Ischemia retinica: studi morfologici e funzionali
Author(s)
Catalani, Elisabetta
Date Issued
April 4, 2008
Type
Doctoral Thesis
Abstract
Ischemia is a primary cause of neuronal death in retinal diseases. The repertoire of
expressed transmitter receptors would determine the neuron’s responses to ischemic damage,
and peptidergic receptors may be involved. With a new in vitro model of the ischemic mouse
retina, we investigated whether an altered expression of somatostatin receptors could
modulate retinal responses to ischemia. We used retinas of somatostatin receptor 1 (sst1)
knock out (KO) mice, where sst2 are over-expressed and over-functional, and of sst2 KO
mice. TUNEL analysis of ischemic retinas showed a marked reduction of cell death in sst1
KO retinas, while there were no differences between wild-type (WT) and sst2 KO retinas. In
addition, caspase-3 mRNA expression was also reduced in sst1 KO as compared to WT
retinas. An immunohistochemical analysis demonstrated that different cell populations
responded differently to the ischemic insult, and that the persistence of some
immunohistochemical markers was greater in sst1 KO than in WT or in sst2 KO retinas. In
particular, rod bipolar cell survival was markedly improved in sst1 KO retinas, while it was
dramatically decreased in sst2 KO retinas. Furthermore, consistent with a role of glutamate
excitotoxicity in ischemia-induced neuronal death, retinal glutamate release was observed to
increase under ischemic conditions, but this increase was significantly reduced in sst1 KO
retinas. These observations demonstrate that an increased presence of functional sst2 protects
against retinal ischemia, thus implementing the background for the use of sst2 analogs in
therapies of retinal diseases such as glaucoma or diabetic retinopathy. On the basis of this
evidence, we tested the hypothesis that stimulation of over-expressed sst2 with exogenous
ligands may guarantee greater protection against ischemia. Isolated retinas from both WT and
sst1 KO mice were incubated in vitro for 1h in an ischemic solution and were treated with 10-
5 M SRIF, octreotide (a sst2 agonist) or SOM230 (a SRIF analog). As expected, in the absence
of pharmacological treatments, cell death was reduced in sst1 KO ischemic vs WT ischemic
retinas. In addition, SRIF, octreotide or SOM230 reduced cell death in ischemic WT retinas.
Surprisingly, instead of inducing larger rescue of retinal neurons, these treatments in ischemic
sst1 KO retinas resulted in significant increase of cell death throughout the retina. Our data
indicate that, although sst2 activation is beneficial against retinal ischemia, a limit exists to
this action, and the effects may turn detrimental. This limit may be due to receptor
desensitization or changes in the transduction pathways. Sst2, as the other SRIF receptors, are
G protein coupled receptors, therefore we investigated the molecules that regulate the activity
of G proteins in WT and in sst1 KO ischemic retinas. In particular, we investigated the
mRNA expression of G protein-coupled receptor kinases (GRKs) and that of regulators of G
protein signaling (RGSs). WT mouse retinas express GRK1 and GRK2, RGS1, RGS2, RGS5
and RGS9 subtypes. GRK1 and RGS1 mRNAs show a significant increase of their expression
after ischemic insult, therefore we investigated the expression of subtypes in ischemic retinas
following octreotide administration. WT ischemic retinas treated with octreotide showed a
significant reduction of both GRK1 and RGS1 mRNA expression. In octreotide-treated,
ischemic sst1 KO retinas, a similar reduction of GRK1 mRNA was observed, however, RGS1
mRNA expression remained unchanged with respect to untreated sst1 KO ischemic retinas.
The persistence of high expression levels of regulatory factors in octreotide-treated sst1 KO
retinas may inhibit sst2 responses and be responsible, at least in part, of the detrimental
effects of octreotide observed in sst1 KO ischemic retinas.
Hypoxia is the primary cause of retinal ischemia and it also causes a neoangiogenetic
response which is responsible of serious retinal illness. Recent data indicate that retinal
neoangiogenesis is contrasted by SRIF through its sst2 receptor. Using an in vivo mouse
model of hypoxia-induced retinopathy, we investigated the possibility that inhibition of
adenylyl cyclase (AC) is involved in SRIF anti-angiogenic actions. Hypoxia increased AC
responsiveness in wild type (WT) retinas and in retinas lacking sst2, but not in sst2-overexpressing
retinas. Hypoxia also altered AC isoform expression, but with different patterns
depending on sst2 expression level. Among the nine AC isoforms, AC VII isoform mRNA
and protein resulted the most affected. Indeed, in hypoxia AC VII expression was
significantly enhanced in WT retinas and it was further increased in sst2-lacking retinas, but
not in sst1 KO retinas. These data suggest an involvement of AC/cAMP in mediating both
hypoxia-evoked retinal neoangiogenesis and SRIF protective actions. In addition, the AC VII
isoform is a candidate to a main role in these mechanisms.
In conclusion, these studies contribute to the knowledge of the effects exerted by SRIF
in retinal disease and support the pharmacological use of SRIF, or SRIF analogues, in the
treatment of retinal pathologies.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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