Download Elevated mRNA expression of brain-derived neurotrophic factor in

Elevated mRNA Expression of Brain-Derived Neurotrophic
Factor in Retinal Ganglion Cell Layer After Optic
Nerve Injury
Hua Gao* Xiaoxi Qiao* Franz Hefti* Joe G. Hollyfield,^ and Beat Knusel*
Purpose. Recent studies show that exogenous brain-derived neurotrophic factor (BDNF) can
promote retinal ganglion cell survival in vivo and in vitro. BDNF is expressed by a subpopulation of cells in the ganglion cell layer (GCL). To investigate whether endogenous BDNF may
play a role in neuronal protection after ganglion cell trauma, BDNF expression in the retina
was examined after optic nerve (ON) injury.
Methods. The optic nerve in Sprague-Dawley rats was crushed intraorbitally posterior to the
optic disc. For controls, the optic nerve on the opposite side in each animal was similarly
exposed but was not crushed. After intervals of 6 hours to 6 weeks, eye tissues were processed
for in situ hybridization, Northern blot, and RNase protection assay using radiolabeled rat
riboprobes.
Results. After ON injury, BDNF expression was significantly elevated in cells restricted to the
GCL, and more cells demonstrated expression of BDNF than were observed in the controls.
Elevated BDNF expression was first observed at 24 hours, peaked at 48 hours, and declined
to the basal level 2 weeks after ON injury. Quantitative analysis showed a fivefold to sixfold
increase in the number of BDNF-positive cells and a 54% increase in BDNF signal intensity
in individual cells in the GCL 48 hours after ON injury. In control retinas without ON injury,
BDNF expression was localized to some cells in the GCL, as was observed in normal eyes
without surgery. Northern blot and RNase protection assay demonstrated a 38% elevation in
BDNF expression above control levels 48 hours after ON injury.
Conclusions. These results indicate that cells in the GCL can upregulate gene expression of
BDNF in response to ganglion cell axonal injury and suggest that endogenous BDNF may
contribute to a natural neuroprotective process after ON injury. Invest Ophthalmol Vis Sci.
1997;38:1840-1847.
J\l eurotrophic factors are key elements in the regulation of neuronal development and are required for
maintenance in the adult nervous system. Among
them is the family of the neurotrophins that includes
nerve growth factor, brain-derived neurotrophic factor (BDNF), and neurotrophins 3, 4/5, and 6. BDNF
From the *Andrus Gerontology Center, University of Southern California, IJJS
Angeles, California, find the ^Division of Ophthalmology, Research Institute/FFb,
The Cleveland Clinic Foundation, Cleveland, Ohio.
Supported by National Institutes of Health grants EY02362, NS22933, AG09793,
AC 10480; by research grants from National Parkinson Foundation (Miami,
Florida); by the Foundation Fighting Blindness (Baltimore, Maryland); by Retina
Research Foundation (Houston, TX); and by Research to Prevent Blindness (New
York, NY;JGH).
Submitted for publication August 27, 1996; revised January 29, 1997; accepted
March IS, 1997.
Proprietary interest category: N.
Reprint requests: Hua Gao, Andrus Gerontology Center, University of Southern
California, 3715 McClintock Avenue, Los Angeles, CA 90089-0191.
1840
is the most abundant neurotrophin in the adult
brain.1'2 Many studies have shown that exogenous
BDNF can promote survival and prevent neuronal
death of retinal ganglion cells (RGCs) after axotomy
in the optic nerve3"6 or in cell culture.7"9 Exogenous
BDNF can enhance optic axon branching and remodeling in vivo10 and can protect ganglion cells from
retinal ischemic injury.11
Although these studies suggest that exogenous
BDNF plays important roles in RGC integrity, little is
known about the function or regulation of endogenous BDNF expression in the retina. Our previous
study12 and others13 showed that a population of RGCs
express BDNF. In the current study, we examined
BDNF mRNA expression in the retina after optic nerve
(ON) injury. By identifying changes in BDNF expression in the retina after ON injury, insights into a func-
Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9
Copyright © Association for Research in Vision and Ophthalmology
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Optic Nerve Injury-Induced BDNF Upregulation in Retina
1841
tional role of endogenous BDNF in ganglion cell
maintenance and protection may be forthcoming.
Our previous studies showed that basic fibroblast
growth factor (bFGF), as a retinal endogenous growth
factor in the photoreceptors, is significantly upregulated by the photoreceptors when they are stressed by
either inherited disorders14'15 or environmental insults
such as intense or constant light.15 Another study
showed an increase of bFGF expression after mechanical injury to the retina.16 Because administration of
exogenous bFGF into eyes could delay photoreceptor
degeneration in some mutant (Royal College of Surgeons) or light-damaged rats,17"19 it is suggested that
endogenous bFGF may function as a natural photoreceptor protection or rescue factor that is activated in
response to photoreceptor stress.
An obvious question arises as to whether other
retinal neurons could also increase the expression of
their endogenous neurotrophic factor when they encounter insults—in other words, whether upregulation of neurotrophic factor in neurons is not only a
feature of the photoreceptors in the outer retina, but
also a characteristic of neurons in the inner retina.
To determine whether ganglion cells also respond to
cellular insults by increasing their BDNF expression
in a manner similar to the photoreceptors' upregulation of bFGF in response to stress, BDNF mRNA expression was examined in the retina after optic nerve
injury.
rior to the optic disc using a microclip (Baby Dieffenbach Serrefines, RS-5471; Roboz Surgical Instrument,
Rockville, MD). The crush was applied for 30 seconds
between the tips of the microclip. After a 60-second
interval, the optic nerve was crushed again at the same
site for another 30 seconds, as described previously.20
The eye was not pulled nasally when the crush was
applied. The opposite eye of each animal was used as
surgery control: an identical operation was performed
on the other eye, except that the optic nerve was only
exposed, not crushed.
After surgery, animals were killed at 6 hours, 24
hours, 48 hours, 72 hours, 1 week, 2 weeks, 4 weeks,
and 6 weeks. Two animals were used for each postinjury stage.
MATERIALS AND METHODS
Animals
Sprague-Dawley albino rats were obtained from
Charles River Laboratories (Wilmington, MA). Animals were fed ab libitum with Purina lab chow and
water with room lighting consisting of a 12-hour light/
12-hour dark cycle. All animals were maintained and
handled in accordance with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research.
Optic Nerve Crush Surgery
Rats 6 to 7 weeks old were anesthetized with intraperitoneal injections of pentobarbital (Nembutal, 75 mg/
kg). Lateral canthotomy was performed and temporal
conjunctiva and Tenon's capsule were opened along
the limbus to allow access to the abductus muscle. The
abductus was then severed so that the eye could be
pulled nasally to expose the posterior pole of the eye.
The optic nerve was approached along the temporal
scleral surface. Dull dissection was used and close attention was paid to avoid damaging blood vessels
around the posterior pole of the eye. When it was
exposed, the optic nerve was crushed 2 to 3 mm poste-
Tissue Preparation
Animals were killed with an overdose of pentobarbital
(Nembutal). Eyes were enucleated, an incision was
made in the cornea, and eyes were fixed immediately
in 4% formaldehyde in 0.1 M phosphate buffer (pH
= 7.4). After 15 minutes in the fixative, the lenses
were removed and eyes were cut along the corneaoptic nerve axis into two halves. Tissues were further
fixed and cryoprotected overnight in 4% formaldehyde, 0.5% glutaraldehyde, and 20% sucrose in 0.1 M
phosphate buffer (pH = 7.4). Tissues were embedded
in Tissue-Tek OCT compound (Miles, Elkhart, IN)
and cryosectioned at a thickness of 10 fim at —21°C.
Tissue sections were cut from areas within 1 mm of the
optic nerve. Thus, each section contained the entire
central and peripheral retina in one meridian. The
tissue sections from the ON-injured and control eyes
were mounted on the same slide and processed identically so that sections could be directly compared with
as litde processing variability as possible.
In Situ Hybridization
A rat BDNF cDNA clone was obtained as a generous
gift from Genentech (San Francisco, CA). It consists
of 460 bases of coding region and was inserted into
plasmid pGEM-4Z.21 For generation of antisense and
sense BDNF riboprobes, the plasmid was linearized
with restriction enzyme EcoR I and Hind III, respectively. 35S-labeled antisense and sense riboprobes were
transcribed using the Riboprobe Gemini System according to the manufacturer's instructions (Promega,
Madison, WI). The tissue sections were pretreated
with 10 /ig/ml proteinase K at 37°C for 20 minutes
and 0.25% acetic anhydrite and 0.1 M triethanolamide
for 10 minutes. The tissue sections were then incubated at 50°C on a slide warmer for 18 ± 2 hours with
the probe solutions containing 5 X 106 cpm/ml 35Slabeled probes, 50% formamide, 10% dextran sulfate,
300 mM NaCl, 0.5 mg/ml tRNA, 10 fjM dithiothreitol,
0.02% Ficoll, 0.02% polyvinyl-pyrolidone, 0.02% bo-
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Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9
1842
vine serum albumin, and 1 mM EDTA in 10 mM TrisHC1 (pH = 8.0). After hybridization, the slides were
rinsed in 4 X SSC (SSC: 150 mM NaCl and 15 mM
NaAc), digested with 20 /zg/ml RNase A at 37°C for
30 minutes, and washed through descending concentrations of SSC to 0.1 X SSC at 60° to 70°C. The slides
were then dehydrated in ethanol, dried, and coated
with NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). After exposure in the dark for 4 weeks, the
emulsion was developed and sections were counterstained with hematoxylin and eosin.
Image Quantitation and Statistical Analysis
The number of BDNF-positive cells in the retinas was
determined using a Nikon image processor interfaced
with a light microscope through an Hitachi video camera (Hitachi Denshi, Tokyo, Japan). Total cell numbers and BDNF-positive cell numbers in the ganglion
cell layer (GCL) were counted separately. Total cells
in the GCL in the entire central and peripheral retina
in one meridian on each tissue section were counted
and used as a denominator; BDNF-positive cells were
identified as cells over which silver grains from in situ
hybridization exceeded five times the background
value. Areas of the inner plexiform layer were used as
background. Thus, the percentage of BDNF-positive
cells was determined in the GCL for each section.
Three or four sections were counted for each eye,
and two animals were used for each postinjury stage.
Analysis of variance (nested effects model) was used
to determine the difference between ON-injured and
control groups in the percentage of BDNF-positive
cells in the GCL.
To compare signal intensities in individual BDNFpositive cells between ON-injured and control groups,
animal eyes at 48 hours after surgery were used.
Twenty-five BDNF-positive cells were randomly selected from each tissue section, and three sections
were used from each of two animals. Silver grain densities over individual cells were determined using a computer-enhanced video densitometer (Southern Micro
Instruments, Atlanta, GA). The means of the densities
were used as the average BDNF signal intensities of
individual cells for either the ON-injured or the control group. Student's Mest was used to determine the
difference between the two groups.
Northern Blot and Ribonuclease Protection
Assay
Total RNA was isolated from six retinas of either ONinjured or control eyes 48 hours after surgery, as described previously.14 RNA was also isolated from six
normal retinas without surgery. The antisense BDNF
RNA probe was synthesized as described above using
32
P-CTP. Northern blot analysis was performed using
standard methods. The total RNA of 30 /ig was separated on 1% agarose formaldehyde-denaturing gel.
The RNA was then blotted to 0.2 fim neutral nylon
FIGURE l. Brain-derived neurotrophic factor (BDNF) mRNA expression in the rat retina
after optic nerve (ON) injury using in situ hybridization. (A) In normal retina without any
surgery, BDNF hybridization signals were present in a subpopulation of cells in the ganglion
cell layer (GCL; arrowheads). (B) Six hours after ON injury. BDNF signals were observed in
a small population of cells in the GCL, and no significant difference was detected when
compared to the normal retina. (C) Twenty-four hours after ON injury. More BDNF-expressing cells were observed in the GCL compared to the control retina without ON injury (I). (D)
Forty-eight hours after ON injury. Maximal number of BDNF-expressing cells was observed at
this time after ON injury. Significandy increased silver grain density was observed over
individual BDNF-positive cells in the GCL. More BDNF-positive cells were present in the
central retina {left) than in the more peripheral retina (right). (E) Seventy-two hours after
ON injury. More BDNF-expressing cells were present in the GCL compared to the control
retina without ON injury (I). However, the number of BDNF-positive cells and the silver
grain density over individual cells were lower at 72 hours than at 48 hours after ON injury
(D). (F) One week after ON injury. There were still more BDNF-positive cells than in the
control (I), but the silver grain density over individual cells was much lower than at 48 or
72 hours after injury (D, E). No significant difference was detected in silver grain density
between ON-injured (F) and control retinas (I). (G) Two weeks after ON injury. Only a
few cells were positive for BDNF, as observed in the control retinas without ON injury (I).
(H) Four weeks after ON injury, no BDNF signal was detected in the retina. However, in
the control retina (I), BDNF signals were present in a small population of cells, as observed
in other controls. (I) BDNF signals in control retinas without ON injury. A small population
of cells in die GCL was positive for BDNF, similar to those observed in normal retinas (A).
(J) Retinas with or without ON injury labeled with sense rat BDNF riboprobe. No signal
was detected in die retinas. All micrographs are at identical magnifications. Scale bar = 200
fj,m.
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Optic Nerve Injury-Induced BDNF Upregulation in Retina
1843
membranes (Schleicher & Schuell, Keene, NH) and
hybridized to 32P-labeled BDNF probe (3 X 106 cpm/
ml). The membrane then was washed in graded SSC,
dried, and exposed to Phosphorlmager plate (Molecular Dynamics, Eugene, OR). Relative abundance of
mRNA was quantified by reading the plate. For accurate quantification, the same blot was stripped off and
hybridized to 32P-labeled /3-actin probe. The ratio of
BDNF to actin densities then was used for comparison
between ON-injured and control groups.
Because the abundance of BDNF mRNA in the
retinas was very low, RNase protection assay, a more
sensitive technique to detect the signal, was used.
RNase protection assay was performed using the RPA
II system (Ambion, Austin, TX) according to the manufacturer's procedure. Briefly, total RNA of 30 fig isolated from retinas 48 hours after surgery (with or with-
out ON injury) and from normal retinas was hybridized with 32P-labeled antisense rat BDNF cRNA probes
(5 X 105 cpm) at43°C for 18 ± 2 hours. Unhybridized
probe was then degraded with 2.5 U/ml of RNase A
and 100 U/ml of RNase Tl for 30 minutes at 37°C.
The samples were then separated on 5% acrylamide/
8 M urea gel and visualized by autoradiography on xray film. Full-length major protected bands were quantitated using the Phosphorlmager plate (Molecular
Dynamics). /3-actin mRNA expression was also examined and used as an internal control.
RESULTS
In the normal adult rat retina, BDNF mRNA expression was present in a subpopulation of cells in the
GCL (Fig. 1A). When quantitated, BDNF-positive cells
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Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9
30
-i
•— Optic N crush
n
Control
o
CD
CD
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20
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co
©
O
5 10
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-Q
D
Q
OQ
FIGURE 2. Percentage of brain-derived neurotrophic factor (BDNF)-positive cells in the ganglion cell layer (GCL) after optic nerve (ON) injury. Analysis of variance (nested effects
model) was used to determine the difference between ON-injured and control groups at
various stages. No significant difference was found at 6 hours or 2 weeks after ON injury.
There were significantly more BDNF-positive cells in the GCL in the ON-injured group than
in the control group at 24 hours, 48 hours, 72 hours, 1 week, and 4 weeks, with P < 0.025,
0.001, 0.001, 0.01, and 0.025, respectively (mean ± SD, n = 2 for each postinjury stage).
accounted for 5% to 6% of the cells in the GCL (Fig.
2) and were randomly distributed throughout the retina (Fig. 1A). Six hours after ON injury, no significant
change of BDNF expression was observed in the retina
(Fig. IB). The number of BDNF-positive cells and signal intensities in individual cells were very similar between ON-injured and control groups (Fig. II).
Twenty-four hours after ON injury, BDNF expression
was elevated (Fig. 1C). The number of BDNF-positive
cells in the GCL increased to 10%, whereas in the
control group only 5% to 6% of the cells were labeled
(Fig. 2). The BDNF signal intensity was also significantly higher in individual cells in the ON-injured
group, as evidenced by increased density of silver
grains (Fig. 1C).
BDNF expression peaked 48 hours after ON injury
(Fig. ID). The number of BDNF-positive cells in the
GCL increased to 28% (24% to 31%) compared to
the control group, in which only 5% to 6% were labeled with BDNF probe (Fig. 2). More BDNF-positive
cells were present in the central retina than in the
peripheral retina (Fig. ID). To confirm that elevated
BDNF expression was caused not only by the presence
of more BDNF-expressing cells after ON injury but
also by increased expression in individual cells, BDNF
signal intensities in individual cells were quantitated
(see Materials and Methods), and ON-injured and
control groups were compared. Quantitative analysis
showed a 54% increase in silver grain density over
individual cells in the ON-injured group than in the
control group at 48 hours after ON injury (Fig. 3).
These results indicate that BDNF expression levels increase in individual GCL cells after ON injury.
BDNF expression in ON-injured retinas declined
from peak levels at 48 hours. Seventy-two hours after
ON injury, BDNF expression was lower than at 48
hours but still significantly elevated (Fig. IE) when
compared to the control retinas (Fig. II). The number
of BDNF-positive cells in the GCL was 23%; in the
control retinas, only 5% to 6% of cells in the GCL
were positive for BDNF (Fig. 2), comparable with that
in normal retinas. The signal intensity in individual
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1845
Optic Nerve Injury-Induced BDNF Upregulation in Retina
cells at this stage was lower than at 48 hours but still
significantly higher than in control retinas (Fig. IE).
BDNF-positive cells were more abundant in the central
than in the peripheral retinas, as observed at 48-hour
stage.
One week after ON injury, the number of BDNFpositive cells and signal intensity in individual cells
continued to decline (Fig. IF), but there were still
more cells positive for BDNF than in the control
group. When quantitated, 12% of cells in the GCL
were positive for BDNF in the ON-injured group; in
the control group, only 7% of cells were labeled (Fig.
2). However, the signal intensity in individual cells was
much lower at this time than at 48 or 72 hours after
ON injury (Figs. ID to IF). No significant difference
was observed in the signal intensity in individual cells
between ON-injured and control retinas (Figs. IF, II).
Two weeks after ON injury, BDNF expression returned to basal level (Fig. 1G). Fewer than 4% (3.4%)
of cells in the GCL were positive for BDNF, comparable to the 3.3% figure in the control group (Fig. 2).
The signal intensities in individual cells were also comparable between the two groups (Figs. 1G, II).
BDNF expression in the retinas continued to decline after 2 weeks after ON injury. Four weeks after
ON injury, BDNF expression declined to a level where
no obvious signal could be detected in the retinas
using the in situ hybridization technique (Fig. 1H).
However, in the control retinas, BDNF expression was
still present in a small population of cells, as observed
in other control stages (Fig. II). Similar findings were
noticed at the 6-week stage, in which no BDNF signal
was observed in ON-injured retinas (data not shown)
but the signal was detected in the control retinas and
was comparable to other control stages.
Analysis of variance (nested effects model) was
used to determine the difference between ON-injured
and control groups in the number of BDNF-positive
cells in the GCL. No significant difference was found
at 6 hours and 2 weeks after ON injury. There were
significantly more BDNF-positive cells in the ON-injured group than in the control group at 24 hours, 48
hours, 72 hours, 1 week, and 4 weeks, with P <0.025,
0.001, 0.001, 0.01, and 0.025, respectively.
To confirm the in situ hybridization results,
Northern blot and RNase protection assay were performed. The stage of 48 hours, in which retinas
showed peak levels of BDNF mRNA expression after
ON injury in in situ hybridization, was chosen to confirm the upregulation of this neurotrophic factor.
RNAs were isolated from retinas with or without ON
injury and used in the assay. RNAs from normal adult
retinas without any surgery were also used as a control.
Analysis of variance showed a significant 38% increase
of BDNF mRNA expression in the retinas with ON
injury than in the control retinas without ON injury;
BDNF expression levels were comparable in normal
retinas and in the control retinas without ON injury
(Fig. 4). /?-actin mRNA expression was also examined
and used as an internal control. No difference was
detected in actin expression among the three groups.
These results are consistent with the in situ hybridization study results and show that BDNF mRNA expression levels were significantly elevated in the retinas
after ON injury.
DISCUSSION
Control
ON injury
FIGURE 3. Brain-derived neurotrophic factor (BDNF) hybridization signal intensity in individual cells 48 hours after
optic nerve (ON) injury. From ON-injured or control retinas, 25 BDNF-positive cells in the GCL were randomly selected from each of three tissue sections. Silver grain densities over each individual cell were determined using a computerized densitometer (see methods). Two animals were
used for ON-injured or control group. Student's Hest, used
to determine the difference between the two groups, showed
a 54% increase in BDNF signal intensity in individual GCL
cells 48 hours after ON injury.
The results of this study clearly demonstrate that
BDNF mRNA expression undergoes significant elevation in a subpopulation of cells in the GCL after ON
crush injury. The upregulation of BDNF expression
was first detected at 24 hours (Fig. 1C), peaked at 48
hours (Fig. ID), lasted about 1 week (Fig. IF), and
returned to basal level 2 weeks after ON injury (Fig.
1G). Increased BDNF expression was confirmed by
Northern blot and RNase protection (Fig. 4). This
increase was the result of more BDNF-expressing cells
and increased signal intensity in individual cells after
ON injury (Fig. 4).
Because it has been reported that as many as
40% to 50% of cells in the GCL are displaced amacrine cells in the adult rat retina,22 an obvious question arises as to the type of cells involved with BDNF
expression in the GCL. If some BDNF-positive cells
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1846
Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9
in the GCL were amacrine cells, it is reasonable to
expect that some amacrine cells in the inner nuclear
layer should also be labeled with BDNF probe, but
this was not observed: No BDNF hybridization signal
was detected in the inner nuclear layer. To our
knowledge, there has been no report that ON injury
can cause changes in amacrine cells, whereas ganglion cell changes after ON injury have been well
documented.
Because ON crush may cause occlusion of the
central retinal artery, it is possible that transient
ischemia may contribute to the change in BDNF
expression observed in the GCL. This study cannot
exclude this possibility. However, previous studies
do not support the possibility that ischemia plays a
major role in this BDNF upregulation. Other studies
showed that a 60-minute period of complete occlusion of the central retinal artery is required to demonstrate mild histologic ischemic damage in the rat
retina; a 30-minute occlusion did not result in any
histologic change.23"25 Our study used two 30-second episodes of ON crush intercalated by a 60-second noncrush period, not long enough to produce
significant ischemic damage to the GCL when compared to those other studies.20 Another analysis
showed that bilateral common carotid artery occlusion for 2 minutes resulted in increased BDNF expression in dentate gyrus granule .cells but not in
any other area in the brain.26 Therefore, it seems
unlikely that the increased BDNF expression in our
study was due to an ischemic effect.
To determine whether BDNF upregulation is a
unique phenomenon in the retina after ON injury,
we also examined nerve growth factor, neurotrophin 3, and trkB receptor mRNA expressions using in situ hybridization. No significant change was
detected in the expression of these growth factors
or receptor after ON injury (data not shown). Thus,
BDNF is the only neurotrophin examined that demonstrated increased mRNA expression in this study.
What is the significance of elevated BDNF expression after ON injury? A recent study showed that
after intraorbital ON transection in the adult rat, all
RGCs survive for 5 days, but 90% of them die by 2
weeks.27 The time course of RGC survival period
after ON transection correlates well with the BDNF
upregulation time in our study. Because exogenous
BDNF could promote RGC survival and delay RGC
death in vivo3'50 and in vitro,7"9 it is reasonable to
speculate that elevated expression of endogenous
BDNF may play a similar role. Increased BDNF expression may reflect the initiation of a natural protective pathway soon after ON injury. Endogenous
BDNF may eventually fail to protect RGCs because
axon damage is too severe to be altered by the protective effects of increased BDNF production, or be-
^
200
CO
100
-
o
<
Q
CD
FIGURE 4. Northern blot and RNase protection assay of brainderived neurotrophic factor (BDNF) mRNA expression in
normal retinas, control retinas without optic nerve (ON)
injury and retinas with ON injury (48 hours). /?-actin mRNA
expression was used as an internal control. Analysis of variance was used to determine the difference among these
three groups. Retinas with ON injury (*) contained 48% and
38% more BDNF mRNA than normal and control retinas,
respectively. No significant difference was observed between
control and normal retinas (based on two independent analyses) .
cause BDNF produced in the GCL does not provide
sufficient protection, because exogenous BDNF
could delay RGC death. 4 " 6
In summary, this study provides the first evidence that cells in the GCL respond to ON injury
by upregulating BDNF expression. These results
suggest that endogenous BDNF may act as a natural
survival or protection factor in the ganglion cells
when they encounter injury. Because the expression
of bFGF, an endogenous growth factor in the photoreceptors, is upregulated when the photoreceptors
encounter either inherited or environmental insults, 14 " 16 we propose that upregulation of endogenous neurotrophic and neuroprotective factors is a
general phenomenon in both the outer and inner
retinas during neuronal degeneration.
Key Words
brain-derived neurotrophic factor, neurotrophins, optic
nerve injury, retina, retinal ganglion cells
Acknowledgments
The authors thank Dr. Sandra K. Kostyk at Massachusetts
General Hospital, Boston, for technical suggestion on ON
injury, Genentech Co. in San Francisco for rat BDNF cDNA
clone, and Dr. David J. Stone at University of Southern California for generous statistical assistance.
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Optic Nerve Injury-Induced BDNF Upregulation in Retina
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