| Abstract |
The discovery of neuroprotective agents for the treatment of retinal
disorders, such as diabetic retinopathy and age-related macular
degeneration, remains an important target for investigation. Retinal ischemia
leads to neovascularization and neurodegeneration of retinal neurons. Over
the years, therapies for ischemic neovascular diseases have been focused on
the regulation of the aberrant proliferation of blood vessels and involve laser
treatment (photocoagulation) and most recently the use of drugs that target
the VEGF system. However, there are no therapeutics that target the
neurodegenerative component. For the more efficacious treatment of
ischemic retinopathies and the preservation of vision, both the vascular and
neural elements of the retina must be treated.
Somatostatin is a cyclic neuropeptide with diverse actions in the
central and peripheral nervous system. It was discovered by Brazeau in 1973,
as the main inhibitor of growth hormone release from the pituitary. In the
retina, somatostatin is localized primarily in amacrine cells with processes that
ramify in the inner plexiform layer and in displaced amacrine cells in the
ganglion cell layer. Although the functional mapping of somatostatin
receptors (sst1-5) in the retina has been established, its role must be further
studied. Due to its inhibitory actions on the secretion of growth hormone, as
well as other growth factors important players in the neovascularization
process, somatostatin’s actions as an antivascular agent were investigated.
The results from these studies support that somatostatin can be a potential
treatment for ocular neovascularization. In addition, studies in brain suggest
that somatostatin and its sst2/5 analogues afford neuroprotection against
NMDA excitoxicity via a mechanism involving cGMP.
The main aim of this thesis was to investigate the neuroprotective role
of somatostatin and its analogues against retinal ischemia. The following
specific objectives were posed: a) to study the functional role of sst1 and sst2
somatostatin receptors, b) to establish a retinal model of ischemia, c) to
ascertain the neuroprotective actions of somatostatin and its analogues in
this model, and d) to elucidate the mechanism involved in the
neuroprotection.
The results of this thesis suggest that activation of sst2 receptors
regulates cGMP levels in the rat retina by increasing NO., suggesting an
important role of the sst2 receptor in the regulation of NO/cGMP signaling. In
addition, we demonstrate that somatostatin release is potassium and
calcium-dependent, thus neuronally released in rat retina. This release is
regulated in a negative manner by the activation of the sst1 receptor,
suggesting an autoreceptor role for somatostatin. These results revealed for
the first time the existence of a peptide autoreceptor in the retina.
In order to investigate the neuroprotective role of somatostatin and its
analogues in retinal ischemia, we developed the model of chemical
ischemia in the rat retina. This model was first used in hippocampal slices. It
includes the use of sodium cyanide and iodo-acetic acid, substances that
mimic conditions of anoxia and hypoglycemia. Incubation of the retina for
one hour in a mixture of chemical ischemia (sodium cyanide 25mM / iodoacetic
acid 5mM) in conditions of 5% CO2/95% air at 37°C resulted in an
attenuation of the number of amacrine cells containing
cholineacetyltransfarase (cholinergic), tyrosine hydroxylase (dopaminergic)
and NO. synthase and rod-bipolar cells, as determined by
immunohistochemical studies. These results were confirmed by TUNEL assays
depicting extensive cell death in the chemical ischemia treated samples.
Co-incubation of somatostatin and the chemical ischemia mixture did not
protect the retina. However, it was demonstrated with radio-immuno-analysis
(RIA) studies that under the conditions of chemical ischemia somatostatin was
strongly metabolized. However, the use of selective agonists of the sst2
subtype BIM23014 and MK678 as well as cortistatin provided neuroprotection
to the retina in a concentration dependent manner.
To study the mechanism via which somatostatin analogues protect the
retina from chemical ischemia, we investigated the involvement of
intracellular signaling pathway NO. / cGMP. As mentioned above, studies in
brain suggest that cGMP is involved in the neuroprotective actions of
somatostatin against NMDA excitotoxicity. Results from the present thesis as
well as from previous studies of our laboratory suggested an increase in cGMP
and NO. levels, respectively, after sst2 receptor activation in the retina. These
results prompted us to initially study the possible neuroprotective action of NO.
in the model of chemical ischemia and subsequently investigate whether this
mechanism is involved in the retinal neuroprotection afforded by the
somatostatinergic analogs.
NO. donors, NONOate and SIN-1 protected the retina from chemical
ischemia in a concentration-dependent manner. Similar results were
observed using the 8-Br-cGMP, an analogue of cGMP with high membrane
permeability. The blockade of NO. synthase and soluble guanylate cyclase,
the enzymes that catalyze the synthesis of nitric oxide and cGMP,
respectively, reversed the neuroprotective effects of the sst2 agonist
BIM23014.
The results of this study suggest new roles for sst2 and sst1 receptors in
the retina. They support that the model of chemical ischemia could be
adapted to the retina and will be useful in the study of new neuroprotective
targets of retinal disease. The sst2 somatostatin analogues provide retinal
neuroprotection via a mechanism involving the signaling pathway NO. /
cGMP.
The present data support future studies to probe further the
downstream mechanisms of somatostatin’s neuroprotection. In addition, the
data support the study of the neuroprotective properties of new somatostatin
analogues with a better pharmacokinetic profile (longer half life, better
bioavailability) and greater efficacy that may be effective in in vivo
preclinical and clinical studies and be beneficial as therapeutics in retinal
disease.
|
Search
