February 2012
Volume 53, Issue 2
Free
Glaucoma  |   February 2012
Long-Term Rescue of Rat Retinal Ganglion Cells and Visual Function by AAV-Mediated BDNF Expression after Acute Elevation of Intraocular Pressure
Author Affiliations & Notes
  • Ruotong Ren
    From the State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; and
  • Ying Li
    From the State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; and
  • Zhiping Liu
    From the State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; and
  • Kegao Liu
    the Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China.
  • Shigang He
    From the State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; and
  • Corresponding author: Shigang He, Institute of Biophysics, Chinese Academy of Sciences, 15 Da-tun Road, Beijing 100101, P.R. China; shiganghe@moon.ibp.ac.cn
Investigative Ophthalmology & Visual Science February 2012, Vol.53, 1003-1011. doi:10.1167/iovs.11-8484
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      Ruotong Ren, Ying Li, Zhiping Liu, Kegao Liu, Shigang He; Long-Term Rescue of Rat Retinal Ganglion Cells and Visual Function by AAV-Mediated BDNF Expression after Acute Elevation of Intraocular Pressure. Invest. Ophthalmol. Vis. Sci. 2012;53(2):1003-1011. doi: 10.1167/iovs.11-8484.

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

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Abstract

Purpose.: To evaluate the ability of increased expression of brain-derived neurotrophic factor (BDNF) using adenoassociated viral (AAV) vector to prevent the loss of rat retinal ganglion cells (RGCs) and visual function after acute elevation of intraocular pressure (IOP).

Methods.: AAV vectors (expressing BDNF or GFP) were injected into the vitreous 6 hours after a transient IOP elevation to 130 mm Hg for 45 minutes. Protective effects were evaluated by counting RGCs retrogradely labeled with fluorogold (FG) from the superior colliculus, measuring the amplitude and the latency of the P1 component of the visual evoked potential (VEP), and observing the visual acuity and contrast sensitivity in awake and behaving animals.

Results.: RGC numbers decreased continuously to 9 weeks after the elevation of IOP. FG-positive RGC loss was significantly decreased in the retinas treated with AAV-BDNF at 3, 6, and 9 weeks after the insult, with corresponding improvements in VEP parameters. Supplementing BDNF protein once to compensate for the slow onset of AAV-mediated gene expression rescued a larger number of RGCs and the parameters of the VEP. Visual acuity and contrast sensitivity were significantly improved in all treated groups, with the largest improvement in the combined-therapy group, and were maintained for up to 70 weeks. The authors further demonstrated that BDNF rescued the RGCs by activating TrkB receptors through both autocrine and paracrine mechanisms.

Conclusions.: AAV-mediated BDNF expression in the rat retina achieved a sustained rescue of RGCs and visual function after an acute elevation of IOP.

Retinal ganglion cell (RGC) loss has been reported in glaucoma, 1 ischemia, 2 age-related macular degeneration, 3 diabetic retinopathy, 4 and optic neuritis. 5 Because RGCs transmit the information processed by the retina to higher visual centers, loss of RGCs inevitably leads to impairment of visual function. 
Treatments generally can be divided into two categories—protection and rescue—but to be clinically relevant, protection as a preventive measure before the onset of symptoms does not justify invasive approaches. Numerous studies have been reported about the protection and rescue of RGCs by adding neuroprotective reagents and by suppressing apoptosis (see Refs. 6, 7 for reviews). 
Brain-derived neurotrophic factor (BDNF) is one of the most potent and frequently used neurotrophic factors in both protection and rescue. 8 Direct application of recombinant proteins or viral vector–mediated expression have been reported in Parkinson's disease, 9 Huntington's disease, 10,11 Alzheimer's disease, 12 and optic neuropathies as glaucoma 13 and optic nerve trauma. 14 16  
Adeno-associated virus (AAV) is by far the most suitable viral vector for gene therapy because of its sustained expression 17,18 and safety. 19,20 The most serious limitation of AAV is the slow onset of exogenous gene expression. 21,22 AAV-mediated BDNF expression has been used to protect RGCs in the high intraocular pressure (IOP) model 13 or to rescue RGCs in the optic nerve section model. 16 Surprisingly, it has never been performed with a protocol analogous to clinical treatment, namely, with the AAV vector delivered after the acute elevation of IOP. 
In this study, we asked whether AAV-mediated BDNF expression can rescue RGCs after the acute elevation of IOP and whether supplementing gene therapy with exogenous recombinant BDNF protein can compensate for the slow onset of AAV-mediated expression. 
Methods
Animals
Adult male Sprague-Dawley rats, each weighing 180 to 280 g and obtained from Charles River Laboratories, were used in this study. Animals were handled strictly in accordance with the ARVO Statement for the Care and Use of Laboratory Animals, with the policies of the Society for Neuroscience, and with institutional guidelines on the use of animals and human subjects in neuroscience research. 
Experimental Elevation of IOP
IOP was elevated experimentally as previously described. 23 Detailed description of the method is provided in the Supplementary Methods
Retrograde Labeling of RGCs
Seven days before the quantification of surviving RGCs, animals were anesthetized with intraperitoneal injection of chloral hydrate (400 mg/kg) and were mounted in a stereotactic apparatus. A solution of 4% fluorogold (Biotium, Hayward, CA) was injected into two adjacent sites in the superior colliculus (SC) of each hemisphere (5.9 mm and 6.7 mm behind bregma, 1.0 mm lateral, and 5.0 mm [first release] and 4.0 mm [second release] deep). Injections were made over a 10-minute period, and the cannula was left in place for another 10 minutes to avoid dye leakage from the injection track. The animals were allowed to recover in solitary cages for the subsequent experiments. 
Recombinant Viruses
The procedure for virus packaging has been described previously. 24,25 Detailed description of this method is provided in the Supplementary Methods
Intraocular Injections
We adopted the procedure previously described for recombinant AAV intraocular injection. 13 Detailed description of this method is provided in the Supplementary Methods
Immunohistochemistry
Animals were euthanized, and retinas were isolated and fixed with 4% paraformaldehyde in 0.1 M PB for 2 hours and then rinsed with 0.01 M PBS. Tissue was incubated in the primary antibody, rabbit anti–green fluorescence protein (GFP) antibody (1:400 dilution; Chemicon, Temecula, CA), or rabbit anti–His-tag antibody (1:200 dilution; Biosynthesis Biotechnology Co., Ltd., Beijing, China), for 2 days at 4°C in a blocking solution of 1% bovine serum albumin and 0.3% Triton X-100 in 0.01 M PBS. The retinas were rinsed with 0.01 M PBS, then incubated in TRITC secondary antibody (Chemicon, Temecula, CA) overnight at 4°C, mounted in mounting medium (Vectashield; Vector Laboratories, Inc., Burlingame, CA), and coverslipped. 
Quantification of RGCs
Images were captured from flat-mounted retinas with an epifluorescence microscope (E800; Nikon, Tokyo, Japan), equipped with a charge-coupled device camera (Cascade; Photometrics, Tucson, AZ). Either a 20× plan apochromat objective, NA 0.75, or a 10× plan apochromat objective, NA 0.45, was used. Images were processed with ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html) and commercial image editing software (Photoshop CS2; Adobe Systems Inc., San Jose, CA). Using a 10× objective, four images were acquired 500 to 1000 μm from the optic nerve head, and four images were acquired 3000 to 3500 μm from optic nerve head in each retinal quadrant. Cell densities of the four images closer to the optic nerve head from each quadrant were averaged and taken as the RGC density of the central retina. Cell densities from the four images farther from the optic nerve head were averaged and taken as the peripheral RGC density. All 32 images were averaged and taken as the RGC density of the whole retina. 
Western Blot Analysis
Retinas from control or treated eyes were homogenized and lysed (150 mM NaCl, 50 mM Tris, pH 8.0, 2% NP-40, phenylmethylsulfonyl fluoride, leupeptin, and aprotinin) for 45 minutes. After centrifugation, the concentration of soluble protein was determined using a kit (Biosynthesis Biotechnology Co., Ltd.). Twenty micrograms of retinal protein per lane was loaded and separated electrophoretically on a 12% SDS-PAGE gel and then was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Rabbit anti–GFP, rabbit anti–BDNF polyclonal antibody (Chemicon, Temecula, CA), and rabbit anti–β-actin polyclonal antibody (Biosynthesis Biotechnology Co., Ltd.) were used as primary antibodies for the detection of GFP and BDNF, and β-actin was used as an internal control. Horseradish peroxidase (HRP)–conjugated goat-anti–rabbit antibody (Biosynthesis Biotechnology Co., Ltd.) was used as a secondary reagent. Immunopositive bands were revealed with enhanced chemoluminescence (Millipore). Image processing and semiquantification were performed with Image J software. 
VEP Recording
After overnight dark-adaptation, animals were anesthetized with chloral hydrate (400 mg/kg) intraperitoneally. Pupils were dilated with 1% atropine. VEP signals were recorded in the scalp covering the visual cortex using a stainless steel recording electrode placed subcutaneously 1 cm anterior to the midpoint of a line connecting the two back ear edges. A stainless steel reference electrode was placed in the mouth, and a stainless steel grounding electrode was placed in the tail. After covering the contralateral eye, VEPs were recorded with flash stimulus (3.5 cd · s/m2; 1.3 Hz) on a commercial VEP system (RETIport 32 System; Roland Consult GmbH, Wiesbaden, Germany) and then amplified (1000×), filtered (1 and 100 Hz), and averaged (100 events) with the corresponding software (RETIport 32 System; Roland Consult GmbH). The amplitude and latency of the VEP P1 component was measured for subsequent statistical analysis. 
Behavioral Test of Visual Function
The apparatus and methodology used to measure both visual acuity and contrast sensitivity in this study have been described previously. 26,27 Detailed description of these methods is provided in the Supplementary Methods
Statistical Analysis
Results are presented as mean ± SEM. Data were analyzed by ANOVA (OriginPro 8.0; OriginLab Corp., Northampton, MA), with P < 0.05 considered significant. 
Results
Acute Elevation of IOP Leads to Continual RGC Loss
When we increased the IOP to 130 mm Hg for 45 minutes, the number of RGCs retrogradely labeled by fluorogold (FG) exhibited a continuous decrease (Figs. 1a–d). By the end of the first week, 54.1% ± 3.0% RGCs remained, and a small but significant decrease continued, with only 20.2% ± 0.8% RGCs remaining until the end of the ninth week (Fig. 1e). Analyzing the central and peripheral retina separately, 59.7% ± 1.3% and 44.0% ± 3.4% RGCs were present at the end of the first week, and 26.2% ± 1.8% and 12.1% ± 0.6% were present by the end of ninth week (Fig. 1f). 
Figure 1.
 
Continuous loss of FG-positive RGCs after acute elevation of the IOP. (ad) RGCs retrogradely labeled with FG from the superior colliculus in the control and 3 and 7 days and 3 weeks after acute IOP elevation. (e, f) The number of FG-positive RGCs in the whole retina (e) and the central and peripheral retina (f). Small but significant reduction was still observable between the sixth to ninth weeks after the increase in IOP. The number of retinas is given in parentheses. Data are mean ± SEM. *P < 0.05. Scale bar, 50 μm.
Figure 1.
 
Continuous loss of FG-positive RGCs after acute elevation of the IOP. (ad) RGCs retrogradely labeled with FG from the superior colliculus in the control and 3 and 7 days and 3 weeks after acute IOP elevation. (e, f) The number of FG-positive RGCs in the whole retina (e) and the central and peripheral retina (f). Small but significant reduction was still observable between the sixth to ninth weeks after the increase in IOP. The number of retinas is given in parentheses. Data are mean ± SEM. *P < 0.05. Scale bar, 50 μm.
Transfection of Retinal Neurons and Expression Time Course
We constructed an AAV vector to express GFP and BDNF in neurons in the ganglion cell layer (GCL). Many neurons in the GCL, including RGCs and displaced amacrine cells, were transfected after intravitreous injection of 2 μL AAV (Figs. 2a, 2b). The number of cells expressing the exogenous genes and level of protein expression increased with the interval between transfection and observation (Figs. 2c–f). Western blot analysis was used to quantify protein levels 1, 3, and 6 weeks after transfection. Both GFP and BDNF expression exhibited a very similar time course; the level of exogenous protein began to increase significantly 1 week after injection and kept increasing to a much higher level by 3 and 6 weeks after injection (Figs. 2g–j), consistent with earlier reports. 28 Only a low level of BDNF was observed in the retinas transfected with GFP (Fig. 2i). 
Figure 2.
 
Expression of exogenous genes. (a) BDNF expression visualized using antibody against the His-tag. (b) RGCs retrogradely labeled with FG from the SC. Arrows: untransfected RGCs (FG-positive/BDNF-negative); arrowheads: transfected amacrine cells (FG-negative/BDNF-positive). (ce) Immunohistochemical staining of GFP showing an increasing number of cells expressing the exogenous gene from 3 days to 3 weeks after transfection. Scale bar, 25 μm. (f) Population data as shown in (c) to (e). Data are mean ± SEM, with six retinas in each group. *P < 0.05. (g, h) Western blot analysis showing the expression level of GFP and BDNF 1, 3, and 6 weeks after transfection. (i, j) Quantitative data from (g) and (h). Relative intensity (%) means the grayscale ratio of BDNF or GFP to β-actin.
Figure 2.
 
Expression of exogenous genes. (a) BDNF expression visualized using antibody against the His-tag. (b) RGCs retrogradely labeled with FG from the SC. Arrows: untransfected RGCs (FG-positive/BDNF-negative); arrowheads: transfected amacrine cells (FG-negative/BDNF-positive). (ce) Immunohistochemical staining of GFP showing an increasing number of cells expressing the exogenous gene from 3 days to 3 weeks after transfection. Scale bar, 25 μm. (f) Population data as shown in (c) to (e). Data are mean ± SEM, with six retinas in each group. *P < 0.05. (g, h) Western blot analysis showing the expression level of GFP and BDNF 1, 3, and 6 weeks after transfection. (i, j) Quantitative data from (g) and (h). Relative intensity (%) means the grayscale ratio of BDNF or GFP to β-actin.
Time of Intervention
Theoretically, intervention should be implemented at the earliest opportunity, as soon as the high IOP is terminated. However, because of the possibility of ischemic and nonischemic injuries caused by high IOP, retinal neurons might suffer from edema and other symptoms of reperfusion. Very few RGCs were transfected when AAV was injected immediately after the termination of high IOP (Fig. 3). More RGCs were transfected when the interval was lengthened, reaching a plateau at 6 hours (Fig. 3). Therefore, we chose 6 hours after the termination of high IOP as the time of intervention. 
Figure 3.
 
Time of intervention. The number of transfected cells increases with the interval between the release of high IOP and the intraocular injection, reaching a plateau at approximately 6 hours. Data are mean ± SEM, with six retinas in each group. *P < 0.05.
Figure 3.
 
Time of intervention. The number of transfected cells increases with the interval between the release of high IOP and the intraocular injection, reaching a plateau at approximately 6 hours. Data are mean ± SEM, with six retinas in each group. *P < 0.05.
Significant Rescue of RGCs by BDNF
AAV-mediated BDNF expression significantly reduced RGC loss after the acute elevation of IOP. More FG-positive RGCs were present in the AAV-BDNF group than in the AAV-GFP group at all time points examined (Fig. 4). Reduction in the total number of FG-positive RGCs stopped 6 weeks after treatment (Fig. 4), remaining at 37.6% ± 0.7% of control in the AAV-BDNF group, whereas in the AAV-GFP group, the number of remaining cells continued to decrease from 27.5% ± 0.6% at the end of sixth week to 20.2% ± 0.8% at the end of ninth week. In the peripheral retina, the reduction stopped after 3 weeks in the group treated with AAV-BDNF (data not shown). 
Figure 4.
 
BDNF significantly rescues RGCs after acute IOP elevation. After elevation of IOP, FG-positive cells exhibited a remarkable decrease. With AAV-BDNF, more FG-positive cells were present 3 weeks after IOP elevation. The number of cells remained unchanged 6 weeks later. When BDNF protein was administered alone or with AAV-GFP, similar results were obtained. A dramatic increase in FG-positive cells was observed, but a reduction in FG-positive cells was also clear from the end of the third week to the end of ninth week in these groups. When recombinant BDNF protein was given together with AAV-BDNF, significantly more FG-positive cells were present at the end of the third week, and no reduction was observed until the ninth week. The number of animals tested is shown in the middle of each bar. Data are the mean ± SEM. *P < 0.05.
Figure 4.
 
BDNF significantly rescues RGCs after acute IOP elevation. After elevation of IOP, FG-positive cells exhibited a remarkable decrease. With AAV-BDNF, more FG-positive cells were present 3 weeks after IOP elevation. The number of cells remained unchanged 6 weeks later. When BDNF protein was administered alone or with AAV-GFP, similar results were obtained. A dramatic increase in FG-positive cells was observed, but a reduction in FG-positive cells was also clear from the end of the third week to the end of ninth week in these groups. When recombinant BDNF protein was given together with AAV-BDNF, significantly more FG-positive cells were present at the end of the third week, and no reduction was observed until the ninth week. The number of animals tested is shown in the middle of each bar. Data are the mean ± SEM. *P < 0.05.
The slow onset of AAV-mediated gene expression could have seriously limited this intervention because approximately half the FG-positive RGCs were lost within 1 week of IOP elevation (Fig. 1). Therefore, we administered 500 ng recombinant BDNF protein with AAV vector in the hope that direct application of the BDNF protein would rescue RGCs before the onset of AAV-mediated BDNF expression. 
The effect of BDNF protein alone was remarkable. By the end of the third week, the number of remaining FG-positive RGCs more than doubled, reaching 77.9% ± 1.4% of the control level compared with 33.1% ± 0.7% in the AAV-GFP group and 40.7% ± 0.9% in the AAV-BDNF group (Fig. 4). However, the FG-positive RGCs underwent similar reductions in the BDNF alone and the BDNF+AAV-GFP groups, from 77.9% ± 1.4% and 76.4% ± 1.5% at the end of the third week to 65.9% ± 1.4% and 65.2% ± 1.3% at the end of the ninth week (Fig. 4), respectively. When BDNF and AAV-BDNF were coadministered, more FG-positive RGCs were present at the end of third week (86.8% ± 1.6%), and no significant RGC loss was observed in following weeks (Fig. 4). 
Sustained Improvement of Visual Function
To assess the function of the surviving RGCs, we first recorded VEPs and measured the amplitude and latency of the P1 component (Fig. 5a). After elevated IOP, the amplitude decreased and the latency increased in the AAV-GFP group (Figs. 5b, 5c). In the AAV-BDNF group, significant protection was observed, although the amplitude was still much smaller and the latency longer than in the healthy controls (Figs. 5b, 5c). 
Figure 5.
 
Visual function. (a) Recording traces of the VEP, from the control animals and animals receiving elevated IOP and treated with AAV-GFP and AAV-BDNF for 3 weeks, respectively. (b) The amplitude of the P1 component significantly decreased after transient elevation of the IOP. Across all time points examined, the amplitude of the VEP in animals receiving AAV-BDNF was much larger than in animals receiving AAV-GFP. When BDNF protein was administered, the amplitude of the P1 component was further increased in all three groups. In the BDNF alone group and the BDNF+AAV-GFP group, the amplitude clearly decreased from the end of the third week to the end of the sixth week. No further reduction was observed at the end of the ninth week. In the BDNF+AAV-BDNF group, no reduction was observed, even at the end of the ninth week. (c) The latency of the P1 component significantly increased 3, 6, and 9 weeks after IOP elevation. However, the group treated with AAV-BDNF showed significantly shorter latency than the AAV-GFP group. In the BDNF alone and the BDNF+AAV-GFP group, latency increased between the third and the sixth weeks. No increase was detected in the BDNF+AAV-BDNF group. The number of animals tested is shown in the middle of each bar. Data in (b) and (c) are mean ± SEM. *P < 0.05. (d, e) Visual acuity and contract sensitivity in different groups. The value displayed in (d) is the highest spatial frequency at which >70% correct choices were made. The number of animals tested is shown in the middle of each bar. Data are mean ± SEM. *P < 0.05. (f) Visual acuity at 52 weeks after insult. There was no difference between the untreated group and the AAV-GFP group, or the BDNF alone and the BDNF+AAV-GFP groups. Clear differences were seen between groups treated by AAV-BDNF, BDNF alone, and BDNF+AAV-BDNF. Data are mean ± SEM. The number of animals tested is indicated in parentheses. (g) Comparison of visual acuity of the control, untreated, and BDNF+AAV-BDNF–treated animals at 12, 20, and 52 weeks after insult. Data are mean ± SEM. Data for the intact control group were collected at 12 and 52 weeks.
Figure 5.
 
Visual function. (a) Recording traces of the VEP, from the control animals and animals receiving elevated IOP and treated with AAV-GFP and AAV-BDNF for 3 weeks, respectively. (b) The amplitude of the P1 component significantly decreased after transient elevation of the IOP. Across all time points examined, the amplitude of the VEP in animals receiving AAV-BDNF was much larger than in animals receiving AAV-GFP. When BDNF protein was administered, the amplitude of the P1 component was further increased in all three groups. In the BDNF alone group and the BDNF+AAV-GFP group, the amplitude clearly decreased from the end of the third week to the end of the sixth week. No further reduction was observed at the end of the ninth week. In the BDNF+AAV-BDNF group, no reduction was observed, even at the end of the ninth week. (c) The latency of the P1 component significantly increased 3, 6, and 9 weeks after IOP elevation. However, the group treated with AAV-BDNF showed significantly shorter latency than the AAV-GFP group. In the BDNF alone and the BDNF+AAV-GFP group, latency increased between the third and the sixth weeks. No increase was detected in the BDNF+AAV-BDNF group. The number of animals tested is shown in the middle of each bar. Data in (b) and (c) are mean ± SEM. *P < 0.05. (d, e) Visual acuity and contract sensitivity in different groups. The value displayed in (d) is the highest spatial frequency at which >70% correct choices were made. The number of animals tested is shown in the middle of each bar. Data are mean ± SEM. *P < 0.05. (f) Visual acuity at 52 weeks after insult. There was no difference between the untreated group and the AAV-GFP group, or the BDNF alone and the BDNF+AAV-GFP groups. Clear differences were seen between groups treated by AAV-BDNF, BDNF alone, and BDNF+AAV-BDNF. Data are mean ± SEM. The number of animals tested is indicated in parentheses. (g) Comparison of visual acuity of the control, untreated, and BDNF+AAV-BDNF–treated animals at 12, 20, and 52 weeks after insult. Data are mean ± SEM. Data for the intact control group were collected at 12 and 52 weeks.
Three weeks after BDNF protein was administered, the amplitude of the P1 component of the VEP was similar in the BDNF alone, the BDNF+AAV-GFP, and the BDNF+AAV-BDNF groups, all much larger than the AAV-BDNF group, consistent with the previous observation that many more FG-positive RGCs were present in former groups. However, in the BDNF alone and the BDNF+AAV-GFP groups, P1 amplitude exhibited a reduction in subsequent time points (Fig. 5b), also consistent with the previous results showing a reduction in the number FG-positive RGCs by the end of sixth and ninth weeks. In the BDNF+AAV-BDNF group, no such reduction was observed (Fig. 5b). 
P1 latencies in all treated groups were much more reduced than in the AAV-GFP group, whereas no significant differences were observed between groups treated with AAV-BDNF, BDNF alone, BDNF+AAV-GFP, or BDNF+AAV-BDNF (Fig. 5c). The BDNF alone and the BDNF + AAV-GFP groups showed significant increases in latency between the third and the ninth weeks (Fig. 5c). No significant difference was observed between different time points within the AAV-BDNF and the BDNF+AAV-BDNF groups (Fig. 5c). The improved amplitudes and latencies demonstrated that the rescue of RGCs did significantly improve visual function. 
We tested visual acuity and contrast sensitivity in awake and behaving animals to further examine visual function. Significant improvements in visual acuity and contrast sensitivity were observed in the AAV-BDNF group. Larger improvements were seen in the BDNF alone and the BDNF+AAV-GFP groups, with the largest improvement seen in the BDNF+AAV-BDNF group. Improvements in visual acuity and contrast sensitivity were maintained for up to 70 weeks (Figs. 5d, 5e). In the untreated and the AAV-GFP groups, visual acuity deteriorated between the 12th and 20th weeks and then stabilized (Fig. 5d). Significant reductions in contrast sensitivity were seen in the BDNF only and the BDNF+AAV-GFP groups between the 12th and 20th weeks (Fig. 5e), suggesting that the loss of RGCs continued 12 weeks after the insult, with the system stabilizing after 20 weeks. Although no difference in visual acuity was observed up to 52 weeks (Fig. 5d), a small but significant reduction in visual acuity was seen in the control, the untreated, and the BDNF+AAV-BDNF groups at 52 weeks (Fig. 5g), possibly because of age-related changes given that the animals were then approximately 60 weeks old. Similar reductions in contrast sensitivity were also seen in the untreated, the BDNF alone, and BDNF+AAV-GFP groups, but not in the BDNF+AAV-BDNF group (Fig. 5e). 
Comparing visual performance at 52 weeks after insult, the visual acuity of the untreated group and the group treated with AAV-GFP fell below threshold at 0.2 cyc/deg, the group treated by AAV-BDNF was above threshold at 0.4 cyc/deg, the groups treated by BDNF alone and BDNF+AAV-BDNF were above 0.6 cyc/deg, and the group treated by BDNF+AAV-BDNF was very similar to the intact group, remaining above threshold at 0.8 cyc/deg and falling below threshold at 0.9 cyc/deg (Fig. 5f). Increasing resolution at the high end revealed that a slight decrease in visual acuity could still be seen as time went by, falling below threshold at 0.85 cyc/deg at 52 weeks (Fig. 5g). 
Autocrine and Paracrine BDNF Activating TrkB Receptors
We analyzed the data to see whether rescue was limited to the cells transfected by AAV. Two weeks after the acute insult, we compared the number of RGCs double labeled with FG and BDNF or GFP for the transfected RGCs. Significant rescue was observed for the RGCs expressing BDNF (Fig. 6a). We also compared cells positive for FG but negative for BDNF or GFP for the untransfected RGCs. Untransfected RGCs in the retinas expressing BDNF were also significantly rescued (Fig. 6b), indicating that BDNF synthesized by RGCs and displaced amacrine cells can rescue neighboring neurons through a paracrine mechanism, consistent with previous reports. 16,29,30 Application of the BDNF receptor TrkB antagonist K252a blocked rescue in a dose- dependent manner. A lower concentration blocked rescue through paracrine mechanisms, and a higher dose blocked all rescue effects (Figs. 6c, 6d). 
Figure 6.
 
Autocrine and paracrine effects through TrkB receptors. (a) Comparison of cells positive for FG and BDNF/GFP. For directly transfected RGCs, significant rescue was obvious. (b) Comparison of cells positive for FG but negative for BDNF/GFP. Untransfected RGCs residing in retinas treated with AAV-BDNF were significantly rescued compared with untransfected RGCs residing in the retinas treated with AA-GFP. (c, d) TrkB receptor antagonist K252a blocked the rescue effects; 15 μM was needed to block the rescue effects of directly transfected RGCs, whereas 10 μM was sufficient to block the rescue effects of untransfected RGCs. Data are mean ± SEM, with six retinas in each group. *P < 0.05.
Figure 6.
 
Autocrine and paracrine effects through TrkB receptors. (a) Comparison of cells positive for FG and BDNF/GFP. For directly transfected RGCs, significant rescue was obvious. (b) Comparison of cells positive for FG but negative for BDNF/GFP. Untransfected RGCs residing in retinas treated with AAV-BDNF were significantly rescued compared with untransfected RGCs residing in the retinas treated with AA-GFP. (c, d) TrkB receptor antagonist K252a blocked the rescue effects; 15 μM was needed to block the rescue effects of directly transfected RGCs, whereas 10 μM was sufficient to block the rescue effects of untransfected RGCs. Data are mean ± SEM, with six retinas in each group. *P < 0.05.
Discussion
The acute elevated IOP model we used in this study is an ischemic rather than a glaucoma model 31 33 because the IOP was raised to a level close to or higher than the arterial pressure. 34 The acute ischemic insult, and likely nonischemic injuries induced by high IOP, resulted in a rapid phase and a slower phase of FG-positive RGC loss. 23,35 37 The slower phase is probably more similar to the gradual loss of RGCs observed in the chronic glaucoma model. 38 44  
The number of FG-positive RGCs continued to decrease up to 9 weeks after the insult. In behavioral tests, however, visual acuity and contrast sensitivity decreased significantly between the 12th and 20th weeks, indicating that RGC loss might continue for more than 12 weeks. No further decrease in visual acuity and contrast sensitivity was observed 20 weeks after the insult, indicating the system finally reached a steady state. This slower phase of RGC loss can be effectively prevented by AAV-mediated BDNF expression. In the groups treated with gene therapy, no RGC loss or deterioration in the VEP amplitude or latency was observed 6 weeks after the insult, nor was there any reduction in visual acuity or contrast sensitivity from the 12th week to the 30th week after insult. The small reduction subsequently seen in visual acuity and contrast sensitivity is probably related to aging because similar reductions were also seen in the control group. Although we cannot separate the loss of RGCs resulting from ischemic or nonischemic causes, the results indicated that the damage resulting from acutely elevated IOP was alleviated. In the case of chronic glaucoma, the loss of RGCs is much more gradual; therefore, the slow onset of AAV-mediated gene expression is less a limitation than in this acute model. We therefore anticipate that gene therapy alone should be sufficient to achieve significant long-term rescue of RGCs in the chronic glaucoma model, in addition to the short-term rescue reported in a previous study. 13  
The rapid loss of RGCs in this ischemic model required immediate treatment with a protective agent, and the application of recombinant BDNF protein produced remarkable rescue with up to 87% of RGCs present at the end of the third week compared to 33% in the AAV-GFP group and 41% in the AAV-BDNF group. Without gene therapy, the number of RGCs decreased continuously, as did contrast sensitivity. These results indicated that the effective duration of exogenous BDNF action is limited. One earlier study 45 indicated that the effective duration of BDNF protein might be not much longer than 2 days: only slightly more RGCs survived in a group receiving BDNF injections 2 days before and 4 days after an ischemic insult than in a group receiving a single BDNF treatment 3 days after. This conclusion is further supported by the results in another group receiving BDNF 2 days before and 5 days after ischemic insult. Almost no rescue was seen in this group. Therefore, BDNF given 2 days before insult might be only slightly effective, though it has been reported that the protective effect of BDNF could be sustained for approximately 1 week. 15,46 In contrast, BDNF recombinant protein given 6 hours after insult was extremely effective in rescuing RGCs in the present study. When combined with gene therapy, no reduction was seen relative to the application of BDNF protein alone, indicating that AAV-mediated BDNF expression began to rescue RGCs before or shortly after the protective effects of the recombinant protein had ceased. 
Using a combination of FG and DiI, it has been shown that the sequence of changes in RGC degeneration was disruption of axonal transport, disconnection from targets, and loss of RGCs. 47 Our results showed that without treatment, 61% of RGCs with functional axons survived from the end of the third week to the end of the ninth week. With AAV-BDNF, 91.3% survived, indicating that in the presence of BDNF, either few functional axons were disconnected or transport was restored in some axons that were not functional at the end of the third week. With BDNF+AAV-BDNF, the number increased to 97.4%. 
In many diseases affecting RGCs, axon transport seems to be one of the most vulnerable processes. Disruption of axon transport has been reported in glaucoma (DBA mice), 47,48 diabetic retinopathy, 4 age-related macular degeneration, 49 and the ischemic model we used in this study. We showed that BDNF activated TrkB receptors through autocrine and paracrine mechanisms, likely improved axon transport and promoted the survival of RGCs, and achieved long-term rescue of visual function. It has been reported that BDNF rescues RGCs by binding the TrkB receptor to activate mitogen-activated protein kinase. 50,51 The ERK pathway might be involved in maintaining mitochondrial membrane potential 52,53 and ATP production, 52 which provides energy for axon transport. One study 54 reported that the application of BDNF downregulated TrkB expression. However, the amount of BDNF used was large, exceeding the amount showing protective effects in one of their earlier reports, 15 which described a dose-dependent manner of BDNF-induced protection in the optic nerve crush model, corresponding with the result of an earlier in vitro study. 55 The amount of BDNF used in this study is 0.5 μg (approximately 0.01 μg/μL vitreal volume), consistent with the doses that promoted the survival of RGCs after optic nerve crush. 15 If BDNF promotes survival by restoring axonal transport, it may have more general applications in other optic neuropathies 4,47 49,56 that involve the disruption of axonal transport. Even in stroke, ischemic insults frequently take place. 57,58 It is conceivable that axonal transport in affected neurons is also disrupted; restoring axonal transport may promote survival and functional rescue in many conditions. 
Supplementary Materials
Text s1, DOC - Text s1, DOC 
Footnotes
 Supported by Natural Science Foundation of China Key Project Grant 30530280, Natural Science Foundation of China-National Research Foundation of Korea Grant 30811140333, and National Basic Research Program of China Grants 2007CB512208 and 2006CB911003 (SH). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Footnotes
 Disclosure: R. Ren, None; Y. Li, None; Z. Liu, None; K. Liu, None; S. He, None
The authors thank Sebastian Kügler (University of Göttingen) for the kind gift of the AAV-2 vector construct pSWEB; Juergen A. Kleinschmidt (German Cancer Research Center) for the kind gift of the adenovirus-independent helper plasmid pDP2; Peixin Zhu (Friedrich Miescher Institute for Biomedical Research) for help in packaging the AAV vector; and Ian Morgan for help with English grammar and style. 
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Figure 1.
 
Continuous loss of FG-positive RGCs after acute elevation of the IOP. (ad) RGCs retrogradely labeled with FG from the superior colliculus in the control and 3 and 7 days and 3 weeks after acute IOP elevation. (e, f) The number of FG-positive RGCs in the whole retina (e) and the central and peripheral retina (f). Small but significant reduction was still observable between the sixth to ninth weeks after the increase in IOP. The number of retinas is given in parentheses. Data are mean ± SEM. *P < 0.05. Scale bar, 50 μm.
Figure 1.
 
Continuous loss of FG-positive RGCs after acute elevation of the IOP. (ad) RGCs retrogradely labeled with FG from the superior colliculus in the control and 3 and 7 days and 3 weeks after acute IOP elevation. (e, f) The number of FG-positive RGCs in the whole retina (e) and the central and peripheral retina (f). Small but significant reduction was still observable between the sixth to ninth weeks after the increase in IOP. The number of retinas is given in parentheses. Data are mean ± SEM. *P < 0.05. Scale bar, 50 μm.
Figure 2.
 
Expression of exogenous genes. (a) BDNF expression visualized using antibody against the His-tag. (b) RGCs retrogradely labeled with FG from the SC. Arrows: untransfected RGCs (FG-positive/BDNF-negative); arrowheads: transfected amacrine cells (FG-negative/BDNF-positive). (ce) Immunohistochemical staining of GFP showing an increasing number of cells expressing the exogenous gene from 3 days to 3 weeks after transfection. Scale bar, 25 μm. (f) Population data as shown in (c) to (e). Data are mean ± SEM, with six retinas in each group. *P < 0.05. (g, h) Western blot analysis showing the expression level of GFP and BDNF 1, 3, and 6 weeks after transfection. (i, j) Quantitative data from (g) and (h). Relative intensity (%) means the grayscale ratio of BDNF or GFP to β-actin.
Figure 2.
 
Expression of exogenous genes. (a) BDNF expression visualized using antibody against the His-tag. (b) RGCs retrogradely labeled with FG from the SC. Arrows: untransfected RGCs (FG-positive/BDNF-negative); arrowheads: transfected amacrine cells (FG-negative/BDNF-positive). (ce) Immunohistochemical staining of GFP showing an increasing number of cells expressing the exogenous gene from 3 days to 3 weeks after transfection. Scale bar, 25 μm. (f) Population data as shown in (c) to (e). Data are mean ± SEM, with six retinas in each group. *P < 0.05. (g, h) Western blot analysis showing the expression level of GFP and BDNF 1, 3, and 6 weeks after transfection. (i, j) Quantitative data from (g) and (h). Relative intensity (%) means the grayscale ratio of BDNF or GFP to β-actin.
Figure 3.
 
Time of intervention. The number of transfected cells increases with the interval between the release of high IOP and the intraocular injection, reaching a plateau at approximately 6 hours. Data are mean ± SEM, with six retinas in each group. *P < 0.05.
Figure 3.
 
Time of intervention. The number of transfected cells increases with the interval between the release of high IOP and the intraocular injection, reaching a plateau at approximately 6 hours. Data are mean ± SEM, with six retinas in each group. *P < 0.05.
Figure 4.
 
BDNF significantly rescues RGCs after acute IOP elevation. After elevation of IOP, FG-positive cells exhibited a remarkable decrease. With AAV-BDNF, more FG-positive cells were present 3 weeks after IOP elevation. The number of cells remained unchanged 6 weeks later. When BDNF protein was administered alone or with AAV-GFP, similar results were obtained. A dramatic increase in FG-positive cells was observed, but a reduction in FG-positive cells was also clear from the end of the third week to the end of ninth week in these groups. When recombinant BDNF protein was given together with AAV-BDNF, significantly more FG-positive cells were present at the end of the third week, and no reduction was observed until the ninth week. The number of animals tested is shown in the middle of each bar. Data are the mean ± SEM. *P < 0.05.
Figure 4.
 
BDNF significantly rescues RGCs after acute IOP elevation. After elevation of IOP, FG-positive cells exhibited a remarkable decrease. With AAV-BDNF, more FG-positive cells were present 3 weeks after IOP elevation. The number of cells remained unchanged 6 weeks later. When BDNF protein was administered alone or with AAV-GFP, similar results were obtained. A dramatic increase in FG-positive cells was observed, but a reduction in FG-positive cells was also clear from the end of the third week to the end of ninth week in these groups. When recombinant BDNF protein was given together with AAV-BDNF, significantly more FG-positive cells were present at the end of the third week, and no reduction was observed until the ninth week. The number of animals tested is shown in the middle of each bar. Data are the mean ± SEM. *P < 0.05.
Figure 5.
 
Visual function. (a) Recording traces of the VEP, from the control animals and animals receiving elevated IOP and treated with AAV-GFP and AAV-BDNF for 3 weeks, respectively. (b) The amplitude of the P1 component significantly decreased after transient elevation of the IOP. Across all time points examined, the amplitude of the VEP in animals receiving AAV-BDNF was much larger than in animals receiving AAV-GFP. When BDNF protein was administered, the amplitude of the P1 component was further increased in all three groups. In the BDNF alone group and the BDNF+AAV-GFP group, the amplitude clearly decreased from the end of the third week to the end of the sixth week. No further reduction was observed at the end of the ninth week. In the BDNF+AAV-BDNF group, no reduction was observed, even at the end of the ninth week. (c) The latency of the P1 component significantly increased 3, 6, and 9 weeks after IOP elevation. However, the group treated with AAV-BDNF showed significantly shorter latency than the AAV-GFP group. In the BDNF alone and the BDNF+AAV-GFP group, latency increased between the third and the sixth weeks. No increase was detected in the BDNF+AAV-BDNF group. The number of animals tested is shown in the middle of each bar. Data in (b) and (c) are mean ± SEM. *P < 0.05. (d, e) Visual acuity and contract sensitivity in different groups. The value displayed in (d) is the highest spatial frequency at which >70% correct choices were made. The number of animals tested is shown in the middle of each bar. Data are mean ± SEM. *P < 0.05. (f) Visual acuity at 52 weeks after insult. There was no difference between the untreated group and the AAV-GFP group, or the BDNF alone and the BDNF+AAV-GFP groups. Clear differences were seen between groups treated by AAV-BDNF, BDNF alone, and BDNF+AAV-BDNF. Data are mean ± SEM. The number of animals tested is indicated in parentheses. (g) Comparison of visual acuity of the control, untreated, and BDNF+AAV-BDNF–treated animals at 12, 20, and 52 weeks after insult. Data are mean ± SEM. Data for the intact control group were collected at 12 and 52 weeks.
Figure 5.
 
Visual function. (a) Recording traces of the VEP, from the control animals and animals receiving elevated IOP and treated with AAV-GFP and AAV-BDNF for 3 weeks, respectively. (b) The amplitude of the P1 component significantly decreased after transient elevation of the IOP. Across all time points examined, the amplitude of the VEP in animals receiving AAV-BDNF was much larger than in animals receiving AAV-GFP. When BDNF protein was administered, the amplitude of the P1 component was further increased in all three groups. In the BDNF alone group and the BDNF+AAV-GFP group, the amplitude clearly decreased from the end of the third week to the end of the sixth week. No further reduction was observed at the end of the ninth week. In the BDNF+AAV-BDNF group, no reduction was observed, even at the end of the ninth week. (c) The latency of the P1 component significantly increased 3, 6, and 9 weeks after IOP elevation. However, the group treated with AAV-BDNF showed significantly shorter latency than the AAV-GFP group. In the BDNF alone and the BDNF+AAV-GFP group, latency increased between the third and the sixth weeks. No increase was detected in the BDNF+AAV-BDNF group. The number of animals tested is shown in the middle of each bar. Data in (b) and (c) are mean ± SEM. *P < 0.05. (d, e) Visual acuity and contract sensitivity in different groups. The value displayed in (d) is the highest spatial frequency at which >70% correct choices were made. The number of animals tested is shown in the middle of each bar. Data are mean ± SEM. *P < 0.05. (f) Visual acuity at 52 weeks after insult. There was no difference between the untreated group and the AAV-GFP group, or the BDNF alone and the BDNF+AAV-GFP groups. Clear differences were seen between groups treated by AAV-BDNF, BDNF alone, and BDNF+AAV-BDNF. Data are mean ± SEM. The number of animals tested is indicated in parentheses. (g) Comparison of visual acuity of the control, untreated, and BDNF+AAV-BDNF–treated animals at 12, 20, and 52 weeks after insult. Data are mean ± SEM. Data for the intact control group were collected at 12 and 52 weeks.
Figure 6.
 
Autocrine and paracrine effects through TrkB receptors. (a) Comparison of cells positive for FG and BDNF/GFP. For directly transfected RGCs, significant rescue was obvious. (b) Comparison of cells positive for FG but negative for BDNF/GFP. Untransfected RGCs residing in retinas treated with AAV-BDNF were significantly rescued compared with untransfected RGCs residing in the retinas treated with AA-GFP. (c, d) TrkB receptor antagonist K252a blocked the rescue effects; 15 μM was needed to block the rescue effects of directly transfected RGCs, whereas 10 μM was sufficient to block the rescue effects of untransfected RGCs. Data are mean ± SEM, with six retinas in each group. *P < 0.05.
Figure 6.
 
Autocrine and paracrine effects through TrkB receptors. (a) Comparison of cells positive for FG and BDNF/GFP. For directly transfected RGCs, significant rescue was obvious. (b) Comparison of cells positive for FG but negative for BDNF/GFP. Untransfected RGCs residing in retinas treated with AAV-BDNF were significantly rescued compared with untransfected RGCs residing in the retinas treated with AA-GFP. (c, d) TrkB receptor antagonist K252a blocked the rescue effects; 15 μM was needed to block the rescue effects of directly transfected RGCs, whereas 10 μM was sufficient to block the rescue effects of untransfected RGCs. Data are mean ± SEM, with six retinas in each group. *P < 0.05.
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