J .-S. Choi et al. Brain Research 883 2000 60 –68
61
death suggest that axotomized RGCs undergo apoptic cell intraperitoneal injection of chloral hydrate 400 mg kg.
death [12,27]. All animals were treated in accordance with the ARVO
The cell death after axotomy seems to be a slow process statement for the Use of Animals in Ophthalmic and
because a significant number of RGCs still survive several Vision Research. The ON optic nerve transection was
days after axotomy. It has been hypothesized that RGC performed at 5 mm from the posterior pole of the eye
death is not an immediate result of axotomy injury, and without damaging the retinal blood vessel.
other factors pro-apoptotic factors may play a role in RGC death [18,29]. Levin suggests that the destructive
molecular events by axotomy lead to the functional failure 2.2. Electron microscopy
of intrinsic protective mechanisms, which in turn brings cell death [18]. Thus, the interplay between pro- and
The retina was fixed in 0.1 M sodium phosphate buffer anti-apoptotic factors may determine the motility of cells.
PB, pH 7.4 containing 4 glutaraldehyde. After washing NF-kB, a transcription factor, is one of the molecules
with the same buffer, the retina was fixed in 1 osmium involved in cell death. NF-kB exists in the cytoplasm as
tetroxide. The retinal tissues were washed and dehydrated heterodimers or homodimers of Rel related proteins. The
using a series of ethanol, then subsequently cleared in amino terminal region of the Rel homology domain
propylene oxide and embedded in epon mixture. The tissue contains DNA binding, dimerization, and nuclear localiza-
preparation was sectioned at 90 nm by ultramicrotome, and tion domains. The predominant form of NF-kB is com-
observed under a transmission electron microscope. posed of NF-kB1 p50 and RelA p65, and is associated
with inhibitor-kB IkB as an inactive form. Upon stimula- tion, I-kB is phosphorylated by IkB kanases, ubiquitinated,
2.3. Immunohistochemistry for NF-kB and Thy-1 and subsequently processed to proteolytic degradation. The
freed NF-kB translocates from the cytoplasm to the The isolated retina tissue was washed in 0.1 M phos-
nucleus by exposing the nuclear localization signal and phate buffered saline PBS, pH 7.4, and then dehydrated
then binds to the target genes to activate transcription through a graded series of ethanol, cleared in xylene, and
[22,25]. NF-kB is activated by various stimuli, including embedded in paraffin. Eight-micrometer-thick vertical sec-
stress or injury. So far, the known inducers for NF-kB tions were made with a microtome Leica, Nussloch,
activation are interlukin IL-1, TNF alpha, bacterial Germany, and the following immunohistochemical pro-
lipopolysaccharides LPS, sphingomyelinase, oxygen free cedure was performed at room temperature. Briefly, the
radicals, ultraviolet light and g-irradiation [9,26,34]. The endogeneous peroxidase was blocked by incubating the
activation of NF-kB as a pro-apoptotic factor has been specimens in 0.3 hydrogen peroxide Sigma, St. Louis,
shown in neuronal cell death induced by TNF alpha, MO, USA for 10 min and rinsed with PBS prior to
glutamate, and reactive oxygen species in vitro [7,8,20,28], antibody application. Nonspecific binding was blocked by
while the anti-degenerative function also has been sug- incubation with 20 normal horse serum Santa Cruz
gested. The mechanism for anti-apoptotic role of NF-kB is Biotech., Santa Cruz, CA, USA for 5 min. The retina
suggested as the elevation of Bcl-2 expression by NF-kB. sections were incubated consecutively with primary anti-
Furthermore, p50 knockout mice showed increased apop- body, rabbit anti-p65 Santa Cruz Biotech. or anti-Thy-1
tosis and the survival pathway by growth factors or Santa Cruz Biotech., biotinylated goat anti-rabbit IgG
cytokines also activate NF-kB through PI-3 kinase path- and
streptavidin conjugated
peroxidase Santa
Cruz way [17,23,31,36,38,41,43].
Biotech.. The immunoreactivity was detected using 3,39- In our previous report, NF-kB was observed in the
diaminobenzidine detection system Boehringer Mann- ganglion cell layer after axotomy with immunohistochem-
heim, Mannheim, Germany and observed under a micro- istry [1]. However, the activation or the role of NF-kB has
scope with a DIC filter Olympus, Tokyo, Japan. not been explored. In this study, we investigated the role of
NF-kB in axotomized ganglion cells by showing whether the cell death is accelerated or blocked by the inhibition of
2.4. Retrograde labeling NF-kB activation.
The degeneration was assessed morphometrically by retrograde labeling of the cell bodies. Application of the
dye involved re-exposure of the nerve without damaging
2. Materials and methods the retinal blood supply. One percent of fast blue, neuro-
tracer dye, was deposited 1 mm from the distal border of 2.1. Animal model
the injury site. Non-injured optic nerves were similarly labeled. Forty-eight hours later, the retinas were prepared
Adult male Sprague–Dawley rats 200–250 g were as flattened whole mounts and the labeled ganglion cells
used for this study. The animals were anesthetized by were counted using fluorescence microscopy.
62 J
2.5. Western blot analysis assessed using one-way ANOVA, and comparisons be-
tween groups were performed using a Student’s t-test. To detect the NF-kB transloction, the cytoplasmic and
nuclear extracts were separated. After axotomy, the retina was isolated and stored at 2708C until further use. The
3. Results
retina was lysed in 10 ml of lysis buffer 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
3.1. Optic nerve transection causes apoptotic cell death dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride
in retinal ganglion cell layer PMSF and incubated on ice for 15 min. Six microliters
of 10 Nonidate P-40 was added to the suspension. After It has been shown that RGCs undergo apoptotic cell
centrifugation at 2000 rev. min for 5 min, the supernatant death after optic nerve transection [29,33]. To demonstrate
was collected for cytoplasmic proteins. The nuclear pellet that our model system, an axotomized retina, is prepared
was resuspended in 10 ml of extract buffer 20 mM without damage to blood vessels near optic nerve, we
HEPES, pH 7.9, 400 mM KCl, 1 mM EDTA, 1 mM observed the retina with a light and an electron microscope
EGTA, 1 mM dithiothreitol, and 1 mM PMSF and Fig. 1. If the blood vessels were damaged, the ganglion
incubated for 30 min at 48C. The suspension was incubated cells die for necrosis rather than apoptosis, and also the
for 20 min at 48C and centrifuged at 15 000 rev. min for cells in inner nuclear layer are also damaged in a day,
20 min. The supernatant was dialyzed for 3 h in dialysis which is a typical ischemic retina model [9]. The light
buffer 10 mM HEPES, 1 mM EDTA, 50 mM KCl, 20 Fig. 1A microscopic observation showed that there are
glycerol, 1 mM dithithreitol, and 1 mM PMSF. The still dying cells in ganglion cell layer 14 days after
supernatant was collected after centrifugation at 15 000 axotomy, which suggests that cell death seemed to be a
rev. min for 20 min at 48C as nuclear protein and stored at slow process rather than a sudden cell death necrosis,
2708C. The amount of protein was determined using BCA which occurs mostly in a few days. Also, the cells in inner
protein assay kit Sigma, St. Louis, MO, USA. nuclear layer were still intact at 14 days after axotomy. In
Thirty grams of protein from each sample was loaded on addition, electron microscopic observation showed that
sodium dodecyl sulfate SDS polyacrylamide gel electro- most of the dying ganglion cells showed apoptotic appear-
phoresis. After running the gel, the proteins were trans- ance, such as chromatin condensation and nuclear frag-
ferred to nitrocellulose membranes hybond-C, Amersham mentation Fig. 1B. The result showed that the RGC
Phamacia Biotech at 300 mA for 1 h. Western blot degeneration started between 7 and 10 days after axotomy.
analysis was performed after blocking with 5 skim milk. These results showed that our model system was well
The membrane was incubated with polyclonal rabbit anti- prepared and did not cause damage to the blood vessels
NF-kB p65 1:1000 or anti-IkB 1:1000 Santa Cruz next to the optic nerve.
Biotechnology, Santa Cruz, CA, USA for 2 h and washed three times in PBST PBS containing 0.1 Twin 20
3.2. Activated NF-kB co-localized with surviving cells buffer. Then the membrane was incubated with horseradish
peroxidase HRP conjugated anti-rabbit IgG. After three NF-kB plays a role in cell death as a pro- or anti-
times of 10 min washing with PBST buffer, the HRP apoptotic transcription factor, depending on cell type or the
activity was visualized by applying chemiluminescent nature of injury [8,7,17,19,28,38,41]. It has been hypoth-
substrate ECL, Amersham, Arlington Heights, IL, USA esized that NF-kB is activated to transcribe the essential
followed by exposing the membrane to X-ray film. genes for cell death or survival [23]. Since whether or not
NF-kB is involved in axotomized retina has not been explored, we determined the correlation between the NF-
2.6. Quantification of cell density in the ganglion cell kB activation and the cell death caused by axotomy. To
layer address the question, we performed immunohistochemistry
with the anti-NF-kB antibody and ethidium bromide For each time point, five retinas from five animals were
arrows in Fig. 2B and D. Ethidium bromide EtBr stains used to assess the density of the ganglion cells. The
for the condensed chromosome, which is a marker for number of cells was counted in 600 mm length of the
apoptotic cell death. ganglion cell layer. Twenty sections per retina were
The results showed that the cells stained with EtBr were observed for the cell counting. To inhibit the activation of
not stained with NF-kB. On the other hand, the EtBr NF-kB, 10 ml of 5 mM sulfasalazine Sigma, St. Louis,
negative cells were positively stained with the anti-NF-kB MO, USA in 25 dimethyl sulfoxide DMSO was
antibody arrowheads in Fig. 2B and D. This indicates injected into the vitreous body 30 min prior to axotomy.
that the surviving cells after axotomy were stained with The control animals were injected with 25 DMSO only.
NF-kB, but the dead or dying cells were not stained with Data are given as mean6S.D. Statistical significance was
NF-kB. Therefore, NF-kB activation seems to make cells
J .-S. Choi et al. Brain Research 883 2000 60 –68
63
Fig. 1. A Light microscopy of axotomized retina. Fourteen days after axotomy, the retinas were collected and subjected to the light microscopic observation. Non-treated retina was healthy and normal a, while axotomized retina b showed pyknotic nucli arrows in ganglion cell layer, but the cells
in inner nuclear layer were intact. Bar520 mm. B Electron microscopy of axotomized retina. Seven days after axotomy, the retinas were collected and subjected to the electron microscopic observation. Non-treated retinal ganglion cells were healthy and normal a, while axotomized cells b showed the
apoptotic cell death such as chromatin arrows condensation, nuclear fragment formation asterisks, insert. Bar52 mm.
more resistant to cell death when the retina is injured by to the translocation from the cytoplasm to the nucleus. We
axotomy. This suggests that NF-kB may play an anti- concluded that NF-kB is activated when the retina is
apoptotic role in axotomized retinas. injured by axotomy.
3.3. NF-kB activation 3.4. Inhibition of NF-kB activation
Since the translocation of NF-kB from the cytoplasm to In order to ascertain the role of NF-kB activation in
the nucleus is essential for NF-kB activation, we examined degenerating RGCs, we used sulfasalazine to inhibit the
its activation with Western blot analysis after separation of translocation of NF-kB [4,40]. The inhibitory effect of
nucleic extract from cytoplasmic extract Fig. 3. Fig. 3 sulfasalazine was confirmed with Western blot Fig. 3C, D
shows that a greater amount of NF-kB was observed in the and immunohistochemistry Fig. 4C, D. The Western blot
nucleus than in the cytoplasm with the injury. The results showed that the treatment of axotomized retina with
translocated NF-kB increased maximally 3 days after 5 mM sulfasalazine did not increase the NF-kB in the
axotomy, and the activation of NF-kB persisted until 14 nuclear compartment while the axotomized retina showed
days after axotomy, even though the activation gradually a dramatic increase of NF-kB in the nuclear extract. The
decreased data not shown. These results confirm that the immunohistochemistry
results showed
that the
sul- positively stained NF-kB in immunohistochemistry is due
fasalazine treatment resulted in a decreased number of
64 J
Fig. 2. Immunohistochemistry of retina with anti-NF-kB antibody. The retina was collected 7 days after optic nerve transection. Positive immunoreactivity for NF-kB arrow heads appeared as dark staining in the cells A, B, and EtBr arrows stained for the condensed chromosome which was shown by
bright white dots C, D. Neither the NF-kB activation, nor the nuclear condensation A, C was observed in the control. In ON transected retina, NF-kB activation B or nuclear condensation D was clearly observed. NF-kB activated cells were not morphologically changed yet in 7 days after the ON
transection B, D. GCL: Ganglion cell layer, Bar520 mm.
NF-kB labeled ganglion cells by 20 of the non-sul- fasalazine exhibited a marked decrease in the number of
fasalazine treated cells. These results showed that sul- Thy-1 labeled ganglion cells after axotomy Fig. 5A.
fasalazine inhibits NF-kB activation in our model system. The difference in the number of Thy-1 positive ganglion
cells between the control and the sulfasalazine-treated 3.5. Role of NF-kB in axotomized retina
group was observed from 10 days after axotomy. The sulfasalazine-treated group contained less than 50 of the
To understand the role of NF-kB in degenerating number of Thy-1 positive cells found in the non-treated
retinas, we quantified the surviving cells with or without group. The most significant difference in the number of
sulfasalazine after axotomy. We stained the retina with the Thy-1 positive ganglion cells was shown at 14 days after
anti-Thy-1 antibody, specific for retinal ganglion cells. The the ON transection, when only 16.264.1 of Thy-1
Thy-1 labeled ganglion cells were counted at various time positive ganglion cells remained in the sulfasalazine-
points 0, 7, 10, and 14 days after the ON transection. treated group compared to 39.366.4 in the non-treated
Compared to the control, the retinas treated with sul- group Fig. 5B.
Fig. 3. Western blot analysis for translocation of NF-kB. Nuclear and cytoplasmic extracts were isolated from the retinas with or wothout sulfasalazine treatment 3 days after axotomy. Thirty micrograms of protein was loaded on the 10 SDS polyacrylamide gel. The immunoreactive proteins were assessed
by Western blot analysis using anti-NF-kB antibody or anti-IkB antibody.
J .-S. Choi et al. Brain Research 883 2000 60 –68
65
Fig. 4. Inhibition of NF-kB translocation by sulfasalazine 3 days after axotomy. A DMSO only; B sulfasalazine only; C DMSO treated axotomized retina; and D sulfasalazine treated axotomized retina. The translocation of NF-kB was inhibited by the treatment of sulfasalazine 3 days after optic nerve
transection. GCL: Ganglion cell layer, INL: Inner nuclear layer, Bar520 mm.
Even though Thy-1 stains only in RGCs, there is a or anti-apoptotic factor is determined by cell type or stress
¨ report that Thy-1 stains in Muller cells in some instances
conditions [19,25,38]. [6,24]. To confirm the Thy-1 staining result, we tried
Recently, an anti-apoptotic function of NF-kB has been retrograde staining Fig. 6, which stains neurons only in
described by Van Antwerp et al., Wang et al. and central nervous system. The data showed a similar result to
Kaltshmidt et al. [13,23,41]. Anti-apoptotic role of NF-kB the Thy-1 staining experiment, where the NF-kB active
is largely substantiated by the report that Rel A NF-kB cells were shown to survive longer than the NF-kB
p65 2 2 knockout mice die at embryonic day 15–16 inactive cells.
with extensive apoptosis in the liver and in tumor cells [41]. Also, NF-kB inhibition by the overexpression of I-kB
potentiates amyloid-beta induced cell death [13]. In addi- tion, NF-kB inhibition by a proteasome inhibitor was
4. Discussion reported to induce neuronal cell death in the hippocampus
