Inaugural Charles River World Congress on Animal Models in Drug Discovery and Development

https://deepblue.lib.umich.edu/bitstream/2027.42/138112/1/12967_2017_Article_1274.pd

Impaired performance of the Q175 mouse model of Huntington’s disease in the touch screen paired associates learning task

Cognitive disturbances often predate characteristic motor dysfunction in individuals with Huntington’s disease (HD) and place an increasing burden on the HD patients and caregivers with the progression of the disorder. Therefore, application of maximally translational cognitive tests to animal models of HD is imperative for the development of treatments that could alleviate cognitive decline in human patients. Here, we examined the performance of the Q175 mouse knock-in model of HD in the touch screen version of the paired associates learning (PAL) task. We found that 10–11-month-old heterozygous Q175 mice had severely attenuated learning curve in the PAL task, which was conceptually similar to previously documented impaired performance of individuals with HD in the PAL task of the Cambridge Neuropsychological Test Automated Battery (CANTAB). Besides high rate of errors in PAL task, Q175 mice exhibited considerably lower responding rate than age-matched wild-type (WT) animals. Our examination of effortful operant responding during fixed ratio (FR) and progressive ratio (PR) reinforcement schedules in a separate cohort of similar age confirmed slower and unselective performance of mutant animals, as observed during PAL task, but suggested that motivation to work for nutritional reward in the touch screen setting was similar in Q175 and WT mice. We also demonstrated that pronounced sensorimotor disturbances in Q175 mice can be detected at early touch screen testing stages, (e.g., during “Punish Incorrect” phase of operant pretraining), so we propose that shorter test routines may be utilised for more expedient studies of treatments aimed at the rescue of HD-related phenotype

Detailed genetic and functional analysis of the hDMDdel52/mdx mouse model.

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level

Cerebral blood volume alterations in the perilesional areas in the rat brain after traumatic brain injury—comparison with behavioral outcome

In the traumatic brain injury (TBI) the initial impact causes both primary injury, and launches secondary injury cascades. One consequence, and a factor that may contribute to these secondary changes and functional outcome, is altered hemodynamics. The relative cerebral blood volume (CBV) changes in rat brain after severe controlled cortical impact injury were characterized to assess their interrelations with motor function impairment. Magnetic resonance imaging (MRI) was performed 1, 2, 4 h, and 1, 2, 3, 4, 7, and 14 days after TBI to quantify CBV and water diffusion. Neuroscore test was conducted before, and 2, 7, and 14 days after the TBI. We found distinct temporal profile of CBV in the perilesional area, hippocampus, and in the primary lesion. In all regions, the first response was drop of CBV. Perifocal CBV was reduced for over 4 days thereafter gradually recovering. After the initial drop, the hippocampal CBV was increased for 2 weeks. Neuroscore demonstrated severely impaired motor functions 2 days after injury (33% decrease), which then slowly recovered in 2 weeks. This recovery parallelled the recovery of perifocal CBV. CBV MRI can detect cerebrovascular pathophysiology after TBI in the vulnerable perilesional area, which seems to potentially associate with time course of sensory-motor deficit

Deposits of mHtt in retinal sections of R6/2 mice in different age groups.

<p>Scale bar = 50 µm.</p

Apoptotic cells were found exclusively in the outer nuclear layer (ONL) of R6/2 mice as revealed by TUNEL staining (TUNEL positive cells are marked with red arrows).

<p>No corresponding staining was observed in WT mice. NFL – nerve fiber layer, RGCL - retinal ganglion cell layer, IPL – inner plexiform layer, INL – inner nuclear layer, OPL – outer plexiform layer, ONL – outer nuclear layer, OS – photoreceptor outer segments. Scale bar = 200 µm.</p

Representative images from semi-thin (1 µm-thick) optic nerve cross-sections of WT and R6/2 mice showing intact morphology of axons in 18-week old R6/2 mice.

<p>Scale bar = 15 µm.</p

Representative ERG traces from one wild-type and two R6/2 mice for each recorded stimulus parameter at 8 weeks of age.

<p>Column A: Normal ERGs derived from a wild-type mouse. Column B: The least affected R6/2 mouse showing decreased amplitudes but preserved waveforms. Column C: A severely affected R6/2 mouse having only a hint of a minor positive going intrusion after a-wave for log 0.5 cd*s/m² flashes, and a minor b-wave for the –log 1.5 cd*s/m^-2 flash. Note: animals having no b-waves were cut out from further statistical analysis.</p

Progressively deteriorating ERG in R6/2 mice (in red) compared to their wild-type littermates (in blue).

<p>The uppermost rows represent the averaged ERG responses recorded at three different ages. Note that also wild-type responses change until animals reach a mature age. Solid lines represent a-wave statistics (mean±SEM) and dashed lines b-wave statistics in amplitude and latency graphs. B/a ratios presented on the lowest rows were calculated by dividing b-wave amplitudes by corresponding a-wave amplitudes. Statistically significant differences from t-test, or U-test in cases of unequal variances, are marked as *p<0.05; **p<0.01; ***p<0.001.</p

Images of β3-tubulin (in red) immunostained retinal sections from WT and R6/2 mice.

<p>The sections were counterstained for DAPI (in blue). Scale bar = 40 µm. NFL – nerve fiber layer, RGCL - retinal ganglion cell layer, IPL – inner plexiform layer, INL – inner nuclear layer, OPL – outer plexiform layer, ONL – outer nuclear layer.</p