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. 2017 Oct 9;12(10):e0185970.
doi: 10.1371/journal.pone.0185970. eCollection 2017.

Behavioral Testing of Minipigs Transgenic for the Huntington gene-A Three-Year Observational Study

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Free PMC article

Behavioral Testing of Minipigs Transgenic for the Huntington gene-A Three-Year Observational Study

Verena Schuldenzucker et al. PLoS One. .
Free PMC article

Abstract

Background: Large animal models of Huntington's disease (HD) may increase the reliability of translating preclinical findings to humans. Long live expectancy offers opportunities particularly for disease modifying approaches, but also challenges. The transgenic (tg) HD minipig model assessed in this study exhibits a high genetic homology with humans, similar body weight, and comparable brain structures. To test long-term safety, tolerability, and efficacy of novel therapeutic approaches in this model reliable assessments applicable longitudinally for several years are warranted for all phenotypical domains relevant in HD.

Objective: To investigate whether the tests proposed assessing motor, cognitive and behavioral domains can be applied repetitively over a 3-year period in minipigs with acceptable variability or learning effects and whether tgHD minipigs reveal changes in these domains compared to wildtype (wt) minipigs suggesting the development of an HD phenotype.

Methods: A cohort of 14 tgHD and 18 wt minipigs was followed for three years. Tests applied every six months included a tongue coordination and hurdle test for the motor domain, a color discrimination test for cognition, and a dominance test for assessing behavior. Statistical analyses were performed using repeated ANOVA for longitudinal group comparisons and Wilcoxon-tests for intra-visit differences between tgHD and wt minipigs.

Results: All tests applied demonstrated feasibility, acceptable variance and good consistency during the three-year period. No significant differences between tgHD and wt minipigs were detected suggesting lack of a phenotype before the age of four years.

Conclusions: The assessment battery presented offers measures in all domains relevant for HD and can be applied in long-term phenotyping studies with tgHD minipigs. The observation of this cohort should be continued to explore the timeline of phenotype development and provide information for future interventional studies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Arrangement of stables.
Arrangement of the stables (each~12m2) in the ZTE showing all six groups with their individual distribution of genotypes (tg = transgenic, wt = wildtype). The assessment area is located in the middle of the stables and hosts the variable setups outlined in Figs 2 and 3.
Fig 2
Fig 2. Setup.
Basic experimental setup used for the assessment battery, which can be modified to the needs of the individual tests. The startboxes SB-A and SB-B of 1.20m2 are located at the end of the 3 m long walkway. The walkway is accessible through trapdoors A and B and holds variable setups as outlined in Fig 3.
Fig 3
Fig 3. Setup of behavioral tests.
(A) In the tongue coordination test, pigs had to enter the walkway and approach the tongue board (TB). The ability to recover the rewards (cornflakes) from holes with continuously increasing depth from left to right was assessed. (B) The hurdle test aimed to assess gait coordination under challenge compared to normal walking. (C) The discrimination test was designed to evaluate the cognitive domain. Minipigs had to explore all boxes and were supposed to learn and remember that only the blue box could be opened. (D) The dominance test was applied to assess behavior. Two animals entered the setup from opposite sides and the animal pushing the opponent backwards was considered dominant. Calculation of an index after exposure of each animal to all group mates was used to determine hierarchy within groups (modified from [27]).
Fig 4
Fig 4. Tongue test.
Results of the Tongue coordination test (means of three runs, compared between tgHD and wt minipigs). (A) “Initiation time” i.e. time the pigs need to enter the walkway. (B) “Investigation time”, i.e. time needed to enter the walkway and start investigating (nose contact) the tongue board. (C) “Exploration time”, i.e. time needed to recover all reachable treats and return to the startbox A. (D) “Depth of holes”, i.e. maximal depth recovering treats from tongue board (TB). [* = p≤0.05, ** = p≤0.01, *** = p≤0.001]
Fig 5
Fig 5. Hurdle test.
Results of the Hurdle test (means of six runs, compared between tgHD and wt minipigs per visit). (A) “Initiation time”, i.e. time the pigs need to enter the walkway. (B) “Run time”, i.e. time to complete the walk and arrive in startbox B.
Fig 6
Fig 6. Discrimination test.
Results of the blue box Discrimination test at each visit v1 –v6 (means of six runs, compared between tgHD and wt minipigs). (A) “Initiation and exploration time”, i.e. time needed to enter the walkway and open the correct (blue) box. (B) and (C) show the number of attempts to open the red or the yellow box before opening the correct box, respectively.
Fig 7
Fig 7. Discrimination test—Reversal learning.
Results of the yellow box reversal learning Discrimination test for each visit v1 –v6 (means of six runs, compared between tgHD and wt minipigs). (A) “Initiation and exploration time” needed to enter the walkway and open the correct (yellow) box. Figs (B) and (C) show the number of attempts to open the red or the blue box before opening the correct box. Measures in Figs A-C show a significant decrease in time and number of attempts during the course of the study. (D) Negative correlation between age and “initiation and exploration time” (***). [* = p≤0.05, ** = p≤0.01, *** = p≤0.001]
Fig 8
Fig 8. Dominance test.
Results of the Dominance test. The figures shows the Clutton-Brock-Index (CBI) [27]. (A) Mean CBI compared between tgHD and wt minipigs at each visit (v1-v6). (B), (C) and (D) sample CBIs of individual pigs in groups 1, 4 and 6 (v1-v6).

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Grant support

The George-Huntington-Institute has received grant support from the CHDI Foundation (www.chdifoundation.org) to conduct the work reported in this study to RR. We are grateful for additional relevant support provided by private donations of families affected by HD that would like to remain anonymous. The authors are not aware of any competing interests of these donors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no other financial interests related to this manuscript.
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