Anti-inflammatory effect of pigment epithelium-derived factor in DBA/2J mice

Animals

The DBA/2J and C57BL/6J mice used in this study were purchased from Jackson Laboratory (Bar Harbor, ME). All animals were born and raised in a 12 h on versus 12 h off cyclic light environment at an illumination of 50–60 lx. Animals were cared for and handled according to the Association for Research in Vision and Ophthalmology statement for the use of animals in vision and ophthalmic research and with approval of the University of Oklahoma Health Sciences Center (Oklahoma City, OK) IACUC.

Identification of retinal ganglion cells by retrograde labeling

The neuronal tracer 4-di-10-ASP, a long-chain dialkylcarbocyanine, is widely used to label living tissues, and with injection of 4-di-10-ASP into the superior colliculus, will label RGC by retrograde axoplasmic transport [24]. It has been shown that 7 days after injection of 4-di-10-ASP, more than 80% of the RGC are labeled [25]. We used 4-di-10-ASP (Molecular Probes, Eugene, OR) to label RGC as previously described [21]. Briefly, 4 weeks-old mice were anesthetized by intraperitoneal administration of a mixture of xylazine and ketamine. The skin over the cranium was incised, and the skull was exposed. Holes ~1 mm in diameter were drilled in the skull 4 mm posterior to the bregma and 1 mm lateral to the midline on both sides of the midline raphe. These positions correspond to the superior colliculi as determined from a stereotactic mouse brain atlas. Retinal ganglion cells project to the superior colliculus and dorsal lateral geniculate nucleus. 4-di-10-ASP (3 μl of a 25 mg/ml concentration) was injected through both holes in the superior colliculi region by inserting a syringe 2 mm deep. The skull openings were then sealed with a petroleum-based antibiotic ointment. The overlying skin was sutured and antibiotic ointment was applied externally. The mice were euthanized by CO₂ inhalation at 1, 6, and 15 months following 4-di-10-ASP administration. Whole retina flat mounts were prepared by detachment of the retina at the ora serrata followed by eight radial relaxing incisions. Imaging of the labeled retinas was performed using a digital camera and a fluorescence microscope (Nikon Instruments, Inc., Melville, NY) at 20X magnification.

Measurement of visual acuity

The Virtual Optomotor System (OptoMotry; CerebralMechanics, Lethbride, Alberta, Canada) was used to measure visual acuity as an index of visual function, as previously shown [26], at 1 to 11 months of age. The grating spatial acuity was measured starting with a low spatial frequency (0.1 cyc/deg) sine wave grating at the same mean luminance and 100% contrast. The spatial frequency of the grating was incrementally increased until the animal failed to respond. Each test was performed at least 5 times. The threshold was identified as the highest spatial frequency that mice could track.

Preparation and intravitreous delivery of adeno-associated virus expressing human PEDF

Adeno-associated virus expressing human PEDF (AAV-PEDF) has been used to protect neurons in animal models [27-29]. AAV-PEDF was constructed by cloning a full-length human PEDF cDNA under the control of the CMV promoter. The construct sequence was confirmed by DNA sequencing (Oklahoma Medical Research Foundation, Oklahoma City, OK). A control virus containing a green fluorescent protein cDNA under the same promoter (AAV-GFP) was obtained from a commercial source (InVivoGen, San Diego, CA). AAV-PEDF and AAV-GFP vector titers were 2.0×10¹³ particles/ml, and 2.5×10¹² particles/ml, respectively. Intravitreal injection was performed under general anesthesia after intramuscular injection of ketamine (85 mg/kg) and xylazine (14 mg/kg). Anesthetic drops (0.5% proparacaine hydrochloride; Alcon, Fort Worth, TX) were also applied topically to each eye before injections. A 30 gauge hypodermic needle was used to perforate the sclera 1 mm behind the limbus. AAV-PEDF (1 μl) was then injected into the left vitreous and AAV-GFP (1 μl) was injected into the right vitreous using a 10 μl Hamilton Syringe (Hamilton Co, Reno, NV) under an ophthalmic operating microscope (Carl Zeiss Meditec, Inc., Thornwood, NY). Care was taken not to damage the lens. Following intraocular injections, the needle was held in place for one min and withdrawn slowly. Animals with retinal bleeding or lens injury following the injection procedure were excluded from the study. Animals were euthanized nine months after the injection, and eyes rapidly enucleated. Retinas were dissected free from the choroid and sclera, and whole mounted on a glass slide. They were fixed briefly in 4% (w/v) paraformaldehyde in PBS. Whole retina was mounted with Vectorshield mounting medium (Vector Laboratories, Inc., Burlingame, CA).

Semiquantitative reverse transcription (RT)-PCR

Reverse transcription RT–PCR was performed as described previously [30,31]. For RT–PCR, 4 µg RNA was mixed in water with random hexamers (50 ng/µl) and 10 mM dNTPs heated to 65 °C for 5 min. The 20 µl RT reaction, containing RNA, primers, 5 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM dNTPs, 40 units of RNase inhibitor (RNase OUT), and 50 units of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), was assembled, heated at 42 °C for 1 h and then at 70 °C for 15 min. After chilling on ice, 2 units of RNase H were added to each reaction and the mixture was incubated at 37 °C for 20 min. PCR was performed in 50 µl reaction volumes, containing 2 µl of cDNA, 2 units of TaqDNA polymerase (Promega, Madison, WI), 0.2 mM dNTPs, 10 µM each of forward and reverse primers, and 1.5 mM MgCl₂. Amplification cycles were: one cycle at 94 °C for 3 min, followed by 35 cycles of 94 °C for 20 s, 58 °C for 45 s, and 72 °C for 1 min, terminating with 72 °C for 10 min. The amplification products were run on 1% agarose gel containing 10ng/ml ethidium bromides and visualized under UV light. PEDF, TNF, and IL-18 gene expression were examined by semi-quantative RT–PCR using the following primers: PEDF (AF017057) Forward: TGA TCA CCA ACC CTG ACA TCC ACA, Reverse: GCT GGG CAA TCT TGC AGT TGA GAT; TNF (NM_013693) Forward: ACA CCG TCA GCC GAT TTG CTA TCT, Reverse: TGG ACA TTC GAG GCT CCA GTG AAT; IL-18 (NM_008360) Forward: GAC AAC TTT GGC CGA CTT CAC TGT, Reverse: CAC AAG GCG CAT GTG TGC TAA TCA.

Western blot analysis

Western blot analysis was performed as described previously [32,33]. Retinal tissues were sonicated in 0.0625 M Tris-HCl, pH 6.8, then centrifuged for 15 min and the supernatants assayed for protein using a Bradford assay. Aliquots (10 µg) of the sonicated supernatant were loaded onto SDS–PAGE mini-gels, electrophoresed, and transferred to nitrocellulose paper. After transferring, blots were washed for 2X 10 min in TTBS (0.1% Tween-20 in 20 mM Tris-HCl, pH 7.4, and 410 mM NaCl) and blocked with 10% BSA in TTBS with 5% milk for 2 h at room temperature or overnight at 4 °C. Blots were incubated with rabbit anti-IL-18 polyclonal antibody (Millipore, Billerica, MA), rabbit anti-TNF polyclonal antibody (Millipore, Billerica, MA), rabbit anti-PEDF polyclonal antibody (Santa cruz, Delaware, CA), or rabbit anti-GFAP polyclonal antibody (Abcam, Cambridge, MA) for 2 h at room temperature. Following primary antibody incubations, blots were washed three times for 5 min each with TTBS, then incubated for 1 h with HRP-linked secondary antibodies, washed four times for 10 min each with TTBS, and developed by enhanced chemiluminescence. In some instances, membranes were stripped by incubation in stripping buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol) for 30 min at 50 °C and reused.

Histopathology

Mouse eyes were enucleated, rinsed in PBS, and fixed with 10% neutral buffered formalin for 24 h at room temperature. After paraffin embedding, whole eyes were cut into 5 µm thick sections, mounted on positively charged slides and air dried overnight. After deparaffinization and rehydration, slides were stained with hematoxylin and eosin.

Immunohistochemistry

Immunohistochemistry staining was performed as described previously [34]. Briefly, Eyes were enucleated and then fixed with 4% paraformaldehyde in phosphate-buffered saline for 4 h. The whole eye was cut along the vertical meridian, and tissue sections incubated in primary antibodies. The secondary antibody (Vector Laboratories, Burlingame, CA) was labeled with fluorescein isothiocyanate. In control experiments, the primary antibody was incubated for 2 h at 4 °C with its blocking peptides before being applied to the section. Sections were photographed and analyzed with a confocal laser scanning microscope (IX81-FV500; Olympus, Melville, NY).

Statistical analysis

Each experiment was performed at least three times. Mean values were calculated and expressed as means±SD. Statistical analyses were performed using Student's t-test, or ANOVA with the Scheffé multiple comparison test. A statistical difference was considered significant at p<0.05.

Loss of retinal ganglion cells in eyes of DBA/2J mice

RGC density was determined by counting 4-di-10-ASP-labeled RGC in flat whole-mount retinas. Significant loss of RGC was observed in flat whole-mount retina from DBA/2J mice at 6 months of age (Figure 1B) in comparison to age-matched control C57BL/6J mice (Figure 1D). This loss of RGC appeared to worsen at 15 months of age (Figure 1C). The morphological difference between ganglion cell layers in the retina of DBA/2J mice and C57BL/6J control animals at matched age was also compared by histology. Loss of RGC was apparent in DBA/2J mice at 6 months, and appeared to have dramatically worsened at 15 months of age (Figures 1F,G, respectively), as compared with retina from C57BL/6J mice (Figure 1H).

Reduction in PEDF gene and protein expression with age in eyes of DBA/2J mice

It was previously shown that PEDF levels in eyes with advanced glaucoma are significantly lower than in eyes with cataract alone. It has also been suggested that the lower levels of PEDF in eyes with neuroretinal dystrophy may be related to the loss of the retinal ganglion or pigment epithelial cells that synthesize PEDF [22]. In DBA/2J mice, semi-quantitative RT–PCR revealed significantly decreased retinal PEDF gene expression with age (Figure 2A,B). A decrease of PEDF protein expression with age in retina from DBA/2J mice was also detected by western blot; the expression of PEDF was significantly reduced at the age of 4 months, and this decrease continued to the age of 6 months (Figure 2C,D). Western blot analysis also showed a significant reduction of PEDF expression in the optic nerve of DBA/2J mice at 6 months of age (Figure 2E,F). Immunohistochemistry demonstrated substantial expression of PEDF in the RGC and retinal pigment epithelial (RPE) layers of DBA/2J mice by 1 month of age (Figure 3A). At 6 months of age, decreased PEDF expression in the RGC layer with residual staining in the RPE layer of DBA/2J mice (Figure 3B) was detected when compared with the age-matched C57BL/6J controls (Figure 3C).

Inflammatory markers in retina and the optic nerve of DBA/2J mice

TNF is an important inflammatory mediator and is expressed in the retina and optic nerve head of patients with glaucoma [15,16,35]. To investigate whether changes in TNF expression occur in DBA/2J mice, we isolated total RNA and protein from retina and optic nerve from 1 to 6 months old mice, and performed RT–PCR and ELISA, respectively. TNF gene and protein expression increased significantly by 3 months of age in both the retina and optic nerve of DBA/2J mice compared with control tissue from one month old mice (Figure 4A-D), and remained elevated through age 6 months. Significant increases in TNF protein expression was also seen in optic nerve of DBA/2J mice from 3 to 6 months of age (Figure 4E,F).

We have previously reported that IL-18 gene expression in the iris/ciliary body and IL-18 protein in the aqueous humor of DBA/2J mice are significantly increased, and further demonstrated that these increases occur before the development of elevated IOP and loss of RGC [21]. To determine whether IL-18 is upregulated in the retina and optic nerve of DBA/2J mice, we performed semi-quantitative RT–PCR and western blot. mRNA for IL-18 was elevated by 3 months of age, and increased almost twofold by 6 months of age as compared to IL-18 expression at 1 month of age (Figure 5A,B). By western blot analysis, IL-18 protein increased approximately twofold at 4–6 months of age (Figure 5C,D). Upregulation of IL-18 in the optic nerve of DBA/2J mice at 5 and 6 months of age was also found (Figure 5E,F).

Increased GFAP expression in retina and the optic nerve of DBA/2J mice

Glial cell activation has been proposed as an important factor contributing to RGC death in glaucoma [36,37]. Chronic IOP elevation results in significantly increased GFAP expression in the rat retina and optic nerve [38]. By immunohistochemistry in DBA/2J mice at 1 month of age, GFAP expression was mainly localized to the vicinity of the inner limiting membrane and nerve fiber layer (Figure 6A,B). However at 6 months of age, GFAP expression was significantly increased in both retina (Figure 6C) and optic nerve (Figure 6D). Western blot analysis demonstrated significantly increased GFAP protein in retina and optic nerve of DBA/2J mice by 3 months of age (Figures 6E-H).

Ocular PEDF gene transfection in DBA/2J mice leads to increased intraocular PEDF expression, improved retinal ganglion cell survival, and better vision

In situ expression of GFP can be observed from cross-section or whole-mount techniques, whereas the expression of PEDF is detected using western blot. Injections of AAV vectors encoding GFP or PEDF were performed in mice at 2 months of age. As demonstrated by assessment of GFP expression in retinal whole mount and cross section (Figures 7A,B, respectively) at 11 months of age (9 months after transfection), the retina can be successfully transduced by intravitreal injection. Transfection of AAV-PEDF resulted in a significant increase in PEDF protein level in retina from DBA/2J mice at 11 months of age (Figure 7C). The long-term effect of PEDF gene transfer on RGC survival was also examined. AAV-PEDF transfected eyes appeared to retain more RGC at 11 months of age (Figure 7D) than age-matched AAV-GFP transfected eyes (Figure 7E). As an index of visual function, we compared optomotor acuity in AAV-GFP, AAV-PEDF, and control transfected mice from 1 to 11 months of age (until 9 months post transfection; Figure 7F). Through 3 months of age, average acuity was approximately 0.33 cyc/deg in all 3 groups. In AAV-GFP transfected and control DBA/2J mice, acuity thresholds decreased rapidly thereafter, dropping to near 0.02 cyc/deg at 11 months of age. In contrast, AAV-PEDF transfected mice maintained near normal acuity throughout the study (Figure 7F).

Ocular PEDF transfection reduces TNF and IL-18 expression in retina and optic nerve of DBA/2J mice

To explore a possible link to inflammation in understanding the salutary effect of PEDF on morphology and visual function in DBA/2J mice, we next determined levels of TNF and IL-18 in retina and optic nerve after AAV-PEDF transfection. For these experiments, eyes were analyzed at 6 months of age (4 months after transfection). By RT–PCR, mRNA and protein expression of both TNF and IL-18 were significantly reduced in the retina and optic nerve of AAV-PEDF transfected mice as compared to AAV-GFP mice (Figure 8A-F), suggesting an anti-inflammatory effect of PEDF in the DBA/2J mice.

PEDF inhibits GFAP overexpression in retina and optic nerve of DBA/2J mice

GFAP expression has been implicated in the pathogenesis of glaucoma, and shown to increase with progression of glaucoma [39]. We examined the effects of PEDF transfection on GFAP expression in retina (Figures 9A,C) and optic nerve (Figures 9B,C) of DBA/2J mice at 6 months of age. As demonstrated by western blot and subsequent densitometry, GFAP protein expression in retina and optic nerve of PEDF transfected DBA/2J mice was significantly reduced compared to that of GFP transfected mice.