Brain Research 883 2000 60–68 www.elsevier.com locate bres
Research report
Failure to activate NF-kB promotes apoptosis of retinal ganglion cells following optic nerve transection
a a
a b
d
Jun-Sub Choi , Jeong-a. Kim , Dong-Hwan Kim , Myung-Hun Chun , Byung J. Gwag ,
c a ,
Sungjoo Kim Yoon , Choun-Ki Joo
a
Department of Ophthalmology and Visual Science , College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku,
Seoul 137-701, South Korea
b
Department of Anatomy , College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul 137-701, South Korea
c
Research Institutes of Medical Science , College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul 137-701,
South Korea
d
Department of Pharmacology , Ajou University School of Medicine, Suwon, South Korea
Accepted 22 August 2000
Abstract
NF-kB is a transcription factor, which is activated by various stimuli. One of the well-known activators of NF-kB is oxidative stress, which is a cause of cell death in some tissue, or cell types. Optic nerve transection, axotomy, results in retinal cell death, because of
oxidative stress, deprivation of neurotrophic factors, etc. Since it has been hypothesized that the retinal ganglion cell death after axotomy is due to the generation of reactive oxygen species, we investigated whether NF-kB is involved in the retinal cell death after axotomy.
This study was performed to investigate the role of NF-kB in retinal ganglion cell death after optic nerve transection. We used double staining experiment by using anti-NF-kB antibody and ethidium bromide to observe the correlation of NF-kB activation and the cell
death. NF-kB was observed only in the surviving cells. NF-kB translocation was observed 3 days after the optic nerve transection. The NF-kB inhibitor, sulfasalazine, was used to block the activation of NF-kB in the axotomized retina, and the number of ganglion cells was
quantified using retrograde in the presence or absence of sulfasalazine after axotomy. Inhibition of NF-kB by sulfasalazine accelerated the degeneration of ganglion cells in the retina. The results suggest that the activated NF-kB plays a protective role from the cell death in the
injured ganglion cells.
2000 Elsevier Science B.V. All rights reserved.
Theme : Sensory systems
Topic : Retina and photoreceptors
Keywords : NF-kB; Ganglion cell; Retina; Degeneration; Axotomy
1. Introduction There have been numerous studies on the regeneration
of the injured optic nerves by applying neuroprotective Optic nerve ON transection causes irreversible degene-
reagents or by nerve transplantation [11,42]. Neurotrophic ration of the retinal ganglion cells RGC [29,39]. The
factors such as BDNF, NGF, or NT3 4 can delay the cell optic nerve provides the retina with various neurotrophic
death, but cannot make them regenerate [33,42]. In con- factors BDNF, NGF, and NT3 4 that are essential for the
trast, treatment of CNTF or a peripheral nerve graft seems RGCs to survive. After axotomy, the ganglion cells
to help regeneration of the RGCs, but those treatments ultimately die due to the deprivation of neurotrophins [33],
cannot make the RGCs survive [5]. Recently, it has been altered gene expression [18,30,39], and various reactive
reported that optic nerve transection showed typical apo- oxygen species [15,16].
ptotic cell death pattern e.g. activation of caspase, DNA fragmentation, TUNEL positive, and chromatin condensa-
tion, etc. [14,29], and also the previous report showing that Bcl-2 overexpression and the introduction of bax
Corresponding author. Tel.: 182-2-590-2613; fax: 182-2-533-3801. E-mail address
: ckjoocmc.cuk.ac.kr C.-K. Joo.
antisense RNA protected the axotomized retina from cell
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 8 6 - 9
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-
