Abstract
purpose. To examine the impact of experimental ischemia and interruption of
glutamate transport on retinal neuronal cell, especially retinal
ganglion cell (RGC), survival in vitro.
methods. Cell cultures were prepared from adult pig retinas and maintained under
different experimental conditions of increasing hypoglycemia,
environmental hypoxia (delayed postmortem period or atmospheric
PO2 <2%), or chemical hypoxia (potassium cyanide), or in
the presence of glutamate transporter blockers l-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and l(−)-threo-3-hydroxyaspartic acid (THA), or the glutamine
synthetase inhibitor methionine sulfoximine (MS). After 48 hours, cells
were returned to standard culture conditions and allowed to develop for
5 days, when they were fixed and immunostained with different retinal
neuronal phenotypic markers.
results. Control normoxic cultures contained large numbers of
immunocytochemically identified photoreceptors (PRs), bipolar cells
(BCs), amacrine cells (ACs), and RGCs after 7 days in vitro. A 24-hour
postmortem delay before culture led to significant reductions in all
types (40%–70%), proportionately greater in ACs and RGCs. Lowering
of sugar levels also led to increased losses in all cell types, whereas
potassium cyanide treatment deleteriously affected only ACs and RGCs.
Ambient hypoxia led to consistent reductions only in the number of
RGCs, which were exacerbated by addition of high concentrations of
glutamate. Inclusion of glutamate receptor antagonists had a partial
protective effect against RGC loss. Treatment with tPDC and THA also
led to selective RGC death, but MS had no effect on any cells.
conclusions. Different components of the ischemic pathologic process (hypoxia,
hypoglycemia, glutamate transport failure) lead to distinctly different
patterns of neuronal loss in adult retina in vitro. RGCs are especially
vulnerable, corresponding to their in vivo susceptibility. These data
may suggest neuroprotective strategies for limiting retinal damage
during ischemia.
Much evidence suggests that alterations and imbalances in
metabolism of the excitatory neurotransmitter glutamate (Glu) play a
pivotal role in central nervous system (CNS) disease.
1 2 3 Under normal physiological conditions, extracellular levels of this
amino acid are maintained at low levels through active uptake by
glutamate transporters (GluT).
4 At present, five distinct
Na
2+-dependent GluTs have been identified,
localized to either the neuronal or glial processes surrounding
glutamatergic synapses: GLAST (EAAT-1), GLT-1 (EAAT-2), EAAC-1
(EAAT-3), EAAT-4, and EAAT-5.
4 5 Maintaining low
extracellular Glu is essential for returning glutamate receptors (GluR)
to their inactive state to permit physiological synaptic function and
for avoiding pathologic overstimulation of GluR, which leads to
excessive depolarization and massive entry of
Ca
2+ into the neuron, often culminating in death.
This pathologic phenomenon, termed excitotoxicity, is frequently
observed in various disease states, including congestive heart failure,
stroke, neurologic disorders, and diabetes.
1 3 Additional
injurious aspects of ischemia-reperfusion include reductions in oxygen
and sugar delivery, reduced clearance of waste products, and build-up
of free radicals.
6 7
The retina, a peripherally located region of the CNS, exhibits all the
features we have described. Glu is the major excitatory
neurotransmitter, four retinal GluT (GLAST, GLT-1, EAAC-1, and
EAAT-5
8 9 10 have been identified, and excitotoxicity is
thought to be a leading factor in neuronal death in retinal ischemia,
diabetic retinopathy, and glaucoma.
11 12 13 Much data have
been obtained on the molecular mechanisms underlying the pathogenesis
of excitotoxic neuronal death, using the retina as a
model.
11 14 15 Ionotropic GluR are defined
pharmacologically as
N-methyl-
d-aspartate (NMDA)–, kainic
acid (KA)–, or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA)–preferring subtypes. Especially NMDA GluRs have been implicated
as principal mediators of excitotoxic damage,
11 16 although there is also evidence that AMPA-KA GluRs play important
roles.
15 Many of these data have been compiled by using
direct application of Glu or one of its nonphysiological agonists,
either in vivo or in vitro.
14 17 18 Additional support for
Glu imbalances underlying retinal ischemic damage has come from
experimental induction of ischemia in vivo accompanied by treatment
with GluR antagonists.
19 21 The approach of studying the
impact on the sensitive neuronal populations through impeding Glu
transport has been less studied, although recent reports indicate such
treatments lead to Glu release
22 and retinal ganglion cell
(RGC) damage.
23
We have developed a cell culture system derived from adult mammalian
retina in which all major neuronal types are represented in
approximately the same proportions as in vivo.
24 26 This
culture model has recently been used to investigate the effects on
retinal neurones of manipulating Glu levels within the medium, and
these treatments have led to specific, dose-dependent losses in
RGCs.
27 In the present study, we have examined the effects
on identified retinal neurons of mimicking ischemia through chemical or
environmental manipulation. The data show that paradigms involving
hypoglycemia led to widespread cell loss, whereas those involving
hypoxia induced more specific damage to amacrine cells (ACs) and
especially RGCs. Addition of GluR antagonists had only limited
protective effects, suggesting alternative routes of cell death, such
as free radical damage.
Immediately after cell seeding, 1 mM potassium cyanide (KCN) was
added to retinal cells (50 μl/well). Control wells received 50 μl
medium alone. After 48 hours’ treatment, cultures were rinsed twice
with serum-free DMEM, replenished with normal DMEM and maintained in
vitro for a further 5 days (total of 7 days).
Immediately after cell seeding (defined as 0 days in vitro), plates
were transferred to a controlled-atmosphere incubator in which oxygen
levels were reduced to less than 1% normal partial pressure (i.e., a
5% CO2-94% N2-1%
O2 mix). In some experiments, in addition to
maintenance under hypoxic conditions cultures were incubated with the
NMDA GluR antagonist
(+)-5-methyl-10,11-dihydro-5H-dibenzo-(α,β)-cyclohepten-5,10-imine
maleate (MK-801; 10 μM) or the AMPA-KA GluR antagonist
6-cyano-7-nitroquinoxaline-2′3-dione (CNQX; 50 μM) alone, exposed to
high concentrations of Glu (1 mM), or exposed to both 1 mM Glu and
either CNQX or MK-801. Cultures were maintained under hypoxic and
excitotoxic conditions for 48 hours, after which the media were changed
to fresh DMEM-5%FCS and the plates returned to a normoxic (5%
CO2-95% air) atmosphere for a further 5 days.
The cultures were then fixed in 4% paraformaldehyde and processed for
immunocytochemistry.
Cultures were treated with two different competitive
Na-dependent GluT blockers: l-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC)
and l(−)-threo-3-hydroxyaspartic acid (THA;
Tocris Cookson, Ltd., Bristol, UK), at 25 and 50 μM each, added at 0
days in vitro for 48 hours. The cultures were then returned to normal
DMEM and maintained for a further 5 days before fixation.
The glutamine synthetase inhibitor methionine sulfoximine (MS; 500μ
M; Tocris Cookson, Ltd.) was added to cultures at the day of seeding
for 48 hours, after which cells were gently washed twice, replenished
with normal culture medium, and allowed to grow for a total of 7 days.
Statistical analysis was performed by computer with an analysis
of variance (ANOVA) software package followed by the Tukey multiple
comparison test. P < 0.05 was considered statistically
significant. For the statistical treatment of the 24-hour postmortem
delay, Student’s t-test was used. These statistical
treatments were performed on raw data, whereas graphic representation
of results was normalized to control values (expressed as 100%) for
each experimental series, to minimize variation.
For studies on RGC neurite morphology after each experimental
treatment, between 100 and 200 individual immunolabeled RGCs were
examined under high-power microscope optics, and the length of their
longest neurites determined with a calibrated graticule inserted in the
microscope eyepiece. Data are expressed relative to cell body
diameters, placed in bins of increasing size, and calculated as
frequency histograms.