVO statement for the Use of Animal in Ophthalmic and Vision Research. They were anesthetized with intramuscular injections of 1.5 ml/kg of an equal volume mixture of 2% Xylazine (Rompun; Bayer AG, Leverkusen, Germany) and 50 mg/ml Ketamine (Ketalar; Parke-Davis, Morris Plains, NJ). After the rats were anesthetized, pupils were dilated with 1% tropiamide (1% mydriacyl; Alcon Laboratories, Hempstead, UK) and the eyes were gently protruded using a rubber sleeve. The eyes were then covered with sodium hyaluronate (Healon; Pharmacia and Upjohn, Uppsala, Sweden) and a transparent disc, which allowed the fundus to be visible under a surgical microscope. Then a 90° periotomy was made in the temporal quadrant and a sclerotomy was made 1 mm behind the limbus with the tip of a 27 gauge needle. A 33 gauze blunt tip needle (Hamilton, Reno, NV) was inserted into the vitreous cavity, and 3 ml of the viral suspension containing 1.1x1010 viral particles-equal to 1.1x108 infectious units or 1.1x107 transduction units was injected . The needle was left in the vitreous cavity for 1 min after injection to reduce the degree of reflux. Similarly, the contralateral eye of each rat was injected with rAAV-LacZ to serve as the control.
Ischemia and reperfusion injury
The animals were prepared and anesthetized as described above three weeks after intravitreal delivery of the viral particles. After topical application of hydrochloride (Novesin; Novartis, Hettlingen, Switzerland), the anterior chambers of both eyes were cannulated with a 27 gauge infusion needle connected to a reservoir containing a balanced salt solution (Alcon Pharma, Fort Worth, TX). The intraocular pressure was raised to 110 mm of Hg by elevating the reservoir. The infusion needle was removed from the anterior chamber 60 min later. This time point was chosen because obvious and consistent damage to the RGCs and inner retina had previously been observed [40,41]. Retinal ischemia and reperfusion were confirmed by the whitening of the fundus and restoration of the retinal blood flow. Animals that were not reperfused or sustained damage to the lens were discarded to prevent interference with the results.
Samples were fixed in 4% paraformaldehyde for 2 h after removal of the cornea and the lens. Then they were incubated in 30% sucrose (in PBS) overnight at 4 °C, embedded in optimal cutting temperature (OCT) compound, and sectioned using a microtome cryostat (CM1900; Leica, Wetzlar, Germany). The sections were placed on slides that had been coated with 1% gelatin and 0.1% chromium potassium sulfate (Sigma, St. Louis, MO) in distilled water to promote adhesion of the sections to the glass surface. Samples were blocked with 1% goat serum and 1% bovine serum albumin for 30 min after washing in PBS. For the detection of GDNF, samples were incubated with a rabbit polyclonal antibody recognizing GDNF (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Anti-rabbit IgG-fluorescein isothiocyanate (FITC) was used as secondary antibody and the sections were counterstained with propidium iodide (PI; Molecular probes, Eugene, OR). The resulting sections were then viewed on a confocal microscope (TCS SP2; Leica).
Enzyme linked immunosorbent assay (ELISA)
ELISA was used to measure the production of GDNF in the retina. According to published protocol, ELISA was performed three weeks after viral transduction . Total protein concentrations were detected using a protein assay system (Bio-Rad Protein Assay Dye Reagent Concentrate; Bio-Rad Laboratories, Hercules, CA). The final results were expressed as pg of GDNF per μg of total retinal protein.
Thickness of the inner retina
Thinning of the inner retina was apparent after retinal ischemia-reperfusion injury . The protective effect of GDNF was measured by its capability to prevent thinning of the inner retina. One week after reperfusion, the rats were sacrificed with an overdose of anesthetics. After removing the cornea, lens, and vitreous, the eyecup was fixed in 2.5% glutaraldehyde in sodium phosphate buffer for 2 h for morphometric measurements. The tissues fixed in gultaraldehyde were osmicated, dehydrated and embedded in epoxy resin. Retinal sections of 0.5 μm were obtained by cutting along the vertical meridian of the eye and passing through the optic nerve head. The sections were counterstained with toluidine blue and examined by light microscopy. Retinas approximately 100 μm temporal to the midline sagittal cut were sectioned and sampled. Ischemic changes were evaluated by measuring the inner retinal thickness, which was defined as the thickness between the inner limiting membrane and the boundary of the outer nuclear layer and the outer plexiform layer. Measurements were taken at intervals of 100 μm in a range of 800-1200 μm above and below the optic nerve head, according to a previously described method . The data were combined to obtain an average inner retinal thickness.
Cell counts in the ganglion cell layer
One week after ischemia and reperfusion injury, rats were sacrificed in order to count the numbers of cells in RGC layer according to published procedures with modifications . Briefly, after enucleation, the eyeballs were fixed in 4% paraformaldehyde overnight. The cornea, lens, and vitreous were removed after fixation. The retina was detached and flat mounted onto a slide. One drop of 1% gelatin was added to the retina to help adhere it to the slide. After allowing the gelatin to dry, retinal flat mounts were stained with 0.1% cresyl violet for 5 min. The slides were dehydrated in graded alcohols and xylene and mounted with resin-base mounting medium. The numbers of nuclei at the RGC layer of the posterior pole (approximately 500 μm from the center of the optic disc) and the peripheral retina (approximately 4 mm from the center of the optic disc) per unit area were counted with an eyepiece reticule of a microscope at 400x magnification. Counts were taken from comparable areas of the four quadrants of the retinal flat mount. The results were then averaged to give the data for one eye. No attempt was made to distinguish the types of RGCs or displaced amacrine cells. Morphologically distinguishable glial cells, vascular endothelial cells, pericytes, and shrunken neuronal cells with pyknotic nuclei were excluded from the cell counts.
Flash ERG results were recorded (UTAS-E 300; LKC Technology, Gaithersburg, MD) to assess the retinal function of rAAV-GDNF injected eyes and control eyes. Rats were dark-adapted for 1 h before performing the ERG. After being anesthetized, the rats were placed on a heating pad during the procedure. The eyes were then proptosed with a rubber sleeve. The recording electrode, an Ag:AgCl electrode, was placed on the cornea with 0.5% methyl cellulose as a conductive medium. A reference electrode was attached to the shaven skin of the head and a ground electrode clipped to the rat's ear. A single flash light (duration, 100 ms) 30 cm from the eye was used as the light stimulus. Responses were amplified with a gain setting ±500 μV and filtered with low 0.3 Hz and high 500 Hz from an amplifier. Data were acquired, digitized, and analyzed using EM for Windows, version 2.6 running on an IBM compatible 667 MHz PentiumTM computer (LKC Technologies). ERG studies were performed in both eyes before ischemia (baseline), one day, and seven days after reperfusion. The amplitudes and the implicit times of the a- and b-waves were measured and averaged.
In situ TUNEL labeling
Apoptosis of RGCs usually occurs 6 h after ischemia reperfusion injury . Therefore, eyeballs were enucleated 6 h after reperfusion for TUNEL analysis. Eyeballs were sectioned along a perpendicular plane close to the optic nerve head, then fixed in 4% paraformaldehyde at 4 °C overnight, embedded in paraffin, and cut into 10 μm sections. Retinas approximately 100 μm from the optic nerve were sampled for TUNEL positive RGCs. TUNEL was performed using a DNA fragmentation detection kit (Fluorescein-FragEL; Oncogene, Darmstadt, Germany) according to the manufacturer's instructions. The results were viewed with a fluorescent microscope (Eclipse E800; Nikon, Osaka, Japan) after mounting using mounting gel. Apoptotic cells were counted by human eye in 250 μm segments across entire sections of retina and the results were averaged.
The Wilcoxon signed-ranks test was used to test for statistical differences in the thickness of the inner retina, cell counts in RGC layer, and ERG results between rAAV-GDNF and rAAV-LacZ injected eyes. The test was also used to determine differences in GDNF production and TUNEL positive RGCs between rAAV-GDNF and rAAV-LacZ injected eyes. The Mann-Whitney test was used to determine differences in GDNF production, thickness of the inner retina, cell counts in RGC layer, ERG results, and TUNEL positive RGCs between naïve and rAAV-GDNF injected eyes. The data were obtained by statistical software (SPSS version 11.0; SPSS Inc., Chicago, IL) and are expressed as the mean±the standard deviation with p<0.05 considered to be significant.
Synthesis of GDNF in ganglion cells after intravitreal injections of rAAV-GDNF
Three weeks after gene delivery, the eyeballs were enucleated for immunohistochemistry study. Prominent GDNF signals (green) were detected in the retinal ganglion cells (Figure 1A) from eyes injected with rAAV-GDNF, while no expression of GDNF was identified in the control eyes (Figure 1B). These results confirmed that GDNF was expressed in rAAV-GDNF transduced cells.
High level of GDNF detected in rAAV-GDNF transduced retinas
As shown in Figure 2, the amounts of GNDF in normal, uninjected eyes were at background levels (6.1±4.1 pg/μg protein; n=7) and no further induction of GDNF production was noted in eyes injected with PBS (8.0±6.1 pg/μg protein; n=7). The most important finding was that the quantity of GDNF in eyes transduced with rAAV-GDNF (38.3±15.1 pg/μg protein; n=10) was approximately five times that in eyes transduced with rAAV-LacZ (7.9±6.4 pg/μg protein; p=0.005, n=10). This indicated that cells transduced with rAAV-GDNF synthesized significant levels of GDNF in the retina.
Morphology and quantitative analysis of the thickness of inner retina
The right eyes of the rats were injected with rAAV-GDNF and the left with rAAV-LacZ (n=10). A separate group of rats without any treatment served as controls (n=10). Ischemia-reperfusion injury was performed three weeks after viral transduction. Normal data of the thickness of inner retina were derived from four rats. Figure 3 shows representative retinal morphologies examined seven days after reperfusion. When compared with the normal retinas (Figure 3A), the ischemic-reperfused retinas with rAAV-LacZ injection (Figure 3C) and untreated retina (Figure 3D) showed signs of inner retinal loss such as RGC dropout, pkynotic nuclei in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL), vacuoles in the INL, and thinning of the inner plexiform layer (IPL). Retinas treated with rAAV-GDNF (Figure 3B) showed better preservation of the IPL and the INL.
To further document the effect of GDNF gene delivery, we compared the thickness of the inner retina between eyes receiving no treatment, eyes injected with rAAV-LacZ, and eyes injected with rAAV-GDNF. These results are summarized in Figure 4. Significant ameliorative effect (p=0.028) on the thickness of the inner retina was observed in rAAV-GDNF treated eyes (80.9±13.9 μm, 77.8±13.4.9% of normal retina; n=10) when compared rAAV-LacZ treated eyes (66.4±7.1 μm, 63.8±6.8% of normal retina; n=10). There was also significant difference (p=0.006) between rAAV-GDNF treated eyes (80.9±13.9 μm, 77.8±13.4.9% of normal retina; n=10) and untreated eyes (64.6±6.6 μm, 62.1±6.3% of normal retina; n=10).
Cell counts in the ganglion cell layer
One week after ischemic treatment, the loss of cells in RGC layer was apparent. This loss was more prominent in the peripheral retina than in the central area (data not shown). The results of the cell counts in RGC layer are summarized in Figure 5. In the intact retinas, the mean number of cell counts in the RGC layer was 2883.7±239.1 cells/mm2 (n=8). One week after reperfusion, more cells in the RCG layer were retained in the rAAV-GDNF treated eyes (2175.6±331.8 cells/mm2, 75.4±11.5% of normal retina; n=16) than in the rAAV-LacZ treated eyes (1624.3±367.0 cells/mm2, 56.3±12.7% of normal retina; n=16) or in the untreated eyes (1512.1±303.4 cells/mm2, 52.4±10.5% of normal retina; n=16). These results were statistically significant (p<0.001 and p<0.001, respectively).
ERG was performed during the pre-ischemic period, one day after reperfusion and seven days after reperfusion. Except for rats transduced with rAAV, a separate group of rats (n=8) received no ischemic injury and served as normal controls. ERG results are summarized in Figure 6. Figure 6A is a representative figure of ERG recording. Figure 6B is the bar figure of the b-wave amplitude before ischemia, and one day and seven days after ischemia. There was no statistical difference in the latencies of ERG a- and b-waves between rAAV-GDNF and rAAV-LacZ transduced eyes throughout the experiment (data not shown). In addition, there was no statistical difference in the ERG a-waves between rAAV-GDNF and rAAV-LacZ treated eyes (data not shown).
As for ERG b-wave amplitide, one day after reperfusion, ERG b-waves decreased in rAAV-GDNF treated eyes (217.8±74.6 μV, 49.5±16.7% of normal eye; n=28), in rAAV-LacZ treated eyes (205.3±87.6 μV, 46.7±19.9% of normal eye; n=28), and in untreated eyes (208.2±72.2 μV, 47.3±16.4% of normal eye; n=28). There was no statistical difference in the ERG b-waves between rAAV-GDNF treated eyes and rAAV-LacZ treated eyes (p=0.264). There was also no statistical difference in the ERG b-waves between rAAV-GDNF treated eyes and untreated eyes (p=0.577). However, one week after reperfusion, significantly (p=0.003) larger amplitudes of ERG b-waves were measured in rAAV-GDNF treated eyes (299.3±131.3 μV, 68.3±30.0% of normal eye; n=26) than those in rAAV-LacZ treated eyes (232.6±133.5 μV, 53.2±30.5% of normal eye; n=26). Also, significantly (p=0.045) larger amplitudes of ERG b-waves were measured in rAAV-GDNF treated eyes (299.3±131.3 μV, 68.3±30.0% of normal eye; n=26) than those in untreated eyes (221.2±108.8 μV, 50.2±24.7% of normal eye; n=26).
Throughout the experiment, ERG b-waves in normal rats (n=8) were relatively constant (460.7±116.1 μV, 402.8±110.2 μV, 437.7±72.4 μV in pre-ischemic period, one and seven days after reperfusion, respectively). It is also worth noting that there was no statistical difference in ERG b-waves between the rAAV-treated eyes (both rAAV-GDNF and rAAV-LacZ treated eyes; 440.0±124.0 μV, 437.7±72.4 μV, respectively) and eyes without treatment (460.7±116.1 μV) during the pre-ischemic period. This result demonstrates that no obvious ocular toxicity was observed after rAAV transduction.
Prevention of apoptosis by rAAV-GDNF injection
More TUNEL positive RGCs were noted in rAAV-LacZ injected eyes (Figure 7E) and untreated retina (Figure 7F), while only sparse TUNEL positive RGCs were noted in rAAV-GDNF injected eyes (Figure 7D). There was almost no TUNEL positive RGC in normal retina (Figure 7C). Samples treated with DNase stained positive in the TUNEL assay and served as a positive control (Figure 7A). Samples not incubated with the TdT enzyme showed no TUNEL staining and served as a negative control (Figure 7B).
After counting the number of apoptotic RGCs in the retina, there were statistically fewer apoptotic cells (p=0.011; Figure 8) in rAAV-GDNF injected eyes (5.4±2.2 cells per 250 μm length of retina; n=8) than in rAAV-LacZ injected eyes (9.1±3.0 cells per 250 μm length of retina; n=8). Also, there were statistically fewer apoptotic cells (p=0.026) in rAAV-GDNF injected eyes (5.4±2.2 cells per 250 μm length of retina; n=8) than in untreated eyes (9.0±3.2 cells per 250 μm length of retina; n=8). Only a few cells were TUNEL positive in normal retinas (0.2±0.4 cells per 250 μm length of retina; n=8).
By immunohistochemistry, we confirmed that intravitreal injections of rAAV-GDNF resulted in GDNF protein expression. Results from ELISA confirmed that high levels of GDNF were produced in the retina. Histological analysis revealed moderate preservation of the inner retina and the cells in the RGC layer of eyes injected with rAAV-GDNF after ischemia reperfusion injury. Functionally, ERG analyses confirmed moderate protection of the retina from ischemia reperfusion injury. The ameliorative effect of ischemia insult by GDNF is probably through the inhibition of apoptosis. Our results demonstrate the possibility of using gene therapy for treating ischemic retinal injuries.
Recent studies have indicated that the expression of GDNF is associated with neuronal injury. After retinal detachment, the production of GDNF is increased dramatically . Following mechanical injury to the adult mouse striatum, GDNF mRNA expression is increased within 6 h . In addition, systemic administration of kainate, which elicits seizures, induces the expression of GDNF and the GDNF family receptor alpha (GFRα1) . Immediately after ischemia in the brain, GDNF and the receptors for GDNF are upregulated [47,48]. Collectively, these data suggest that the family of GDNF molecules may be endogenous neuroprotective agents that can be activated during ischemia-reperfusion injury. However, the expression of GDNF following ischemia-reperfusion is merely transient [47,48], but the dose of GDNF required for neuroprotection is high . It is possible that the endogenous upregulation of GDNF alone is not sufficient for the rescue of retinal cells. We speculate that sustained expression of GDNF will provide the additional neuroprotection needed to combat ischemia-reperfusion injuries. The observations described in this study support this postulation.
Our ELISA shows the efficiency of rAAV in the transduction of retina. In addition, we found that intravitreal injection of rAAV (data not shown), as well as subretinal injection as revealed in our previous study , did not result in obvious adverse morphologic effects. Furthermore, our ERG results provide further evidence that no obvious ocular toxicity was noted after rAAV transduction. These results show that rAAV is an efficient and safe vector for the transduction of retina tissues.
The anesthetic agents used in this study, Ketamine and Xylazine, have been reported to have neuroprotective effects [50,51]. Ketamine, an N-methyl-D-aspartate (NMDA) antagonist, has been reported to decrease plasma catecholamines and improves outcome from incomplete cerebral ischemia in rats . Xylazine has been reported to induce basic fibroblast growth factor expression in photoreceptors and ameliorate light damage . Therefore, the overall rescue effect we observed may not merely the effect of GDNF, but an additive effect caused by the combination of Ketamine, Xylazine, and GDNF. This also explains why the effect of injury was not so severe when comparing with some studies [40,52]. However, our results show that there is statistical difference between the rAAV-GDNF treated eyes and the control eyes (including the rAAV-LacZ treated eyes and untreated eyes) after ischemic insult. The difference is strictly due to the effect of GDNF. Therefore, the conclusion we reached was not affected by the use of these anesthetics.
GDNF signals through a receptor complex consisting of Ret protein tyrosine kinase and a member of the GDNF receptor family (GFRα) . Previous studies have shown that GFRα and Ret are expressed in the retina [54,55]. GFRα-1 immunoreactivity in the retina is localized in RGCs, Müller cells, and photoreceptors . Ret is localized primarily in the outer segments of photoreceptors, the inner retina, and the ganglion cell layer . Because GDNF signal transduction requires both GFRα-1 and Ret, these results suggest that GDNF is capable of acting directly on RGCs. However, the interaction with other cells or other neurotrophic factors is still unknown.
GDNF exerts its neuroprotective effect on neurons by inhibiting apotosis [34,48,56], but the intracellular signaling involved in mediating GDNF induced neuroprotection is still under investigation. GDNF has been found to activate mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) [56-58]. In fact, the motor axon outgrowth promoting activity of GDNF can be prevented by a MAPK inhibitor (PD98059) and a PI3K inhibitor (LY294002). However, the neuroprotective effect of GDNF is unaffected . These findings suggest that the neuroprotective effects of GDNF are not exclusively dependent on the MAPK or PI3K pathways. GDNF attenuates NMDA induced excitotoxic neuronal death by activation of MAPK, thereby modulating the NMDA receptor and reducing calcium influx . GDNF also exerts protection against apoptosis by upregulating Bcl-2 and Bcl-x, which suppresses the accumulation of oxygen free radicals, following caspase-3 activation . GDNF has been shown to attenuate the activity of nitric oxide synthase (NOS) activity, which is enhanced during ischemia . GDNF also exerts an antiapoptotic effect by recruiting NF-kappaB, which translocates to the nucleus and can activate neuroprotective genes such as superoxide dismutases or inhibitors of apoptosis .
Neuropretective agents can protect the retina from ischemic damage; however, reduction of the retinal ischemia itself is just as important. Most of the current neuroprotective agents exert protection temporarily [18,19,34], but even sustained expression of neurotrophic factors cannot infinitely rescue retinal cells . Therefore, measures that will reduce retinal ischemia itself are still necessary approaches. Rapid reduction of intraocular pressure in acute glaucoma , panretinal photocoagulation in diabetic retinopathy , control of blood pressure in hypertensive retinopathy , and thrombolytic therapy for retinal vascular occlusion  are still important treatment modalities for these diseases. Combined with these treatments, GDNF gene therapy may be a good adjuvant for several retinal diseases including acute glaucoma, acute retinal vascular occlusion, diabetic retinopathy, and hypertensive vascular disease.
In conclusion, gene therapy with GDNF is a safe and effective method in ameliorating retinal damage caused by ischemia. Further studies are needed to optimize the effectiveness of this approach and to explore the role of GDNF and other neurotrophic factors during episodes of retinal ischemia.
The authors thank Yun-Ying Chou for excellent technical support in the process of resin-embedded tissues. The authors also thank Stephen Gee (University of North Carolina at Chapel Hill) for editing this manuscript.
This study was supported by grants from National Science Council, Taiwan (NSC 91-2320-B-182A-009, NSC 92-2311-B-182A-001) and Department of Health, Taiwan (DOH92-TD-1057).
1. Levin LA. Models of neural injury. J Glaucoma 2001; 10:S19-21.
2. Stefansson E, Machemer R, de Juan E Jr, McCuen BW 2nd, Peterson J. Retinal oxygenation and laser treatment in patients with diabetic retinopathy. Am J Ophthalmol 1992; 113:36-8.
3. Tso MO, Jampol LM. Pathophysiology of hypertensive retinopathy. Ophthalmology 1982; 89:1132-45.
4. Hill DW. Fluorescein studies in retinal vascular occlusion. Br J Ophthalmol 1968; 52:1-12.
5. Kuroiwa S, Katai N, Shibuki H, Kurokawa T, Umihira J, Nikaido T, Kametani K, Yoshimura N. Expression of cell cycle-related genes in dying cells in retinal ischemic injury. Invest Ophthalmol Vis Sci 1998; 39:610-7.
6. Osborne NN, Herrera AJ. The effect of experimental ischaemia and excitatory amino acid agonists on the GABA and serotonin immunoreactivities in the rabbit retina. Neuroscience 1994; 59:1071-81.
7. Roth S. Role of nitric oxide in retinal cell death. Clin Neurosci 1997; 4:216-23.
8. Bonne C, Muller A, Villain M. Free radicals in retinal ischemia. Gen Pharmacol 1998; 30:275-80.
9. Lam TT, Siew E, Chu R, Tso MO. Ameliorative effect of MK-801 on retinal ischemia. J Ocul Pharmacol Ther 1997; 13:129-37.
10. Osborne NN, Larsen AK. Antigens associated with specific retinal cells are affected by ischaemia caused by raised intraocular pressure: effect of glutamate antagonists. Neurochem Int 1996; 29:263-70.
11. Shibuki H, Katai N, Kuroiwa S, Kurokawa T, Yodoi J, Yoshimura N. Protective effect of adult T-cell leukemia-derived factor on retinal ischemia-reperfusion injury in the rat. Invest Ophthalmol Vis Sci 1998; 39:1470-7.
12. Nayak MS, Kita M, Marmor MF. Protection of rabbit retina from ischemic injury by superoxide dismutase and catalase. Invest Ophthalmol Vis Sci 1993; 34:2018-22.
13. Osborne NN, Wood JP, Cupido A, Melena J, Chidlow G. Topical flunarizine reduces IOP and protects the retina against ischemia-excitotoxicity. Invest Ophthalmol Vis Sci 2002; 43:1456-64.
14. Toriu N, Akaike A, Yasuyoshi H, Zhang S, Kashii S, Honda Y, Shimazawa M, Hara H. Lomerizine, a Ca2+ channel blocker, reduces glutamate-induced neurotoxicity and ischemia/reperfusion damage in rat retina. Exp Eye Res 2000; 70:475-84.
15. Ostwald P, Goldstein IM, Pachnanda A, Roth S. Effect of nitric oxide synthase inhibition on blood flow after retinal ischemia in cats. Invest Ophthalmol Vis Sci 1995; 36:2396-403.
16. Geyer O, Almog J, Lupu-Meiri M, Lazar M, Oron Y. Nitric oxide synthase inhibitors protect rat retina against ischemic injury. FEBS Lett 1995; 374:399-402.
17. Junk AK, Mammis A, Savitz SI, Singh M, Roth S, Malhotra S, Rosenbaum PS, Cerami A, Brines M, Rosenbaum DM. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc Natl Acad Sci U S A 2002; 99:10659-64.
18. Ogata N, Wang L, Jo N, Tombran-Tink J, Takahashi K, Mrazek D, Matsumura M. Pigment epithelium derived factor as a neuroprotective agent against ischemic retinal injury. Curr Eye Res 2001; 22:245-52.
19. Unoki K, LaVail MM. Protection of the rat retina from ischemic injury by brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor. Invest Ophthalmol Vis Sci 1994; 35:907-15.
20. Shibuki H, Katai N, Kuroiwa S, Kurokawa T, Arai J, Matsumoto K, Nakamura T, Yoshimura N. Expression and neuroprotective effect of hepatocyte growth factor in retinal ischemia-reperfusion injury. Invest Ophthalmol Vis Sci 2002; 43:528-36.
21. Siliprandi R, Canella R, Carmignoto G. Nerve growth factor promotes functional recovery of retinal ganglion cells after ischemia. Invest Ophthalmol Vis Sci 1993; 34:3232-45.
22. Bennett J, Wilson J, Sun D, Forbes B, Maguire A. Adenovirus vector-mediated in vivo gene transfer into adult murine retina. Invest Ophthalmol Vis Sci 1994; 35:2535-42.
23. Di Polo A, Aigner LJ, Dunn RJ, Bray GM, Aguayo AJ. Prolonged delivery of brain-derived neurotrophic factor by adenovirus-infected Muller cells temporarily rescues injured retinal ganglion cells. Proc Natl Acad Sci U S A 1998; 95:3978-83.
24. Flannery JG, Zolotukhin S, Vaquero MI, LaVail MM, Muzyczka N, Hauswirth WW. Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus. Proc Natl Acad Sci U S A 1997; 94:6916-21.
25. Isenmann S, Klocker N, Gravel C, Bahr M. Short communication: protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo. Eur J Neurosci 1998; 10:2751-6.
26. Miyoshi H, Takahashi M, Gage FH, Verma IM. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci U S A 1997; 94:10319-23.
27. Kitagawa H, Hayashi T, Mitsumoto Y, Koga N, Itoyama Y, Abe K. Reduction of ischemic brain injury by topical application of glial cell line-derived neurotrophic factor after permanent middle cerebral artery occlusion in rats. Stroke 1998; 29:1417-22.
28. Miyazaki H, Okuma Y, Fujii Y, Nagashima K, Nomura Y. Glial cell line-derived neurotrophic factor protects against delayed neuronal death after transient forebrain ischemia in rats. Neuroscience 1999; 89:643-7.
29. Sakurai M, Abe K, Hayashi T, Setoguchi Y, Yaginuma G, Meguro T, Tabayashi K. Adenovirus-mediated glial cell line-derived neurotrophic factor gene delivery reduces motor neuron injury after transient spinal cord ischemia in rabbits. J Thorac Cardiovasc Surg 2000; 120:1148-57.
30. Frasson M, Picaud S, Leveillard T, Simonutti M, Mohand-Said S, Dreyfus H, Hicks D, Sabel J. Glial cell line-derived neurotrophic factor induces histologic and functional protection of rod photoreceptors in the rd/rd mouse. Invest Ophthalmol Vis Sci 1999; 40:2724-34.
31. McGee Sanftner LH, Abel H, Hauswirth WW, Flannery JG. Glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. Mol Ther 2001; 4:622-9.
32. Yan Q, Wang J, Matheson CR, Urich JL. Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of axotomized retinal ganglion cells in adult rats: comparison to and combination with brain-derived neurotrophic factor (BDNF). J Neurobiol 1999; 38:382-90.
33. Klocker N, Braunling F, Isenmann S, Bahr M. In vivo neurotrophic effects of GDNF on axotomized retinal ganglion cells. Neuroreport 1997; 8:3439-42.
34. Koeberle PD, Ball AK. Effects of GDNF on retinal ganglion cell survival following axotomy. Vision Res 1998; 38:1505-15.
35. Schmeer C, Straten G, Kugler S, Gravel C, Bahr M, Isenmann S. Dose-dependent rescue of axotomized rat retinal ganglion cells by adenovirus-mediated expression of glial cell-line derived neurotrophic factor in vivo. Eur J Neurosci 2002; 15:637-43.
36. Politi LE, Rotstein NP, Carri NG. Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid. Invest Ophthalmol Vis Sci 2001; 42:3008-15.
37. Tsai TH, Chen SL, Chiang YH, Lin SZ, Ma HI, Kuo SW, Tsao YP. Recombinant adeno-associated virus vector expressing glial cell line-derived neurotrophic factor reduces ischemia-induced damage. Exp Neurol 2000; 166:266-75.
38. Xiao X, Li J, Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 1998; 72:2224-32.
39. Xiao X, Li J, McCown TJ, Samulski RJ. Gene transfer by adeno-associated virus vectors into the central nervous system. Exp Neurol 1997; 144:113-24.
40. Hughes WF. Quantitation of ischemic damage in the rat retina. Exp Eye Res 1991; 53:573-82.
41. Selles-Navarro I, Villegas-Perez MP, Salvador-Silva M, Ruiz-Gomez JM, Vidal-Sanz M. Retinal ganglion cell death after different transient periods of pressure-induced ischemia and survival intervals. A quantitative in vivo study. Invest Ophthalmol Vis Sci 1996; 37:2002-14.
42. Wu WC, Lai CC, Chen SL, Xiao X, Chen TL, Tsai RJ, Kuo SW, Tsao YP. Gene therapy for detached retina by adeno-associated virus vector expressing glial cell line-derived neurotrophic factor. Invest Ophthalmol Vis Sci 2002; 43:3480-8.
43. LaVail MM, Battelle BA. Influence of eye pigmentation and light deprivation on inherited retinal dystrophy in the rat. Exp Eye Res 1975; 21:167-92.
44. Lam TT, Abler AS, Kwong JM, Tso MO. N-methyl-D-aspartate (NMDA)--induced apoptosis in rat retina. Invest Ophthalmol Vis Sci 1999; 40:2391-7.
45. Liberatore GT, Wong JY, Porritt MJ, Donnan GA, Howells DW. Expression of glial cell line-derived neurotrophic factor (GDNF) mRNA following mechanical injury to mouse striatum. Neuroreport 1997; 8:3097-101.
46. Humpel C, Hoffer B, Stromberg I, Bektesh S, Collins F, Olson L. Neurons of the hippocampal formation express glial cell line-derived neurotrophic factor messenger RNA in response to kainate-induced excitation. Neuroscience 1994; 59:791-5.
47. Miyazaki H, Nagashima K, Okuma Y, Nomura Y. Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats. Brain Res 2001; 922:165-72.
48. Wang Y, Chang CF, Morales M, Chiang YH, Hoffer J. Protective effects of glial cell line-derived neurotrophic factor in ischemic brain injury. Ann N Y Acad Sci 2002; 962:423-37.
49. Carwile ME, Culbert RB, Sturdivant RL, Kraft TW. Rod outer segment maintenance is enhanced in the presence of bFGF, CNTF and GDNF. Exp Eye Res 1998; 66:791-805.
50. Hoffman WE, Pelligrino D, Werner C, Kochs E, Albrecht RF, Schulte am Esch J. Ketamine decreases plasma catecholamines and improves outcome from incomplete cerebral ischemia in rats. Anesthesiology 1992; 76:755-62.
51. Wen R, Cheng T, Li Y, Cao W, Steinberg RH. Alpha 2-adrenergic agonists induce basic fibroblast growth factor expression in photoreceptors in vivo and ameliorate light damage. J Neurosci 1996; 16:5986-92.
52. Weber M, Mohand-Said S, Hicks D, Dreyfus H, Sahel JA. Monosialoganglioside GM1 reduces ischemia--reperfusion-induced injury in the rat retina. Invest Ophthalmol Vis Sci 1996; 37:267-73.
53. Jing S, Wen D, Yu Y, Holst PL, Luo Y, Fang M, Tamir R, Antonio L, Hu Z, Cupples R, Louis JC, Hu S, Altrock BW, Fox GM. GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF. Cell 1996; 85:1113-24.
54. Jomary C, Thomas M, Grist J, Milbrandt J, Neal MJ, Jones SE. Expression patterns of neurturin and its receptor components in developing and degenerative mouse retina. Invest Ophthalmol Vis Sci 1999; 40:568-74.
55. Koeberle PD, Ball AK. Neurturin enhances the survival of axotomized retinal ganglion cells in vivo: combined effects with glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor. Neuroscience 2002; 110:555-67.
56. Nicole O, Ali C, Docagne F, Plawinski L, MacKenzie ET, Vivien D, Buisson A. Neuroprotection mediated by glial cell line-derived neurotrophic factor: involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway. J Neurosci 2001; 21:3024-33.
57. Ho TW, Bristol LA, Coccia C, Li Y, Milbrandt J, Johnson E, Jin L, Bar-Peled O, Griffin JW, Rothstein JD. TGFbeta trophic factors differentially modulate motor axon outgrowth and protection from excitotoxicity. Exp Neurol 2000; 161:664-75.
58. Sawada H, Ibi M, Kihara T, Urushitani M, Nakanishi M, Akaike A, Shimohama S. Neuroprotective mechanism of glial cell line-derived neurotrophic factor in mesencephalic neurons. J Neurochem 2000; 74:1175-84.
59. Hayashi H, Ichihara M, Iwashita T, Murakami H, Shimono Y, Kawai K, Kurokawa K, Murakumo Y, Imai T, Funahashi H, Nakao A, Takahashi M. Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor. Oncogene 2000; 19:4469-75.
60. Soltau JB, Zimmerman TJ. Changing paradigms in the medical treatment of glaucoma. Surv Ophthalmol 2002; 47 Suppl 1:S2-5.
61. Agarwal T, Gupta M. Treatment of hypertensive retinopathy. Surv Ophthalmol 2002; 47:513.
62. Vallee JN, Paques M, Aymard A, Massin P, Santiago PY, Adeleine P, Gaudric A, Merland JJ. Combined central retinal arterial and venous obstruction: emergency ophthalmic arterial fibrinolysis. Radiology 2002; 223:351-9.