We further investigated whether α
2-SAs
would be neuroprotective when administered after retinal ischemia. When
administered 1 hour after the onset of reperfusion, BMD was also
effective in preventing the death of approximately 19%, 12%, and 22%
of the RGC population by 7, 14, or 21 days, respectively
(Fig. 4) .
Moreover, the densities of FG-labeled RGCs did not decrease further
between 14 and 21 days in the BMD-treated groups, but there were
significant reductions in the vehicle-treated groups. Taken together,
these results show that, when administered 1 hour after ischemia, BMD
partially prevented RGC death and diminished the slow loss of RGCs
observed between 7 and 21 days. In the group of animals treated with
BMD 2 hours after ischemia there were also significant neuroprotective
effects. However, the neuroprotective effect was lost when BMD was
injected 4, 24, or 72 hours after onset of reperfusion, presumably
because it were administered at a time when a large proportion of RGCs
were already committed to die.
48
RGC death after transient ischemia of the retina may result in both
acute excitotoxic necrotic cell death and delayed apoptotic cell
death.
44 45 46 47 48 Because posttreatment with anα
2-SA resulted in increased RGC survival after
ischemia and diminished the secondary slow loss of RGCs, it is possible
that α
2-SAs have antiapoptotic effects. Recent
studies, in neuronally differentiated PC12 cells deprived of serum and
nerve growth factor (NGF) and in cerebellar granule neurons placed in
low-K
+ medium, indicate thatα
2-SAs may halt apoptotic degradation by
maintaining the mitochondrial permeability and avoiding release of
degradation-signaling factors, suggesting thatα
2-SAs act early on the cascade of events
leading to apoptosis.
63 The absence of neuroprotection ofα
2-SAs against axotomy-induced RGC death
observed in this study
(Table 4) may be interpreted as an indication
that the pathogenic pathways activated by transient ischemia or axotomy
differ,
17 although leading to a common cell death
mechanism, apoptosis. An inhibitor of a late event in apoptosis, the
irreversible wide-range CPP32-like caspase inhibitor Z-DEVD-cmk, was
found to be neuroprotective against both axotomy- and ischemia-induced
RGC death.
17 An effect on the early events that lead to
ischemia-induced apoptosis could explain whyα
2-SAs were effective against ischemia- but not
axotomy-induced RGC death.
Finally, it is possible that the neuroprotective effects ofα
2-SAs are due to specific upregulation of
survival pathways in the retina through activation of Müller
cells.
64 Systemic administration of anα
2-SA induces activation in Müller cells
of extracellular signal-regulated kinases (ERKs),
64 which
are classic members of the mammalian mitogen-activated protein kinases
(MAPKs). The ERK signaling pathway appears to mediate survival
effects.
65 For example, brain-derived neurotrophic factor
(BDNF)–mediated neuroprotection in ischemic–hypoxic brain injury in
vivo is known to be mediated through activation of ERKs.
66 In addition to ERKs, two other types of MAPK, known as stress-activated
protein kinases, are thought to be involved in the signaling pathways
to apoptosis induced by various stress and injury stimulus, including
glutamate excitotoxicity
17 67 and cerebral
ischemia.
68 The observation thatα
2-SAs selectively activate MAPKs in
Müller cells may be taken as an indication that these cells,
which maintain integrity and normal function of the retina, may play an
important role in protecting retinal neurons.
64
The authors thank Larry Wheeler for helpful discussions during the
course of this work, Constantino Sotelo for critical comments on the
manuscript, Marcelino Avilés for advice, and Sergio
Mayor-Torroglosa, Diego Martínez-Pérez, and M. Elena
Aguilera-Meseguer for technical support.