0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

CAMPUS.1 – Analysis of developmental interactions between reelin haploinsufficiency, male…
performed in subset of experiments. In subgroups of mice from each treatment
group sacrificed at P21 we will measure Hg content in cerebellum using
HPLC.
Animals and treatments
One male mouse a time will be kept with 3 females for 2-3 days. Thereafter
the females will be removed and each one of them will be kept in a single
cage. The females will be checked out daily for delivery and day of delivery
will be considered P0. Mice will be weaned at P21.
Exposure to methyl mercury (Me Hg) will be through the drinking water.
The concentration of MeHg in the bottle will be as follows: a) 0 ppm (“ controls
” group); b) 2.0 ppm (“ low MeHg ” group); c) 4.0 ppm (“ intermediate
MeHg ” group). These doses have been extrapolated from the existing literature
in rodents as potentially sub-threshold for neurotoxic effects in the cerebellum
(2.0 ppm) or slightly toxic (4.00 ppm). However, in light of the difficulty
of extrapolating data from different species and mice strains, we will
need to validate such findings in our mice. Depending on the results from this
experimental step we might decide to add further groups of treatment with
different doses of MeHg.
A group of mothers will be treated pre-delivery (“ prenatal exposure ”). In
particular, after mating they will be treated daily with MeHg in the water bottle
mixed with tap water, starting from P0 they will receive regular water
without MeHg. A second group of mothers will be exposed to the same doses
of MeHg as above, but only in the lactating phase, i.e. from P0 to P21, which
is approximately the day in which pups start sucking directly from the bottle
(“ postnatal exposure ”).
For each mother only 8 pups will be allowed (the remaining ones will be
sacrificed; some of them for data collection, too, such as Hg level measurement
at P0-see below). Mice will be genotyped starting from DNA extracted
from tails fragments collected at P0. They will be genotyped for the rl jx
mutation by a standard method slightly modified from D’Arcangelo et al.
[1996], and for the expression of EGFP by the method by Tomomura et al.
[2001].
Concerning male mice submitted to castration at P0, the male offspring
will be either bilaterally gonadectomized or sham-operated under cryogenic
anesthesia according to standard methods [see for instance Lisciotto et al.
1990]. Gonadectomy will be performed within 2 hours after birth, since
serum testosterone in newborn male mice increases more than 2 times within
2 hours after delivery, to reach levels that are comparable to adult males, and
thereafter rapidly declines [Corbier et al. 1992].
Animals will be sacrificed at P21. Briefly, mice will be deeply anaesthetized
by i.p. injection of a mixture of 2mg/ml ketamine, 0.2 ml/10 g body
weight [Ketavet, Gellini Farmaceutici, Italy] and 0.23 mg/ml medetomidine,
0.24 ml/10 g body weight [Domitor, Orion Corp., Espoo, Finland] and perfused
via the left ventricle with a phosphate-buffered 4% paraformaldehyde
solution for 10 minutes. The brains will be removed, placed in the same fixative
over-night, cryo-protected with 30% sucrose and then frozen at -80 °C.
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