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(Investigative Ophthalmology and Visual Science. 2002;43:2401-2405.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Rescue of Axotomized Retinal Ganglion Cells by BDNF Gene Electroporation in Adult Rats

Xiaofen Mo1, Akiko Yokoyama1, Toshiyuki Oshitari1, Hisanari Negishi1, Mari Dezawa2, Atsushi Mizota1 and Emiko Adachi-Usami1

1 From the Department of Ophthalmology and Visual Science, Graduate School of Medicine, Chiba University, Chiba, Japan; the 2 Department of Anatomy, Yokohama City University School of Medicine, Yokohama, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine whether the brain-derived neurotrophic factor (BDNF) gene can be transfected into retinal ganglion cells (RGCs) by electroporation and whether axotomized RGCs can be rescued after transfection by BDNF in adult rats.

METHODS. Mouse BDNF cDNA was injected intravitreally followed by in vivo electroporation in adult rats. The expression of BDNF in RGCs was confirmed by Western immunoblot analysis and immunohistochemistry. After introduction of BDNF cDNA, the survival of axotomized RGCs was estimated by the TdT-dUTP terminal nick-end labeling (TUNEL) method and measured by counting the number of RGCs that were labeled retrogradely by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyamine percholorate (diI) applied to the superior colliculus (SC).

RESULTS. Eyes with injection of the BDNF gene followed by in vivo electroporation showed a significantly higher level of expression of BDNF in the RGC layer, a higher rescue ratio, and a lower number of TUNEL-positive cells than the control samples.

CONCLUSIONS. These findings demonstrate that electroporation is an effective method for the direct delivery of genes into RGCs, and that the BDNF gene transferred into RGCs by in vivo electroporation can protect axotomized RGCs against apoptosis.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Injury to the optic nerve (ON) causes rapid degeneration of the axons and more than 90% of the retinal ganglion cell (RGCs) die by apoptosis by 2 weeks.1 2 Attempts have been made to rescue the axotomized RGCs by changing the environment or by upregulating intrinsic growth factors surrounding the RGCs.3

Among the different factors that have been shown to enhance the survival of RGCs during the early stage of injury, brain-derived neurotrophic factor (BDNF)1 2 4 5 6 7 appears to have interesting potential. Mansour-Robaey et al.1 reported that an intravitreal injection of BDNF protein can rescue axotomized RGCs with a survival percentage (SP) of 42% at 2 weeks and 15% at 4 weeks, and repeated injections increase the percentage rescued to 28% at 4 weeks. Unfortunately, the retinal tissue was damaged by the repeated injections of BDNF.

To overcome the damage induced by repeated injections, it has been suggested that BDNF could be delivered by use of gene therapy techniques. For example, Castillo et al.8 genetically modified astrocytes to secrete BDNF and showed a significant 15-fold increase in the survival of cultured RGCs by coculturing the astrocytes and RGCs. Different techniques, such as in vivo viral delivery systems9 10 and lipofection,11 12 have been used for the direct transfer of genes into retinal cells. For example, an intravitreal injection of adenovirus containing the BDNF gene temporarily extended the survival of axotomized RGCs of adult rats by the mediation of transfected Müller cells.9

We devised an in vivo electroporation method and showed that a green fluorescent protein gene can be delivered into RGCs without detectable damage to the RGCs.13 The results indicate that in vivo electroporation is a practical method of delivering transgenes into RGCs. In the current study, we applied in vivo electroporation to transfect RGCs with BDNF cDNA, and examined whether the presence of BDNF cDNA would rescue axotomized RGCs.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Nine-week-old male rats (Wister strain; weight 200–250 g) were housed in a temperature-controlled room. The animals were kept on a 12-hour light–dark schedule and had free access to food and water. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

BDNF Plasmid Preparation and Electroporation
The expressing plasmid vector pRc/CMV, carrying the mouse BDNF cDNA in the XbaI site, was provided by Yves-Alain Barde (Max-Planck Institute of Neurobiology, Martinsried, Germany).14 15 The vector was transformed into Escherichia coli JM109 competent cells (Takara Shuzo, Kyoto, Japan) by heat shock, multiplied by culturing the host E. coli cells, and isolated with use of a kit (EndoFree Plasmid Maxi Kit; Qiagen, Santa Clarita, CA). The isolated host E. coli were suspended in 10 mM Tris-HCl buffer (pH 7.4) with 1 mM EDTA (TE buffer; Qiagen) at a concentration of 2.5 µg/µL, as determined by a spectrophotometer (model DU640; Beckman Instruments, Fullerton, CA). For confirmation, the product was digested by XbaI and electrophoresed on a 0.6% agarose gel before use. The BDNF gene was approximately 700 to 800 bp, as measured by a 100-bp DNA ladder (data not shown).

In rats under deep anesthesia (intraperitoneal injection of 500 mg urethane, 11 mg ketamine, and 14 mg xylazine per kilogram body weight), the left eye and ON were exposed. The intraocular pressure was first reduced by paracentesis. A small hole was made with a 31-gauge needle 0.5 to 1.0 mm posterior to the limbus, and while the tip of the needle was viewed as it entered the vitreous at a 40° to 50° angle through the dilated pupil, 10 µg plasmid DNA in 4 µL TE buffer was injected into the vitreous.16 17

The eye was then immediately grasped between forceps-type electrodes, with the cathode attached to the corneal electrode and the anode attached to the scleral electrode, as reported.13 17 Square-wave pulses of 99-ms duration were applied with five pulses of 12-V/cm field strength, administered twice with a 5-minute interval (Electro Square Porter T820; BTX, San Diego, CA). The rats receiving the BDNF gene by electroporation are referred to as the BDNF E(+) group. Rats injected with 4 µL phosphate-buffered saline (PBS) or plasmid pRc/CMV with electroporation but containing no BDNF cDNA are referred to as the PBS group and Plasmid group, respectively, and those receiving 4 µL of BDNF gene without electroporation are the BDNF E(-) group. Rats that did not receive any intravitreal injection served as the control and are referred to as the NO group.

Immunohistochemistry
To confirm the transfection of BDNF into RGC, immunohistochemistry was performed on the rats in the BDNF E(+) (n = 3), BDNF E(-) (n = 3), PBS (n = 3), and NO (n = 3) groups. All rats were killed by an intracardiac perfusion of 4% paraformaldehyde in 0.1 M PBS, while under deep ether anesthesia, 1 week after in vivo electroporation. The retina was removed from the left eye, and postfixed in the same fixative. Frozen retinal sections of 14-µm thickness (six sections for each retina in each group) were stained with an affinity-purified rabbit polyclonal antibody raised against amino acids 128-147 of human BDNF (N-20; 4 µg/mL; Santa Cruz Biotechnology, Santa Cruz, CA) as the primary antibody and the anti-rabbit IgG-fluorescein isothiocyanate (FITC) as the secondary antibody. The sections were then counterstained with propidium iodide (PI; Molecular Probes, Eugene, OR) to label all the nuclei and were examined with a fluorescence microscope (Carl Zeiss, Jena, Germany).

To quantify the BDNF expression, six sections for each retina and approximately 200 to 300 RGCs per section were counted. For analysis, photomicrographs were taken with the same magnification and exposure time for each slide. Positive signal intensity was measured with NIH Image digital analyzing software ((NIH Image is provided in the public domain by the National Institutes of Health, Bethesda, MD, and is available at http://rsb.info.nih.gov/nih-image/).

Western Blot Analyses
To further confirm the overexpression of BDNF in the retina, we dissected the left retinas (L group) with BDNF transfection and the contralateral control retinas (R group) 1 week after BDNF cDNA transfection and homogenized them by ultrasound in lysis buffer, as reported.18 After centrifugation, the protein concentration was determined by the Bradford method with the spectrophotometer (Beckman Instruments). Samples (100 µg retinal extract, for the L and R groups), human recombinant (hr)BDNF (75 ng; Diaclone Research, Besançon, France) and prestained standards (Kaleidoscope; Bio-Rad, Hercules, CA) were resolved on 10% to 20% (wt/vol) SDS-polyacrylamide gels (Tris-Glycine Ready Gels; Bio-Rad) and transferred to polyvinylidene difluoride membrane (PVDF, Immobilon-P; Millipore, Bedford, MA). Nonspecific protein-binding sites were blocked for 2 hours in blocking solution containing 5% dry milk and 0.1% Tween 20 in TBS at pH 7.6. Membranes were incubated for 16 to 18 hours at 4°C with the affinity-purified rabbit polyclonal anti-human BDNF (N-20; 2.5 µg/mL; Santa Cruz Biotechnology), and after three washes in TBS-T (0.1% Tween 20 in TBS), the membranes were incubated with secondary antibody (peroxidase labeled anti-rabbit antibody; Amersham Pharmacia Biotech, Piscataway, NJ) at a dilution of 1:500 in TBS-T for 1 hour and washed again in TBS-T. Detection was performed by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).

Transection of the ON
To induce apoptosis of RGCs, the ONs were transected as described earlier.2 The ON was exposed by making a longitudinal incision in the retractor muscle and perineurium, and was transected 1 mm posterior to the bulbar exit, with special care taken to avoid cutting the blood vessels. To ensure that the retinal circulation was not interrupted, the fundus was observed by indirect ophthalmoscopy through dilated pupils.

TUNEL Staining
A TUNEL assay was performed to detect in situ DNA fragmentation arising from apoptosis, necrosis, and autolytic cell death. However, most studies agree that the death of most RGCs after axotomy occurs by apoptosis.19 20 The apoptosis in RGCs after ON transection was measured in rats in the BDNF E(+), BDNF E(-), Plasmid, PBS, and NO groups. Five animals were used in each group. Seven days after the in vivo electroporation, the left ON was transected, and the rats were killed 7 days later.21 22 Three intact retinas were processed in the same way and examined as the negative control.

Five cryostat sections (9 µm) of each retina were stained with an in situ apoptosis detection kit (Trevigen, Gaithersburg, MD), and the labeled nuclei were detected by streptavidin-FITC. The sections were then counterstained with PI and examined under a fluorescence microscope. The number of TUNEL-positive cells among all the cells in the RGC layer was counted in 25 sections for each group at 2 mm from the optic disc. Approximately 5000 to 7500 RGCs were counted for each group, except in the intact group, in which 3000 to 4500 RGCs were counted.

Counting of Living RGCs after ON Transection
Seven microliters of the lipophilic tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyamine percholorate (diI; Molecular Probes, Eugene, OR) was placed on both superior colliculi for retrograde labeling of RGCs in all study groups,23 and the in vivo electroporation was performed on the same day.

One week later, the left ON was transected. Five rats were killed at 2, 4, and 6 weeks after the transection in each group. Wholemounted retinas were examined under a confocal laser microscope (Radians 2000; Bio-Rad, Hertfordshire, UK) within 3 hours of enucleation. Three selected areas (0.307 mm x 0.307 mm) at 2, 3, and 4 mm from the optic disc were photographed in the four retinal quadrants. The number of diI-labeled cells was counted in the photographs and the density of labeled RGCs/mm2 was calculated by averaging the 12 counts in each retina.

The Mann-Whitney test was used for statistical analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemistry
Photomicrographs of the ganglion cell layer (GCL) of rats in the BDNF E(+), BDNF E(-), PBS, and NO groups that were immunohistochemically stained for BDNF expression are shown in Figure 1 . BDNF-positive cells were stained green, and the nuclei of all cells were stained red by PI. In the BDNF E(+) group, cells in the GCL showed strong immunoreactivity (Fig. 1A) , but in the BDNF E(-) and PBS groups, only a trace of BDNF-positive cells were found. Statistical analysis showed that the mean number of BDNF-positive cells in the BDNF E(-) and PBS groups was not statistically different from the mean number in the NO group. The positively stained cells were observed mainly in the GCL, and occasionally in the inner border of the inner plexiform layer (IPL), which indicates that electroporation was selective in transfecting the RGCs.



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  		Figure 1. Cells with the BDNF protein appear green (FITC) and the nuclei in the RGCs red (PI), and double-stained cells yellow. One week after electroporation, the retina of the BDNF E(+) group (A) were strongly stained with antibody to BDNF, but in the control retinas—the BDNF E(-) (B), PBS (C), and NO (D) groups—BDNF staining was weak. Scale bar, 20 µm.

 
Western Blot Analyses
Immunoblots probed with anti-BDNF N-20 identified a 14-kDa protein in both the L and R groups, which had identical mobility with human recombinant BDNF (hrBDNF; Fig. 2 ). The signal intensity in the BDNF band, measured with NIH Image digital image-analysis software, showed that the treated retina had a statistically higher quantity of BDNF than the contralateral control retina. We thus concluded that BDNF had been overexpressed in the gene-transfected retina.



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  		Figure 2. Western immunoblot analysis of BDNF in solubilized extracts of left (BDNF transfected) and right (untreated) retina in rats. The left eyes were transfected with BDNF cDNA by electroporation. Bands at ~14-kDa correspond to hrBDNF and were detected with anti-BDNF N-20 in both the left and right retinal extracts. However, the signal intensity in the left group was significantly higher than in the right group.

 
TUNEL Staining
The mean percentage of TUNEL-positive to -negative cells in the RGC layer/mm2 7 days after ON transection was 25.52% ± 9.32% (mean ± SD) in the BDNF E(+) group, 86.92% ± 22.31% in the BDNF E(-) group, 89.20% ± 2% in the Plasmid group, 80.17% ± 14.49% in the PBS group, and 78.15% ± 27.16% in the NO group (Fig. 3) . The difference between BDNF E(+) and BDNF E(-) group was statistically significant (P < 0.01). Thus, the injection of the BDNF gene alone (BDNF E(-) group) had no effect in reducing apoptosis. Because no statistically significant difference was found between the PBS, BDNF E(-) and NO groups, we concluded that the electric pulses alone did not reduce the apoptosis. There was no statistically significant difference between the PBS, Plasmid, and NO groups, which showed that the vector alone also did not reduce the percentage of apoptotic cells.



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  		Figure 3. Percentage of TUNEL-positive cells to all cells in the GCL 7 days after ON transection. In the BDNF E(+) group, the percentage of TUNEL-positive cells was significantly lower than in the BDNF E(-), Plasmid, PBS, and NO groups (P < 0.01).

 
DiI Retrograde Labeling
The numbers of DiI-labeled RGCs at 2, 4, and 6 weeks after ON transection are shown in Table 1 (n = 5, at each time-point in each group). The mean number of labeled RGCs in the intact retinas was 2271 ± 241 cells/mm2 (mean ± SD). Two weeks after ON transection, the mean number of RGCs was significantly reduced to 114 ± 36 cells/mm2 for an SP of 5.01% ± 1.57% in the PBS group and 142 ± 72 cells/mm2 (SP, 6.34% ± 3.16%) in the BDNF E(-) group (Table 1) . However, the number was 1429 ± 638 cells/mm2 and the SP was 62.93% ± 28.10% in the BDNF E(+) group at 2 weeks. This is significantly higher than that in PBS and BDNF E(-) groups (P < 0.01).


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  		Table 1. Number of RGCs/mm2 and Survival Ratio 2, 4, and 6 Weeks after ON Transection

 
At 4 weeks after ON transection, the SP was reduced in all groups, but the BDNF E(+) group maintained a labeled-cell density of 1169 ± 415/mm2, with a significantly higher SP of 51.5% ± 18.3%, compared with the BDNF E(-) (0.4 ± 0.5%), PBS (0.2% ± 0.1%), and Plasmid (0.6% ± 0.3%) groups. At 6 weeks after ON transection, there were few RGCs labeled in the three control groups, whereas in the BDNF E(+) group, there were 879 ± 436/mm2 RGCs labeled with an SP of 38.7% ± 19.2%.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results demonstrated that the transfection of BDNF cDNA into RGCs can promote the survival of RGCs that have been axotomized. These observations are similar to the rescue effect of RGCs that have undergone ischemic injury.1 2 4 6 7 BDNF mRNA and the trkB receptor mRNA have been reported to be expressed and colocalized in the RGC layer.24 25 With blockage of the main retrograde uptake pathway by axotomy, the trkB-mediated response26 to RGC-expressed BDNF, acting in an autocrine fashion, is consistent with our finding of a neurotrophic effect of the overexpression of BDNF protein in the GCL.

The results of the immunohistochemical study showed that there was strong staining for the BDNF protein only in the BDNF E(+) group, suggesting that the BDNF gene enters the RGCs with the assistance of electroporation. The data from the Western immunoblot confirmed the observation of overexpression of BDNF in BDNF cDNA-transfected retinas. Compared with the PBS and the BDNF E(-) groups, only the BDNF E(+) group showed a significantly higher SP and number of RGCs and a significantly lower percentage of apoptotic cells after ON transection. These results indicate that electroporation alone and intravitreal injection of the BDNF gene alone were not sufficient for the rescue of RGCs.

Electrical pulses of high voltage and short duration induce transient and reversible changes in cell membranes, so that they become permeable to a number of molecules.27 We suggest that this electropermeabilization28 is the best method for transfection of DNA into the targeted cells. In this experiment, the anodic electrode was positioned on the scleral side and the cathodic electrode on the corneal side, and the negatively charged BDNF cDNA injected into the vitreous migrated to the anode during the application of the electrical current. Such a directional force is expected to increase the DNA’s delivery into the RGCs. Because the BDNF E(-) group did not show strong staining of BDNF protein compared with the PBS group and showed no protective effect on the axotomized RGCs, only a limited number of BDNF genes entered the RGCs without the electric current. The fact that the percentage of TUNEL-positive cells in the PBS group did not differ significantly from the transection-alone group indicates that the electric current alone did not affect the RGCs.

The survival effect produced by the techniques used in this study with the BDNF gene suggests that these methods may be useful for treating diseases involving the RGCs.


   Acknowledgements
 
The authors thank Duco I. Hamasaki (Bascom Palmer Eye Institute, Miami, Florida) for editing assistance and Jun Nomura (Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University) for technical direction.


   Footnotes
 
Supported by the Ministry of Education, Science, Sports, and Culture of Japan (NO.10357015), Strategic System for Brain Science from the Ministry of Science and Technology, and the Study Group of Chorio-retinal Degeneration from the Ministry of Health, Labor and Welfare of the Japanese Government.

Submitted for publication August 13, 2001; revised January 22, 2002; accepted February 11, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Xiaofen Mo, Department of Ophthalmology and Visual Science, Graduate School of Medicine, Chiba University, Inohana 1-8-1 Chuo-ku, Chiba 260-8670, Japan; xfmo{at}hotmail.com.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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