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BDNF Diminishes Caspase-2 but Not c-Jun Immunoreactivity of Neurons in Retinal Ganglion Cell Layer after Transient Ischemia

¹ From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto; and ² Sumitomo Pharmaceuticals Research Center, Osaka, Japan.

	Abstract

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PURPOSE. Retinal ischemia-reperfusion injury induces apoptosis of retinal neurons. The purpose of this study was to examine the association of c-Jun, caspase-1, -2, and -3 immunoreactivities and neuronal apoptosis in the retinal ganglion cell layer (GCL) and to study the effects of intravitreal brain-derived neurotrophic factor (BDNF) on the expression of these gene products in a rat model of retinal ischemia-reperfusion injury.

METHODS. After 60 minutes of ischemia, eyes were enucleated after 3, 6, 12, 24, and 168 hours of reperfusion. The numbers of c-Jun-, caspase-1-, caspase-2-, caspase-3, and TdT-dUTP terminal nick-end labeling (TUNEL)–positive cells in the GCL were counted. Recombinant human BDNF (5 µg) or vehicle was injected intravitreally immediately after reperfusion. At 6, 24, and 168 hours, the numbers of immunoreactive cells in BDNF- and vehicle-treated groups were compared.

RESULTS. Expression of c-Jun and caspase-2 was found in dying cells in flat-mounted retinas. The numbers of caspase-1– and caspase-3–positive cells were fewer than c-Jun– or caspase-2–positive cells. Cell death in the retinal GCL was suppressed by an intravitreal injection of BDNF. The numbers of TUNEL- and caspase-2–positive cells were lower in the BDNF-treated group at 6 hours after reperfusion (P < 0.01). The number of c-Jun–positive cells in the treated retinas was not altered by the treatment.

CONCLUSIONS. Expression of c-Jun and caspase-2 is associated with neuronal cell apoptosis in the GCL. Suppression of caspase-2 expression may explain the neuroprotective effects of BDNF.

	Introduction

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Brain-derived neurotrophic factor (BDNF) is a neurotrophin that suppresses neuronal death from various injuries. Although little is known about how BDNF protects neurons from such injuries, it is likely that it modifies some mechanism(s) in the pathway of cell death. Retinal ganglion cell (RGC) death occurs in several ocular and systemic diseases including glaucoma, ischemic optic neuropathy, and Alzheimer’s disease. The RGC death in these diseases has been shown to be by apoptosis.¹ In neuronal apoptosis, the caspase family proteases play an important role in the execution phase. Although BDNF is known to suppress RGC death, it is not clear how BDNF prevents apoptosis and whether BDNF suppresses the activation of caspase family proteases.

To investigate the mechanism of RGC death, experiments have been carried out on the rat axotomy and on the retinal ischemia-reperfusion models. In these models, the expression of immediate early gene products such as c-Jun is found in dying cells.² We have shown previously that expression of cell cycle–related gene products such as c-Jun and cyclin D1 takes place in the dying neurons, especially those in the inner nuclear layer.³

The retinal ischemia-reperfusion model is known to induce apoptosis of cells in all layers of the retina, and we used this model to study the mechanism of cell death. We chose to study the expression of cell cycle–related gene products that have been shown to be associated with cell death and to study only cells (RGCs and amacrine) in the retinal ganglion cell layer (GCL). We shall show that cell death after ischemia-reperfusion injury in the retinal GCL is associated with the expression of c-Jun and caspase family proteases and describe the neuroprotective effects of BDNF.

	Materials and Methods

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Animal Ischemia Model
Adult female Sprague–Dawley rats (Japan SLC, Hamamatsu, Japan) weighing 160 to 180 g (n = 350) were used. The experiments were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Rats were anesthetized by intraperitoneal pentobarbital (50 mg/kg), and retinal ischemia was induced by cannulating the anterior chamber with a 27-gauge needle connected to a bag containing normal saline.⁴ The intraocular pressure was increased to 110 mm Hg for 60 minutes by elevating the bag. The temperature of the rats was kept at 37°C by a heating pad. Sham operations were performed without increasing the intraocular pressure.

Antibodies
The following antibodies were used: rabbit anti-mouse c-Jun/AP-1 (N) antibody raised against a peptide that corresponds to amino acids 91–105 mapping within the amino terminal domain; goat anti–caspase-1 (M-19) antibody against amino acids 276–294 mapping to the carboxyl terminus of the 20-kDa subunit of caspase-1 of mouse origin; goat anti–caspase-2(N-19) antibody against amino acids 3–21 mapping to the amino terminus of the precursor of caspase-2 of mouse origin; and goat anti–caspase-3 (L-18) antibody against amino acids 157–174 mapping to the carboxyl terminus of the 20-kDa subunit of the protease precursor of human origin. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Tissue Processing and Immunohistochemical Studies
At 3, 6, 12, 24, and 168 hours after reperfusion, rats were deeply anesthetized and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The eyes were enucleated, and the retinas were separated from the eyecups in plastic petri dishes containing PBS. The retinas were then fixed for 2 hours in 4% paraformaldehyde. After rinsing in PBS, retinas were incubated for 30 minutes at room temperature with 0.2% Triton-X in PBS in plastic petri dishes.

For the detection of caspase-1 and caspase-3 immunoreactivities, the retinas were incubated with NeuroPore (Trevigen, Gaithersburg, MD) for 30 minutes (caspase-1) or 3 hours (caspase-3) at room temperature. The retinas were rinsed with PBS and then incubated with rabbit anti-mouse c-Jun antibody (1 µg/ml), goat anti–caspase-1 antibody (20 µg/ml), goat anti–caspase-2 antibody (20 µg/ml), or goat anti–caspase-3 antibody (20 µg/ml) for 48 hours at 4°C. The working concentrations of antibodies were determined after preliminary experiments with various concentrations. The retinas were then rinsed with PBS and incubated with fluorescein isothiocyanate (FITC)–conjugated anti-rabbit or anti-goat IgG antibody for 75 minutes.

To count the numbers of cells in the retinal GCL, the retinas were stained with propidium iodide (PI, 20 µg/ml) for 10 minutes at room temperature, and the retinas were flatmounted vitreous side up on glass slides. The retinas in which the primary antibodies were omitted were used as negative controls. Positive controls were generated by using retinal sections or flatmounted retinas in which these proteins were known to be expressed.^{3 5}

TdT-dUTP Terminal Nick-End Labeling
After fixation, the retinas were subjected to three cycles of freezing and thawing and then incubated overnight with NeuroPore in plastic petri dishes. The DNA nick-end labeling was performed by a Mebstain apoptosis kit (MBL, Nagoya, Japan). The retinas were incubated with TdT and biotinylated dUTP in TdT buffer for 2 hours at 37°C, then rinsed with termination buffer containing 30 mM sodium chloride and 3 mM sodium citrate for 30 minutes at room temperature followed by rinsing with PBS. The retinas were incubated with avidin–FITC for 2 hours at 37°C and rinsed with PBS. After PI staining, the retinas were flatmounted on glass slides. Positive controls were generated by incubating the specimens with DNase1 in water (1 µg/ml, for 1 hour at 37°C) before incubation with TdT and biotinylated dUTP. The control eyes without ischemia-reperfusion injury were used as negative controls.

Quantitative Analysis
The flatmounted retinas were photographed with a scanning laser confocal microscope (model LSM 410; Zeiss, Oberkochen, Germany) using a green filter to detect FITC and a red filter for PI; the focus was in the retinal GCL plane. The numbers of FITC-labeled c-Jun– and caspase-positive cells, TdT-dUTP terminal nick-end labeling (TUNEL)–positive cells and PI-stained cells were counted in six areas (0.2 x 0.2 mm), 1 and 2 mm away from optic disc and every 30° of the circle in each quadrant. Thus, data from 24 areas from one eye were obtained. Regions with thick nerve fiber or blood vessels were avoided, and a more central or peripheral area was chosen. Of the PI-stained cells, white blood cells, which have fragmented nuclei, and red blood cells, which have oval-shaped and spindle-shaped endothelial cells, were excluded from cell counts. The cell count was done by two examiners in a masked fashion. Data are expressed as the number of cells per square millimeter at each time point, and results are expressed as mean ± SEM.

Retrograde Labeling of RGCs
To label the RGCs, rats were anesthetized and placed in a stereotaxic frame (Narishige, Tokyo, Japan). A part of the skull and cerebral cortex was removed to expose the superior colliculi bilaterally. Multiple injections of neurotracer, FluoSpheres, or 1% DiI (Molecular Probe, Eugene, OR) were made in different regions of each superior colliculus with a glass micropipette attached to a 1-µl Hamilton syringe, at a depth of 0.5 or 1.0 mm. Seven days later, ischemia-reperfusion injury was induced, and the eyes were treated as described for TUNEL and immunostaining.

Intravitreal Injection of Brain-Derived Neurotrophic Factor
Recombinant human brain-derived neurotrophic factor (BDNF; N-terminal methionine-free, 2.0 mg/ml in 10 mM sodium phosphate and 150 mM NaCl, pH 7.0) was obtained from Regeneron Pharmaceuticals (Tarrytown, NY).⁶ Immediately after reperfusion, 2.5 µl (5.0 µg) of BDNF was injected into the vitreous cavity with a 5-µl Hamilton microsyringe attached to a 30-gauge needle. In the control group, 2.5 µl of vehicle was injected. Care was taken not to injure the lens and retinal vessels, and eyes that exhibited any complications were excluded. At 6, 24, and 168 hours after reperfusion, rats were deeply anesthetized, and the eyes taken for immunohistochemical and TUNEL studies as described.

Statistical Analysis
Statistical analysis was done using a two-way ANOVA followed by Fisher’s post hoc test. P < 0.05 was considered statistically significant.

	Results

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Cell Numbers in the Retinal GCL
The number of cells in the retinal GCL decreased from 3250.2 ± 131.3 cells/mm² in sham-operated eyes (mean ± SEM, n = 5) to 2098.2 ± 45.3 at 6 hours, to 1858.5 ± 63.2 at 24 hours, and 1439.2 ± 50.8 at 168 hours after reperfusion (Fig. 1) .

View larger version (27K): [in this window] [in a new window]   Figure 1. Quantitative analysis of dying cells. (A) Total number of cells stained with PI (open columns) and TUNEL-positive cells (gray columns) in the retinal GCL. (B) The number of c-Jun– (open columns), caspase-1– (gray columns), caspase-2– (hatched columns), and caspase-3– (filled columns) positive cells at different times after reperfusion. Data are mean ± SEM (n = 5 for each time point).

View larger version (58K): [in this window] [in a new window]   Figure 2. (A through C) Immunohistochemistry of retrogradely labeled RGCs. (A) Red cells are RGCs labeled by FluoSpheres or DiI, and green cells are TUNEL-positive. (B) c-Jun immunoreactivity. (C) Caspase-2 immunoreactivity at 6 hours after reperfusion. Staining is seen on labeled RGCs (large arrows) and also on unlabeled cells (small arrows). Scale bar, 10 µm. (D through G) Magnified microphotographs of c-Jun and caspase-2 immunoreactivities at 6 hours after reperfusion. (D and F) PI staining. (E and G) Double staining with PI and anti–c-Jun (E) or caspase-2 (G). Red cells are PI stained, and green cellsare immunostained. c-Jun and caspase-2 immunoreactivities are seen in cells with condensed chromatin. Scale bar, 5 µm.

View larger version (43K): [in this window] [in a new window]   Figure 4. Quantitative analysis of dying cells. Open columns, buffer-treated control group; filled columns, BDNF-treated group. Results are mean ± SEM (n = 5) for each time point. Asterisks indicate a statistically significant difference between buffer-treated group and BDNF-treated group (*P < 0.05, **P < 0.01; two-way ANOVA followed by Fisher’s post hoc test). (A) Total number of cells in the retinal GCL stained with PI. (B) TUNEL-positive cells. (C) c-Jun–positive cells. (D) caspase-2–positive-cells.

View larger version (73K): [in this window] [in a new window]   Figure 3. Cells in the retinal GCL in a flatmounted retina stained with PI. (A) Sham operated followed by intravitreal injection of buffer. (B) Intravitreal injection of BDNF. (C through H) 6 hours after reperfusion. Green represents TUNEL staining (C and D), c-Jun immunostaining (E and F), and caspase-2 immunostaining (G and H). Colabeling of condensed PI and immunoreactivity yielded yellow color as found in (C) and (D). The number of c-Jun–positive cells is not different between the vehicle-treated control group (E) and BDNF-treated group (F). The number of TUNEL-positive or caspase-2–positive cells was decreased in the BDNF-treated group (D and H) compared with the buffer-treated group (C and G). (I and J) 168 hours after reperfusion. The number of total cells in the retinal GCL in BDNF-treated group (J) is larger than in the untreated group (I). Scale bar, 25 µm.

The number of c-Jun–positive cells in the control group was 226.5 ± 24.3 cells/mm² (n = 5) at 6 hours after reperfusion and decreased to 68.0 ± 14.3 at 24 hours (n = 5; Figs. 3 and 4 ). The number of c-Jun–positive cells in the BDNF-treated group was 224.8 ± 24.9 cells/mm² (n = 5) at 6 hours and 82.0 ± 18.1 at 24 hours (Figs. 3 and 4) . These values were not significantly different from those of the controls.

The number of caspase-2–positive cells in the vehicle control also showed a peak at 6 hours after reperfusion (244.6 ± 15.7 cells/mm² ; n = 5) and decreased to 57.8 ± 12.6 at 24 hours (Fig. 4) . In the BDNF-treated groups, there were fewer caspase-2–positive cells than in the vehicle group: 124.4 ± 35.4 cells/mm² (n = 5) at 6 hours and 35.4 ± 11.2 at 24 hours (Figs. 3 and 4) . The difference between the control and BDNF-treated groups at 6 hours was statistically significant (P < 0.01). In the quantitative study of caspase-1–positive and caspase-3–positive cells, there were no statistically significant differences between these groups and the BDNF-treated group (graphic data not shown).

	Discussion

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These results showed that c-Jun and the caspase family proteases, especially caspase-2, were expressed in cells in the retinal GCL that had morphologic and histochemical signs of dying cells (Figs. 2 and 3) . Although the observations made with the retrograde labeled retinas suggest that the dying cells were mainly RGCs, we cannot rule out amacrine cells as being part of this group. In any case, these observations suggest an association between the cell death and c-Jun and/or caspase-2 expression. This relationship agrees with the role played by c-Jun in cell death in the central and peripheral nervous systems and in the RGC death in the axotomy model.²

Among the caspase family of proteases, expression of caspase-2 was detected in the highest number of cells, with much fewer cells expressing caspase-1 and -3. There is good evidence that caspase-2 induces apoptosis,^{7 8} and more recently, an independent processing of caspase-2 and upregulation of caspase-3 activity have been shown in trophic factor–deprived PC12 cell death. This type of cell death required caspase-2 activation, and the upregulation of caspase-3–like activity was neither necessary nor sufficient to induce cell death.⁹ Our data are in keeping with these findings.

Expression of c-Jun and caspase-2 was already present at 3 hours after reperfusion (Fig. 1) , and their peak expression was seen at 6 hours. Thus, most of the c-Jun– and caspase-2–related cell death occurred within 24 hours after reperfusion, which also corresponds to the time course of TUNEL-positive cells (Fig. 1) . However, there were a number of TUNEL-positive cells that did not express c-Jun or caspase immunoreactivities. One possible explanation for this discrepancy is that the window for the expression of c-Jun and caspases is shorter than the window to detect dying cells by TUNEL staining. Another possible explanation is that cell death may occur independent of c-Jun expression, caspase expression, or both.

The protective effects of BDNF were evident as early as 6 hours after reperfusion (Fig. 4) . Intravitreal injection of BDNF decreased the number of TUNEL-positive cells in the GCL, but the number of c-Jun–positive cells remained unchanged. This suggests that BDNF does not influence the expression of c-Jun, which agrees with De Felipe and Hunt,¹⁰ who suggested that BDNF does not regulate c-Jun expression in damaged neurons. However, BDNF did suppress the expression of caspase-2, both directly and indirectly. Similar regulation of caspase-2 expression by nerve growth factor occurred in sympathetic neurons and PC12 cells.⁸

The relationship between c-Jun– and caspase-2–dependent pathways is not known: They may be distinct pathways or they may be linked. In this experiment, the number of caspase-2–positive cells was significantly decreased by BDNF but not the c-Jun–positive cells. These findings suggest two hypotheses: One is that c-Jun and caspase-2 are linked and BDNF works between the c-Jun and caspase-2 steps and the other is that c-Jun and caspase-2 have distinct pathways and BDNF only blocks expression of caspase-2. From our experiments, it is not possible to select which of these hypotheses is correct because we do not know whether a single cell expresses c-Jun and caspase-2 sequentially. Expression of c-Jun and caspase-2 was detected in cells with a shrunken cell body and condensed chromatin (i.e., morphological signs of cell death; Figs. 2 and 3 ). Thus, even if BDNF works between c-Jun and caspase-2, it is unlikely that the process of apoptosis can be reversed. We hypothesize that c-Jun–dependent and caspase-2–dependent cell death occurs via distinct pathways and that the cascade associated with caspase-2 is blocked by BDNF but c-Jun–dependent pathways are not.

In this study, retrograde labeling of the RGCs was not done routinely; instead, we used confocal microscopy and counted the number of cells stained with PI in the retinal GCL. Because amacrine cells are also found in the retinal GCL, their presence will alter the overall numbers of cells. Thus, the cell counts presented represent both RGCs and amacrine cells. Even when the RGCs were labeled retrogradely, it was not easy to differentiate cells that lost the dye due to the destruction of cell body from displaced amacrine cells (Fig. 2) . Because our analysis consisted of the cell numbers in the retinal GCL, and because our conclusions depended on changes in the cell numbers, the presence of amacrine cells should not alter our conclusions.

In summary, we have shown that the expression of c-Jun and caspase-2 is associated with cell death in the retinal GCL in a rat ischemia-reperfusion injury model. BDNF had a neuroprotective effect on cell death, possibly by suppressing caspase-2 expression, but had no influence on c-Jun expression. Although the pathway linked to caspase-2 is suppressed by BDNF, there may exist other pathways through which BDNF works.

	Footnotes

 
Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of the Japanese Government (10470567 to NY).

Submitted for publication January 29, 1999; revised June 10, 1999; accepted July 6, 1999.

Commercial relationships policy: P: CN; N: TK, NK, HS, SK, YK, NY.

Corresponding author: Nagahisa Yoshimura, Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto 390-8621, Japan. E-mail: nagaeye{at}hsp.md.shinshu-u.ac.jp

	References

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1. Quigley, HA, Nickells, RW, Kerrigan, LA, Pease, ME, Thibault, DJ, Zack, DJ (1995) Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis Invest Ophthalmol Vis Sci 36,774-786[Abstract/Free Full Text]
2. Robinson, GA (1994) Immediate early gene expression in axotomized and regenerating retinal ganglion cells of the adult rat Brain Res Mol Brain Res 24,43-54[Medline][Order article via Infotrieve]
3. Kuroiwa, S, Katai, N, Shibuki, H, et al (1998) Expression of cell cycle-related genes in dying cells in retinal ischemic injury Invest Ophthalmol Vis Sci 39,610-617[Abstract/Free Full Text]
4. Shibuki, H, Katai, N, Kuroiwa, S, Kurokawa, T, Yodoi, J, Yoshimura, N. (1998) Protective effect of adult T-cell leukemia-derived factor on retinal ischemia-reperfusion injury in the rat Invest Ophthalmol Vis Sci 39,1470-1477[Abstract/Free Full Text]
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