Equine Brain Research Articles

Diagnostic sensitivity and specificity of in situ hybridization and immunohistochemistry for Eastern equine encephalitis virus and West Nile virus in formalin-fixed, paraffin-embedded brain tissue of horses

Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded (FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from 20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was used as the gold standard to confirm infection. All horses that tested positive for EEEV by RT-PCR also tested positive by IHC and ISH, except for 1 case that was false-negative by ISH. In contrast, all horses that tested positive for WNV by RT-PCR tested negative by IHC and only 2 horses tested positive by ISH. No false-positives were detected with either method for both viruses. Both IHC and ISH are highly specific and sensitive diagnostic methods to detect EEEV in equine FFPE brain tissues, although neither appear effective for the diagnosis of WNV in equine neurologic cases.

Role of adhesion molecules and inflammation in Venezuelan equine encephalitis virus infected mouse brain

BackgroundNeuroinvasion of Venezuelan equine encephalitis virus (VEEV) and subsequent initiation of inflammation in the brain plays a crucial role in the outcome of VEEV infection in mice. Adhesion molecules expressed on microvascular endothelial cells in the brain have been implicated in the modulation of the blood brain barrier (BBB) and inflammation in brain but their role in VEEV pathogenesis is not very well understood. In this study, we evaluated the expression of extracellular matrix and adhesion molecules genes in the brain of VEEV infected mice.FindingsSeveral cell to cell adhesion molecules and extracellular matrix protein genes such as ICAM-1, VCAM-1, CD44, Cadherins, integrins, MMPs and Timp1 were differentially regulated post-VEEV infection. ICAM-1 knock-out (IKO) mice infected with VEEV had markedly reduced inflammation in the brain and demonstrated a delay in the onset of clinical symptoms of disease. A differential regulation of inflammatory genes was observed in the IKO mice brain compared to their WT counterparts.ConclusionsThese results improve our present understanding of VEEV induced inflammation in mouse brain.

Miniature kilohertz-frequency ultrasound implant for brain drug delivery.

Glioblastoma multiforme is a highly aggressive brain malignancy currently treated by surgical resection, radiotherapy, and local drug delivery. However, due to heterogeneities in neural tissue and tumor cell migration into surrounding healthy tissue, treatment strategies remain ineffective and patient prognosis poor. We have shown that low intensity focused and nonfocused ultrasound may safely enhance the penetration and distribution of chemotherapeutics delivered locally via convection-enhanced infusions in rodent brain parenchyma in vivo. In scaling the ultrasound-assisted therapy from rodent to large animal and increasing its clinical translatability, we designed a miniature kilohertz (100 and 600 kHz) ultrasound implant that can be placed within the brain tumor resection cavity during patient recovery to assist chemotherapy. The miniature ultrasound system has been tested in a diffusion-based human-sized agarose brain phantom tumor model under various acoustic intensities and pulse sequences. Results indicate that ultrasound increases the volume of tracer distribution by a factor of 2–3 compared with controls. We present the effects of acoustic intensity and pulse interval from the implant on distribution of tracers in vitro and ex vivo in the phantom model and intact equine brain, respectively. The miniature ultrasound implant provides significant drug distribution gains that may improve patient outcome.

Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D

The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.

Diagnostic utility of computed tomography imaging in equine intracranial conditions

The use of computer tomography (CT) and contrast-enhanced CT (CCT) to image the head is common. However, the validity of CT as a neurodiagnostic indicator of intracranial diseases in horses is unknown. To define the validity of CT and CCT in horses with suspected intracranial disorders. The validity of CT imaging was estimated by comparing clinical, clinicopathological and histopathological findings to CT findings in 15 horses presented for intracranial disorders, for which pre- and post contrast CT images and post mortem examination of the brain and skull were reviewed. Post mortem examination (gross and histopathological examination) was considered as the gold standard; and sensitivity, specificity, predictive values, likelihood ratios, and pre- and post test probabilities were calculated. All horses had abnormal neurological examinations on admission. Computer tomography imaging identified intracranial lesions in 8 horses, and included masses (oligodendroglioma, adenocarcinoma and cholesterinic granulomas), acute haemorrhage and skull fractures. Computer tomography imaging failed to identify intracranial lesions in 6 cases, which included meningitis, meningoencephalitis and nonacute haemorrhage. Lesions not recognised by CT were also not evident on gross examination but were identified by histopathological examination of the brain. Post mortem examination of the brain and skull was unremarkable in one horse, for which cranial CT imaging was normal (specificity, 100%). Therefore, the odds of having an intracranial lesion after an abnormal CT were very high. In contrast, there was a moderate sensitivity (57.1%, 95% confidence interval: 29.6-81.2). CT was an excellent neurodiagnostic tool in identifying skull fractures, intracranial space-occupying lesions (e.g. neoplasia) and acute haemorrhage and allows to rule in intracranial disorders. However, CT showed limited sensitivity in identifying inflammatory disorders and small parenchymal lesions in the equine brain, which was not further detectable after contrast administration.

Equine transcriptome quantification using human GeneChip arrays can be improved using genomic DNA hybridisation and probe selection

Affymetrix GeneChip arrays are a powerful tool for transcriptome profiling and have been applied to a wide range of species. A genomic DNA (gDNA)-based probe selection method has been developed which broadens the range of species to which GeneChips may be successfully applied. This study demonstrated that gDNA-based probe selection on the Affymetrix U133+2 GeneChip array can be used to study the equine transcriptome which, to date, has received only limited attention. More than 29,000 transcripts can be detected in equine brain and liver and in primary cultures of equine articular chondrocytes. Gene ontology analysis of differentially expressed genes revealed the presence of expected categories within each tissue. The level of gene expression could also be correlated with the phenotypes and specialised functions of each tissue. The results demonstrated that probe selection on a human chip can be successfully used to study the equine transcriptome.

Parenchymal and Vascular Lesions in Ageing Equine Brains: Histological and Immunohistochemical Studies

Many age-related changes are described in the nervous system of different species, but detailed studies of brain lesions in ageing horses are lacking. The aim of the present study was to systematically characterize lesions in the brains of 60 horses aged from 7 to 23 years. No gross changes were present in any brain. Microscopically, spongiform changes, lipofuscin storage, corpora amylacea, gliosis and satellitosis were common, together with axonal and neuronal swellings. The most important findings were the presence of pseudocalcium-calcium (pCa-Ca) deposits and arterial wall degeneration. Scanning electron microscopical examination of two cases with vascular mineralization revealed marked deposition of an amorphous substance in the vessel walls that was probably formed by a polyanionic protein matrix and a mineral component. Immunohistochemically, numerous axonal spheroids were positively labelled for ubiquitin. No PrPsc was detected in sections with neuronal vacuolation. Neuronal swelling, corpora amylacea, hippocampal Tau-positive neurons and methenamine-positive diffuse (preamyloid) plaques were also detected. Congo red staining failed to detect amyloid deposition. The characterization of age-related lesions in the brains of these horses will allow these changes to be discriminated from pathological processes in future studies. Some lesions described here, including some vascular changes, the presence of diffuse plaques and tau accumulation in hippocampal neurons, have not been described previously in the horse.

Infectious entry of equine herpesvirus-1 into host cells through different endocytic pathways

We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery assay after viral internalization, suggesting that EHV-1 enters E. Derm cells via energy- and pH-dependent endocytosis. These results suggest that EHV-1 utilizes multiple endocytic pathways in different cell types to establish productive infection.

Oligonucleotide array analysis of Toll-like receptors and associated signalling genes in Venezuelan equine encephalitis virus-infected mouse brain

Venezuelan equine encephalitis (VEE) is an emerging infectious disease. VEE virus (VEEV) may cause lethal infection of the central nervous system in horses and humans. The mechanisms underlying the host immune response to VEEV infection in the brain are not fully understood. Toll-like receptors (TLRs) recognize conserved microbial sequences and induce specific biological responses in the form of proinflammatory cytokine induction. TLR expression in blood following VEEV infection has been reported in non-human primates and TLRs are also upregulated in the brains of mice infected with other alphaviruses. In this study, mice (3-5 weeks old) were infected with V3000, a neurovirulent strain of VEEV, and gene expression of TLRs and their associated signalling molecules was evaluated. VEEV infection resulted in upregulation of TLR 1, 2, 3, 7 and 9, chemokines, inflammatory cytokines, interferon (IFN), IFN regulatory factors and genes involved in signal transduction such as Mcp1, Cxcl10, IL12alpha/beta, IFN-beta, IRF-1, IRF-7, Jun, Fos, MyD88, Nfkb, Cd14 and Cd86. These results demonstrate the upregulation of TLRs and associated signalling genes following VEEV infection of the brain, with important implications for how VEEV induces inflammation and neurodegeneration.

Pharmacologic characterization of novel adenosine A2A receptor agonists in equine neutrophils

To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes. Neutrophils isolated from 8 healthy horses. Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A(1), A(2A), and A(3) receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined. Radioligand binding experiments yielded a ranked order of affinity for the brain equine A(2A) receptor on the basis of 50% inhibitory concentrations (IC(50)) of the agonists as follows: ATL307 (IC(50) = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5'-N-ethylcarboxyamidoadenosine > 5'-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A(2A) over A(1) and A(3) receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A(2A) receptors. Results indicated that activation of A(2A) receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A(2A) receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.

Preliminary Anatomic Investigation of Three Approaches to the Equine Cranium and Brain for Limited Craniectomy Procedures

To describe surgical approaches to the equine cranium and brain for limited craniectomy. Descriptive anatomic study. Equine cadavers (n=7). Head and neck sections from cadavers were used to establish techniques for exposing areas of the equine brain. Three basic approaches were used: rostrotentorial, suboccipital, and transfrontal. Techniques were adapted from small animal descriptions and modified to account for anatomic differences. Descriptions of the rostrotentorial, suboccipital, and transfrontal approaches to the equine cranium and brain were defined. Dorsal, caudal, lateral, and rostral aspects of the cranium and underlying cerebral cortex can be accessed using the rostrotentorial, suboccipital, and transfrontal approaches. Access to the dorsal, dorsolateral, and rostral aspects of the cerebral cortex is uncomplicated and more superficial compared with access to the extreme caudolateral cerebrum and cerebellum, which is complicated by the location of the transverse and temporal sinuses and deep exposure. Many cases of equine brain disorders involve intracranial abscessation or hematoma formation because of trauma. Components of therapy may include microbial culture and surgical drainage or decompression of the affected area through limited craniectomy procedures. Increased use of advanced imaging in equine brain disease has resulted in earlier diagnosis and more precise lesion localization. Subsequently, the number of horses likely to improve with limited surgical intervention has increased. When surgery is indicated, descriptions of surgical approaches to the equine cranium and brain provide important information.

Age-related and Non-age—related Changes in 100 Surveyed Horse Brains

Brains from 100 horses, aged 2-25 years, were systematically examined by histopathology at 46 different neuroanatomical sites. The horses were sourced from a slaughterhouse (group A, n = 57), from a kennel that collected dead animals, and from 2 diagnostic laboratories (group B, n = 43). All horses from group A and 26 horses from group B were examined by a veterinarian in the period before death. None of the horses were known to exhibit clinical signs suggestive of neurologic disease. Among the main changes identified were vacuolation in the neuropil (n = 73), neurons (n = 32), white matter (n = 31), and focal perivascular lymphoid cell infiltrates (n = 35). Spheroids were frequently seen (n = 91), and 10 horses each had more than 10 spheroids in the cuneate or gracile nucleus. Statistically significant age-related changes noted included intraneuronal (n = 97) and glial or extracellular lipofuscin deposition (n = 41), hemosiderin deposition around blood vessels (n = 60), and calcium depositions (n = 24). One horse had low-grade nonsuppurative meningoencephalitis; Alzheimer type II cells were detected in the brains of 2 horses. Hyalinized vessel walls in the cerebellum were observed in 1 horse. It was concluded that some histopathologic changes are a frequent feature in equine brains, which has implications for the pathologists involved in equine neurology and disease surveillance.

Zoonoses: A Case Presentation

ISSUE: Certain bacteria can cause disease in animals as well as humans. In some cases the bacteria is transmitted from the animal to the human. A case of Rhodococcus equine brain abscess was identified in a 26 year old intravenous drug user with exposure to horses.Once thought to be exclusively an equine pathogen, Rhodococcus equine infection is occuring in humans with an increasing frequency. It has been found in immunocompromised individuals including those with AIDS, small cell lung carcinoma, lymphoma, or lung and bone marrow transplant recipients. Very little is known about the pathogenesis of the disease except for its mode of transmission which is almost always via the droplet/ respiratory route. PROJECT: A patient was admitted to the hospital with the diagnosis of sinus infection. Her presenting signs and symptoms were right sided weakness, headache, and facial droop. Her mental status progressively declined. Lab studies included a negative HIV test and a CD4 count of 813. A CT of the brain revealed a thalamic brain lesion. Since the differential diagnosis included an infectious process the patient was empirically started on Vancomycin, Ceftriaxone, Metronidazole, Bactrim, Amphotericin B, and Acyclovir. A brain biopsy was performed on hospital day 60. RESULTS: On hospital day 64 brain tissue grew Rhodococcus equine, a bacteria that causes disease in horses. Upon further questioning of the patient's family a possible link was found. The family stated that the patient was known to inject herself with needles previously used on horses. LESSONS LEARNED: Although many zoonotic diseases have been reported this is believed to be the first case of brain abscess caused by Rhodococcus equine. A zoonotic disease should be considered in the differential diagnosis of any patient that works closely with animals. A thorough history is important. The patient's exposure to horses and her intravenous drug use were known, however more intense questioning may have yielded critical information about her injection practices. Clinical correlation is important so as not to disregard a culture result as a contaminant. In this era of bioterrorism it is important to consult with the state and national authorities to determine if an unusual organism has been earmarked as a potential agent of bioterrorism. As of this time Rhodococcus equine is not. When a brain biopsy of unknown etiology is performed consideration should be given to handling the instruments using a protocol for suspected prion disease.

Structural and Functional Diseases of the Equine Brain

Structural and functional diseases of the equine brain may cause a variety of clinical signs depending on both the cause and the precise location of the disease in the brain. Signs of behavioral abnormality and central blindness are two of the more common signs. Structural disorders are most common in adults, whereas a greater number of functional diseases occur in foals. Computed tomography and magnetic resonance imaging are of great diagnostic value for structural diseases. Treatment and prognosis vary considerably depending on the specific cause of the brain disease.

Differential susceptibility of equine and mouse brain microvascular endothelial cells to equine herpesvirus 1 infection

Equine herpesvirus 1 (EHV-1) shows endotheliotropism in the central nervous system (CNS) of infected horses. However, infection of endothelial cells has not been observed in the CNS of infected mice. To explore the basis for this difference in endotheliotropism, we compared the susceptibility of equine brain microvascular endothelial cells (EBMECs) and mouse brain microvascular endothelial cells (MBMECs) to EHV-1 infection. The kinetics of viral growth in EBMECs was typical of a fully productive infection whereas viral infection in MBMECs seemed to be nonproductive. Immunofluorescence microscopy using anti-EHV-1 polyclonal antibody demonstrated viral antigen in infected EBMECs, but not infected MBMECs. EHV-1 immediate early (IE), early (ICP0), and late (gB, gD and gK) transcripts were expressed in infected EBMECs. However, none of these genes was detected in infected MBMECs by reverse transcription-polymerase chain reaction. Electron microscopic examination at the stage of viral entry showed that viral particles were present within uncoated vesicles in the cytoplasm of EBMECs, but absent from those of MBMECs. These results suggest that viral entry is an important determinant of the susceptibility of EBMECs and MBMECs to EHV-1 infection.

Motor and sensory laterality in thoroughbred horses

We investigated lateralisation in horses because it is likely to be important in training and athletic performance. Thoroughbred horses ( n = 106) were observed every 60 s for 2 h, when they were at pasture, and the position of the forelimbs in relation to one another was recorded. There was a population bias skewed to standing with the left forelimb advanced over the right (i.e. directional lateralisation). Using the first 50 observations, the distribution of preferences was 43 significantly left, 10 significantly right with 53 being non-significant (i.e. ambidextextrous). The strength of motor bias increased with age, suggesting maturation or an influence of training. The horses were also presented with an olfactory stimulus (stallion faeces) to score the tendency to use one nostril rather than the other. A significant preference to use the right nostril first was shown in horses under 4 years of age ( n = 61) but not in older horses. Of the 157 horses tested for nostril bias, 76 had been assessed for motor bias and so were used for analysis of the relationship between laterality in the two modalities. There was no significant relationship between direction of foreleg motor bias and first nostril used, total number of inhalations or laterality index of nostril use. The absence of a correlation between laterality of nostril use and motor bias indicates that lateralisation of the equine brain occurs on at least two levels of neural organisation—sensory and motor—a finding that is consistent with other examples of lateralisation in species that have been examined in more detail.

Magnetic resonance imaging of the normal equine brain.

The purpose of this investigation was to define the magnetic resonance (MR) imaging appearance of the brain and associated structures of the equine head. MR images were acquired in oblique dorsal (T2-weighted), sagittal (T1-weighted), and transverse planes (T2-weighted), using a magnet of 1.5 Tesla and a human body coil. Relevant anatomic structures were identified and labeled at each level. The resulting images provided excellent anatomic detail of the cranioencephalic structures. Annotated MR images from this study are intended as a reference for clinical imaging studies of the equine head, specially in the diagnosis of brain diseases in the horse.

Magnetic resonance imaging of two normal equine brains and their associated structures

Magnetic resonance images were obtained from two isolated horses' heads. Ten mm thick, Ti -weighted images were taken with a 1.5 Tesla magnet and a body coil, and compared with...

Development of the equine brain motor system

The purpose of the present study was to demonstrate the structural maturation of the horse brain in the critical period of development emergence of coordinated locomotion. Equine brains from 14 days before expected birth to adulthood were fixed in formaldehyde and embedded in paraffin. After taking the outer parameters of the brains, full series of large-area coronal sections were prepared on a special microtome and stained with Nissl's cresyl violet and Haidenhain's iron-haematoxylin. Microscopic images of sections were digitized and were subjected to computer-aided image analysis. The gross morphology of the brains and the image analysis of histological preparations suggest that in the perinatal period studied there is no substantial increase in brain size and mass, while the amount of Nissl substance and myelin grows rapidly till postnatal day 45. Then a relative decrease of both is observed till adulthood accompanied by a doubling of brain size and mass. It is concluded that during the maturation of the equine brain, decisive changes of the motor system such as up-regulation of protein sysnthesis and full myelination of motor tracts takes place during the critical period of onset of coordinated locomotion.

Diagnosis of Eastern Equine Encephalitis by Immunohistochemistry in Two Flocks of Michigan Ring-Neck Pheasants

The diagnosis of eastern equine encephalitis (EEE) virus infection in avian species is relatively difficult when compared with other species. There are no characteristic histologic lesions in the avian brain that would serve to distinguish EEE from infections with, for example, Newcastle disease or highly pathogenic avian influenza virus. Traditionally, virus isolation (VI) and/or hemagglutination inhibition (HI) has been used for a definitive diagnosis of EEE in birds. Recently, we developed an immunohistochemistry (IHC) technique for confirmatory diagnosis of EEE infection in equine brain. This test also detected EEE virus in formalin-fixed avian brain. VI confirmed IHC finding in two cases of EEE in ring-neck pheasants. IHC is a rapid, sensitive test for confirming and differentiating a histopathologic diagnosis of EEE in avian species and should be considered as an alternative test to VI or HI.