Neurochemistry and circuit organization of the lateral spiriform nucleus of birds: A uniquely nonmammalian direct pathway component of the basal ganglia.

We used diverse methods to characterize the role of avian lateral spiriform nucleus (SpL) in basal ganglia motor function. Connectivity analysis showed that SpL receives input from globus pallidus (GP), and the intrapeduncular nucleus (INP) located ventromedial to GP, whose neurons express numerous striatal markers. SpL-projecting GP neurons were large and aspiny, while SpL-projecting INP neurons were medium sized and spiny. Connectivity analysis further showed that SpL receives inputs from subthalamic nucleus (STN) and substantia nigra pars reticulata (SNr), and that the SNr also receives inputs from GP, INP, and STN. Neurochemical analysis showed that SpL neurons express ENK, GAD, and a variety of pallidal neuron markers, and receive GABAergic terminals, some of which also contain DARPP32, consistent with GP pallidal and INP striatal inputs. Connectivity and neurochemical analysis showed that the SpL input to tectum prominently ends on GABAA receptor-enriched tectobulbar neurons. Behavioral studies showed that lesions of SpL impair visuomotor behaviors involving tracking and pecking moving targets. Our results suggest that SpL modulates brainstem-projecting tectobulbar neurons in a manner comparable to the demonstrated influence of GP internus on motor thalamus and of SNr on tectobulbar neurons in mammals. Given published data in amphibians and reptiles, it seems likely the SpL circuit represents a major direct pathway-type circuit by which the basal ganglia exerts its motor influence in nonmammalian tetrapods. The present studies also show that avian striatum is divided into three spatially segregated territories with differing connectivity, a medial striato-nigral territory, a dorsolateral striato-GP territory, and the ventrolateral INP motor territory.

Protein expression of KIT, BRAF, GNA11, GNAQ and RASSF1 in feline diffuse iris melanomas.

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/µm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.

Effects of three blood derived products on equine corneal cells, an in vitro study.

BACKGROUND: Despite advances in therapy of corneal ulcerative diseases in horses, a vast number of cases require surgical intervention, due to poor response to treatment. Topical application of serum has been used for many years, based on its anticollagenolytic properties and the presence of growth factors promoting corneal wound healing. However, although other blood derived products i.e. platelet rich plasma (PRP), plasma rich in growth factors (PRGF) have been widely used in equine orthopaedics and in human ophthalmology, no reports of the effects of these blood derived products exist in equine ophthalmology. OBJECTIVES: To determine in vitro effects of PRGF and PRP on equine corneal cells compared with serum. STUDY DESIGN: Prospective controlled cohort study. METHODS: Blood from 35 healthy horses was used to produce serum, PRGF (Endoret® ), and PRP (E-PET™). Limbal- and stromal cells were isolated from healthy corneas of six horses and treated with 20% serum, 20% PRGF or 20% PRP. Proliferation rates and migration capacity were analysed in single cell cultures as well as co-culture systems. RESULTS: Cell proliferation increased with PRP treatment, remained constant in PRGF treated cells, and declined upon serum treatment over a period of 48 h. Migration capacity was significantly enhanced with PRP treatment, compared with PRGF treatment. Intact leucocytes, mainly eosinophils, were only detected in PRP. MAIN LIMITATIONS: Due to the study design use of autologous blood products on corneal cells was not possible. CONCLUSIONS: The results demonstrate beneficial effects of PRP on proliferation as well as migration capacity of equine corneal cells in vitro. In vivo studies are warranted to determine further beneficial effects of PRP in horses with corneal ulcers.

Mutation analysis and gene expression profiling of ocular melanomas in cats.

Feline ocular melanomas show a high malignant behaviour, but adjunctive therapies are non-existent. The aim of this pilot study was to determine, whether feline ocular melanomas harbour mutations comparable to mutations in human melanomas and to evaluate the gene expression status of genes known to be involved in initiation and progression of human melanomas. Mutation hotspot regions of several genes of feline ocular melanomas were analysed by DNA sequencing and RNA expression levels of the respective genes and others were evaluated by quantitative real-time polymerase chain reaction (RT-qPCR). Common mutations found in human melanomas are not present in feline tumours. Gene expression analysis revealed a significant upregulation of KIT and LTA4H, as well as a downregulation of GNAQ, GNA11, BRAF and RASSF1 in feline ocular melanomas. As KIT seems to harbour a potential as target gene in human uveal melanomas, future studies should further investigate the potential of KIT as target for adjunctive therapy in feline ocular melanomas.

Impression cytology as diagnostic tool in horses with and without ocular surface disease.

BACKGROUND: Cytology plays a major role in the diagnosis of ocular surface diseases. OBJECTIVE: To compare 2 cytological sampling methods for obtaining corneal and conjunctival cell samples regarding irritation for the patient, invasiveness, sample quality and diagnostic equivalence. STUDY DESIGN: Observational prospective study. METHODS: In 5 healthy horses, conjunctival and corneal samples were taken bilaterally by impression cytology sampling (ICS) and cytobrush sampling (CBS). Irritation and invasiveness were assessed with an eye irritation and an epithelial damaging score system, respectively. Sample quality was evaluated via morphometric analysis and graded by a board certified clinical pathologist. For the assessment of diagnostic equivalence, 15 eyes of 14 client owned horses with ocular surface anomalies were sampled by ICS and CBS. The methods were compared regarding the types of inflammatory cells and/or infectious agents detected and if the correct diagnosis could be achieved. Histopathology served as gold standard when available. RESULTS: ICS was significantly less invasive and less irritating for the horses. Both methods retrieved cells of overall high quality; the cell quantity was significantly higher in IC samples. ICS preserved the natural cellular layout. There was a fair, but no statistically significant agreement between the diagnostic outcomes between sampling methods although CBS resulted in a slightly greater variability of inflammatory cell types compared to ICS. MAIN LIMITATIONS: None. CONCLUSIONS: Because of its low irritability and invasiveness ICS can be recommended for cell sampling in fragile corneas and for experimental studies. ICS is especially useful in cases where preservation of cellular layout is advantageous. CBS samples are easier to obtain because of the small equine palpebral fissure. Additionally, the identification of inflammatory cells within conjunctival cell samples is easier. Overall, CBS is still an appropriate method in clinical settings.

Keratoconjunctivitis in a group of Icelandic horses with suspected γ-herpesvirus involvement.

REASONS FOR PERFORMING STUDY: The role of equid γ-herpesviruses on ocular surface diseases has been disputed, because the diagnosis is usually based on clinical symptoms and detection of viral DNA from samples obtained from live animals. OBJECTIVES: To describe the clinical course, results of polymerase chain reaction (PCR) analysis, in situ hybridisation, cell culture and pathohistological findings of select cases in a presumed outbreak of herpesvirus infection in a group of 15 Icelandic horses. STUDY DESIGN: Case series. METHODS: Pooled ocular and nasal swabs and peripheral blood mononuclear cells of horses diagnosed clinically with herpesvirus-associated keratoconjunctivitis were analysed for presence of equine herpesviruses (EHV)-2 and EHV-5 nucleic acid using real-time PCR. Necropsy specimens from one horse, subjected to euthanasia due to deterioration of clinical symptoms were examined histopathologically, and analysed for presence of EHV-2 and EHV-5 nucleic acid using real-time PCR. In situ hybridisation and cell culture of select samples were performed. RESULTS: All horses with symptoms of severe keratoconjunctivitis were positive for presence of either EHV-2 and/or EHV-5 nucleic acid using real-time PCR. Assessment of necropsy specimens of the most severely affected case, revealed presence of EHV-2 and/or EHV-5 nucleic acid in several ocular and extraocular anatomical locations. The remaining horses responded favourably to symptomatic treatment. CONCLUSIONS: This case series illustrates a severe outbreak of keratoconjunctivitis in a group of Icelandic horses, with suspected γ-herpesvirus involvement. For the first time equid γ-herpesviruses were detected in intraocular anatomical locations.

Encephalitozoon cuniculi causes focal anterior cataract and uveitis in dogs.

Three mongrel dogs, aged 10 months (case 1), 14 months (case 2) and 7.5 years (case 3), were presented because of ophthalmologic disorders of 4 months, 6 months and 7 years duration, respectively. All three dogs were offspring of stray dogs from Hungary and Serbia and had positive serum antibody titres against Encephalitozoon (E.) cuniculi. The two young dogs showed unilateral, the older dog bilateral chronic anterior uveitis with posterior synechia and focal anterior cortical cataract. The fundi that could be evaluated developed focal tapetal hyporeflective lesions in the course of the disease. Dogs 1 and 2 underwent removal of the lens via phacoemulsification. PCR of the lens material was positive for E. cuniculi strains IV and II, respectively. In dog 2 findings suggestive of microsporidia were detected underneath the anterior lens capsule by immunohistochemical staining. In all cases medical treatment consisted of systemic fenbendazole, prednisolone, and topical anti-inflammatory drugs, and additional brinzolamid/timolol for dog 3. For the time being all cases (follow up 23 months, 6 months and 3 months, respectively) are still on topical anti-inflammatory therapy. It is concluded that E. cuniculi infections can cause cataract and chorioretinal lesions in dogs.

Introduction of the use of thermography and thermometry in the diagnosis of uveitis in horses: a pilot project.

AIMS AND OBJECTIVES: To date assessment of changes in ocular temperature, as a hallmark of uveitis in horses has not been determined. Therefore the aim of the current study was to determine whether ocular temperature is increased in acute uveitic eyes compared with non-uveitic eyes, and to compare an affordable thermometry device with a thermography device. MATERIAL AND METHODS: Ocular temperatures of both eyes of five horses with acute unilateral uveitis and 10 normal horses were measured using thermometry and thermography. Presence and absence of acute uveitis were diagnosed through a complete ophthalmological examination. Ambient temperature and core body temperature were also recorded. RESULTS: The difference in temperatures between uveitic eyes and non-uveitic eyes was marked but not statistically significant (mean thermography temperature 34.0°C sd±1.6°C and 32.7°C sd±2.4°C, respectively v mean thermometry temperature 34.0°C sd±1.9°C and 31.6°C sd±3.1°C, respectively). No influence of core body temperature on either method was detected. Thermography was less influenced by ambient temperature than was thermometry. CONCLUSION: In conclusion uveitic eyes are not significantly warmer than non-uveitic eyes. Despite the lack of significance, a tendency towards increased ocular temperature in uveitic eyes, compared with non-uveitic eyes was noted. Therefore more research on this topic is warranted.

A retrospective survey of ocular abnormalities in pugs: 130 cases.

OBJECTIVES: To determine the types and frequency of ophthalmic findings in pugs. MATERIALS AND METHODS: Retrospective analysis of case records of pugs presented to an ophthalmology unit between 2001 and 2012. Ophthalmological findings were correlated with age, gender, presenting signs and time of onset of disease. RESULTS: In total, 130 pugs (258 eyes) with a mean (±sd) age of 2 · 8 (±2 · 87) years were examined. Ocular abnormalities identified included keratoconjunctivitis sicca (n = 39 eyes), macroblepharon (n = 258 eyes), entropion (n = 258 eyes), distichiasis (n = 56 eyes), ectopic cilia (n = 8 eyes), conjunctivitis (n = 88 eyes), corneal pigmentation (n = 101 eyes), opacity (n = 63 eyes), ulceration (n = 46 eyes), vascularisation (n = 35 eyes), iris-to-iris persistent pupillary membranes (n = 21 eyes) and cataract (n = 18). Keratoconjunctivitis sicca was significantly associated with the presence of corneal pigmentation (P = 0 · 007 for left eyes; P = 0 · 043 for right eyes). However corneal pigmentation was also identified in pugs (n = 61) without keratoconjunctivitis sicca. There was a significant influence of ectopic cilia on corneal ulceration (P < 0 · 001). Younger dogs (mean age, 1 · 28 (±0 · 45) years) were significantly more affected by distichiasis. CLINICAL SIGNIFICANCE: The high number of cases of corneal pigmentation without keratoconjunctivitis sicca suggests that there may be additional yet undetermined factors involved in the development of corneal pigmentation in pugs.

Prevalence of linear keratopathy in a herd of Lipizzaners over an 18-month period.

The prevalence of linear keratopathy with progressing age in a closed population of a single horse breed is reported. All Lipizzaners in three federal states in Austria underwent complete ophthalmic examination four times over a period of 18 months, with six-month intervals. Findings consistent with linear keratopathy were recorded, and associated with factors such as sex, location, boarding system and level of performance throughout the study period. Logistic regression was applied to determine the influence of age on ophthalmic findings. On the first, second, third and fourth examinations, 0.8 per cent, 3.1 per cent, 4.4 per cent and 4.8 per cent (of 266, 261, 249 and 230 horses, respectively) of the study population, were diagnosed with linear keratopathy. This finding was consistently identified in the same horses, and once identified, no further progression was noted. Horses with this finding had no history of previous ocular problems or concurrent ocular abnormalities. Statistical analysis did not reveal any influence of sex, location, boarding, or level of performance; however the prevalence of linear keratopathy was found to increase with progressive age (P<0.5). The results of this study indicate that linear keratopathy was not congenital and was non-progressive in the Lipizzaner over a period of 18 months.

Patterns of convergence in the central nucleus of the inferior colliculus of the Mongolian gerbil: organization of inputs from the superior olivary complex in the low frequency representation.

Projections to the inferior colliculus (IC) from the lateral and medial superior olivary nuclei (LSO and MSO) were studied in the gerbil (Meriones unguiculatus) with neuroanatomical tract-tracing methods. The terminal fields of projecting axons were labeled via anterograde transport of biotinylated dextran amine (BDA) and were localized on series of horizontal sections through the IC. In addition, to make the results easier to visualize in three dimensions and to facilitate comparisons among cases, the data were also reconstructed into the transverse plane. The results show that the terminal fields from the low frequency parts of the LSO and MSO are concentrated in a dorsal, lateral, and rostral area that is referred to as the "pars lateralis" of the central nucleus by analogy with the cat. This region also receives substantial input from both the contralateral and ipsilateral cochlear nuclei (Cant and Benson, 2008) and presumably plays a major role in processing binaural, low frequency information. The basic pattern of organization in the gerbil IC is similar to that of other rodents, although the low frequency part of the central nucleus in gerbils appears to be relatively greater than in the rat, consistent with differences in the audiograms of the two species.

Ophthalmological findings in a closed herd of Lipizzaners.

REASONS FOR PERFORMING THE STUDY: There are no studies on the ophthalmic health status in the Lipizzaner breed. OBJECTIVE: To perform an ophthalmological survey in a closed herd in 3 housing systems. MATERIALS: All horses at the Federal Stud Piber, Heldenberg and the Spanish Riding School Vienna were subjected to ophthalmological examination. Findings were documented and correlated with factors such as age, gender, location and housing situation. The interrelation of abnormalities of different regions was determined by calculating accuracy. Pedigree analysis of horses affected by cataract was performed. RESULTS: A total of 267 horses met the inclusion criteria of this study. Of these, 172 horses were stabled in Piber, 72 in Vienna and 23 in Heldenberg. Ophthalmological examination revealed 203 cases with ocular findings, predominantly involving the conjunctiva (44.6%), cornea (21.7%) and fundus (37.5%). In 24 (8.9%) horses a diagnosis of cataract was made. There was no interrelation between different eye diseases. Horses stabled at Piber were significantly more affected by conjunctival and fundic abnormalities. Fewer abnormalities were found in horses stabled in open boxes. Access to free stalls resulted in more cases of conjunctivitis. Pedigree analysis showed no evidence of inbreeding as a cause of cataract formation. CONCLUSION AND POTENTIAL RELEVANCE: Acquired corneal abnormalities were over-represented in this study. This survey may serve as a basis for further research on the pathogenesis of ophthalmic diseases in Lipizzaners.

Bovine papillomavirus type 1 and Equus caballus papillomavirus 2 in equine squamous cell carcinoma of the head and neck in a Connemara mare.

In January 2010, 18 months after excision of an ocular squamous cell carcinoma (SCC), a Connemara mare presented with anorexia and periorbital/parotideal lesions. Post mortem examination revealed these lesions as forming one entity, with 2 additional growths in the retropharyngeal region and the left jugular groove, respectively. The lesions were confirmed histopathologically as SCCs. Using PCR, peripheral blood mononuclear cells (PBMCs) from 2008 and 2010, tumour tissue, intact skin and vulval mucosa were screened for Equus caballus papillomavirus type 2 (EcPV-2) and bovine papillomavirus types 1 and 2 (BPV-1/2) DNA. Whereas PBMCs from 2008 scored negative, EcPV-2 DNA was present in PBMCs and SCCs from 2010. Furthermore, reverse transcription PCR revealed EcPV-2 E6 transcripts in these samples. BPV-1/2 DNA, but not RNA, was demonstrated in the periorbital/parotideal mass, the SCC of the jugular groove, vulval mucosa and intact skin, but not in the pharyngeal SCC and PBMCs. Sequencing revealed a 99% similarity of EcPV-2 amplicons with the published EcPV-2 sequence. BPV-1/2 amplicons corresponded to BPV type 1. This report is the first to describe co-presence of BPV-1 and EcPV-2 DNA in a pony affected by an uncommon form of nongenital SCC, and the detection of EcPV-2 transcripts in lesions and PBMCs.

Posterior segment diseases.

Diseases of the vitreous, fundus and optic nerve are described and illustrated according to their ophthalmological appearance. Vitreal alterations are commonly of developmental, degenerative, age related, traumatic or inflammatory origin and of minor clinical relevance. In contrast, those affecting the fundus, may be accompanied by visual deficits or blindness. Fundic lesions of inflammatory (multifocal or peripapillary chorioretinitis, haemorrhage, retinal detachment) and traumatic origin have to be differentiated from congenital (congenital stationary night blindness, colobomatous defects, retinal dysplasia), degenerative (cystic retinal degeneration), metabolic (equine motor neuron disease), neoplastic and age related alterations (senile retinopathy). Inflammation (acute neurochorioretinitis) and ischaemia (ischaemic neuro-chorioretinopathy) involving the optic nerve will lead to blindness. The optic disc can be aplastic/hypoplastic or excavated due to coloboma or glaucoma, affected by ligation of the carotid and palatine arteries. Optic disc masses may arise due to inflammation, severe systemic haemorrhage, neoplasia or trauma. Lesions of the retrobulbar optic nerve can be caused by Borna virus infection, fungal granuloma, toxic insult, halicephalobiasis, trauma, severe blood loss and infection of the sphenopalatine sinus.

The ventral nucleus of the lateral lemniscus of the gerbil (Meriones unguiculatus): organization of connections with the cochlear nucleus and the inferior colliculus.

The spatial organization of projections from the ventral cochlear nucleus (VCN) to the ventral nucleus of the lateral lemniscus (VNLL) and from the VNLL to the central nucleus of the inferior colliculus (CNIC) was investigated by using neuroanatomical tracing methods in the gerbil. In order to label cells in the VNLL that project to the CNIC, focal injections of biotinylated dextran amine (BDA) were made into different CNIC regions. Retrogradely labeled cells were distributed throughout the dorsal-to-ventral axis of the VNLL in all cases. In contrast, the distribution of labeled cells across the lateral-to-medial dimension of the VNLL was related to the location of the injection site along the dorsolateral to ventromedial (frequency) axis of the CNIC. Cells projecting to dorsolateral (low-frequency) regions of the CNIC were located peripherally in the VNLL, mainly laterally and caudally, whereas those projecting to ventromedial (high-frequency) regions of the CNIC tended to be clustered centrally. Projections to the VNLL were labeled anterogradely following injections of BDA in the VCN. The distribution of terminal fields in the VNLL closely paralleled the topographic arrangement of cells projecting to the CNIC; projections from ventrolateral (low-frequency) areas of the VCN terminated mainly along the lateral and caudal borders of the VNLL, whereas projections from dorsomedial (high-frequency) areas terminated in more central regions. The results demonstrate a topographic organization of the major afferent and efferent connections of the gerbil VNLL.

Severe chemosis caused by nephrotic syndrome in a goat: a case report.

A 3-year old female goat with nephrotic syndrome was presented because of severe bilateral chemosis. The diagnosis was based on clinical findings, decreased packed cell volume, increased serum urea and creatinine, decreased serum protein and protein loss via the urinary tract. At post mortem examination, a chronic glomerulonephritis and interstitial nephritis were found. In addition to severe conjunctival oedema, ascites and subcutaneous oedema in the region of the larynx were noted. Immunohistochemistry for the detection of chlamydial antigen in the conjunctiva was negative. This is the first report of severe chemosis caused by nephrotic syndrome in a goat.

Multiple topographically organized projections connect the central nucleus of the inferior colliculus to the ventral division of the medial geniculate nucleus in the gerbil, Meriones unguiculatus.

The ventral division of the medial geniculate nucleus (MGv) receives almost all of its ascending input from the ipsilateral central nucleus of the inferior colliculus (CNIC). In a previous study (Cant and Benson [2006] J. Comp. Neurol. 495:511-528), we made injections of biotinylated dextran amine into the CNIC of the gerbil and demonstrated that it can be divided into two parts. One part (zone 1) receives almost all of its ascending input from the cochlear nuclei, the nuclei of the lateral lemniscus, and the main nuclei of the superior olivary complex; the other part (zone 2) receives inputs from the cochlear nuclei and nuclei of the lateral lemniscus but few or no inputs from the main olivary nuclei. Here we show that these two parts of the CNIC project differentially to the MGv. Axons labeled anterogradely by injections in zone 1 project throughout the rostral two-thirds of the MGv, whereas axons from zone 2 project to the caudal third of the MGv. Throughout much of their extent, the terminal fields do not appear to overlap, although both parts of the CNIC project to medial and dorsal parts of the MGv, and there may be overlap in the most ventral part as well. The results indicate that two parallel pathways arising in the CNIC remain largely separate in the medial geniculate nucleus of the gerbil. It seems most likely that the neurons in the two terminal zones in the MGv perform different functions in audition.

Myotonic dystrophy in two European grey wolves (Canis lupus).

Two related European Grey wolves (Canis lupus) with the history of muscle stiffness beginning at 2 weeks of age were examined in this study. Muscle tone and muscle mass were increased in both animals. Muscle stiffness was worsened by stress so that the animals fell into lateral recumbency. Blood chemistry revealed mildly increased serum creatine kinase activity. Abnormal potentials typical of myotonic discharges were recorded by electromyography. Cataract, first-degree atrioventricular (AV) block and inhomogeneous myocardial texture by ultrasound suggested extramuscular involvement. Myopathology demonstrated dystrophic signs in the muscle biopsy specimen. The presumptive diagnosis based on the in vivo findings was myotonic dystrophy. Immunochemistry of the striated muscles revealed focal absence of dystrophin 1 and beta-dystroglycan in both cases. Cardiac and ophthalmologic involvement suggested a disorder very similar to a human form of myotonic dystrophy. This is the first description of myotonic dystrophy in wolves.

Wisteria floribunda lectin is associated with specific cell types in the ventral cochlear nucleus of the gerbil, Meriones unguiculatus.

The cochlear nucleus is made up of a number of diverse cell types with different anatomical and physiological properties. A plant lectin, Wisteria floribunda agglutinin, that recognizes specific carbohydrate residues in the extracellular matrix binds to some cell types in the ventral cochlear nucleus but not to cells in the dorsal cochlear nucleus. In the ventral cochlear nucleus, the most intensely labeled cells are octopus cells, a subset of multipolar cells and cochlear root neurons. The multipolar cells that are labeled may correspond to the population that projects to the inferior colliculus.