Equine Brain Research Articles

Impact of equine herpesvirus-1 ORF15 (EUL45) on viral replication and neurovirulence

Equine herpesvirus 1 (EHV-1) causes respiratory illness, fetal loss, perinatal mortality, and myeloencephalopathy. This study investigated ORF15's impact on virus infectivity and neurovirulence. The Ab4p neurovirulent strain of EHV1 was used as a backbone to create Ab4p attB, Ab4p∆ORF15, and Ab4p∆ORF15R chimeras via BAC DNA transfection into RK-13 cells. Viral growth kinetics, plaque size, transcription, and growth were assessed in MDBK cells, mouse neurons, and fetal equine brain cells. Neurovirulence was evaluated post-intranasal inoculation into male CBA/N1 SPF mice, measuring signs, virus titers, and histopathological changes. Deletion of EUL45 (Ab4p-∆EUL45) reduced viral replication efficiency, resulting in decreased release and smaller plaques. EUL45 deletion also upregulated neighbouring genes (EUL46 and EUL44). Ab4p-∆EUL45 exhibited reduced virulence and poor growth in neural cells compared to wild-type viruses. This study sheds light on EUL45's role in EHV-1, viral replication, and regulation of EUL46 and EUL44 expression, suggesting potential as a vaccine candidate.

An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

Detection of Venezuelan Equine Encephalitis Virus from Brain Samples of Equines with Encephalitis

Alphavirus species are globally distributed zoonoses primarily transmitted by arthropods. The Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) are endemic in Costa Rica. This study aims to detect these viruses in brain samples from equines displaying nervous signs. For this purpose, four published Universal RT-PCR methods were compared. The most sensitive and specific RT-PCR method was used to test a total of 70 brain samples, including 40 from bovines and 30 from equines, all exhibiting nervous signs. In the positive cases, eight different brain regions were extracted and tested using this RT-PCR. Positive cases were confirmed through sequencing. Torii RT-PCR demonstrated the highest sensitivity and specificity for diagnosing VEEV and EEEV/Sind among the four Universal RT-PCR assays. Not all assessed brain regions showed DNA amplification. None of the bovine brains was positive, and out of the 30 equine brain samples, only four tested positive, and sequencing confirmed two of these samples as VEEV subtype IE. Torii RT-PCR successfully detected VEEV in pools of the hippocampus, spinal cord, and basal nuclei, making these brain regions suitable for diagnosing this virus. None of the samples were positive for EEEV or WEEV.

The Activity of Substance P (SP) on the Corneal Epithelium

In 1931, Von Euler and Gaddum isolated substance P (SP), an undecapeptide from the tachykinin family, from equine brain and intestine tissue extracts. Numerous types of cells, including neurons, astrocytes, microglia, epithelial, and endothelial cells, as well as immune cells including T-cells, dendritic cells, and eosinophils, are responsible for its production. The corneal epithelium, immune cells, keratocytes, and neurons all express the two isoforms of NK1R, which has the highest affinity for SP. The most recent research supports SP’s contribution to corneal healing by encouraging epithelial cell migration and proliferation. Additionally, when applied to the eyes, SP has proinflammatory effects that result in miosis, intraocular inflammation, and conjunctival hyperemia. In this review article, we examine the role of substance P within the eye. We focus on the role of SP with regards to maintenance and healing of the corneal epithelium.

Feasibility of using tissue autolysis to estimate the postmortem interval in horses.

Estimation of the postmortem interval (PMI) is a poorly studied field in veterinary pathology. The development of field-applicable methods is needed given that animal cruelty investigations are increasing continually. We evaluated various histologic criteria in equine brain, liver, and muscle tissue to aid the estimation of PMI in horses, which is central to forensic investigations of suspicious death. After death, autolysis proceeds predictably, depending on environmental conditions. Currently, no field-applied methods exist that accurately estimate the PMI using histology in animals or humans through quantification of autolysis. Brain, liver, and skeletal muscle from 12 freshly euthanized horses were held at 22°C and 8°C for 72 h. Tissues were sampled at T0h, T1h, T2h, T4h, T6h, T12h, T24h, T36h, T48h, T60h, and T72h. For each tissue, we quantified 5 to 7 criteria associated with autolysis, based on the percentage of microscopic field involved. Each criterion was modeled, with temperature and time as independent variables. Changes were most predictable in liver and muscle over the first 72 h postmortem. The criteria for autolysis that were present most extensively at both temperatures were hepatocyte individualization and the separation of bile duct epithelium from the basement membrane. The changes that were present next most extensively were disruption of myofiber continuity, hypereosinophilia, and loss of striation. Brain changes were highly variable. The high statistical correlation between the parameter "autolysis" and the variables "time/temperature", indicates that autolysis is progressive and predictable. Further investigation of these criteria is needed to establish histologic algorithms for PMI.

The contribution of the middle cerebral artery and callosal artery to the vascularisation of the Facies convexa of the brain in horses with reference to the equine-specific cartographic pattern of the neopallium.

The extremely complex surface architecture of the equine brain does not allow a uniform transfer of anatomical data from other mammalian species, e.g., dog or cat. Rather, a special approach is required to elucidate the equine-specific patterns of cerebral vascular ramifications. Therefore, a novel cartographic system was applied. Prior attention was paid to the A. cerebri media (MCA) and to the A. corporis callosi (CA), as they spread over the widest part of the neopallium's Facies convexa (i.e. the lateral and dorsal surface), thus being of particular interest in terms of surgical treatment of trauma of the skull and brain. The brains of 17 adult warmblood horses were studied. The neopallium's Facies convexa was subdivided into 15 sectors clearly delineated by the primary sulci and by related auxiliary lines. The courses and destinations of main branches (= branches of 1st or 2nd order, with a minimum calibre of 0.75 mm) of MCA and CA were topographically analysed by means of superimposed graphical sketches. The MCA had six main branches (numbered in rostrocaudal direction); the CA had seven main branches. The main branches of the MCA spread over the widest part of the Facies convexa, but never reached the rostral pole and the caudal pole of the neopallium. Clearly, the main branches of CA proceeded from the hemisphere's medial side across the Margo dorsalis cerebri, thus supplying rostrodorsal and dorsal sectors of the Facies convexa. The topographical analysis of the vascular ramification patterns (regions of residence) in combination with the cartographic system of sectors of the neopallium respected the equine-specific surface architecture. It highlighted the distinct vascular supply areas and the particular multiple-supply situations especially in sectors presumably related to the equine area motorica.

A Systematic Approach to Dissection of the Equine Brain-Evaluation of a Species-Adapted Protocol for Beginners and Experts.

Introduction of new imaging modalities for the equine brain have refocused attention on the horse as a natural model for ethological, neuroanatomical, and neuroscientific investigations. As opposed to imaging studies, strategies for equine neurodissection still lack a structured approach, standardization and reproducibility. In contrast to other species, where adapted protocols for sampling have been published, no comparable guideline is currently available for equids. Hence, we developed a species-specific slice protocol for whole brain vs. hemispheric dissection and tested its applicability and practicability in the field, as well as its neuroanatomical accuracy and reproducibility. Dissection steps are concisely described and depicted by schematic illustrations, photographs and instructional videos. Care was taken to show the brain in relation to the raters' hands, cutting devices and bench surface. Guidance is based on a minimum of external anatomical landmarks followed by geometric instructions that led to procurement of 14 targeted slabs. The protocol was performed on 55 formalin-fixed brains by three groups of investigators with different neuroanatomical skills. Validation of brain dissection outcomes addressed the aptitude of slabs for neuroanatomical studies as opposed to simplified routine diagnostic purposes. Across all raters, as much as 95.2% of slabs were appropriate for neuroanatomical studies, and 100% of slabs qualified for a routine diagnostic setting. Neither autolysis nor subfixation significantly affected neuroanatomical accuracy score, while a significant negative effect was observed with brain extraction artifacts. Procedure times ranged from 14 to 66 min and reached a mean duration of 23.25 ± 7.93 min in the last of five trials in inexperienced raters vs. 16 ± 2.83 min in experts, while acceleration of the dissection did not negatively impact neuroanatomical accuracy. This protocol, derived analogously to the consensus report of the International Veterinary Epilepsy Task Force in dogs and cats, allows for systematic, quick and easy dissection of the equine brain, even for inexperienced investigators. Obtained slabs feature virtually all functional subcompartments at suitable planes for both diagnostic and neuroscientific investigations and complement the data obtained from imaging studies. The instructive protocol and brain dissection videos are available in Supplementary Material.

Diffusion Tensor Imaging Tractography of White Matter Tracts in the Equine Brain

Tractography, a noninvasive technique tracing brain pathways from diffusion tensor magnetic resonance imaging (DTI) data, is increasingly being used for brain investigation of domestic mammals. In the equine species, such a technique could be useful to improve our knowledge about structural connectivity or to assess structural changes of white matter tracts potentially associated with neurodegenerative diseases. The goals of the present study were to establish the feasibility of DTI tractography in the equine brain and to provide a morphologic description of the most representative tracts in this species. Postmortem DTI and susceptibility-weighted imaging (SWI) of an equine brain were acquired with a 3-T system using a head coil. Association, commissural, and projection fibers, the three fiber groups typically investigated in tractography studies, were successfully reconstructed and overlaid on SWI or fractional anisotropy maps. The fibers derived from DTI correlate well with their description in anatomical textbooks. Our results demonstrate the feasibility of using postmortem DTI data to reconstruct the main white matter tracts of the equine brain. Further DTI acquisitions and corresponding dissections of equine brains will be necessary to validate these findings and create an equine stereotaxic white matter atlas that could be used in future neuroimaging research.

Equine Stereotaxtic Population Average Brain Atlas With Neuroanatomic Correlation.

There is growing interest in the horse for behavioral, neuroanatomic and neuroscientific research due to its large and complex brain, cognitive abilities and long lifespan making it neurologically interesting and a potential large animal model for several neuropsychological diseases. Magnetic resonance imaging (MRI) is a powerful neuroscientific research tool that can be performed in vivo, with adapted equine facilities, or ex-vivo in the research setting. The brain atlas is a fundamental resource for neuroimaging research, and have been created for a multitude animal models, however, none currently exist for the equine brain. In this study, we document the creation of a high-resolution stereotaxic population average brain atlas of the equine. The atlas was generated from nine unfixed equine cadaver brains imaged within 4 h of euthanasia in a 3-tesla MRI. The atlas was generated using linear and non-linear registration methods and quality assessed using signal and contrast to noise calculations. Tissue segmentation maps (TSMs) for white matter (WM), gray matter (GM) and cerebrospinal fluid (CSF), were generated and manually segmented anatomic priors created for multiple subcortical brain structures. The resulting atlas was validated and correlated to gross anatomical specimens and is made freely available at as an online resource for researchers (https://doi.org/10.7298/cyrs-7b51.2). The mean volume metrics for the whole brain, GM and WM for the included subjects were documented and the effect of age and laterality assessed. Alterations in brain volume in relation to age were identified, though these variables were not found to be significantly correlated. All subjects had higher whole brain, GM and WM volumes on the right side, consistent with the well documented right forebrain dominance of horses. This atlas provides an important tool for automated processing in equine and translational neuroimaging research.

Neuroanatomy of the equine brain as revealed by high-field (3Tesla) magnetic-resonance-imaging.

In this study, the morphology of the horse brain (Equus caballus) is decribed in detail using high field MRI. The study includes sagittal, dorsal, and transverse T2-weighted images at 0.25 mm resolution at 3 Tesla and 3D models of the brain presenting the external morphology of the brain. Representative gallocyanin stained histological slides of the same brain are presented. The images represent a useful tool for MR image interpretation in horses and may serve as a starting point for further research aiming at in vivo analysis in this species.

Viral Enrichment Methods Affect the Detection but Not Sequence Variation of West Nile Virus in Equine Brain Tissue.

West Nile virus (WNV), a small, positive sense, single stranded RNA virus continues to encroach into new locales with emergence of new viral variants. Neurological disease in the equine can be moderate to severe in the face of low to undetectable virus loads. Physical methods of virus enrichment may increase sensitivity of virus detection and enhance analysis of viral diversity, especially for deep sequencing studies. However, the use of these techniques is limited mainly to non-neural tissues. We investigated the hypothesis that elimination of equine brain RNA enhances viral detection without limiting viral variation. Eight different WNV viral RNA enrichment and host RNA separation methods were evaluated to determine if elimination of host RNA enhanced detection of WNV and increase the repertoire of virus variants for sequencing. Archived brain tissue from 21 different horses was inoculated with WNV, homogenized, before enrichment and separation. The protocols utilized combinations of low-speed centrifugation, syringe filtration, and nuclease treatment. Viral and host RNA were analyzed using real-time PCR targeting the WNV Envelope (E) protein and equine G3PDH to determine relative sensitivity for WNV and host depletion, respectively. To determine the effect of these methods on viral variation, deep sequencing of the E protein was performed. Our results demonstrate that additional separation and enrichment methods resulted in loss of virus in the face of host RNA depletion. DNA sequencing showed no significant difference in total sequence variation between the RNA enrichment protocols. For equine brain infected with WNV, direct RNA extraction followed by host RNA depletion was most suitable. This study highlights the importance of evaluating viral enrichment and separation methods according to tissue type before embarking on studies where quantification of virus and viral variants is essential to the outcome of the study.

Developing a 3-choice serial reaction time task for examining neural and cognitive function in an equine model

BackgroundLarge animal models of human neurological disorders are advantageous compared to rodent models due to their neuroanatomical complexity, longevity and their ability to be maintained in naturalised environments. Some large animal models spontaneously develop behaviours that closely resemble the symptoms of neural and psychiatric disorders. The horse is an example of this; the domestic form of this species consistently develops spontaneous stereotypic behaviours akin to the compulsive and impulsive behaviours observed in human neurological disorders such as Tourette’s syndrome. The ability to non-invasively probe normal and abnormal equine brain function through cognitive testing may provide an extremely useful methodological tool to assess brain changes associated with certain human neurological and psychiatric conditions. New methodAn automated operant system with the ability to present visual and auditory stimuli as well as dispense salient food reward was developed. To validate the system, ten horses were trained and tested using a standard cognitive task (three choice serial reaction time task (3-CSRTT)). ResultsAll animals achieved total learning criterion and performed six probe sessions. Learning criterion was met within 16.30±0.79 sessions over a three day period. During six probe sessions, level of performance was maintained at 80.67±0.57% (mean±SEM) accuracy. Comparison with existing method(s)This is the first mobile fully automated system developed to examine cognitive function in the horse. ConclusionsA fully-automated operant system for mobile cognitive function of a large animal model has been designed and validated. Horses pose an interesting complementary model to rodents for the examination of human neurological dysfunction.

Rabies direct fluorescent antibody test does not inactivate rabies or eastern equine encephalitis viruses

An examination using the routine rabies direct fluorescent antibody test was performed on rabies or Eastern equine encephalitis positive mammalian brain tissue to assess inactivation of the virus. Neither virus was inactivated with acetone fixation nor the routine test, thus laboratory employees should treat all samples as rabies and when appropriate Eastern equine encephalitis positive throughout the whole procedure.

Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue.

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3+ (pAb A0452, Dako) T-lymphocytes, CD79αcy+ B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+ neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+ astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

Comparative Study of the Major White Matter Tracts Anatomy in Equine, Feline and Canine Brains by Use of the Fibre Dissection Technique

The spatial anatomy of the white matter tracts is a subject of growing interest not only for researchers but also for clinicians. Imagistic methods have some limitations so that they should be confronted with dissection studies. The aim of this paper was to provide a three-dimensional view of the major white matter tracts in equine, feline and canine brains by use of the fibre dissection technique. Twenty cerebral hemispheres (six equine, four feline and 10 canine brains) were prepared according to the Klingler method. Stepwise mediolateral and lateromedial blunt dissections were performed using wooden sticks and spatulas. The lateromedial dissection was followed by the opening of the lateral ventricle. The use of the same multi-stage procedures resulted in a comparable exposure of the major association, projection and commissural fibres and their spatial relation with the lateral ventricle. To conclude, the proposed techniques are reproducible in equine, feline and canine brains and they can be successfully used for teaching, training or research in the field of neurobiology.

Comparison and Clinical Application of CT and MRI for Evaluation of the Equine Cranial Nerves

Reasons for performing studyAdvanced imaging modalities enable assessment of the equine skull and brain. Horses are susceptible to neurological dysfunction of the cranial nerves; however, our understanding of these structures’ imaging anatomy is limited.HypothesisMagnetic resonance imaging (MRI) will be superior to computed tomography (CT) for the identification of cranial nerves, but optimal assessment may depend on both modalities.ObjectivesIdentify cranial nerves and compare and contrast the utility of MRI and CT images of cadaver and clinical material. Interpret images from both modalities and determine the cranial nerve imaging anatomy.Study designProspective cadaver anatomical study combined with retrospective clinical case study.MethodsThe head of a neurologically normal 9‐year‐old Thoroughbred gelding was scanned immediately following euthanasia (performed for reasons unrelated to this study). High resolution MRI (1.5 Tesla) and CT examinations were conducted over a 12 h interval following euthanasia. Images obtained were compared with selected clinical cases which were scanned during anaesthesia (MRI; approximately 30–60 min) or standing sedation (CT; approximately 30 s).ResultsOn a high resolution MRI scan of a cadaver equine skull, each of the 12 cranial nerves and their topographic location was readily appreciated. Cranial nerves 1, 2, 3, 5, 7 and 8 were more easily identified in clinically relevant MRI scans. CT allowed visualisation of the stylomastoid foramen, the inner and middle ear and cranial nerves 2, 3, 5 and 7.ConclusionsHigh field MRI allows for excellent visualisation of equine cranial nerves. CT allows for detailed visualisation of the osseous canals and foramina. This study advances anatomical knowledge of the normal equine cranial nerves to aid interpretation in horses that display neurological dysfunction localising to the brain and brainstem.Ethical animal research: Horse owners gave their consent for their animals to be included in the study. Sources of funding: Institutional. Competing interests: None.

Development of a method for analysis of ketamine and norketamine enantiomers in equine brain and cerebrospinal fluid by capillary electrophoresis.

Ketamine and norketamine are being transported across the blood brain barrier and are also entering from blood into cerebrospinal fluid (CSF). Enantioselective distributions of these compounds in brain and CSF have never been determined. The enantioselective CE based assay previously developed for equine plasma was adapted to the analysis of these compounds in equine brain via use of an acidic pre-extraction of interferences prior to liquid/liquid extraction at alkaline pH. CSF can be treated as plasma. With 100 mg of brain tissue and 0.5 mL of CSF or plasma, assay conditions for up to 30 nmol/g and 6 μM, respectively, of each enantiomer with LOQs of 0.5 nmol/g and 0.1 μM, respectively, were established and the assays were applied to equine samples. CSF and plasma samples analyzed stemmed from anesthetized patient horses and brain, CSF and plasma were obtained from anesthetized horses that were euthanized with an overdose of pentobarbital. Data obtained indicate that ketamine and norketamine enantiomers are penetrating into brain and CSF with those of ketamine being more favorably transported than norketamine, whereas metabolites of norketamine are hindered. More work is required to properly investigate possible stereoselectivities of the ketamine metabolism and transport of metabolites from blood into brain tissue and CSF.

3-Tesla magnetic resonance imaging of the equine brain in healthy horses – Potentials and limitations

3-Tesla magnetic resonance imaging of the equine brain in healthy horses – Potentials and limitations

Diergeneeskundig gebruik van cardiogene biomarkers : focus op het gebruik bij paarden

Cardiale biomarkers, zoals natriuretische peptiden en troponinen, worden reeds routinematig in de humane geneeskunde gebruikt voor de diagnose, prognose en monitoring van hartaandoeningen. Hoewel ze ook in de kleine huisdierensector regelmatig bepaald worden om cardiologische en niet-cardiologische aandoeningen van elkaar te onderscheiden, is de literatuur omtrent het gebruik bij paarden beperkt. Bij paarden werd reeds een significante correlatie aangetoond tussen het ‘atrial natriuretic peptide’ (ANP), N-terminal ANP (NT-proANP) en de voorkamergrootte. Specifieke equine testen voor ‘brain natriuretic peptide’ (BNP) of ‘N-terminal BNP’ (NT-proBNP) bepaling ontbreken echter nog steeds. Troponinen (vooral troponine I) zijn meer uitgebreid bestudeerd bij het paard. Ze worden reeds gebruikt voor de diagnose van myocardschade en vervangen de minder specifieke lactaatdehydrogenase en creatinekinase iso-enzymen, waardoor het gebruik van deze laatste niet meer gerechtvaardigd is. Een laatste mogelijk belangrijke biomarker is aldosteron, maar aangezien slechts één studie bij paarden een correlatie aangetoond heeft tussen de aldosteronconcentratie en hartaandoeningen is uitgebreider onderzoek nodig.

Cross-modal recognition of human individuals in domestic horses (Equus caballus)

This study has shown that domestic horses are capable of cross-modal recognition of familiar humans. It was demonstrated that horses are able to discriminate between the voices of a familiar and an unfamiliar human without seeing or smelling them at the same moment. Conversely, they were able to discriminate the same persons when only exposed to their visual and olfactory cues, without being stimulated by their voices. A cross-modal expectancy violation setup was employed; subjects were exposed both to trials with incongruent auditory and visual/olfactory identity cues and trials with congruent cues. It was found that subjects responded more quickly, longer and more often in incongruent trials, exhibiting heightened interest in unmatched cues of identity. This suggests that the equine brain is able to integrate multisensory identity cues from a familiar human into a person representation that allows the brain, when deprived of one or two senses, to maintain recognition of this person.