Animal models of human disease provide insights into pathophysiology at a molecular level, and gene discovery in dogs has become an important resource. In human medicine, neuroimaging techniques have helped to improve the definitions of hindbrain and cerebellar malformations. The classification of midbrain, cerebellar, and hindbrain malformations has evolved from several image-based classification systems to a classification system that is based on embryonic development (1–4). This system was produced by modern neuroimaging techniques as well as increased understanding of correlations between gene mutations and certain brain imaging phenotypes. The recognition of phenotypic heterogeneity of certain gene mutations and the fact that different gene mutations may result in nearly identical neuroimaging findings has further contributed to the understanding of these mutations (4, 5). Many of these data are derived from animal studies (2).

Genetic cerebellar hypoplasia (CH) in Eurasier dogs represents the first genetically defined CH that has been described in dogs. It is caused by a fully penetrant recessive mutation in the very-low-density-lipoprotein-receptor gene (VLDLR) (6, 7). Genetic testing for breeders is now available (6, 7). The VLDLR is part of the Reelin signaling pathway and influences cell migration and positioning during embryonic development, especially in the cerebellum (8–10). The neuroanatomic changes are absence of the caudal aspects of the cerebellar vermis and the cerebellar hemispheres in association with large retrocerebellar fluid accumulations (11, 12). Initial observations suggested an abnormal configuration of the caudal (cranial) fossa and an enlarged caudal fossa as an additional imaging feature in some puppies with VLDLR-associated CH resembling a Dandy–Walker like malformation [Figure 1; reported in Table 3 of Ref. (7)]. The evaluation was problematic, however, because of the lack of reference data in the literature related to caudal fossa size in puppies and the different imaging modalities used.

Therefore, the aim of this study was to validate the use of the caudal fossa ratio (CFR) from area measurements of midsagittal brain images to assess caudal fossa size in dogs of various age groups, body weights, and breeds. Specifically, we aimed to explore the application of CFR to puppies with and without VLDLR-associated CH to define the animal model in more detail.

Caudal fossa ratio was calculated from midsagittal brain images of 130 dogs. Study design was retrospective and observational. The data of the dogs in this study were used with the consent of their owners and the breed club. The study was approved by the institutional research review board (#09-06-2014).

The caudal cranial fossa henceforth in this manuscript referred to as caudal fossa for ease of reading, which is called the posterior fossa in humans, is the inner surface of the skull that surrounds the brainstem and cerebellum. Its rostral and caudal borders are the tentorium cerebelli and the foramen magnum (13, 14). A variety of brainstem and cerebellar malformations are associated with disturbed growth of the cranium (2, 3). Consequently, assessment of caudal fossa size should be part of the description and definition of animal models of brain malformations. A precise description of congenital malformations enhances comparisons with analogous diseases in humans.

Our findings establish CFR as a reliable parameter for assessment of the relative size of the caudal fossa. The measurements demonstrate excellent agreement within and between raters (intra-, inter-rater agreement). There is only a minor influence of imaging modality on the ratio and results are in the same range as reported from other authors (13, 16).

An important finding is the lack of impact of weight on CFR (Figure 6). This finding supports the use of CFR as a parameter that is independent of the size of the dog and can be applied to large dog breeds as well as small dog breeds and over a wide range of weights. Dog breeds also differ according to skull morphology. A review of groups of dogs from different breeds indicated an influence of breed on CFR that should be addressed in future studies. While we failed to find overall influence of a brachycephalic or a mesaticephalic skull, there were visible differences in CFR between small and large brachycephalic breeds.

We postulate that area measurements are precise parameters and reflect the basic configuration and the size of the caudal fossa in relation to the whole skull. It has been stated that three-dimensional assessments (volume measurements) are more accurate for assessment of caudal fossa size than midsagittal area measurements in dogs (17). However, this statement occurred in the context of an investigation of pathomechanisms in Chiari malformation/syringomyelia in Cavalier King Charles Spaniels in which assessment of overcrowding requires precise measurements of the proportions of brain parenchyma and CSF space. Our investigations in 64 dogs with various skull conformations showed a close correlation between midsagittal caudal fossa area and caudal fossa volume. This result confirms the validity of CFR and provides further support for the use of CFR for the characterization of animal models. It should be noted, however, that previous studies in humans showed variable results and either weak (18) or no (17) correlation between area and volume measurements. Others have successfully applied a brainstem/cerebellar ratio for diagnosis of cerebellar atrophy and cerebellar degeneration in dogs (19). They also showed close correlation between area and volume measurements. Both brainstem/cerebellar ratio and CFR may be used for phenotyping cerebellar and hindbrain malformations in dogs.

Our data show that puppies up to 4 months (19 weeks) of age have a smaller CFR than adult dogs (Figure 4). This observation has not appeared in the literature to date. Some investigators excluded dogs younger than 12 months of age from measurements based on their assumption of an influence of incomplete skull growth (20, 21). Other researchers considered dogs younger than 12 months of age for their measurements (13, 22) or dogs as young as 4 months of age and assumed that there was no influence of skull growth on their results (23). Early diagnosis of brain malformations is advantageous to breeders and clients. Furthermore, neuroimaging of affected dogs and their littermates may be necessary for confirmation and precise definition of the phenotype for genetic investigations (7). A smaller CFR in puppies up to 4 months of age reflects discordant growth of the skull in puppies in which the growth of the caudal fossa is smaller. Disproportionate growth started at 50 days in German Shepherd puppies. Excessive growth of the caudodorsal cranium with development of the external sagittal crest and occipital protuberance occurred from days 70 to 107 (15).

We used CFR to validate caudal fossa size in an animal model of a genetic CH. A variant in the very-low density lipoprotein receptor gene (VLDLR:c.1713delC) is the cause of inherited CH in the Eurasier dog breed. Inheritance is recessive and genetic testing is now available for breeders to avoid the disease (6). Clinically, homozygous dogs present with non-progressive ataxia which is first evident when the animals start to walk. VLDLR is part of the Reelin signaling pathway and involved in neuronal migration and cerebellar development (8–10). The analogous human disease has been described as disequilibrium syndrome and involves mutations in VLDLR. To date, there have been 12 reports from 15 families and 45 involved persons (24–30). Both humans and dogs present non-progressive cerebellar (truncal) ataxia as the main complaint. Neuroanatomic changes are strikingly similar between the two species. Caudal aspects of the cerebellar vermis and the cerebellar hemispheres are absent and replaced by large retrocerebellar fluid accumulations. Our measurements of CFR in Eurasier dogs with VLDLR-associated CH and appropriate controls confirm the presence of two variants: variant 1 with a grossly enlarged caudal fossa and variant 2 with a small to normal-sized caudal fossa. This observation has several implications. First, it proves phenotypic variability with regard to the size of the caudal fossa in dogs with VLDLR-associated CH. Wide variability in the size of the posterior fossa from normal to large is also seen in humans and mice with FOXC1-associated Dandy–Walker and related malformations (5). Furthermore, our observations also support an interaction between the cerebellum and the overlying mesenchyme (meninges, skull) during development. The concept that cerebellar and posterior skull development are closely linked through interactions between the rhombic lip and the overlying mesenchyme (3, 31) arose from observations in FOXC1 mice with Dandy–Walker malformations. These mice express FOXC1 only in the mesenchyme overlying the cerebellum and not in the cerebellum itself (5). Finally, variability in genetic background and interaction between different genes could also contribute to the conformation of the caudal fossa in this dog breed. We observed slightly more variability in caudal fossa size in unaffected Eurasier dogs compared to other dog breeds. Finally, there could be an impact of environmental or vascular factors during development. Limitations of this study are obviously the lack of MRI from breed- and age-matched controls as well as absence of measurements on T1-weighted midsagittal images and that we combined images from a 1.5 and 1.0 T MR. Furthermore, the size of the breed groups was too small to asses the influence of breed and larger cohorts with more breeds representatives of their skull type and separating large and small brachycephalic dogs (and other skull types) into different control groups need to be examined. However, CFR in some pupies with vldlr-associated CH was even larger than in adult Eurasier dogs and clearly exceeded any of our control dogs.

In summary, our data show that CFR is a reliable parameter with a negligible influence of imaging modality, examiner, and weight of the dog and a minor influence of breed. The CFR in healthy adult dogs lies within 0.255 and 0.330, but CFR is smaller in puppies. Furthermore, the midsagittal area of the caudal fossa is positively correlated with caudal fossa volume. Measurements in Eurasier dog puppies with genetic VLDLR-associated CH demonstrated the presence of a variant with an enlarged caudal fossa. This observation further substantiates the application of CFR for characterization of translational animal models of cerebellar and hindbrain malformations.

The study was carried out in accordance with the German Animal Protection law. The protocol was approved by the institutional research review board of the Clinic of Small Animal Medicine; #09-06-2014. Dog owners approved use of their dogs’ data for scientific purposes by signature in clinical records. Some images from diseased dogs were contributed by the breed club with the consent of the owners for research purposes.

AL, AF, AB, and ALM designed and coordinated the study. AL and AB determined the measurements. JF and AK performed the statistical analysis. MS contributed in acquisition of the data. AL, AF, and JF wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.