In light of the dysfunctional consequences of the mutant PrP
association with α2δ-1, disrupting their binding might represent a means for therapeutic intervention. The production of Tg mice expressing wild-type, PG14, and D177N/V128 mouse PrPs with an epitope for the monoclonal antibody 3F4 has already been reported by Chiesa et al. (1998) and Dossena et al. (2008). In this study we LY2157299 used Tg mice of the Tg(WT-E1+/+) line, which expresses about four times the endogenous PrP level, referred to throughout the text as Tg(WT); we also used Tg(PG14-A3+/−) and Tg(D177N/V128-A21+/−) mice expressing Tg PrP at approximately one time, referred to as Tg(PG14) and Tg(CJD), respectively. These mice were originally generated on a C57BL/6J X CBA/J hybrid and were then bred with the Zurich I line of Prnp0/0 mice ( Büeler et al., 1992) with a pure C57BL/6J background (European Mouse Mutant Archive, Monterotondo, Rome; EM:01723). C57BL/6J mice were purchased from Charles River Laboratories. All procedures involving animals were conducted according to European Union (EEC Council Directive 86/609, OJ L 358,1;
December 12, 1987) and Italian (D.L. n.116, G.U. suppl. 40, February 18, 1992) laws and policies, and were in accordance with the United States Department of Agriculture Animal Welfare Act and the National Institutes of Health Policy on Humane Care and Use of Laboratory Animals. They were reviewed and approved by the Mario Negri Institute Animal Care and Use Committee that includes ad hoc members for ethical ever issues (18/01-D, see more 18/01-C). Animal facilities meet international standards and are regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, experimental protocols, and review of procedures. Animals were anesthetized with 1% isoflurane in a 30%:70% O2:N2O gas mixture and imaged in a horizontal bore 7-Tesla USR preclinical MRI system (BioSpec 70/30; Bruker BioSpin, Germany) with a shielded gradient insert (BGA 12, 400 mT/m; rise time, 110 us). A 7 mm birdcage resonator for RF transmission and a 10 mm diameter single-loop receiver coil were used to receive the signal. T2-weighted anatomical
images of the mouse brain were acquired with the following parameters: TR 2500 ms, TE 50 ms, RARE factor 16, FOV 3 × 1.5 × 1.5 cm, Matrix 256 × 102 × 102, voxel 0.147 × 0.117 × 0.147. The scan time was approximately 25 min. The cerebellar volume was quantified using the ImageJ software (http://rsbweb.nih.gov/ij/). We used an accelerating Rotarod 7650 model (Ugo Basile). Juvenile mice were tested starting from 19 days of age (P19) for 7 consecutive days. On the first day a training session was done during which each mouse was placed on the Rotarod at a constant speed (4 rpm) for a maximum of 60 s. Then they were assessed in three consecutive test sessions with a 10 min intertrial resting period. They were positioned on the rotating bar and allowed to become acquainted with the environment for 30 s.
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