Purpose: A
dysregulation of blood flow has been proposed to play a role in the
pathogenesis of glaucoma. We have previously shown that simulated ischaemia
results in retinal ganglion cell (RGC) death in human organotypic retinal
cultures (HORCs). The aim of these experiments was to investigate the effects
of graded glucose deprivation on HORCs.
Methods:
The
East Anglian Eye Bank provided donor human eyes within 24 hours post mortem,
with full ethical approval and consent. Five retinal explants (HORCs), 4mm in
diameter, were taken from the paramacular region of each retina. The HORCs
were cultured in serum free (SF) DMEM at different glucose concentrations.
Oxygen deprivation was induced using a custom-made, computer-controlled,
environmental chamber. Medium was removed for LDH analysis. HORCs were fixed
in 4% paraformaldehyde and RGC number assessed using neuronal nuclei (NeuN)
immunohistochemistry and apoptosis by TUNEL assay. Glutamine synthetase
(GLUL) and Na+-glutamate transporter (SLC1A3) mRNA levels were measured using
QRT-PCR. All experiments were carried
out to at least n=4.
Results: Total
deprivation of glucose and oxygen for 24 hours caused an approximate 4-fold
increase in LDH release from HORCs. Deprivation of glucose alone also caused
an increase in LDH release, whereas there was no observed increase in LDH
release from HORCs as a result of oxygen deprivation alone. Graded glucose
deprivation alone (0-5.55mM) showed a progressive increase in LDH release
with decreased glucose concentration, with increases of approximately 2.5-,
3- and 3.5-fold seen at 1.11, 0.56 and 0mM (20%, 10% and 0% control glucose
concentrations). An approximate 40% decrease in the number of RGCs was
observed in HORCs cultured for 24 hours in 1.11mM glucose with an approximate
2-fold increase in TUNEL-positive RGCs. Glucose deprivation caused an
increase in expression of GLUL in HORCs, with a 3-fold increase observed
following total glucose deprivation at 24 hours. Increases in expression of SLC1A3 after
total glucose deprivation for 24 hours were also observed.
Conclusions:
Deprivation of glucose, but not of oxygen, caused RGC death in human retinal
explants. Glucose deprivation caused increased expression of genes involved
in regulation of extracellular glutamate concentrations, suggesting that
following glucose deprivation glutamate excitotoxicity may be involved in the
observed loss of RGCs.
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