0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
will be likely to be independent from the prenatal/post-natal differences in
sex-hormonal.
III. In the case we find no specific sex-difference in behavioral/morphological
effect of Hg exposure [i.e. all groups will show similar degeneration/impairment]:
we will have been able to demonstrate that Hg exposure by
our approach is by far the most relevant factor in determining the autism-like
phenotype, i.e. massively overwhelm the weight of the other two independent
factors (i.e. sex hormones and reelin expression level). Thus, we will test in
additional experiments lower dosages of Hg to determine the threshold level
for Hg-induced neuroanatomical and neurofunctional alterations.
IV. In the case we will not find any behavioral/morphological alteration
in any Hg exposure group (2.00 or 4.00 ppm) as compared to respective controls
(0 ppm): we will test the effect of higher Hg dosages.
In particular, for the above mentioned experiments the behavioral/morphological
parameters to be measured as outcome indices will be the ones for
which an inter-group difference had been found in the first two years: all of
them have been described in the methods section.
The precise definition of the number of the animal used in the third year
would be settled on the basis of the results of the first two years of the study.
Technical notes
– Every two days the volume of water intake by the mother will be measured,
and the approximate daily dose will be extrapolated at the end of the
experiment.
– It has been demonstrated that the MeHg aqueous solution does not
need to be freshly prepared throughout the experiments: a concentration
modification of less than 3% has been demonstrated three days after modification
[Rasmussen and Newland 2001].
– Castration at P0 will be performed according to standard experimental
procedures [Lisciotto et al. 1990]. Since surgical stress per se has been shown
to affect brain development in several areas (see for instance) the control
groups throughout the whole study will be sham-operated as well.
DATA ANALYTIC STRATEGIES AND POWER ANALYSES
We have calculated the need of approximately 250 pups for the first two
years. This number of animals is compatible with the foreseen behavioral and
morphological assessments (we plan to process blocks of 14 animals each 3
weeks). Animal numbers in the third year will depend on the results of the
first two years. Statistical analysis will be performed by three-way ANOVA
with gender (including gonadectomized males), genotype (+/+ and rl/+) and
treatment (MeHg 0 ppm, 2 ppm, and 4 ppm) as independent variables, using
the Sigma Stat software package (version 3.1 for Windows); individual differences
will be tested by the Bonferroni-corrected t-test.
592 2009