0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
Morphological studies on purkinje cells
Alternate sections from the brain specimens will be used for stereological
and other morphological analysis. In fact for stereology and regional mapping
we have previously used 1 out of 8 sections for the StereoInvestigator system
to calculate the total number of PCs [Biamonte et al. 2006], meaning that the
remaining sections can be used for other morphological analyses.
Stereological counts of Purkinje cells – For stereological counts of total number
of PCs for each animal, we will use an optical fractionator stereological design
[West et al. 1991] to produce unbiased estimates of total PC number using the
Stereo Investigator system [Stereo Investigator software, Version 4.04© 2000,
MicroBrightField Europe, Magdeburg, Germany]. In our set-up, a stack of
MAC 5000 controller modules [Ludl Electronic Products, Ltd., Hawthorne,
NY, USA] is configured to interface an Olympus BX 50 microscope with a
motorized stage and a HV-C20 Hitachi color digital camera with a Pentium II
PC workstation. A three-dimensional optical dissector counting probe (x, y, z
dimension of 30 × 30 × 10 mm respectively) is applied to a systematic random
sample of sites in coronal sections of the cerebellar cortex over the entire cerebellum.
For total PC counts for each animal we will evaluate every 8 th section
(beginning randomly with section 1-8) from the approximately 60 sections
taken through the cerebellum. The PC layer will be outlined through the
Stereo Investigator software using the 10× objective, while the 100× oil
immersion objective will be used for the PC counts.
Total PC number will be estimated according to the formula given below:
where SQ represents the total number of neurons counted in all optically sampled
fields of the cerebellum, ssf is the section sampling fraction, asf is the
area sampling fraction and tsf is the thickness sampling fraction [West et al.
1991]. We will count at least 5 cerebella per group; to obtain this, each group
will be planned for N= 7.
Regional analysis of PC loss – One specific question we will address is whether
the PC loss in rl/+ mice is more pronounced in the lateral hemispheres than in
the cerebellar vermis, as observed in some human autistic patients.
For the regional analysis of PC loss, one representative cerebellar sagittal
section will be used for the vermis and lateral hemisphere regions per experimental
animal. The number of EGFP-positive PCs in each section will be
counted manually in each lobule belonging to the anterior, central, posterior
and inferior lobes of the vermis and in the lobulus simplex, crus I and crus II
of the ansiform lobule, and paramedian lobule of the lateral hemispheres,
according to previously described procedures [Marti et al. 2002]. All counts
will be made at 20x magnification using a Leitz DMRB fluorescence light
microscope [Leica Microsystems, Milano, Italy]. We will use at least 5 mice
per group; to obtain this each group will be planned for N= 7.
Measurements indices of neuronal damage in the cerebellum
Neuronal DNA fragmentation – DNA fragmentation will be detected by
TUNEL-assay method using In Situ Cell death Detection kit, POD [Roche].
TUNEL staining will then be developed by DAB and 0.01% H 2
O 2
[Sigma Fast
588 2009