0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

MERCK.1 – Effects of interferon b-1a and cladribine on synaptic neurodegenerative damage…
(degenerative) phases of EAE. The effects of both compounds will be compared
with those of corticosteroids. We will select the striatum for our investigation
since this subcortical brain area is particularly prone to develop neurodegenerative
damage in the course of MS and EAE [Centonze et al. 2009].
Our objectives will be pursued:
a) By studying the effects of interferon b-1a or cladribine treatments on
clinical score in EAE mice.
b) By studying if interferon b-1a or cladribine treatments are able to prevent
the synaptic defects of glutamate transmission measured by means of
whole-cell patch-clamp recordings in corticostriatal brain slices prepared
from EAE mice in the acute and chronic phases of the disease.
c) By studying if interferon b-1a and cladribine are able to reduce neuronal
damage and dendritic spine loss measured through the Golgi technique.
The final goal of the study is to provide a reliable mechanism at the basis
of the putative neuroprotective action of interferon b-1a and cladribine in
experimental MS.
METHODS
The experiments will be performed in Rome at the IRCCS Fondazione
Santa Lucia.
As described [Centonze et al. 2009], chronic-progressive EAE will be
induced in 6-8 week old C57BL/6 female mice by subcutaneous immunization
with 300 ml of 200 mg major oligodendrocyte glycoprotein(MOG)35-55
(Espikem, Florence) in incomplete Freund’s adjuvant supplemented with
8 mg ml -1 Mycobacterium tuberculosis (strain H37Ra; Difco). Pertussis toxin
(Sigma) (500 ng) will be injected on the day of the immunization and again
two days later. EAE mice will be compared with two control (HC) groups, one
composed of naïve, untreated mice, and a second of mice treated with complete
Freund’s adjuvant without MOG, and pertussis toxin. Body weight and
clinical score (0=healthy; 1=limp tail; 2=ataxia and/or paresis of hindlimbs;
3=paralysis of hindlimbs and/or paresis of forelimbs; 4=tetraparalysis;
5=moribund or death) will be recorded daily. Drug or vehicle administration
will be started 1 week after the immunization and continued for the next 4-5
weeks. All efforts will be made to minimize animal suffering and to reduce the
number of mice used, in accordance with the European Communities Council
Directive of November 24, 1986 (86/609/EEC). All procedures involving animals
will be performed according to the guidelines of the Institutional Animal
Care and Use Committee of the Fondazione Santa Lucia, Rome.
To study synaptic transmission, we will perform in vitro whole-cell patch
clamp electrophysiological recordings in corticostriatal brain slices prepared
from EAE mice at different (acute and chronic) stages of their disease. As a
measure of striatal synaptic functioning, in these models we will study the
intrinsic membrane properties of the recorded neurons (membrane potential,
input resistance, firing activity and current-voltage relationship), evoked
(eEPSCs), spontaneous (sEPSCs) and miniature glutamate-mediated excita-
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