Modelling Neurological Diseases in Large Animals: Criteria for Model Selection and Clinical Assessment

Abstract

Issue: The impact of neurological disorders is recognised globally, with one in six people affected in their lifetime and few treatments to slow or halt disease progression. This is due in part to the increasing ageing population, and is confounded by the high failure rate of translation from rodent-derived therapeutics to clinically effective human neurological interventions. Improved translation is demonstrated using higher order mammals with more complex/comparable neuroanatomy. These animals effectually span this translational disparity and increase confidence in factors including routes of administration/dosing and ability to scale, such that potential therapeutics will have successful outcomes when moving to patients. Coupled with advancements in genetic engineering to produce genetically tailored models, livestock are increasingly being used to bridge this translational gap. Approach: In order to aid in standardising characterisation of such models, we provide comprehensive neurological assessment protocols designed to inform on neuroanatomical dysfunction and/or lesion(s) for large animal species. We also describe the applicability of these exams in different large animals to help provide a better understanding of the practicalities of cross species neurological disease modelling. Recommendation: We would encourage the use of these assessments as a reference framework to help standardise neurological clinical scoring of large animal models.

Full text

Citation: Eaton, S.L.; Murdoch, F.;
Rzechorzek, N.M.; Thompson, G.;
Hartley, C.; Blacklock, B.T.;
Proudfoot, C.; Lillico, S.G.;
Tennant, P.; Ritchie, A.; et al.
Modelling Neurological Diseases in
Large Animals: Criteria for Model
Selection and Clinical Assessment.
Cells 2022,11, 2641. https://doi.org/
10.3390/cells11172641
Academic Editor: Zhongde Wang
Received: 25 July 2022
Accepted: 22 August 2022
Published: 25 August 2022
Publisher’s Note: MDPI stays neutral
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Copyright: © 2022 by the authors.
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4.0/).
cells
Review
Modelling Neurological Diseases in Large Animals: Criteria for
Model Selection and Clinical Assessment
Samantha L. Eaton 1, *,† , Fraser Murdoch 1, †, Nina M. Rzechorzek 2, † , Gerard Thompson 3,4, Claudia Hartley 1,
Benjamin Thomas Blacklock 1, Chris Proudfoot 5, Simon G. Lillico 1, Peter Tennant 5, Adrian Ritchie 5,
James Nixon 5, Paul M. Brennan 6, Stefano Guido 1,7, Nadia L. Mitchell 8, David N. Palmer 8,
C. Bruce A. Whitelaw 1, Jonathan D. Cooper 9and Thomas M. Wishart 1,*
1
Royal (Dick) School of Veterinary Studies and Roslin Institute, University of Edinburgh, Easter Bush Campus,
Roslin, Midlothian EH25 9RG, UK
2
Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
3
Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent,
Edinburgh EH16 4SB, UK
4Department of Clinical Neurosciences, NHS Lothian, 50 Little France Crescent, Edinburgh EH16 4TJ, UK
5
The Large Animal Research & Imaging Facility, Royal (Dick) School of Veterinary Studies and Roslin Institute,
University of Edinburgh, Easter Bush Campus, Roslin, Midlothian EH25 9RG, UK
6Translational Neurosurgery, Centre for Clinical Brain Sciences, University of Edinburgh,
Edinburgh EH16 4SB, UK
7
Bioresearch & Veterinary Services, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent,
Edinburgh EH16 4SB, UK
8Faculty of Agriculture and Life Sciences, Lincoln University, P.O. Box 85084, Lincoln 7647, New Zealand
9
Departments of Pediatrics, Genetics, and Neurology, Washington University School of Medicine in St. Louis,
660 S Euclid Ave, St. Louis, MO 63110, USA
*Correspondence: sam.eaton@roslin.ed.ac.uk (S.L.E.); t.m.wishart@roslin.ed.ac.uk (T.M.W.);
Tel.: +44-(0)-131-651-9125 (S.L.E.); +44-(0)-131-651-9233 (T.M.W.)
† These authors contributed equally to this work.
Abstract:
Issue: The impact of neurological disorders is recognised globally, with one in six people
affected in their lifetime and few treatments to slow or halt disease progression. This is due in part to
the increasing ageing population, and is confounded by the high failure rate of translation from rodent-
derived therapeutics to clinically effective human neurological interventions. Improved translation is
demonstrated using higher order mammals with more complex/comparable neuroanatomy. These
animals effectually span this translational disparity and increase confidence in factors including routes
of administration/dosing and ability to scale, such that potential therapeutics will have successful
outcomes when moving to patients. Coupled with advancements in genetic engineering to produce
genetically tailored models, livestock are increasingly being used to bridge this translational gap.
Approach: In order to aid in standardising characterisation of such models, we provide comprehensive
neurological assessment protocols designed to inform on neuroanatomical dysfunction and/or
lesion(s) for large animal species. We also describe the applicability of these exams in different large
animals to help provide a better understanding of the practicalities of cross species neurological
disease modelling. Recommendation: We would encourage the use of these assessments as a reference
framework to help standardise neurological clinical scoring of large animal models.
Keywords: neurological disease; large animal model; clinical assessment; model selection criteria
1. Introduction
Neurological disorders affect one in six people globally and are the leading cause of
disability-adjusted life-years (DALY) that accounts for the combined burden of disease,
health risks, and premature death [
1
–
3
]. This equates to nearly 1 billion people worldwide
with conditions caused by a broad range of factors which ultimately result in the altered
Cells 2022,11, 2641. https://doi.org/10.3390/cells11172641 https://www.mdpi.com/journal/cells

Cells 2022,11, 2641 2 of 27
structure and/or function of the nervous system. These conditions can vary greatly in
terms of aetiology, age of onset, and duration, but collectively represent a significant
healthcare burden. The United Kingdom’s publicly funded National Health Service spends
approximately GBP 4.4 billion annually on the treatment and management of neurological
conditions, the vast majority of which are currently incurable [
1
]. Brain disorders in the
United Kingdom alone are estimated to exceed a cost of GBP 100 billion per year [
4
].
Effective therapeutic strategies are therefore in high and urgent demand.
In the field of neurodegenerative research, translation of potential therapeutics from
the laboratory to the clinic has a high failure rate. One reason for this failure is an over-
reliance on rodent model systems, which often do not recapitulate key aspects of the
human condition of interest, and are developed in the physiological context of a small,
nocturnal prey species [
5
–
7
]. Such work is critical in advancing our understanding of the
molecular mechanisms governing the function and dysfunction of the mammalian nervous
system. However, it is increasingly accepted that the use of larger, more physiologically
complex model systems is required to effectively assess the translational relevance of novel
therapeutic developments for human interventions. Using more physiologically relevant
model systems can reduce waste in translational research and increase the likelihood of
promising treatments achieving regulatory approval [8,9].
Whilst some might consider the choice of one animal model system over another to
be equally unpalatable, it matters when this has the potential to efficiently generate more
clinically relevant data, with a smaller number of animals used in fewer, more targeted
experiments. It also matters when the same outputs can be used to enhance life quality and
advance treatment for veterinary patients with similar neurological disorders. A ‘better’
model, coupled with careful study design and species–specific ethical considerations
guided by specialists trained in comparative neuroanatomy, behaviour, physiology, and
neuropathology has the power to truly embrace 3Rs objectives [
10
]. ‘Larger’ does not
necessarily mean ‘better’, and ‘better’ requires evidence and validation.
Any model system is limited not only by its physiological representation of the condi-
tion in question, but also by our understanding of that disease. From the outset, a detailed
and robust characterisation is necessary to determine how effectively the model repro-
duces the pathophysiological hallmarks of the corresponding human condition, but also
to define ‘biomarkers’ for therapeutic intervention. Biomarkers of disease could include
behavioural, imaging-based, or molecular markers, or (ideally) a combination of these
features, that should track with disease progression and provide an insight to the ideal
time when therapies should be administered to maximise their efficacy. Sometimes, the
distinct consequences of a human mutation in a non-human animal can be as insightful
for understanding the human disease as any similarities—it is therefore vital that any
observations diverging from those expected are reported with as much rigour and care as
the similarities, and not simply discarded as an aspect of ‘model failure’.
Given the complexity and composition of the nervous system, behavioural or neu-
rological tests are often the first line of assessment in diagnosing neurological disorders
in human patients [
11
]. Assessment of species–species behaviour and mental status also
forms an important and early aspect of managing patients in the veterinary clinical set-
ting [
12
]. Followed by a complete general physical and systematic neurological evaluation
(some aspects of which must be tailored to the species), these examinations can confirm
whether a neurological deficit exists, and provide an accurate neuroanatomical diagnosis
or limit the possible causes of clinical signs [
13
]. Repeating these assessments over time
can monitor how a neurological disease progresses and importantly, the effectiveness of
any therapeutic intervention [14].
Recent developments in genome editing have given us the ability to generate bespoke
mutations in almost any species required in order to fill translational gaps with physio-
logically relevant models [
15
]. The most ethically viable and physiologically appropriate
systems for neurological modelling are of livestock origin (sheep and pig) [
16
–
19
]. Yet,
there are a limited number of publications that cover neurological assessment specifically

Cells 2022,11, 2641 3 of 27
in livestock [13,14,20–23]. This reflects the reality that most livestock presenting with neu-
rological disease would usually be culled prior to or as a result of in-depth examinations,
since treatment of these cases is rarely justifiable on welfare, biosecurity, and/or financial
grounds [
24
]. This in turn leads to fewer veterinary neurologists who specialise in produc-
tion animal neurology, since the demand for this expertise is substantially lower than in
companion animal medicine.
Here, we discuss the key factors relevant to model selection with appropriate options
for premortem assessments that would enable comprehensive and longitudinal monitoring
of a neurological phenotype in large animal models. Species–specific considerations (such
as size and demeanour) with implications for their implementation will also be highlighted,
with a focus on production animals. Postmortem extraction, processing, preservation,
and neuropathological analysis of tissues are critical aspects to optimise when validating
any disease model, but these are covered well in other texts [
25
–
27
] and are beyond the
scope of this work. Likewise, although several human neurological conditions have their
counterparts in equids [
13
,
28
], for financial and safety reasons, these species are not a
practical choice for comparative study beyond the veterinary clinical research setting and
are therefore not covered in any detail here.
2. Model Selection
What is the need?
Research underpinning therapeutic development, particularly in
the field of neurodegenerative conditions, requires animal models. Some fundamental
research questions can be answered using culture-based systems, but the complexity of
the human nervous system, coupled with the (now accepted) multi-systemic nature of
many of these conditions, means that appropriate and effective therapeutic development
mandates
in vivo
systems [
29
–
32
]. This includes, but is not limited to, considerations
such as scaling up, routes of administration (especially important with viral-mediated
therapeutics), distribution and persistence, immune response, and physiological coupling
to the day–night cycle [33].
What are the options?
In order to address research needs for clinical development
there are a few options in which large animals can be used where appropriate:
Using diseases of large animals to inform on human disease
—when assessing natu-
rally occurring neurological conditions in domestic species, veterinarians typically classify
neurological diagnoses according to the ‘VITAMIN D’ acronym (Table 1). For each of these
categories, there are notable examples of translational relevance. There are obvious gains to
be made from studying these clinical cases as they arise, and in considering the potential for
human and veterinary medical developments based on repurposing of successful animal
intervention trials. However, the sporadic nature of neurological case presentation and
the inherent challenges of veterinary clinical research (with ethical considerations and
breed-related factors) reduce the opportunities to study clinical populations with adequate
control and statistical power to meaningly inform human medicine and clinical practice on
a desirable timescale.

Cells 2022,11, 2641 4 of 27
Table 1. Classification of neurological conditions using VITAMIN D acronym.
Neurological Disease
Category Large Animal Example Translational Relevance Reference(s)
Cerebrovascular accident Ischaemic stroke [34]
Vascular Amyloid angiopathy with cerebral haemorrhage
in dogs Haemorrhagic stroke [35]
Listeriosis in sheep (infection of the trigeminal nerve
and associated brainstem nuclei by
Listeria monocytogenes
Listeriosis [36]
Meningoencephalitis of unknown origin (presumed
immune-mediated) in dogs Autoimmune encephalitis [37]
Primary progressive
multiple sclerosis [38]
Anti-NMDAR encephalitis in dogs and bears Anti-NMDAR encephalitis aka
“brain on fire” [39–41]
Infectious/
inflammatory
Toxoplasma-induced encephalitis in utero leading to
abortion due to livestock feed contamination with
cat faeces Toxoplasma encephalitis [42,43]
Vertebral osteomyelitis in sheep and cattle Vertebral osteomyelitis [44]
Rabies Rabies [45]
West Nile encephalomyelitis in horses West Nile meningoencephalitis [46]
Cysticercosis/taeniasis Cysticercosis/taeniasis [47]
Hydatid disease Hydatid disease [48]
Louping ill Louping ill [49]
Lyme disease Lyme disease [50]
Tick-borne encephalitis Tick-borne encephalitis [51]
Vertebral fracture Vertebral fracture [52]
Peripheral nerve injury Peripheral nerve injury [53]
Traumatic Traumatic brain and spinal cord injury Traumatic brain and spinal
cord injury [54]
Cerebellar hypoplasia (inherited or infectious origin) Dandy-Walker syndrome [55,56]
Chiari-like malformation Chiari-malformation [57]
Spina bifida Spina bifida [58]
Anomalous Hydrocephalus (infectious, inherited, nutritional or
toxic origin) Hydrocephalus [59]
Hypoxic-ischaemic encephalopathy in foals or birth
asphyxia in calves Neonatal hypoxic ischaemic
encephalopathy [60]
Equine nigropallidal encephalomalacia (chewing
disease) causes a Parkonsonian-like phenotype due to
ingestion of toxic Russian knapweed Movement disorder [54]
Metabolic/toxic Closantel toxicity Demyelinating disorders [21,61]
Tetanus and botulism Tetanus and botulism [62]
Organophosphate intoxication Organophosphate intoxication [63]
Spastic paresis in cattle (hereditary) Spastic paresis [64]
Idiopathic Epilepsy Epilepsy [65,66]
Narcolepsy/cataplexy Narcolepsy/cataplexy [67]
Brain tumours (intra and extra axial)
e.g., glioma, meningioma Brain tumours (intra and
extra axial) [54]
Lymphosarcoma leading to spinal ataxia
(caused by bovine leukaemia virus) Spinal lympoma [68]
Neoplastic/nutrional Swayback (enzootic ataxia) in lambs due to copper
deficiency in the ewe during pregnancy Menkes syndrome [69]
Thiamine deficiency in ruminants leading to
polioencephalomalacia (cerebrocortical necrosis) Wernicke-Korsakoff syndrome [70]
Prion disease (scrapie in sheep; bovine spongiform
encephalopathy in catlle)
Creutzfeld-Jakob disease (CJD),
variant CJD (vCJD), Kuru,
Gerstmann–Sträussler–
Scheinker (GSS)
[71,72]
Degenerative Cerebellar abiotrophy (often hereditary in ruminants) Cerebellar abiotrophy [73]
Lysosomal storage diseases (CLN5 & 6 in sheep, CLN2
in dogs, Sandhoff disease in cats, Tay-Sachs disease) Lysosomal storage diseases [74–78]
Canine degenerative myelopathy (multi-system central
and peripheral axonopathy in senior dogs 8 years+)
Amyotrophic lateral sclerosis
(ALS) and motor neurone
disease (MND) [79]
Spinal muscular atrophy in brown swiss calves Spinal muscular atrophy [80]

Cells 2022,11, 2641 5 of 27
Spontaneous mutations in animal populations
—animal models of neurological dis-
ease that exist naturally are an obvious study choice, especially for those conditions where
there are multiple genetic and environmental factors which impact on disease phenotype.
These models have worked successfully in certain conditions where scientists have been
notified of neurological phenotypes and, following appropriate genetic testing, a specific
breeding cohort has been established for future studies (this is true of canine narcolepsy,
muscular dystrophy in Golden Retriever dogs; and CLN5 and CLN6 neuronal ceroid lipo-
fuscinosis (NCL) in South Hampshire and Merino sheep, respectively [
81
–
83
]). However,
due to the low incidence of animals which harbour mutations causing these conditions
naturally, their identification and ability to generate a breeding nucleus is problematic.
This is compounded by late presentation and/or diagnosis by which point breeding be-
comes unethical, or the animals have already been neutered such as in CLN1 NCL disease
in canines [84,85].
Sporadic cases can be developed as a research tool. For example, a promising gene-
editing strategy to restore dystrophin expression was developed in dogs with a canine
version of Duchenne muscular dystrophy [
86
]. Similarly, enzyme replacement therapy
trials in the naturally occurring CLN2-deficient Dachshund model of NCL directly led
to successful implementation of cerliponase alfa (Brineura) treatment in patients with
early results indicating a cessation or slowing of disease progression [
87
–
89
]. Trials in
these animals has informed on clinical trial design, dosage, and routes of administration
in patients producing successful outcomes [
88
]. This led to Brineura obtaining Food
and Drug Administration (FDA) approval in a significantly shorter timeframe [
88
]. In
another example, studies in sheep with endemic scrapie have determined that blood is
infectious in animals devoid of clinical symptoms, which has fundamentally changed blood
donation protocols and legislation in light of the Variant Creutzfeldt–Jakob disease (vCJD)
epidemic [
89
,
90
]. Moving forward, collaborations are combining expertise in human and
veterinary neurology to elucidate molecular mechanisms of immune-mediated encephalitis
for the benefit of both human and feline patients [91–93].
However, given the limitations around the sporadic nature of these useful mutations
appearing and the fact that some therapeutic interventions require the exact mutation
as identified in the patient population, it is often necessary to generate bespoke models.
This approach is beneficial for identification of any altered molecular pathway(s) and/or
cellular functions that are perturbed
in vivo
yet are not present in transgenic knockout or
spontaneously arising animals that are phenotypically similar to its human counterpart,
but with a distinct genetic aetiology [94].
Bespoke model systems
—Tailor-made models of human disease are now well estab-
lished [
15
,
95
]. Genetic engineering of livestock places specific disease-causing gene into
species that can more effectively model human physiology than rodents [
17
–
19
]. However,
the generation of a new model for any neurodegenerative condition is a decision which
should not be taken lightly.
Benefits of generating models of neurological disorders in large animals have already
been reported [
8
]. A new model of ataxia telangiectasia in pigs has given novel insights into
cerebellar Purkinje cell loss at birth, which was not observed in the murine model, as well as
producing a multi-systemic phenotype as documented in human disease [
19
,
96
]. An ovine
model of CLN1 disease produced a lifespan, clinical presentation, and duration that more
closely resembles the human disease. Cerebellar density is altered similarly to humans;
a feature not observed in the rodent models [
17
]. A porcine model of neurofibromatosis
type 1 mimics more of the hallmarks of human disease than the heterozygous mouse
models including the classical caféau lait macules and neurofibroma phenotypes [
18
].
Such improvements in clinical phenotypes observed in large animal models can not only
provide a better understanding of disease but importantly an opportunity for increased
translational therapeutic efficacy on this intermediary platform [
9
]. There are also practical
and translational advantages that result from using large animal models; due to their more

Cells 2022,11, 2641 6 of 27
complex brain and body size they are better suited to testing drug delivery systems and
carry out therapeutic biodistribution studies [97].
Considerations:
Ethical
—must be a priority when modelling any disease with either ‘spontaneous’/
naturally occurring systems or bespoke engineered animals (for which, a critical need that
cannot otherwise be satisfied must exist). This is especially important for neurological
disorders which often have a devastating phenotype and/or result in neurological deficits
that impact life quality long before a humane endpoint is reached. The potential clinical
benefit needs to be weighed against the lived experience cost to the individual animal; the
combined expertise of experienced animal handlers, veterinarians with specialist skills both
in neurology and the species of interest, and personnel trained in animal welfare and ethics
are essential. Appropriate experimental end points must be established during the study
design and are typically much earlier than would necessitate euthanasia in the veterinary
clinical setting. Ultimately, clinical success in neurological patients will be best achieved
by early disease detection and early intervention. As our ability to identify and treat the
earliest stages of disease in appropriate animal models increases, the need and justification
for progressing research animals to a humane end point will diminish.
Legislation and guidelines
—Appropriate legislative and welfare measures must be
in place when defining humane end points and this will differ from country to country.
In the United Kingdom, for example, horses, dogs, and cats (in addition to non-human
primates) are specially protected and subject to even stricter regulations that other species.
The establishment of a National Centre for the Replacement Refinement and Reduction
of Animals in Research [
10
] further encourages best practice through dissemination of the
ARRIVE and PREPARE Guidelines [
10
,
98
,
99
]. In the UK, the use of animals for scientific
purposes is regulated by the Animals (Scientific Procedures) Act 1986; however, every
country will have their own regulations, which should be considered when deciding on
model selection.
Costs
—the cost of large animal model generation and upkeep is considerable but the
benefits for appropriately bridging the translation gap leading to effective therapies cannot
be underestimated. For example, when modelling a neurological condition that correlates
with advanced age, the lifespan of large animal models is more readily suited to late
onset phenotypes due to their longevity. However, such model systems require significant
financial support for their long-term maintenance, assessment, and sample collection.
The real term cost of large animal model generation is in the region of 25
×
greater
than the cost to create a bespoke rodent model [
10
,
100
] and may only generate a handful of
founders. Founders will then need to be fully characterised to understand if the engineered
disease model has recapitulated phenotypic traits observed in patients. Investment is
vital; a successful model can become cost effective by decreasing time from drug trial to
regulatory approval, and it increases confidence that therapy or interventions have a better
chance of translating to humans [97,101].
Practicalities
—if using multiple cohorts for longitudinal studies, practical consid-
erations regarding the number of offspring that can be produced from a species, and
seasonality of breeding, should be noted. For example, pigs can have two litters per year
independent of the season, with each litter comprising 10–12 piglets, but this is generally
lower in minipigs 6–8 [
102
]. By contrast, sheep are typically seasonal breeders and have in
the region of 1–3 lambs per year, although some breeds, e.g., Poll Dorsets can lamb up to
three times in a 2-year period. Importantly, when selecting a specific species in which to
develop a model, it is essential to consider the practicalities of carrying out specific neuro-
logical and neuroimaging assessments in that large animal model. For example, certain
breeds of sheep are not ideal for Magnetic Resonance (MR)-based neuroimaging and/or
surgical delivery of therapeutics due to the presence of horns that prevent application of a
suitable head coil or optimal positioning of a craniotomy, respectively. Unless well-trained
and habituated in advance, pigs are not amenable to clinical assessment of certain facial
sensation parameters, due to fear responses or aggression. Viral or pharmaceutical delivery

Cells 2022,11, 2641 7 of 27
trials can be successful in pigs if the condition they are modelling presents at young ages,
but are limited in adults (excluding mini pigs) due to their gross size and weight presenting
challenges to imaging, (e.g., manoeuvring into a scanner) (see Table 2). Finally, behavioural
assessment requires experience in recognising and accounting for behaviours that are
normal/abnormal for the species within the environment in which they are being tested.
Table 2.
Validity of clinical assessment procedures to determine site-specific neurological deficits in
different large animal models.
Species Tests Are Applicable
Test Target Area Feline Canine Ovine Porcine *
Head & Neck / Cranial Nerves
Menace response Cranial nerve II & VII, visual
& motor cortex, cerebellum + + + +
Pupillary light reflex (PLR) Cranial nerve II & III + + + +
Vestibulo-ocular reflex (VOR) Cranial nerve III, IV, VI & VIII + + + +
Startle response Cranial nerve VIII,
auditory cortex + + + +
Dazzle reflex Cranial nerve II & VII + + + +
Jaw tone Cranial nerve V + + + +
Nasal sensation Cranial nerve V, forebrain + + + +
Facial sensation Cranial nerve VII + + + +
Swallow reflex Cranial nerve IX, X, XI + + + +
Tongue tone Cranial nerve XII + + + +
Spinal cord & proprioception
Cutaneous trunci Spinal cord (afferent T3-L1)
(efferent C8/T1) + + + +
Step/visual and tactile placing test Visual system and cerebellum + + + –
Knuckling/paw or hoof
placement test
Spinal cord forelimb (C1-5)
Hindlimb (C6-T2) + + + –
Patellar reflex Spinal cord segments L4 & L5 + + + –
Hop test Visual cortex, cerebellum, and
spinal cord + + + –
Push test Somatosensory
cortex, cerebellum + + + –
Withdrawal (flexor) reflex: Forelimb
Spinal cord (C6-T2), lower
motor neurons + + + –
Withdrawal (flexor) reflex:
Hindlimb
Spinal cord (L4-S2), lower
motor neurons + + + –
Perineal (anal) reflex Spinal cord S1-3 + + + –
* Minipigs should be treated as ovine only if they have been well-handled.
In summary, there is a clear need for larger animal model systems to bridge transla-
tional gaps and build on existing work using simpler model systems; however, as with any
experimental paradigm, there are a number of considerations which must be taken into
account when planning longer term characterisation and interventional assessments.
3. Clinical Assessment
In order to fully diagnose and/or characterise any neurological condition, it is im-
perative to assess the clinical signs the patient is exhibiting, and to provide evidence of
location, severity, and progression of disease [
11
]. Similar assessment criteria are applicable
to animals which model any given disorder, but aspects of the exam will need to be tailored
to the species of interest, and cognitive testing must be limited to the objective analyses
currently available in veterinary medicine. In essence, symptomatic information can only
be obtained from and communicated by patients. It is prudent to assume that some im-
pacts on life quality will be missed in veterinary species simply because we are unable to
recognise them, and this is an important welfare consideration.
Understanding exactly what examinations can be undertaken in different species
and their comparative translation from to human patients is fundamental for obtaining

Cells 2022,11, 2641 8 of 27
robust characterisation of a human neurological disease model. It is good practice to use a
system that gives an appropriate range of scores to easily differentiate severity and give
margins for improvement or decline. For instance, depending on the expected neurological
progression, a scale of 0–8 is likely more appropriate than scoring 0–3 (0 = normal to
3 = severe
). Furthermore, a more comprehensive scoring system allows for subtle changes
to be recorded with the caveat that this would be observer-dependent and continuity by
consistently using the same scorer is preferred to maintain an equivalent level of subjectivity.
Interpretation of reflex tests is best approached more cautiously, limiting the range of scores
to what can be observed objectively and reproducibly in the given species. Pilot testing
should be performed to determine the expected intra- and inter-observer variability in
clinical scoring before embarking on any experimental study with live animals.
Neurological examinations are designed primarily to detect the presence of neuro-
logical dysfunction and then to determine the neuroanatomical location(s) that has been
affected by damage or disease [
103
,
104
]. In the clinical setting, neurolocalisation is es-
tablished prior to generating a list of differential diagnoses (Table 1), primarily because
different diseases can affect different parts of the nervous system in a focal, multifocal, or
even diffuse manner [
105
]. For these examinations to have meaningful results, they should
be undertaken by an experienced veterinarian to score the experimental animals, and
appropriate age-matched controls. This may require a specialist in veterinary neurology
and/or a veterinarian that specialises in the species under test (ideally both). Some of the
earliest signs of disease may also be detected by the personnel who manage the animals on
a daily basis; their experience and observations are invaluable and should be documented
carefully. The animals should be handled often enough that routine examinations are
tolerated as a non-stressful experience, but not in such a way that habitual responses to
touch mask true deficits in other sensory modalities. It is good practice for the personnel
involved in husbandry and the examiner to be ‘blinded’ to the disease status of the animal,
where possible. If behavioural testing of each animal is needed away from the flock/herd,
the presence of a familiar handler is paramount [106].
Whilst it is best practice to perform the most aversive aspects of the neurological
exam last, the order in which tests are presented below is not intended to dictate the
order in which they should be carried out—this will vary by species, experimental design,
and preference of the examiner. The exact sequence of tests is less important than being
systematic, comprehensive, and keeping the sequence consistent across animals, and across
repeated measures for an animal, in any given experiment. It is highly recommended
to document key elements of the examination (particularly behaviour, posture, and gait)
using a standardised video recording protocol. Alongside documenting progression of
neurological signs, video enables additional ‘hands off’ assessment by multiple observers
who can be blinded to disease status and stage; moreover, slowing of the viewing speed
permits subjective (and sometimes objective) evaluation of subtle deficits.
Here, we describe specific assessments to determine the neuroanatomical location of
of dysfunction, highlighting some practical aspects most relevant to livestock species. As
with any veterinary species, ‘hands off’ evaluations should be performed prior to tests that
involve progressively more handling.
3.1. Mental Status and Behaviour
This initial assessment can provide valuable additional information to the owner/
handler’s management history. The examiner should observe the animal in situ, first from
a distance (without being disturbed) and then closer (without being handled) in order to
judge how it responds and interacts with everyday environmental stimuli [
13
,
22
]. The level
and quality of consciousness should be assessed, with the level of consciousness being
classified as alert, obtunded, stuporous, or comatose (in order of increasing deficit) [
12
].
An animal which is bright and/or quiet, alert, and attentive to its environment would
require an intact cerebrum and ascending reticular activating system, which extends over
the entire length of the brainstem to activate the cortex [
12
,
107
]. Unusual interaction or

Cells 2022,11, 2641 9 of 27
dulled responses to the surroundings therefore indicate a lesion to the forebrain and/or
brainstem [20]. Notably, large chronic forebrain lesions in livestock can be clinically silent
and may therefore correlate poorly with neuroimaging results [
13
]. The quality or ‘content’
of consciousness refers to inappropriate responses to stimuli or abnormal behaviours that
can occur even in the presence of a high level of consciousness [12].
Head pressing or ‘getting stuck in corners’, compulsive wandering, head/neck turning
and circling (typically towards the worst affected side), and signs associated with blindness
(such as tripping over or bumping into obstacles), suggest problems associated with the
thalamocortex, often with a metabolic, traumatic, toxic, or inflammatory
cause [13,20,104]
.
Seizure activity, whether generalised or focal, establishes forebrain dysfunction and typi-
cally comprises a humane end point. Sometimes seizure activity can be elicited by auditory
or tactile stimuli [
13
]. In the absence of classic generalised tonic–clonic activity with auto-
nomic signs, however, other types of seizure activity can be challenging to detect and to
differentiate from stereotypy, syncope, movement disorders, or sleep disorders without
continuous video monitoring and electroencephalography.
3.2. Posture and Stance
Posture is evaluated systematically in three anatomical regions: (1) head, (2) body, and
(3) limbs [
22
]. Careful assessment of an animal’s muscle tone and condition in each region
(1, 2, and 3), identifying any muscle asymmetry and/or atrophy through observation and
palpation, can provide clues for further investigation [
108
]. If tolerated, straightening the
head and neck along the midline can help with assessment of any head/neck deviation,
for which musculoskeletal problems must be ruled out [
13
]. Otherwise, abnormalities in
attitude or positioning of the head (e.g., head tilting) and/or limbs in relation to the body
(e.g., leaning, broad-based stance, one or more limbs are not situated in their normal axial
position) indicate a lesion of the vestibular system [
105
]. The vestibular system’s primary
function is maintenance of balance and positioning of the eyes, neck, body, and limbs
relative to the movement of the head [14].
Importantly, a head tilt must be distinguished from a head turn; the former specifies a
lesion affecting the vestibular system, whereas the latter is a sign of forebrain dysfunction.
It is possible however for a severe vestibular lesion to produce a head tilt together with a
head and neck turn [
13
]. Severe forebrain disease, myotonia, and visual loss can all result in
a ‘star-gazing’ posture with opisthotonos of the head and neck [
13
]. Star-gazing may be part
of decerebrate rigidity, but other acute neurological postures such as decerebellate rigidity,
and Schiff–Sherrington posture are far more common in small companion animals [
12
].
Kyphosis, lordosis, scoliosis, torticollis, and abnormal limb postures such as palmigrade
or plantigrade stance are rarely observed in livestock beyond congenital disorders and
acquired trauma [13].
3.3. Gait Analysis
Gait analysis can help track the effect of disease progression on vestibular, motor, and
proprioceptive function as well as visual capacity [
109
]. Along with mentation, gait should
be initially assessed (within reason) in the animal’s normal environment prior to more
hands-on elements of the neurological exam. That said, for some species (especially equids),
the most informative gait assessment usually results from a combination of examiner-led
maneuvers using halter and lead rope, with and without tests of strength, balance, and
proprioception through tail pulling and circling [
13
]. Nonetheless, key first steps in all
cases are to establish which (if any) limbs are abnormal and to rule out non-neurological
gait deficits such lameness due to orthopaedic disease. As a standard, observation should
be made of the animal walking in a straight line, viewed from the side, front, and back,
ideally with video recording. Stride length, trajectory, pattern of foot placement, and
any evidence of toe dragging or head nodding must be carefully assessed, noting that a
short-strided gait can arise from pain, weakness (neurogenic or non-neurogenic in origin),
or incoordination. Ataxia, by definition, comprises incoordinated (typically irregular and

Cells 2022,11, 2641 10 of 27
unpredictable) motor activity that does not result from weakness or musculoskeletal issues,
and is therefore neurological in origin [
12
]. It can affect movements of the head, neck, trunk,
limbs, eyes, or a combination thereof, and involves elements of hypermetria, hypometria
or both (dysmetria) [
13
]. Ataxia can indicate a lesion affecting the brain stem, cerebellum,
spinal cord or peripheral nerves [
21
,
22
] and is categorised as either vestibular, cerebellar,
or proprioceptive.
Vestibular ataxia typically includes a hypermetric, wide-based, staggering gait with
exaggerated extension of the limbs and leaning and falling towards the side of the lesion as
a result of relative hypertonia on the unaffected side (in the case of unilateral vestibular
disease) [
20
,
105
]. Mild signs are exacerbated when blindfolding the animal. Unilateral
vestibular lesions usually also produce a head tilt and abnormal nystagmus (see below).
Bilateral vestibular dysfunction (central or peripheral) is characterised by swinging head
and neck ataxia in the absence of a clear head tilt, and a crouched posture with reluctance to
move [
12
,
13
]. Cerebellar ataxia features alterations in the rate, range, and force of movement
(a dysfunction of subconscious proprioception) manifesting in dysmetria (classically a jerky,
hypermetric, high-stepping gait along with intention tremor or titubation of the head and
trunk) [
13
]. This type of ataxia is caused by a lesion of the cerebellum (sometimes with asso-
ciated deficits in the menace response) or spinocerebellar tracts [
12
]; affected animals may
also adopt a broad-based stance but typically show no signs of weakness (paresis). Lastly,
sensory or proprioceptive (spinal) ataxia results from lesions affecting ascending pathways for
subconscious proprioception in the spinal cord and brainstem and is best described as an
irregularly irregular (and therefore highly unpredictable) gait [
13
]. Scuffing, toe dragging,
or knuckling with hypometria in the thoracic limbs, alongside hyperflexion of the pelvic
limbs is often observed, together with a wide-based stance, a delayed-onset stride with
swaying or floating swing phase, and abduction or adduction during foot placement [
13
].
Circumduction, crossing of the limbs, or stepping on the opposite foot may also be seen.
Abrupt changes in direction, circling, backing the animal up, or walking it up or down a
slope with the head elevated can help identify proprioceptive ataxia, which can be very
subtle in the early stages of disease [
20
]. Proprioceptive ataxia is often accompanied by
varying levels of paresis.
Paresis or weakness involves partial loss of voluntary movement; there may be reduced
ability to initiate a gait, to maintain a posture, to support body weight, and/or to resist
gravity [
12
,
13
]. Typically, a decreased rate or range of motion will be observed (hypoflexion
and hypometria), sometimes manifesting as a short-strided bunny hopping gait, alongside
increased fatigability, hypotonia, low head and neck carriage, and sometimes postural
tremor [
12
]. A ‘two-engine’ gait with short strides in the thoracic limbs and long strides
in the pelvic limbs is characteristic of, but not pathognomonic for, a C6-T2 spinal cord
lesion involving grey and white matter [
13
]. Paralysis (plegia) is defined as complete loss
of voluntary movement.
Sophisticated electronic systems such as GAITRite or Zeno walkway from ProtoKinet-
ics have been used to quantify gait in to infer disease and treatment status in a broad range
of conditions from Parkinson’s disease, multiple sclerosis, and stroke, to residual deficits
following complex orthopaedic surgeries [
109
–
111
]. This system, and similar derivatives,
have been adopted in some large animal studies which use a 5 m electronic walkway to mea-
sure cadence, speed, stride length and width as well as individual pad pressure [
96
,
112
] in
a similar fashion to the catwalk systems that are commonly used in rodents [
113
]. GAITrite
was able to detect subtle gait changes in CLN5 disease sheep but not the ovine model of
Huntington Disease [106].
If gait analysis systems are not readily available, gait can be measured by using more
crude techniques developed in rodent models in which the feet are painted and the animals
walk along a length of paper [
114
]. It is advisable to paint the front and hind feet in different
colours to easily discriminate horizontal distances between the front to front and hind to
hind, and also, the stride length and pattern (Figure 1). This manual form of gait analysis
will be limited to species in which the size of the animal permits painting of the underside

Cells 2022,11, 2641 11 of 27
of the feet. Animals should also be habituated to the process of walking on the paper prior
to any testing occurring to ensure a ‘normal’ gait is recorded.
Cells 2022, 11, x FOR PEER REVIEW 11 of 28
foot may also be seen. Abrupt changes in direction, circling, backing the animal up, or
walking it up or down a slope with the head elevated can help identify proprioceptive
ataxia, which can be very subtle in the early stages of disease [20]. Proprioceptive ataxia
is often accompanied by varying levels of paresis.
Paresis or weakness involves partial loss of voluntary movement; there may be re-
duced ability to initiate a gait, to maintain a posture, to support body weight, and/or to
resist gravity [12,13]. Typically, a decreased rate or range of motion will be observed
(hypoflexion and hypometria), sometimes manifesting as a short-strided bunny hopping
gait, alongside increased fatigability, hypotonia, low head and neck carriage, and some-
times postural tremor [12]. A ‘two-engine’ gait with short strides in the thoracic limbs and
long strides in the pelvic limbs is characteristic of, but not pathognomonic for, a C6-T2
spinal cord lesion involving grey and white matter [13]. Paralysis (plegia) is defined as
complete loss of voluntary movement.
Sophisticated electronic systems such as GAITRite or Zeno walkway from ProtoKi-
netics have been used to quantify gait in to infer disease and treatment status in a broad
range of conditions from Parkinson’s disease, multiple sclerosis, and stroke, to residual
deficits following complex orthopaedic surgeries [109–111]. This system, and similar de-
rivatives, have been adopted in some large animal studies which use a 5 m electronic
walkway to measure cadence, speed, stride length and width as well as individual pad
pressure [96,112] in a similar fashion to the catwalk systems that are commonly used in
rodents [113]. GAITrite was able to detect subtle gait changes in CLN5 disease sheep but
not the ovine model of Huntington Disease [106].
If gait analysis systems are not readily available, gait can be measured by using more
crude techniques developed in rodent models in which the feet are painted and the ani-
mals walk along a length of paper [114]. It is advisable to paint the front and hind feet in
different colours to easily discriminate horizontal distances between the front to front and
hind to hind, and also, the stride length and pattern (Figure 1). This manual form of gait
analysis will be limited to species in which the size of the animal permits painting of the
underside of the feet. Animals should also be habituated to the process of walking on the
paper prior to any testing occurring to ensure a ‘normal’ gait is recorded.
Figure 1. Manual acquisition, analysis, and assessment of gait. Feet are painted in two different
colours to differentiate between front and hind limbs (light and dark grey respectively) and animals
Figure 1.
Manual acquisition, analysis, and assessment of gait. Feet are painted in two different
colours to differentiate between front and hind limbs (light and dark grey respectively) and animals
are made to walk over approximate 5 m length of paper once habituated to the process. (
a
) Overlay
of hoof prints from a photograph and (
b
) measurements of stride length (left and right), forelimb and
hindlimb distances.
3.4. Cranial Nerve (CN) Tests
It should be noted that CN I and XI cannot be adequately tested in livestock species
in routine practice [
20
]. Whilst normal olfactory nerve (CN I) function may be suggested
by the sniffing response of a blindfolded animal to the presence of a food item near
the nostril, this almost certainly invokes additional stimulation of sensory afferents of
the trigeminal nerve (CN V) supplying the nasal mucosa [
12
,
13
]. The spinal accessory
branch of the accessory nerve (CN XI) provides motor innervation to trapezius and cranial
sternocephalicus muscles which are rarely atrophied in isolation, and thus lesions are
difficult to detect [13] (Figure 2).
3.4.1. Pupillary Size and Symmetry
The optic nerve (CN II) can be assessed through evaluating pupil size and symme-
try, performing pupillary light reflex and menace response tests, and by monitoring for
behaviours consistent with visual loss. Pupillary size should be noted bilaterally and any
anisocoria (asymmetrical pupil size) documented [
12
]. Subtle anisocoria is often of no
significance and is easily missed in livestock because the lateral globe positioning makes it
difficult to examine both pupils simultaneously. Looking through a direct ophthalmoscope,
an arms-length away from the animal’s head, on a low light setting, is ideal for assessing
resting pupil size. Healthy pupillary size is ultimately determined by a balance between
parasympathetic oculomotor nerve (CN III) input to pupillary constrictor muscles via the
ciliary ganglion, and sympathetic innervation of the pupillary dilator muscles arriving via
the cranial cervical ganglion [
13
]. Anisocoria has many potential causes, however, some of
which are non-neurological in origin, and the reader is referred to a detailed discussion of
this elsewhere [14].

Cells 2022,11, 2641 12 of 27
Cells 2022, 11, x FOR PEER REVIEW 12 of 28
are made to walk over approximate 5 m length of paper once habituated to the process. (a) Overlay
of hoof prints from a photograph and (b) measurements of stride length (left and right), forelimb
and hindlimb distances.
3.4. Cranial Nerve (CN) Tests
It should be noted that CN I and XI cannot be adequately tested in livestock species
in routine practice [20]. Whilst normal olfactory nerve (CN I) function may be suggested
by the sniffing response of a blindfolded animal to the presence of a food item near the
nostril, this almost certainly invokes additional stimulation of sensory afferents of the tri-
geminal nerve (CN V) supplying the nasal mucosa [12,13]. The spinal accessory branch of
the accessory nerve (CN XI) provides motor innervation to trapezius and cranial sterno-
cephalicus muscles which are rarely atrophied in isolation, and thus lesions are difficult
to detect [13] (Figure 2).
Figure 2. Selection of commonly examined reflexes and reactions to assess neurological function.
(A–D) illustrates cranial nerve assessments with (A) pupillary light reflex (PLR), (B) palpebral re-
flex, (C) nasal sensation, (D) facial symmetry. (E–H) illustrates spinal reflex exams with (E) patellar
reflex, (F) withdrawal reflex, (G) perineal reflex and (H) cutaneous trunci reflex. (I–L) demonstrates
proprioceptive positioning with (I) knuckling/hoof placement test, (J) hop test, (K) step/visual and
tactile placing test and (L) push test.
3.4.1. Pupillary Size and Symmetry
The optic nerve (CN II) can be assessed through evaluating pupil size and symmetry,
performing pupillary light reflex and menace response tests, and by monitoring for be-
haviours consistent with visual loss. Pupillary size should be noted bilaterally and any
anisocoria (asymmetrical pupil size) documented [12]. Subtle anisocoria is often of no sig-
nificance and is easily missed in livestock because the lateral globe positioning makes it
difficult to examine both pupils simultaneously. Looking through a direct ophthalmo-
scope, an arms-length away from the animal’s head, on a low light setting, is ideal for
assessing resting pupil size. Healthy pupillary size is ultimately determined by a balance
between parasympathetic oculomotor nerve (CN III) input to pupillary constrictor mus-
cles via the ciliary ganglion, and sympathetic innervation of the pupillary dilator muscles
arriving via the cranial cervical ganglion [13]. Anisocoria has many potential causes,
Figure 2.
Selection of commonly examined reflexes and reactions to assess neurological function.
(
A
–
D
) illustrates cranial nerve assessments with (
A
) pupillary light reflex (PLR), (
B
) palpebral reflex,
(
C
) nasal sensation, (
D
) facial symmetry. (
E
–
H
) illustrates spinal reflex exams with (
E
) patellar
reflex, (
F
) withdrawal reflex, (
G
) perineal reflex and (
H
) cutaneous trunci reflex. (
I
–
L
) demonstrates
proprioceptive positioning with (
I
) knuckling/hoof placement test, (
J
) hop test, (
K
) step/visual and
tactile placing test and (L) push test.
3.4.2. Pupillary Light Reflex (PLR)
The PLR (Figure 2A) tests involuntary aspects of the light-responsive pathways, i.e.,
those that require no input from higher centres in the brain. The afferent pathway comprises
the retina, optic nerve (CN II), optic chiasm, optic tracts lateral and dorsal to the thalamus,
pretectal nuclei, and finally the parasympathetic motor nuclei of the midbrain, from where
the efferent output is mediated by parasympathetic fibres of CN III [
13
]. The examiner tests
the PLR in each eye by shining a bright light in a left to right direction, toward the maximum
density of rods and cones [
20
]. This causes the pupil of the illuminated eye to constrict
(the direct PLR). At the same time, this should cause constriction of the contralateral pupil
(the indirect or consensual PLR; [
115
]. It should be noted that ruminant pupil constriction
during the PLR is much slower than that of dogs and cats, but is generally faster than that in
horses [
14
,
22
]. The result of the direct reflex is normally greater than that of the consensual
reflex because most optic nerve axons decussate at the optic chiasm and most pretectal
nuclear axons cross back again at the caudal commissure [
14
]. This differential effect is most
notable in livestock species and horses because a greater proportion of fibres (80–90%) cross
over at the optic chiasm [
14
]. Observing the PLR in both eyes simultaneously is challenging
in livestock and so the swinging light test may be the most practical approach [
13
]. Diseases
which progressively affect CNs II or III will typically lead to a loss of vision before a loss
of the PLR [
14
]. The oculomotor nerves are vulnerable to pressure-induced injury in the
context of diffuse brain swelling and space-occupying lesions of the forebrain [13].
3.4.3. Dazzle Reflex
During PLR testing, animals sometimes partially blink or squint, retract the globe
(with protrusion of the nictitating membrane), and/or avert the head. This is known as
the dazzle reflex and is mediated via the retina, optic nerve, (CN II), rostral colliculus
and/or supraoptic nucleus of the hypothalamus, facial nucleus, and facial nerve (CN

Cells 2022,11, 2641 13 of 27
VII); [
14
,
116
,
117
]. Since all processing of this photo-aversion is subcortical, the result is
entirely involuntary and is typically only observed with the intense stimulation of a very
bright light source. It may however be stronger in photophobic animals, or those who
are acclimatised to the dark [
116
,
118
]. The dazzle reflex may be particularly useful when
observation of the pupils during PLR testing is precluded by severe corneal oedema or
hyphema [
117
]. More recent research suggests a contributory role for intrinsically photosen-
sitive retinal ganglion cells (ipRGCs) in the dazzle reflex [
118
]. ipRGCs also regulate light
responses over longer timescales, projecting directly via the retinohypothalamic tract to
the suprachiasmatic nucleus where they help regulate circadian entrainment [
119
]. Given
the increasing evidence of circadian disruption in chronic brain disease [
120
], it may be
prudent to include dazzle reflex testing routinely in neuro-ophthalmic assessments of
animal models of these disorders.
3.4.4. Menace Response
The menace response tests vision through evaluating the ipsilateral optic (CN II) and
facial (CN VII) cranial nerves, as well as the ipsilateral cerebellum and the contralateral
forebrain (thalamocortical system) [
12
]. It requires integrity of the entire visual pathway
including sensory and central processing (retina, optic nerve, optic chiasm, optic tract,
lateral geniculate nucleus of the thalamus, optic radiation, and occipital cortex), as wells
as integration and output pathways via the cerebellum and facial nerve [14,20]. A normal
menace response therefore confirms that all the above structures are functional, but a weak
or absent menace response poorly isolates lesion location. It is recommended to first test
the palpebral and pupillary light reflexes to ensure the animal has capacity to blink and
respond to light, respectively, before testing the menace response [
12
]. Each side should
be tested in isolation, covering the eye that is not being examined—although this is less
important in livestock species in which the eyes are positioned laterally [
20
]. The animal
should be positioned approximately 30–50 cm away from the examiner and a threatening
gesture made by the examiner’s finger being rapidly directed towards the animal’s eye,
testing both nasal and temporal regions of the retina [13,20,22]. Generation of air currents
onto, and any direct contact with, the ocular surface must be avoided to prevent invocation
of the corneal reflex via stimulation of the ophthalmic branch of the trigeminal nerve
(CN V). Similarly, touching any facial hairs or eyelashes will erroneously elicit the palpebral
reflex (Figure 2B) [
14
]. The normal response is to blink, but this is a learned response which
is not present in dogs and cats younger than 12 weeks of age and in foals, piglets, calves,
and lambs younger than 7 to 10 days old [
14
,
115
]. Animals with a facial nerve deficit may
instead retract the globe (noted by protrusion of the third eyelid) and/or withdraw the
head if the remainder of the pathway is intact [12].
It is crucial, however, to account for interspecies differences to allow appropriate
comparisons to be made. For example, prey species such as sheep with laterally directed
axis of vision and wider field of view have comparatively smaller binocular visual coverage
and larger monocular visual coverage than humans, resulting from varying degrees of
decussation at the optic chiasm, and thus different amounts of binocular cortical repre-
sentation in the primary visual cortex [
121
]. Generally, as binocular capacity increases,
decussation at the chiasm decreases; in primates, the extent of decussation is just above 50%,
whilst in livestock it is 80–90% [
14
]. Sensitivity to motion in peripheral vision combined
with hyperopia in the central field must be considered when comparing response to visual
stimuli with humans.
3.4.5. Eye Position
CNs III, IV, and VI together with the vestibular system, determine the position of the
eye in the orbit by providing motor efferents to the extraocular muscles. With the head at
rest, both eyes should point in the same direction. If this is not the case, a strabismus must
be recorded. Abducens nerve (CN VI) lesions will cause a medial strabismus (abnormal
angling of the eye towards the nose) and will potentially affect globe retraction; a deficit

Cells 2022,11, 2641 14 of 27
which may be noted during corneal reflex testing. Trochlear nerve (CN IV) deficits will
produce a rotational strabismus (with dorsal deviation of the medial aspect of the pupil
in ruminants and horses), and oculomotor nerve (CN III) deficits may produce a ventro-
lateral strabismus [
14
]. A positional strabismus may be unmasked by moving the head
into different positions [
12
], a manoeuvre which may also elicit a positional nystagmus.
Whilst dorsomedial rotation of the eyeball is characteristic of diffuse brain disorders in
ruminants and remains controversial in its neurolocalisation (some consider CN IV dys-
function); positional strabismus and/or positional nystagmus are almost always due to
vestibular dysfunction [13].
3.4.6. Vestibulo–Ocular Reflex (VOR)
Together with the vestibular portion of CN VIII; CNs III, IV, and VI are further re-
sponsible for conjugate eye movements [
20
]. Nystagmus is involuntary oscillation of the
eyeball [
14
]. Normal eye movement can be tested by eliciting physiological nystagmus,
also known as the VOR or oculocephalic reflex. Both eyes are observed whilst moving
the head repeatedly in a horizontal and then vertical direction [
12
]. Normal physiologic
nystagmus has a fast phase in the direction of head movement and the extent and direction
of gaze in both eyes should be the same—towards the direction of head movement [
115
].
Note that in unaffected ruminants and horses the eyeballs tend to maintain a horizontal
gaze and thus move ventrally in the orbits when the head and neck are extended [
13
,
14
].
The eyes should stop moving as soon as the head is at rest; continued movement or the
presence of nystagmus at rest is abnormal (pathological or spontaneous nystagmus) and
indicates a vestibular disorder [
20
]. A positional nystagmus is always abnormal and refers
to one that is only observed when the head is elevated, or changes direction when the head
is elevated. In the case of a peripheral vestibular problem, the fast phase of the nystagmus
is directed away from the side of the lesion and the plane of the oscillation is horizontal or
rotatory [
14
]. A consistently vertical nystagmus, or a nystagmus that changes with different
head positions usually indicates a central vestibular problem [14].
3.4.7. Corneal and Palpebral Reflexes
The corneal reflex involves stimulation of the ophthalmic branch of the trigeminal
nerve (CN V) with the efferent mediated by the abducens nerve (CN VI). The test should be
performed carefully (to avoid damage to the ocular surface), either by palpation through
closed eyelids, or by gently touching the cornea in a region away from the pupil aperture
using a cotton bud moistened with saline [
12
,
13
]. The normal result is retraction of the
globe, the fullness of which may depend on the functioning of all extraocular muscles in
livestock and horses [
13
]. The palpebral reflex (Figure 2B) assesses the sensory function of
the ophthalmic and maxillary branches of CN V, via stimulation of the medial and lateral
canthi of the eye, respectively. The motor efferent is via CN VII leading to wrinkling of
the face and blinking [
14
]. A full assessment of facial sensation (but not perception of it)
would include blunt stimulation of the head and face in all key areas of sensory innervation
provided by the major branches of CN V [
13
]. For example, touching the base of the ear
to stimulate the sensory portion of the mandibular branch of CN V should elicit reflexive
eyelid closure ipsilaterally by CN VII [20].
3.4.8. Nasal Sensation
Evaluation of nasal sensitivity assesses if there are abnormalities in the ipsilateral
ophthalmic or maxillary branch of the trigeminal nerve (CN V), or in the nociceptive
pathway to the contralateral cerebral hemisphere [
14
]. A normal response is withdrawal
of the head. The examiner would assess sensitivity by touching the nasal septum for the
ophthalmic branch (Figure 2C) or the external parts of the nostril for the maxillary branch
of CN V [12].

Cells 2022,11, 2641 15 of 27
3.4.9. Facial Symmetry
The examiner should look at the ears, eyelids, lip commissures, and nostrils to check
for symmetry (Figure 2D) [
20
,
21
,
115
]. A narrow palpebral fissure (loss of function of the
levator anguli oculi medialis muscle) or any other asymmetry such as unilateral drooping
of the ear or lip, indicates facial nerve (CN VII) motor dysfunction [
14
]. In horses and sheep,
deviation of the nose and upper lip toward the normal side will be evident, whereas in
cattle, this deviation is prevented by the large planum nasolabiale [
14
]. Loss of buccinator
tone may lead to residual food in the cheek pouches [
13
,
122
]. CN VII is a mixed nerve
with parasympathetic fibres that supply mandibular and sublingual salivary glands and
others that mediate lacrimation—this can be evaluated using the Schirmer tear test [
12
].
Additional motor fibres of CN VII supply the caudal belly of the digastricus muscle and the
stapedius muscle of the middle ear, whilst afferent CN VII fibres provide taste sensation
to the rostral two-thirds of the tongue. None of these functions are readily tested in the
neurological exam.
3.4.10. Jaw Tone
The examiner should test the resistance of the animal to having their mouth opened. A
flaccid or dropped jaw with inability to chew indicates total bilateral loss of trigeminal nerve
(CN V) motor function (mandibular branch) [
13
,
115
]. Tongue protrusion and drooling may
be observed, followed by atrophy of the muscles of mastication [13].
3.4.11. Startle Response
This test assesses responses of CN VIII (most likely the vestibular portion according to
recent studies) to a loud and unexpected noise, with resultant body and limb movements
mediated by the tectospinal tract [
13
]. A true startle is thus considered as a reflexive,
subconscious response. As prey species, however, livestock are generally very sensitive
to sound, and the lack of any response to a standard auditory cue might indicate a deficit
in the cochlea of the inner ear, or other region of the auditory pathway via CN VIII and
into the brainstem, from where sound information projects bilaterally up to the midbrain,
thalamus, and auditory cortex [
13
,
115
]. As with humans and other domestic animals, these
pathways are best tested using the brainstem auditory evoked response (BAER).
3.4.12. Swallow (Gag) Reflex
Abnormalities in swallowing are known as dysphagia and could be due to lesions
in the glossopharyngeal (CN IX), vagus (CN X) and/or accessory (CN XI) nerves which
together provide sensory and motor innervation to the pharynx and larynx [
13
]. In order to
determine the efficacy of the animal’s swallow response, the examiner would insert a finger
caudally into the mouth as far as possible until the reflex is initiated. With ruminant pharyn-
geal paralysis, food, water, and saliva may be noted in the nostrils and mouth [
13
]. Rubbing
the ventral larynx can also initiate the swallow reflex. Any change in voice/vocalisation
should also be noted and could be caused by damage to CN IX and/or X [20,104,123].
3.4.13. Tongue Tone
Motor control of the tongue is governed by the hypoglossal nerve, CN XII. To assess
CN XII function, the examiner should open the animal’s mouth to observe the position and
movement of the tongue and assess for any atrophy [
20
]. The tongue can also be grasped
to assess for a normal level of strength/resistance [
115
]. A unilateral lesion will lead to
lateral deviation of the tongue, the direction of which may depend on the stage and extent
of dysfunction. Bilateral dysfunction will impair prehension and swallowing [13].
3.5. Spinal Reflex Tests
3.5.1. Patellar Reflex (a Monosynaptic, Myotatic Reflex)
This tests the reflex arc comprising sensory and motor fibres of the femoral nerve, and
primarily spinal cord segments L4 and L5 [
21
]. The test can be performed in recumbent

Cells 2022,11, 2641 16 of 27
animals or those that are amenable to being positioned in lateral recumbency or turned
onto their back and held in a seated position (e.g., small ruminants; [
115
]). Each pelvic
limb should be tested when uppermost and when dependent and the tested limb should be
supported in a partially flexed position with slight tension in the muscle [13]. The middle
patellar ligament is then tapped with a patellar hammer and the normal response is swift
contraction of the quadriceps muscle with stifle extension. This action must be carried
out with precision to avoid striking the muscle tissue or bone giving rise to erroneous
results (Figure 2E).
3.5.2. Extensor Carpi Radialis (ECR) Reflex
The ECR reflex tests the integrity of the radial nerve and spinal cord segments C7-T1.
The antebrachium is held, keeping the elbow flexed, and the belly of the carpi radialis
muscle is sharply struck just distal to the elbow [
12
]. Normally, contraction of the ECR
results in an extension of the carpus [
21
]. This may be a muscular response rather than
a true myotatic reflex as it has been observed in dogs with a transected radial nerve; the
results of this test may not be reliable in adult livestock [12,13].
3.5.3. Withdrawal (Flexor) Reflexes
The withdrawal reflexes test the function of: (1) C6-T2 spinal cord segments and
the musculocutaneous, axillary, median, radial, and ulnar nerves in the thoracic limbs
and (2) L5-S3 spinal cord segments and the sciatic nerve in the pelvic limbs [
14
,
20
]. The
animal is assessed in lateral recumbency and, using a pair of artery forceps, the examiner
pinches the interdigital skin between the toe(s) in the paw or hoof (Figure 2F). The normal
response is withdrawal of the thoracic limb via flexion of the shoulder, elbow, and carpus,
or withdrawal of the pelvic limb via flexion of the hip, stifle, and hock. A neurologically
normal animal will also display a behavioural response to this noxious stimulus, since it
tests the nociceptive pathway from spinal cord to cerebrum [13].
3.5.4. Perineal (Anal) Reflex
The perineal reflex is performed by lifting the tail and gently prodding the skin on each
side of the perineum with a pen or cotton swab (Figure 2G). A normal reaction includes
reflex contraction of the anal sphincter and clamping down of the tail. The sensory arm of
the reflex involves the perineal branches of the pudendal nerve from S1–S3. Contraction of
the anal sphincter is mediated by caudal rectal branches of the pudendal nerve, and tail
flexion by the sacral and caudal segments and nerves, S1–Ca [
13
]. In adult cows, gentle
stroking of the anus typically causes reflex tail extension a micturition reflex. Sensory
perception of these stimuli may still be present even when the segmental reflex functions
are lost [13].
3.5.5. Cutaneous Trunci Reflex
The cutaneous trunci reflex assesses the sensory integrity of all dermatomes over
the thoracolumbar vertebral column, spinal cord segments C8-T1, and the lateral thoracic
nerve running to the cutaneous trunci muscle of the flank [
12
]. The examiner would
elicit a stimulus by pinching the skin on both sides of the thoracolumbar region which
should make the cutaneous trunci contract bilaterally (Figure 2H) [
115
]. The reflex may
not be elicited caudally to the level of a spinal cord lesion but should be intact cranially
to it and the examiner may need to carry out multiple stimulations for the animal to
respond normally [20,21].
3.6. Postural Reactions
Postural reactions primarily help to identify subtle proprioceptive and motor system
lesions in the absence of obvious gait deficits [
13
,
124
]. These tests will also evaluate the
visual system and the cerebellum [
115
] but are more challenging to perform in large animals
such as horses and pigs [
14
]. Although not all of these tests inform on the anatomical loca-

Cells 2022,11, 2641 17 of 27
tion of a lesion, they do help build a profile of neurological dysfunction with abnormalities
detected in these assessments not necessarily observed in gait analysis [124].
3.6.1. Knuckling/Paw or Hoof Placement Test (Proprioceptive Positioning)
A neurologically normal animal should immediately correct the abnormal positioning
of a foot if it has the strength and attention to do so [
14
]. In a properly supported animal
(which may be difficult in most large species), each paw or hoof can be turned over by the
examiner so that the incorrect (dorsal) surface touches the ground (Figure 2I).
3.6.2. Hop Test
Hopping is a sensitive test for subtle weakness or asymmetry [
12
]; it simultaneously
assesses the function of the visual cortex, cerebellum, and spinal cord and is often con-
sidered the most reliable postural reaction test [
14
]. The examiner should pick up and
support one limb, so that three out of the four limbs are on the ground, and move the
animal laterally sidewards (Figure 2J). This should be carried out for each limb. The normal
animal will hop quickly and smoothly in the direction in which it is being moved in order
to resist gravity and maintain balance [
14
]. Cerebral lesions produce contralateral deficits,
whilst brainstem and spinal cord lesions produce ipsilateral deficits [13].
3.6.3. Step/Visual and Tactile Placing Test
As for Section 3.6.1, the animal should be supported by the examiner and/or assistant
throughout the assessment. With (tactile) and without (visual) blindfolding of the animal,
the foot can be crossed over its contralateral counterpart or slowly abducted by the examiner
until the animal lifts the foot and correctly places it underneath its body (Figure 2K). Use of
a sliding feed sack beneath the foot can help avoid the invocation of a withdrawal reflex
through manual handling of the limb. It should be noted that the utility and interpretation
of Sections 3.6.1 and 3.6.3 in livestock and horses remains controversial; spontaneous
abnormal positioning of the limb(s) after abruptly stopping the movement of a large animal
may be a more informative test of conscious proprioception [13].
3.6.4. Push Test
The push test assesses strength, balance, coordination, and postural stability and is a
test commonly carried out in patients suspected of having Parkinson disease [
125
]. The
examiner should support the animal and push against its body and should feel there is
resistance in the animal to being pushed (Figure 2L). In horses, tail pulling in stance and
during ambulation is more often employed to detect extensor weakness and propriocep-
tive deficits [13].
3.7. Pain Perception (Nociception)
Assessing an animal’s response to pain (nociception) is important to help pinpoint
areas that may have undergone mechanical, soft tissue, or neurological injury evoking an
emotional response. This reaction may be in response to a number of different stimuli and
is a protective behaviour to alert an animal to danger or protect it from further injury by,
for instance, not bearing weight on a damaged limb [
126
,
127
]. Other clinical signs of pain
in an animal may include loss of appetite, withdrawn demeanour, unwillingness to work,
and vocalisation [
126
]. Additional neurological pain can present as hypersensitivity to
touch or temperature. A behavioural response elicited during withdrawal reflex testing is a
good indicator that sensory afferents in the peripheral nerves, spinal cord, and pathways
ascending to the brainstem and forebrain are intact [128].
3.8. Cognitive Assessment Trials
Assessing cognitive decline in patients suffering from neurodegenerative disease is
well established with tests such as the dementia detection test (DemTect) or Montreal
Cognitive Assessment (MoCA) that rely on language for communication [
129
,
130
]. This

Cells 2022,11, 2641 18 of 27
poses an additional challenge for assessment in animals, but tests have been devised that
inform on deficits of memory and spatial awareness in animals. Here, we list two tests that
have been successfully implemented in large animal models, building upon the clinical
data acquired.
3.8.1. Maze Testing
Maze tests help to assess an animal’s memory and spatial awareness, but can also be
used to unmask more subtle visual loss, using non-aromatic, non-auditory visual cues [
13
].
Due to the preference of most livestock species to remain in herds or flocks, separation
from their pen mates can be stressful but re-integration into a familiar group of conspecifics
can also serve as a useful reward. Visual awareness of the pen mate/owner’s location at
the end of a maze or course is often enough motivation for the animal to navigate through
the maze alone. A good example of a successful use of a maze trial was published by
Mitchell et al. [
131
], who have shown that sheep with naturally occurring CLN5 disease
progressively lose the ability to quickly navigate the same maze over time due to disease
advancement. Moreover, this assessment was used to aid determination of the efficacy of
gene therapy treatment and clearly identified treated individuals as ‘out-performing’ those
that had not been treated.
3.8.2. Executive Function
Executive function skills which allow us to focus, memorise, and multitask are vital
cognitive functions which also allow us to plan, organise, and reason [
132
]. These skills are
diminished with progressive neurodegeneration associated with normal healthy ageing but
are markedly lost in a variety of other disorders, including, but not limited to, Alzheimer’s,
Huntington’s, and motor neuron disease [
133
–
135
]. Just as these tests are crucial to assess
advancement of disease in patients it is important to devise similar exams for large animal
models of these diseases. Morton and Avanzo successfully implemented a range of tests
in sheep in order to assess executive function which included discrimination learning,
retention, and reversal learning with the latter used to determine the functional integrity of
striatum and pre-frontal cortex in Huntington patients [
134
]. Follow-up studies have also
objectively measured stop-signal reaction time and facial recognition in this species [136].
4. In Vivo Imaging Assessment(s)
4.1. Central Nervous System (CNS)
Another key tool for diagnosis and assessment of neurological disease is cross-sectional
imaging, most commonly computed tomography (CT) and magnetic resonance imag-
ing (MRI) in clinical settings, with additional low volume specific use of positron emis-
sion tomography (PET). The clinical neurological assessment can determine lateralisa-
tion/localisation within the neuraxis, with additional demographic and temporal features
helping to narrow down potential clinical diagnoses [
137
]. However, imaging is routinely
acquired for confirming or refining the differential diagnosis, and for assessing disease
progression or response to treatment [
138
]. As non-invasive techniques, with minimal,
quantifiable risks from ionising and non-ionising radiation and high magnetic fields, neu-
roimaging represents a unique opportunity to study nervous system structure and function
in a longitudinal setting allowing for direct clinical translation when using the same imag-
ing platforms with both large animals and humans. Given the extensive knowledge of
structure-function relationships in the nervous system, it is possible to relate the imaging
phenotype to the clinical assessment, e.g., measuring optic nerve or chiasmatic thickness
in anterior visual pathway disturbances, or confirming whether a pelvic limb motor dis-
turbance is arising from the cord or primary motor neocortex. Whilst very important in
primates, it should be noted that corticospinal motor pathways do not play a dominant
role in initiating voluntary limb and body movement in domestic mammals; thus, large
lesions of cerebrocortical motor centres in isolation do not typically produce permanent

Cells 2022,11, 2641 19 of 27
gait abnormalities in livestock [
13
]. Figure 3demonstrates how the imaging component
complements and further scrutinises the outcomes from clinical assessment.
Neurological Examination
(a) (b) Impaired motor response to facial
sensation testing (c) Lack of facial sensation:
somatosensory pathway
VII -
Temporozygo
matic branch
VII – Buccal
branch
VII – Mandibulocervical
branch
Parotid gland
Left Postcruciate Gyrus
(Somatosensory Cortex)
Left Thalamocortical
Fibres
Left Thalamus
Lack of facial sensation :
trigeminal nerve
V1 – Ophthalmic
Division
V2 – Maxillary
Division
V3 –
Mandibular
Division
V – Cavum
Trigeminale
(d)
II – Right Optic
Nerve II –Left Optic Nerve
II –
Optic
Chiasm
Left Optic
Tract
Left Lateral Geniculate
Nucleus
Left Lateral Gyrus
(Primary Visual Cortex)
Left
Optic
Radiation
Lack of menace response:
visual pathway
(e)
Poor hop test assessment:
motor cortex
(f)
Left Precruciate Gyrus
(Primary Motor Cortex)
Left Pyramidal Tract
(Corticospinal)
Cranial
Nerves Proprioception
Gait Demeanour
Figure 3.
Use of MRI to enable detection of lesions in any part of the reflex pathway, and can be scored
or quantified for statistical analysis alongside clinical scoring results. Examples of clinically relevant
neuroanatomy from a healthy sheep (Ovis aries). (
a
) Initial neurological assessment by species
specific expert identifies abnormalities in cranial nerve and proprioception exams. (b) MRI analysis
can then determine whether any lesions or abnormalities are present e.g., cranial nerve VII (facial
nerve) when facial motor function is impaired in isolation. If facial motor function is intact, but facial
sensation is impaired, investigation of the thalamus and somatosensory cortex (
c
) and trigeminal
nerve branches (
d
) should be undertaken. (
e
) Abnormalities arising in any of the visual exams can be
further scrutinised in the visual pathway to determine if lesions are localised in the optic nerve/tract
or chiasm as well as the lateral geniculate nucleus and visual cortex. (
f
) Proprioceptive deficit(s)
can also be further investigated with imaging of the spinal cord if the exam pinpoints to a specific
region of interest and/or to the motor pathway. Cortical and subcortical regions of interest (ROI;
solid red or green) are labelled in the native individual animal using an atlas and template-based
approach. Probabilistic tractography is carried out using FSL BEDPOSTX and PROBTRACKX on
diffusion-weighted imaging data using cortical and subcortical ROIs. Tract probabilities are shown
with a heatmap. The left pyramidal/corticospinal tract, left somatosensory thalamocortical fibres,
and left postchiasmatic optic pathway are shown, overlaid on selected orthogonal slices from a
fat-saturated 3D FLAIR structural image.
4.2. Eyes
In addition to the neuro-ophthalmological tests described above, a complete oph-
thalmic examination may be beneficial, especially if any deficits in the ocular reflexes
and responses are detected. The Schirmer Tear Test I can give an indication of basal
and reflex tear production; reference values are available for domesticated species [
139
]
Gelatt K. N et al., 1975. Hand-held slit lamp biomicroscopy (e.g., Kowa SL-17) allows
detailed examination of the ocular adnexa, cornea, anterior chamber, iris, lens and anterior
vitreous, and in most species can be performed with gentle manual restraint only. An indi-
rect ophthalmoscope headset, combined with a 20–40D lens can give an excellent overview

Cells 2022,11, 2641 20 of 27
of the fundus. The direct ophthalmoscope can be used to closely examine any detected
lesions and the optic nerve head at increased magnification but with a smaller field of
view. Intraocular pressure measurements are routinely taken in a non-invasive fashion
with a handheld rebound tonometer (iCare TonoVet). Additional tests can be performed
as required, for example gonioscopy to assess the iridocorneal angle, electroretinography
to interrogate the electrical activity of the retina, fluorescein angiography to examine the
vascular components and perfusion of the fundus, and ocular ultrasound (including high-
resolution ultrasound, contrast-enhanced ultrasound, and ultrasound biomicroscopy) [
139
]
Gelatt N et al. 1975. Some of these examinations require pharmacological pupil dilation,
typically with topical tropicamide, but this is animal model dependent. It is important to
time examinations carefully as bright light stimulation (e.g., retinal photography or indirect
ophthalmoscopy) is likely to impact electroretinography (ERG) results therefore, it is crucial
to allow sufficient recovery time with dark adaptation prior to ERG recording [
140
]. More
advanced imaging modalities, such as SD-OCT (spectral domain ocular coherence tomogra-
phy) and SD-OCT angiography is likely to form a cornerstone of future research, providing
real time
in vivo
analysis of retinal pathology [
141
].
In vivo
confocal microscopy can image
corneal innervation (parameters including corneal nerve fibre length, density, branching
density, tortuosity coefficient and beadings frequency) and repeated examinations allow
demonstration of disease progression if present [142,143].
5. Cerebrospinal Fluid (CSF) Analysis
Techniques for collection and standard analysis of CSF from large animals are de-
scribed in detail elsewhere [
13
,
21
,
144
,
145
]. Sampling in large domestic species is generally
carried out at the lumbosacral space. However, sampling from the atlanto–occipital space is
possible but is more challenging, carries greater risk, and requires stable head positioning
under general anaesthesia or deep sedation [
145
]. It may be that the most informative
diagnostic samples are obtained as close as possible to the site of pathology, which for
most neurological disease models will be the brain and/or spinal cord. In the research
context, the need for CSF analysis needs to be weighed up against the risks of sampling for
the animal, and it is important to consider whether serial longitudinal samples are really
needed for validation of a particular pathological process. Where the disease process is
expected to result in raised intracranial pressure (or where there is evidence of such with
advanced neuroimaging), CSF should not be sampled in a conscious animal, nor under
anaesthesia, unless this is pre-designated as a non-recovery procedure. Whilst 1–2 mL
would be sufficient for routine cytological analysis and determination of protein levels [
145
],
up to 1 mL per 5 kg can be safely removed at one time from companion animal patients [
12
].
In theory, much larger volumes can be safely obtained from livestock species, but even
with cryopreservation of excess sample for future analyses, volumes of more than 5–10 mL
are unlikely to be warranted. Where analysis is planned only at end stage disease, or at
experimental/humane end points, CSF is best obtained immediately postmortem, with
no restrictions on sample site or volume. Certain analytes and standard readouts require
immediate processing to be of worth [
145
]. We do however recommend biobanking of
excess samples at
−
80
◦
C to facilitate future multicentre and multiomics studies, whilst
reducing the number of animals required to answer research questions.
6. Advanced Functional Readouts and Emerging Interdisciplinary Opportunities
Whilst electrodiagnostic testing is fairly routine in companion animal neurology [
12
],
it is rarely conducted in livestock species except in the research or academic setting. Nev-
ertheless, it is possible to perform, e.g., electromyography (EMG), electroretinography
(ERG), nerve conduction velocity (NCV), brainstem auditory evoked response (BAER),
and somatosensory evoked potential tests in these species if relevant to the disease being
modelled [
13
,
144
,
145
]. High temporal resolution longitudinal assessments of general phys-
iology and neurophysiology are now possible in large animals, enabled by radiotelemetry,
non-invasive activity monitors [
106
], and conscious ambulatory electroencephalography

Cells 2022,11, 2641 21 of 27
(EEG) [
33
]. The latter has been able to identify non-convulsive epileptiform activity and
early changes in sleep in a sheep model of CLN5 disease [
33
,
146
]. Likewise, early changes
in metabolism and circadian function have been captured in a sheep model of Hunt-
ington’s disease through metabolic profiling of repeated blood samples and actimetry,
respectively [
147
–
149
]. The increasing importance of detecting more subtle deficits in neu-
rodegenerative disorders such as sleep and circadian disruption underpins the attraction
to modelling these disorders in diurnal species such as sheep [
120
,
150
,
151
]. Moreover,
species–specific behaviours such as flock activity, combined with modern GPS tracking
systems, can be exploited to study more complex social interactions in natural environ-
ments [
106
]. The emerging portability of advanced imaging modalities such as optical
coherence tomography (OCT) may offer ‘windows’ into the brain without the need for
general anaesthesia [
152
,
153
]; such opportunities would undoubtedly benefit from vet-
erinary ophthalmology expertise. As multiomics data tools become more accessible for
comparative studies, and species–specific resources such as brain atlases [
154
–
160
] continue
to develop, there will be few barriers to truly comprehensive clinical phenotyping of large
animal models of neurological disease.
7. Conclusions
In this paper, we have summarised the most important considerations when devel-
oping and validating a large animal model of neurological disease, from species selection,
to practical neurological phenotyping and monitoring. Certain aspects of the standard
neurological examination can be challenging to perform in certain species. Therefore, it is
essential to choose a species in which the key neuroanatomical functions of interest can
be objectively tested prior to engineering a disease state that would modify them. Welfare
must always be at the forefront of any decision to create an induced model of disease. This
is ever more critical for disorders of the nervous system which, by their nature, can have a
devastating impact on life quality. A collaborative approach to clinical phenotyping that in-
corporates the expertise of specialists in veterinary neurology, ophthalmology, anaesthesia,
neuroimaging, and production animal medicine can provide valuable insights into the very
earliest stages of disease; these are features which are often elusive in patients. Applied
sequentially and systematically, clinical assessments and other diagnostic tests can provide
vital biomarkers of disease advancement as well as indices of therapeutic efficacy.
Author Contributions:
Conceptualisation of the review was carried out by S.L.E., T.M.W., N.M.R.
and F.M.; writing was carried out by S.L.E., N.M.R., F.M., G.T., B.T.B., C.H. and T.M.W.; review and
editing was carried out by S.L.E., P.M.B., S.G.L., S.G., N.M.R., T.M.W., F.M., D.N.P., N.L.M., J.D.C.,
C.H., B.T.B., C.B.A.W., C.P., P.T., A.R. and J.N. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was partly funded by Biotechnology and Biological Sciences Research Coun-
cil (BBSRC) Institute strategic funding BB/P013732/1 and BB/J004316/1. N.L.M. and D.N.P. are
funded by Cure Kids New Zealand (grant 6501) and by Batten Disease Support and Research As-
sociation (
BDSRA
). N.M.R. is funded by Medical Research Council Clinician Scientist Fellowship
(MR/S022023/1). This work was partly funded by NINDS National Institutes of Health grant R56
NS117635 and R01 NS124655 (J.D.C., T.M.W.).
Institutional Review Board Statement:
All animal work in the United Kingdom was reviewed and
approved by the Animal Welfare and Ethical Review Board (AWERB) at the Roslin Institute and
conducted under the authority of the UK Home Office (equivalent of IACUC). All animal work in
New Zealand was approved by the Lincoln University Animal Ethics Committee and performed in
accordance with the National Institutes of Health guide for the care and use of Laboratory animals
(NIH Publications No. 8023, revised 1978), the Lincoln University Code of Ethical Conduct for the
Use <