0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

AISLA.1 – Validation of a combined pharmacological treatment to slow progression of disease…
paper tape lining onto the floor of the ramp. Stride length will thus be defined
as the distance between successive right-to-right and left-to-left footprints.
The paw grip strength will be assessed by placing the mouse on a the
wire-lid of a conventional housing cage. For this analysis, the mouse tail is
gently pulled to prompt the mouse to hold onto the grid before the lid is
swiftly turned upside down. The latency until the mouse let go with at least
both hind limbs is timed. Each mouse is given up to three attempts to hold on
to the inverted lid for a maximum of 90 sec and the longest latency is
recorded.
The Rotarod performance will be evaluated as the period (sec.) spent by
the mouse on a rotating axle without falling. The diameter of the rotating bar
is 3 cm and the speed of rotation increases gradually starting from 3,6 rpm
during the first 30 sec until 36 rpm during the last 30 sec of the test, which
ends after 5 min. If the mice fall before the 5 min time-laps they repeat the
test after a 5 min rest. The maximum time spent on rotating bar is taken as
measure for statistical analysis.
Mice will be killed when they will be unable to roll over when laid down
on their side and this time will be considered for the survival analysis.
Biochemical and histopathological analysis
The mice that will be used for these analyses will be sacrificed at the
time when the transgenic SOD1G93A mice that will receive the placebo will
show the 50% decrease in their performance. 20 mice per group will be
examined. For the biochemical analyses, the mice will be anesthetized with
Equithesin (30ml/10g body weight, ip.) and immediately sacrificed by decapitation.
The blood will be collected in normal eppendorf tubes while the
spinal cord and brain will be immediately removed and rapidly frozen on
dry ice and maintained at -80°C until the experiments. Samples from tissues
(spinal cord, brain, blood samples) from groups of 10 mice at different
stages of treatment will be subject to biochemical determinations such as
markers of mitochondrial function (e.g. mitochondrial complexes, ATP
level), oxidative stress (e.g. ROS, GSH/GSSG), level of cytokines (e.g. TNFa
and IL-1b) (U.O. 1).
For the immunohistopathological analyses, the anesthetized mice will
be transcardially perfused with 20 ml saline followed by 50 ml of sodium
phosphate buffered 4% paraformaldehyde solution. After the perfusion the
spinal cords and brains will be rapidly removed, shortly postfixed, transferred
to 20% sucrose solution in PBS overnight, then in 30% sucrose solution
until they sink and finally frozen in 2-methylbutane at -45˚C. Serial
frozen section will then be cut and processed for immunohistochemistry
with antibodies selectively recognizing motoneurons (ChAT) and glial cells
(astrocytes and microglia) in adjacent sections. The material will be
analyzed quantitatively for cell counts (percentage of surviving motoneurons
in the lumbar spinal levels; increase in the number of astrocytes and/
or microglia), and densitometric evaluation of immunosignal. An image
analysis system connected to the microscope will be used to obtain such
sets of data.
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