I identified several phenotypes in the Tc1 brain using the automated pipeline, the first such morphometric analysis of a DS brain model compared with WTs, and revealed local differences not distinguishable between samples by eye, histology, or segmentations alone.
In human DS, BV is reduced by around 18% (Aylward et al., 1999; Pinter et al., 2001a; White et al., 2003). Whereas the whole mandible is reduced in size in human DS, O’Doherty et al. (2005) measured only partial mandible reduction in Tc1s, and no craniofacial malformation or reduction in skull size. Distances between landmarks in
µCT images of Tc1 skulls also exhibited no significant size difference compared with WTs. I observed significantly increased Tc1 TIV, BV, GM and WM volumes (Table 4.1a).
This unexpected global finding was consistent in C1 and C2 – scanned independently and with considerations for gradient scaling – and indicates the utility of whole-brain MR and tissue segmentation over histology and landmark measurements, which are necessarily localised and limited to a few subjects and by rater variability.
All tissues and most parcellated regions displayed greater volume variance in the Tc1s. O’Doherty et al. (2005) reported that approximately 66% of Tc1 brain nuclei retain Hsa21. This mosaicism means Hsa21 levels are unpredictable and vary between organs, mice, and with background, leading to phenotypic variation (Reeves, 2006). Olson et al. (2004) noted: “most DS phenotypes are incompletely penetrant and variable in expressivity – the mechanism(s) by which increased gene dosage causes any specific DS feature is not established”. This could potentially be addressed using morphometry with more selective trisomic animal models.
The ventricles are enlarged in human DS (White et al., 2003) and both VBM and TBM detected their bilateral enlargement in the Tc1s. This is likely underestimated in ex vivo brains, which shrink slightly during fixation as tissues relax and ventricles partially collapse (§5.7; Zhang et al., 2010). Although mitigated in-skull (Sawiak et al., 2013), one would expect the same systematic effect across groups. Brain expansion due to hydrocephalus has been reported in a DS model (Yu et al., 2010); however, these mice died by 10 weeks of age, and the Tc1s did not exhibit gross ventricular enlargement of the same degree. (Also, only 6.5% of those mice exhibited hydrocephalus. They had rounded and enlarged skulls. Although I investigated volume, I did not quantify the shape or roundedness of the Tc1 brains. This would be possible, however, using their principal axes, as calculated in §3.3.) Overall vCSF volumes did differ significantly between groups before and after correction for TIV. However, this measurement is likely an underestimate, due to unpredictable ventricular collapse. Upon visual inspection, one brain exhibited uncollapsed 3rd and 4th ventricles absent of CSF (and therefore appearing
dark), however, this came from a WT littermate. As the ventricular spaces were present without bright vCSF, segmenting this brain required manual attention.
That the UFL atlas-segmented ventricles were significantly different in volume prior to TIV normalisation is likely a result of the atlas registering poorly in these regions, again owing to ventricular collapse in our data. The only tissue segmentation-derived region to exhibit a significant difference in volume surviving Bonferroni correction was GM/WM mixture (not shown). This may be due to increased PV in the Tc1 group, or imprecision in boundary delineation of this PV class (Fig 3.15); corresponding parcellations (broadly, the caudate putamen/striatum; midbrain; superior and inferior colliculi) showed differences. As noted in §3.6, for these reasons of imprecision, I later removed this measurement and replaced it with more smoothly-varying GM and WM segmentations, which better account for PV.
One may safely conclude that the Tc1 mouse exhibits an enlarged brain, in contrast to people with DS. Tc1 mice have some rearrangements of their copy of Hsa21 (Gribble et al., 2013), and it is possible that this contributes to the megaly phenotype; there may also be some non-specific interaction of the human chromosome in the mouse cells that gives rise to larger brains. Finding relatively greater subcortical GM volumes in DS patients compared to the total GM volume, and preservation of GM volume in the parietal cortex, Pinter et al. (2001a) suggested that greater basal ganglia and thalamus volumes in humans may result from insufficient apoptosis.
In humans and mice with deletion or truncation of the Hsa21 gene DYRK1A3, brain size and weight is reduced (Sebrié et al., 2008; Guedj et al., 2012). Through phosphorylation, this gene is thought to be tied to many DS phenotypes, and it is likely modulated by the presence of other genes (Wiseman et al., 2009). It is dose-dependent and hence, in humans and Tc1 mice, it is overexpressed. In two mouse models4 of partial trisomy, overexpressing the DYRK1A gene, Sebrié et al., (2008) and Guedj et al. (2012) found increased brain size (measured via MRI, weight and histology). The thalamus, midbrain
3: dual-specificity tyrosine-phosphorylation-regulated kinase 1A. 4: hYACtgDyrk1a and mBACtgDyrk1a.
and colliculus were preferentially affected. In the thalamus, neuronal density and number increased, while both neuron size and extracellular space decreased. Conversely, cell density was negatively correlated with DYRK1A dosage in the hippocampus and somatosensory and entorhinal cortex (Guedj et al., 2012). This may underlie my TBM results, which showed expansion of the Tc1 midbrain and central thalamus. Guedj et al. noted that Ts65Dn mice, also with three copies of DYRK1A, do not exhibit elevated BVs, and that other genes may compensate.
In subsequent voxel-wise statistical tests I controlled for TIV to reveal differences in the Tc1 group independent of the total volume increase. TBM detected significant bilateral, local volume reductions in the olfactory bulbs. Bianchi et al. (2014) recently observed, via histology, impaired neurogenesis in the olfactory bulbs of 13-month-old Ts65Dn mice, and remarked that this may parallel the loss of smell in older human DS individuals. The reductions seen here suggest there may be a similar functional impairment in Tc1 mice.
I found significant local reductions in GM volume within the cerebellum, using both TBM and VBM, focussed medially in Declive VI, as well as unilaterally within the simple lobule and lobules 4/5, in both granular and molecular cell layers.
Cerebellar GM was reduced in a VBM study of non-demented people with DS, and exhibits reduced overall volume compared with TIV in humans (Raz et al., 1995; White et al., 2003) and the Ts65Dn, Ts1Cje and Tc1 mouse models (Ma et al., 2014; Olson et al., 2004). My TBM analysis reveals that rather than the cerebellum being uniformly reduced in volume, reductions have discrete local foci. There is evidence cerebellar lobules have distinct functional correlates (Stoodley & Schmahmann, 2010). It may be possible to map local volume reductions to functional topography and hence to behaviour in Tc1 mice.
The cerebellum is associated with fine motor control and cognitive processes. In children with DS, cerebellar hypoplasia is implicated in motor and speech difficulties (Pinter et al., 2001a). Galante et al. (2009) found motor learning and coordination deficits in Tc1 mice. Histological staining revealed reduced internal granule layer density in the Tc1
cerebellum compared with WTs, mirroring observations of the Ts65Dn and Ts1Cje models (Baxter et al., 2000; O’Doherty et al., 2005; Olson et al., 2004). I repeated these findings with VBM, showing reduced GM density in the granule cell layer of several lobules (Fig 4.7, 4.8). This supports the utility of VBM for informing histology. VBM also showed bilateral reductions in GM density in the entorhinal cortex, recapitulating the progressive atrophy of this region in human DS (Teipel et al., 2004).
VBM also detected reduced GM proportion in the dentate gyrus (DG). Long-term potentiation in the DG – synaptic plasticity thought to be directly related to long-term memory – was found to be reduced in Tc1 mice (O’Doherty et al., 2005), and behavioural observations demonstrated reduced spatial working memory (Morice et al., 2008). I also observed bilaterally elevated GM proportion in the CA3 region of the Tc1 hippocampus. Insausti et al. (1998) measured elevated neuronal numbers in Ts65Dn CA3, and suggested this may compensate for reductions in DG, although Kurt et al. (2004) found normal neuron density, but reduced synapse density, in both structures. Witton et al. (2015) also recently showed decreased synapse density in the DG, and related this to the poorer performance of Tc1 mice in a radial arm maze, compared with WTs. These cellular changes could underpin the differences in VBM GM signal observed here. Mouse brain tissue classification is complicated by smaller structures and greater PV proportion than is found in humans. Both may be mitigated using higher field strengths, enabling greater SNR, spatial precision, and contrast (Natt et al., 2002). Structural differences between the in vivo, skull-stripped NUS atlas used for tissue segmentation and this ex vivo, in-skull data were resolved with NRR. By employing this atlas, and explicitly modelling background, external tissues, PV and CSF, my tissue classifications included fine WM detail, including the deep WM of the cerebellum and PV regions such as the striatum and midbrain. I averted misclassifications which have befallen previously published TPMs, including the presence of a brain-enveloping ‘rim’ where GM PV is misinterpreted as WM (Fig 2.8).