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Research report
Neuroprotective effects of brain-derived neurotrophic factor in eyes
with NMDA-induced neuronal death
a b ,
*
a a cNoriaki Kido , Hidenobu Tanihara
, Megumi Honjo , Masaru Inatani , Tohru Tatsuno ,
c a
Chikao Nakayama , Yoshihito Honda
a
Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan
b
Department of Ophthalmology, Tenri Hospital, Mishima-cho 200, Nara 632-8552, Japan
c
Sumitomo Pharmaceuticals Research Center, Osaka, Japan Accepted 22 August 2000
Abstract
Purpose: To determine if brain-derived neurotrophic factor (BDNF) has a neuroprotective effect against N-methyl-D-aspartate
(NMDA)-induced cell death in retina. Methods: NMDA was injected into the vitreous of rat eyes. NMDA-induced neuronal death was measured by morphometric analyses on cell counts of ganglion cell layer cells and thickness of retinal layers. Also, we conducted additional experiment using retrograde labeling with a fluorescent tracer (Fluoro-Gold) for exact counting of retinal ganglion cells (RGCs). In addition, intravitreal glutamate levels were measured with the use of a high-performance liquid chromatography (HPLC) system. Results: Morphometric analysis of retinal damage in NMDA-injected eyes showed that BDNF could protect inner retinal cells from glutamate receptor-mediated neuronal death. Also, counts of RGCs labeled with a fluorescent tracer showed that BDNF could protect RGCs from glutamate receptor-mediated neuronal death. Furthermore, measurements of intravitreal glutamate levels indicated an increase in this excitatory amino acid in the vitreous after NMDA injection. Conclusions: Exogenous BDNF can protect inner retinal cells (possible RGCs and amacrine cells) from NMDA-induced neuronal death. However, increased intravitreal glutamate levels in response to NMDA-mediated neurotoxicity may augment retinal degeneration. 2000 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters, and receptors
Topic: Excitatory amino acids: excitotoxicity
Keywords: Glutamate; Retina; Flat mount; Retinal ganglion cell; Cell death; Brain-derived neurotrophic factor; N-Methyl-D-aspartate
1. Introduction this mechanism is hypothesized to at least partially explain
why damage to neurons of the optic nerve results in death Several growth factors and neurotrophic agents are of RGCs. On the other hand, some investigations showed important for regulation of neuronal development and that RGCs receive BDNF mainly from intraretinal sources maintenance of the adult central and peripheral nervous [5] and retrograde BDNF cannot rescue RGCs in the long systems [15]. Brain-derived neurotrophic factor (BDNF) term [8]. Thus, although further studies are needed for supports survival of retinal ganglion cells (RGCs) exact identification of intraocular origin and physiologic [9,10,16,20,30] and has been shown to act as a target- role for BDNF, it is hypothesized to play important roles in derived trophic factor [16]. Thus, it is likely that damage to the development and maintenance of retinal tissue. neural axons in the optic nerve may cause inadequacy of The presence of TrkB, a high affinity BDNF receptor, target-derived trophic factors, and result in RGCs death. In also implies a potential of BDNF as a neuroprotective the pathogenesis of glaucoma and other optic neuropathies, factor against numerous ocular diseases such as glaucoma, ischemic retinal diseases and retinal degenerative diseases [2]. In a number of animal models of ocular diseases,
*Corresponding author. Tel.: 181-743-635-611; fax: 1
81-743-625-BDNF has been reported to protect retinal neuronal cells
576.
E-mail address: [email protected] (H. Tanihara). from injuries such as light exposure [12], ischemia [28],
retinal degeneration [13] and axotomy [10,19,26,30]. Fur- ride and tropicamide and a single dose of 5ml of 40 mM thermore, BDNF is involved in regeneration of axotomized NMDA (total amount, 200 nmol) was injected into the retinal neurons [17,26] and supports survival of RGCs in vitreous space.
culture [9].
The glutamate receptor-mediated neurotoxicity is re- 2.3. Morphometric analysis garded as one important mechanism in the pathogenesis of
neurodegenerative diseases [11,14,18,23,27,29]. Indeed, in Morphometric analysis was carried out in a manner a number of ocular diseases such as glaucoma [4], similar to that described previously [21]. Briefly, 7 days ischemia [3,22,24] and optic neuropathy [32], elevated after NMDA injection, the animals were killed by intra-levels of glutamate have been reported. Thus, elevated peritoneal overdose injection of pentobarbital, and the eyes glutamate concentrations are hypothesized to be a common were enucleated. Eyes were immersed in fixative solution pathway of visual threatening diseases and to be involved containing 2.5% glutaraldehyde and 2% paraformaldehyde in augmentation of neurodegenerative processes. As men- in 0.1 M phosphate buffer (pH 7.4) for 24 h at 48C, tioned above, although BDNF has been regarded to followed by dehydration and paraffin embedding. Trans-possibly be a neuroprotective drug against many ocular verse sections with a thickness of 3mm were made of rat diseases [1], knowledge about its neuroprotective mecha- retinas through the optic disc. The sections were stained nism in animal models of retinal damage is limited with hematoxylin and eosin and subjected to morphometric [13,28,30,31]. In particular, there are no conclusive data analysis. The degree of NMDA-induced retinal neuronal about whether BDNF inhibits primary (and disease-spe- death was quantified by cell counts in the ganglion cell cific) damaging processes in neuronal lesions, or secondary layer (GCL) and by the thickness of the inner plexiform (and probably more extensive) neurodegenerative events. layer (IPL) at 1.0 to 1.5 mm from the optic disc. Data from Among the known mediators involved in secondary neuro- the three sections were averaged for each eye.
degenerative processes, abnormal release and / or uptake of
glutamate and resultant stimulation of some glutamate 2.4. Analysis for the number of fluoro-gold labeled receptors have been regarded as central events in many RGCs
neurodegenerative processes [3,4,22,24,32]. N-methyl-D
-aspartate (NMDA) glutamate receptors are known to be Retrograde labeling was made in a manner similar to involved in neuronal cell death, including retinal neuronal that described by Sawada et al. [25]. Briefly, rats were cell death [11,14,18,27]. Herein, we report that BDNF anesthetized with xylazine and ketamine and then the protects retinal neuronal cells from glutamate receptor- heads were fixed in a stereotaxic apparatus. Fluoro-Gold
mediated neurodegenerative damage. (Fluorochrome, Englewood, CO, USA) was microinjected
bilaterally into the superior colliculi of rats. Three days after Fluoro-Gold injection, the animals were killed by
2. Materials and methods intraperitoneal overdose injection of pentobarbital and the eyes were enucleated. Eyes were fixed with 4%
parafor-2.1. Animals maldehyde. Retinas were divided into six radial cuts and
removed from the sclera and mounted on slides. Analysis Experiments were performed on adult male Sprague for the number of Fluoro-Gold labeled RGCs was carried Dawley rats (6 weeks after birth) which were housed at out. Briefly, the regions for counting the number of RGCs room temperature on a 12-h light / 12-h dark cycle and were selected from two fields of the central area (1 mm which were given water and food ad libitum. The animals from the optic disc) and two fields of the peripheral area (4 were killed by intraperitoneal overdose injection of pen- mm from the optic disc) on each retinal cut from six radial tobarbital. All studies were conducted in accordance with cuts. Thus, in each eye, a total of 24 fields (12 fields from the ARVO Statement for Use of Animals in Ophthalmic the central areas and 12 fields from the peripheral areas)
and Vision Research. were examined for counting the labeled RGCs.
2.2. Animal models of glutamate receptor-mediated 2.5. Amino acid analysis
neuronal death
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 24.061.8mm, 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 ($1mg) 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. 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. 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. 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 (1mg) (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 (10mg) 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 (10mg)-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
trations at the 14 days after treatment day in comparison
Fig. 7. Analysis of neuroprotective effects of BDNF on labeled RGCs after NMDA injection. At 7 days after the NMDA-injection, in NMDA (200
2
nmol)-injected eyes, counts (cell number per mm ) of the labeled cells were counted. Data from the 24 fields in two (central and peripheral) regional areas were added for each eye. The results are shown as the mean6standard error. Statistical analysis showed a statistically significant difference between the cell counts in control and experimental [BDNF (1mg)-pretreated] eyes (P50.0092, ANOVA). NS indicates not significant.
acts as a target-derived trophic factor [16], damage in tered intravitreally 2 days before NMDA injection in the neural axons and resultant inadequacy of retrograde supply present study. In addition, intravitreal injection of BDNF of this neurotrophic factor may play a part in the neurode- (10 mg) results in inhibition in elevations of glutamate generative processes seen in glaucoma and other optic concentrations in the vitreous of eyes with retinal damage neuropathies. This is why BDNF is regarded as a potential including NMDA-mediated neurotoxicity. Similar findings neuroprotective drug against any ocular conditions, such as were previously obtained from the measurements of in-glaucoma, ischemia, traumatic injury and retinal degenera- travitreal glutamate levels in eyes with chemically-induced tive diseases. In these diseases, elevated levels of gluta- retinal ischemia [7]. So, despite these limitations of BDNF mate in the vitreous have been reported [3,4,22,24,32], so as a candidate for a possible neuroprotective drug for it is quite possible that abnormal levels of glutamate result ocular diseases, inhibition of release and / or uptake in in more extensive and serious secondary damage in neural diseased retinas suggests an inhibitory effect against tissues. However, to our knowledge, there were no reports secondary neurodegenerative events, which are common in about the selective neuroprotective effects of BDNF many retinal diseases.
against glutamate receptor-mediated secondary neurode- BDNF has been regarded as a target-derived trophic generation in retina. Our attempt is to examine the factor [16]. However, some investigators have shown that possibility that BDNF protects against retinal neuronal RGCs receive BDNF mainly from intraretinal sources [5]. damage from secondary neurodegenerative processes. In addition, recently, Isenmann et al. have reported that Our morphologic studies demonstrated that injection of retrograde BDNF cannot rescue RGCs on the long term NMDA, a ligand for glutamate receptors related to neuro- [8]. Thus, these findings suggest that the retrograde nal cell death results in statistically significant cell loss in degeneration of RGCs after lesion of the optic nerve may the GCL and thinning of the IPL as described elsewhere be the results of the physical lesion inflicted to the nerve [6], which is in agreement with a previous report [21]. In instead of deprivation from target-derived neurotrophic our previous study [6], in NMDA-injected eyes, up-reg- factors. Also, since intravitreal injection of BDNF is ulated expression of another neurotrophic factor, CNTF, effective for survival of RGCs in our present study, has been found in the retina. Our morphometric studies intravitreal or intraretinal BDNF may be essential for showed the neuroprotective effects of CNTF against RGCs rather than superior colliculi-derived BDNF. NMDA-induced retinal damages. Also, neuroprotective In conclusion, we suggest that exogenous BDNF can effects of BDNF against the retinal neurotoxicity mediated protect retinal neuronal cells in the inner retinal layers by NMDA have been shown by our results in the present from glutamate receptor-mediated neuronal death, and that study. In the present study, we have conducted a series of this neurotrophic factor can be potentially a clinically experiments in an attempt to elucidate protective effects of useful drug for many retinal diseases.
BDNF in NMDA-related retinal damage. Our morphomet-ric analysis showed that intravitreal injection of BDNF (1
mg) results in less cell loss in the GCL and less thinning of Acknowledgements the IPL, but injection of BDNF (0.1 mg) does not.
Interestingly, intravitreal injection of a larger amount (10 Supported in part by a Grant-in-Aid for Scientific
mg) of BDNF does not seem to have a significant additive Research from the Ministry of Education, Science, Sports neuroprotective effect. Furthermore, an additional experi- and Culture, from the Ministry of Health and Welfare of ment using retrograde labeling of RGCs with Fluoro-Gold Japan.
showed that BDNF protects RGCs in the GCL from glutamate receptor-mediated neuronal death. Statistically
significant protective effects of BDNF are seen at 7 days References after the NMDA treatment.
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