N . Kido et al. Brain Research 884 2000 59 –67
61
amino acids using precolumn o-phthaladehyde derivatiza- the mean numbers 6standard error of GCL cells at 0, 3
tion and fluorescence detection CMNA 1200 refrigerated and 7 days after injection of NMDA 200 nmol were
Microsampler, CMA 280 Fluorescence Detector, BAS, 57.761.2, 28.660.9 and 36.962.0, respectively. In con-
Tokyo, Japan. trast, in PBS-injected control eyes, the mean numbers
6standard error of GCL cells at 0, 3 and 7 days after injection of NMDA 200 nmol were 59.261.5, 24.462.3
3. Results and 25.960.9, respectively. The differences between the
number of GCL cells in PBS- and BDNF-injected eyes on 3.1. Morphometric analysis for neuroprotective effects of
the seventh post-treatment day were statistically significant BDNF
P,0.0001, ANOVA, but not on day 0 P50.5651 and day 3 P50.3860 Fig. 4.
In eyes that have undergone intravitreal injection of After pretreatments with intravitreal injection of BDNF,
NMDA 200 nmol, significant cell loss in the GCL and the mean thickness 6standard error of the IPL at 0, 3 and
thinning of IPL were observed after 7 days, as described 7 days after injection of NMDA 200 nmol was 36.261.6,
previously [6]. In an effort to elucidate the neuroprotective 22.261.5 and 24.061.8 mm, respectively. In PBS-injected
effects of BDNF against NMDA-mediated retinal damage, control eyes, however, the mean thickness 6standard
we conducted morphometric analyses. At 2 days after error of the IPL at 0, 3 and 7 days after injection of
injection of 1 ml of BDNF 0.1, 1 or 10 mg into the NMDA 200 nmol was 39.261.0, 21.362.6 and 18.861.4
vitreous, 200 nmol of NMDA was injected into the same mm, respectively. The difference between the thickness of
animals. Phosphate-buffered saline PBS was injected the IPL in PBS- and BDNF-injected eyes was statistically
instead of BDNF-containing solution in some animals as a significant on the seventh post-treatment day P50.0135,
negative control Fig. 1. In NMDA 200 nmol-injected ANOVA, but not on day 0 P50.1691 or day 3 P5
eyes, the mean numbers 6standard error of GCL cells 0.8371 Fig. 5.
were 25.960.9, 33.661.5, 36.962.0 and 37.865.4, in PBS-injected eyes, 0.1 mg BDNF-injected eyes, 1 mg
3.2. Analysis of neuroprotective effects of BDNF in BDNF-injected eyes and 10 mg BDNF-injected eyes,
experiments using retrograde labeling of RGCs respectively. Statistical analysis showed that intravitreal
injection of BDNF of 1 and 10 mg has significant In an effort to investigate if BDNF can actually protect
protective effects against NMDA-induced retinal damage RGCs from NMDA-induced neuronal death, we conducted
[PBS vs. BDNF 0.1 mg, P50.0788; PBS vs. BDNF 1 retrograde labeling of RGCs with Fluoro-Gold Fig. 6. At
mg, P50.0030, PBS vs. BDNF 10 mg, P50.0017, first, in PBS-injected eyes without NMDA 200 nmol, the
ANOVA] Fig. 2. The mean thickness 6standard error mean
density 6standard
error of
RGCs was
2
of the IPL was 18.861.4, 21.061.9, 24.061.8 and 2342.36103.4 mm . Then, 2 days after injection of BDNF
23.961.8 mm, in PBS-injected eyes, 0.1-mg BDNF-in- 1 mg or PBS in control experiments into the vitreous,
jected eyes, 1-mg BDNF-injected eyes and 10-mg BDNF- 200 nmol of NMDA was injected in the same animals. At
injected eyes, respectively. The results demonstrated that 7 days after NMDA-injected eyes, in control PBS-in-
treatments with BDNF 1 mg inhibited thinning of the jected and experimental BDNF 1 mg-pretreated eyes,
IPL in NMDA-injected eyes [PBS vs. BDNF 0.1 mg, the mean density 6standard error of RGCs was
2
P50.4635; PBS vs. BDNF 1 mg, P50.0126; PBS vs. 269.9624.2 and 561.1619.8 mm , respectively, which
BDNF 10 mg, P50.0516, ANOVA] Fig. 3 .
shows a statistically significant difference P50.0092, After the pre-treatment with intravitreal BDNF 1 mg,
ANOVA Fig. 7.
Fig. 1. Light microscopic photographs of representative retinal tissues in control and experimental eyes at 7 days after intravitreal injection of NMDA 200 nmol.
62 N
Fig. 2. Morphometric analysis of protective effects of BDNF in NMDA-injected eyes. In NMDA 200 nmol-injected eyes, counts cell number per mm of cells present in the ganglion cell layer GCL were evaluated. Data from three sections were averaged for each eye. The results are shown as the
mean6standard error. Morphometric analysis showed statistically significant protective effects of BDNF 1 and 10 mg to the decrease in GCL cells P50.0030 and P50.0017, ANOVA. NS indicates not significant.
Fig. 3. Morphometric analysis of protective effects of BDNF in NMDA-injected eyes. In NMDA 200 nmol-injected eyes, the thickness mm of the inner plexiform layer IPL was evaluated. Data from three sections were averaged for each eye. The results are shown as the mean6standard error.
Morphometric analysis showed protective effects of BDNF 1 and 10 mg to the decrease in IPL thickness. NS indicates not significant.
N . Kido et al. Brain Research 884 2000 59 –67
63
Fig. 4. Time course of morphological changes in protective effects of BDNF on NMDA-induced retinal toxicity. With the pretreatments with intravitreal injection of BDNF, counts cell number per mm of cells present in the ganglion cell layer GCL at 0, 3 and 7 days after NMDA 200 nmol injection were
evaluated. Data from three sections were averaged for each eye. The results are shown as the mean6standard error. Morphometric analysis showed statistically significant protective effects of 7 days after NMDA injection to the decrease in GCL cells P,0.0001, ANOVA. NS indicates not significant.
Fig. 5. Time course of morphological changes in protective effects of BDNF on NMDA-induced retinal toxicity. With the pretreatments with intravitreal injection of BDNF, the thickness mm of the inner plexiform layer IPL at 0, 3 and 7 days after the NMDA 200 nmol injection was evaluated. Data
from three sections were averaged for each eye. The results are shown as the mean6standard error. Morphometric analysis showed statistically significant protective effects of 7 days after NMDA injection to the decrease in IPL thickness P50.0135, ANOVA. NS indicates not significant.
64 N
Fig. 6. Fluorescence microscopic photographs of representative retinal sections in control and experimental eyes at 7 days after intravitreal injection of NMDA. At 2 days after injection of BDNF 1 mg or PBS in control eyes into the vitreous, 200 nmol of NMDA was injected in the same animals. In
control PBS-injected eyes, significant cell loss of labeled cells presumably RGCs was observed. On the other hand, in experimental 1-mg BDNF-pretreated eyes, a larger number of the labeled cells were found.
3.3. Amino acid analysis of vitreous humor error concentrations of glutamate in vitreous humor of
eyes pretreated with BDNF 10 mg at 0, 1, 3, 7 and 14 With the use of HPLC, concentrations of glutamate in
days after the NMDA injection were 127.50629.77 n55, vitreous humor from experimental BDNF-injected and
115.48640.92 n54, 141.20631.39 n54, 71.80612.87 control PBS-injected eyes were measured. As shown in
n54 and 109.60612.06 n54 pmol, respectively. There Fig. 8, the mean 6standard error concentrations of
were significant differences between glutamate concen- glutamate in vitreous humor of PBS-injected eyes at 0, 1,
trations in PBS- and BDNF 10 mg-treated eyes at days 7 3, 7 and 14 days after the NMDA injection were
and 14 after the NMDA injection P50.0364 and P5 166.14623.04
n59, 127.00615.16
n59, 0.0010, ANOVA Fig. 8.
120.35618.67 n56,
180.93627.37 n56
and 280.80633.10 n57 pmol, respectively. Statistical analy-
sis showed a significant increase in glutamate concen-
4. Discussion
