ABSTRACT
Neosporosis and toxoplasmosis are major causes of abortion in livestock worldwide, leading to substantial economic losses. Detection tools are fundamental to the diagnosis and management of those diseases. Current immunohistochemistry (IHC) tests, using sera raised against whole parasite lysates, have not been able to distinguish between Toxoplasma gondii and Neospora caninum. We used T. gondii and N. caninum recombinant proteins, expressed in Escherichia coli and purified using insoluble conditions, to produce specific polyclonal rabbit antisera. We aimed to develop species-specific sera that could be used in IHC on formalin-fixed, paraffin-embedded (FFPE) tissue sections to improve the diagnosis of ruminant abortions caused by protozoa. Two polyclonal rabbit sera, raised against recombinant proteins, anti-Neospora-rNcSRS2 and anti-Toxoplasma-rTgSRS2, had specificity for the parasite they were raised against. We tested the specificity for each polyclonal serum using FFPE tissue sections known to be infected with T. gondii and N. caninum. The anti-Neospora-rNcSRS2 serum labeled specifically only N. caninum-infected tissue blocks, and the anti-Toxoplasma-rTgSRS2 serum was specific to only T. gondii-infected tissues. Moreover, tissues from 52 cattle and 19 sheep previously diagnosed by lesion profiles were tested using IHC with our polyclonal sera and PCR. The overall agreement between IHC and PCR was 90.1% for both polyclonal anti-rNcSRS2 and anti-rTgSRS2 sera. The polyclonal antisera were specific and allowed visual confirmation of protozoan parasites by IHC, but they were not as sensitive as PCR testing.
Subject(s)
Antibodies, Protozoan , Coccidiosis , Neospora , Toxoplasma , Toxoplasmosis, Animal , Neospora/immunology , Neospora/isolation & purification , Animals , Toxoplasma/immunology , Coccidiosis/veterinary , Coccidiosis/diagnosis , Coccidiosis/parasitology , Toxoplasmosis, Animal/diagnosis , Toxoplasmosis, Animal/parasitology , Antibodies, Protozoan/blood , Rabbits , Sheep , Species Specificity , Sheep Diseases/diagnosis , Sheep Diseases/parasitology , Immunohistochemistry/veterinary , Cattle Diseases/diagnosis , Cattle Diseases/parasitology , Sensitivity and Specificity , CattleABSTRACT
The aetiology and pathophysiology of many diseases of the motor unit remain poorly understood and the role of the neuromuscular junction (NMJ) in this group of disorders is particularly overlooked, especially in humans, when these diseases are comparatively rare. However, elucidating the development, function and degeneration of the NMJ is essential to uncover its contribution to neuromuscular disorders, and to explore potential therapeutic avenues to treat these devastating diseases. Until now, an understanding of the role of the NMJ in disease pathogenesis has been hindered by inherent differences between rodent and human NMJs: stark contrasts in body size and corresponding differences in associated axon length underpin some of the translational issues in animal models of neuromuscular disease. Comparative studies in large mammalian models, including examination of naturally occurring, highly prevalent animal diseases and evaluation of their treatment, might provide more relevant insights into the pathogenesis and therapy of equivalent human diseases. This review argues that large animal models offer great potential to enhance our understanding of the neuromuscular system in health and disease, and in particular, when dealing with diseases for which nerve length dependency might underly the pathogenesis.
Subject(s)
Axons , Neuromuscular Junction , Animals , Humans , Mammals , Neuromuscular Junction/pathologyABSTRACT
Morphological study of the neuromuscular junction (NMJ), a specialised peripheral synapse formed between a lower motor neuron and skeletal muscle fibre, has significantly contributed to the understanding of synaptic biology and neuromuscular disease pathogenesis. Rodent NMJs are readily accessible, and research into conditions such as amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy (SMA) has relied heavily on experimental work in these small mammals. However, given that nerve length dependency is an important feature of many peripheral neuropathies, these rodent models have clear shortcomings; large animal models might be preferable, but their size presents novel anatomical challenges. Overcoming these constraints to study the NMJ morphology of large mammalian distal limb muscles is of prime importance to increase cross-species translational neuromuscular research potential, particularly in the study of long motor units. In the past, NMJ phenotype analysis of large muscle bodies within the equine distal pelvic limb, such as the tibialis cranialis, or within muscles of high fibrous content, such as the soleus, has posed a distinct experimental hurdle. We optimised a technique for NMJ location and dissection from equine pelvic limb muscles. Using a quantification method validated in smaller species, we demonstrate their morphology and show that equine NMJs can be reliably dissected, stained and analysed. We reveal that the NMJs within the equine soleus have distinctly different morphologies when compared to the extensor digitorum longus and tibialis cranialis muscles. Overall, we demonstrate that equine distal pelvic limb muscles can be regionally dissected, with samples whole-mounted and their innervation patterns visualised. These methods will allow the localisation and analysis of neuromuscular junctions within the muscle bodies of large mammals to identify neuroanatomical and neuropathological features.
Subject(s)
Coloring Agents , Peripheral Nervous System Diseases , Animals , Horses , Mammals , Motor Neurons/pathology , Muscle Fibers, Skeletal , Muscle, Skeletal/pathology , Neuromuscular Junction/pathology , Peripheral Nervous System Diseases/pathologyABSTRACT
Axonal transport ensures long-range delivery of essential cargoes between proximal and distal compartments, and is needed for neuronal development, function, and survival. Deficits in axonal transport have been detected at pre-symptomatic stages in the SOD1G93A and TDP-43M337V mouse models of amyotrophic lateral sclerosis (ALS), suggesting that impairments in this critical process are fundamental for disease pathogenesis. Strikingly, in ALS, fast motor neurons (FMNs) degenerate first whereas slow motor neurons (SMNs) are more resistant, and this is a currently unexplained phenomenon. The main aim of this investigation was to determine the effects of brain-derived neurotrophic factor (BDNF) on in vivo axonal transport in different α-motor neuron (MN) subtypes in wild-type (WT) and SOD1G93A mice. We report that despite displaying similar basal transport speeds, stimulation of wild-type MNs with BDNF enhances in vivo trafficking of signalling endosomes specifically in FMNs. This BDNF-mediated enhancement of transport was also observed in primary ventral horn neuronal cultures. However, FMNs display selective impairment of axonal transport in vivo in symptomatic SOD1G93A mice, and are refractory to BDNF stimulation, a phenotype that was also observed in primary embryonic SOD1G93A neurons. Furthermore, symptomatic SOD1G93A mice display upregulation of the classical non-pro-survival truncated TrkB and p75NTR receptors in muscles, sciatic nerves, and Schwann cells. Altogether, these data indicate that cell- and non-cell autonomous BDNF signalling is impaired in SOD1G93A MNs, thus identifying a new key deficit in ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Axonal Transport , Brain-Derived Neurotrophic Factor , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Brain-Derived Neurotrophic Factor/metabolism , Disease Models, Animal , Mice , Mice, Transgenic , Motor Neurons/metabolism , Superoxide Dismutase-1/geneticsABSTRACT
Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movement was detected in cultured cells and was significantly slower in glial cells compared to DRG-derived neurons. In addition, mitochondria were more elongated in glial cells than those in neurons. Our culture model will be a useful tool to study the contribution of axonal transport defects to specific neurodegenerative diseases in horses as well as comparative studies aimed at evaluating species-specific differences in axonal transport and survival.
Subject(s)
Axonal Transport , Ganglia, Spinal , Animals , Cells, Cultured , Horses , Neurites/physiology , NeuronsABSTRACT
CASE DESCRIPTION: Three dogs were presented for investigation of chronic nasal discharge and epistaxis 141, 250, and 357 days after undergoing transfrontal craniotomy to treat an intracranial meningioma (2 dogs) or a meningoencephalocele (1 dog). CLINICAL FINDINGS: CT findings were consistent with destructive rhinitis and frontal sinusitis in all 3 dogs, with results of histologic examination and fungal culture of samples obtained during frontal sinusotomy confirming mycotic infection. Frontal sinusotomy revealed fungal plaques covering a combination of bone and residual surgical tissue adhesive at the site of the previous craniotomy in all 3 dogs. Aspergillus spp were identified in all 3 dogs, and Chrysosporium sp was also identified in 1 dog. TREATMENT AND OUTCOME: Surgical curettage was followed by antifungal treatment (topical clotrimazole in 2 dogs and oral itraconazole for 3 months in 1 dog). Nasal discharge improved in the short-term but recurred in all dogs 99, 118, and 110 days after frontal sinusotomy. One dog received no further treatment, 1 dog received an additional 8.5 months of oral itraconazole treatment, and 1 dog underwent 2 additional surgical debridement procedures. At last follow-up, 2 dogs were alive 311 and 481 days after frontal sinusotomy; the third dog was euthanized because of status epilepticus 223 days after frontal sinusotomy. CLINICAL RELEVANCE: Sinonasal mycosis should be considered as a potential complication in dogs developing persistent mucopurulent nasal discharge, intermittent epistaxis, and intermittent sneezing following transfrontal craniotomy. The pathophysiology may be multifactorial, and potential risk factors, including use of surgical tissue adhesive in the frontal sinus, require further investigation.
Subject(s)
Aspergillosis , Dog Diseases , Mycoses , Animals , Aspergillosis/veterinary , Craniotomy/adverse effects , Craniotomy/veterinary , Dog Diseases/diagnosis , Dogs , Mycoses/veterinary , Neoplasm Recurrence, Local/veterinaryABSTRACT
BACKGROUND: Horses are affected by various peripheral nerve disorders but defining their aetiology and pathophysiology is hampered by limited understanding of associated morphological and pathological changes and involvement of specific axonal types. OBJECTIVES: To investigate the hypothesis that selected antibody markers, used in conjunction with various tissue processing methods, would enable identification of axons with different functional modalities within a range of equine peripheral nerves. STUDY DESIGN: Optimisation and validation study. METHODS: A range of antibodies were evaluated immunohistochemically via fluorescence confocal microscopy in cadaver equine nerve samples of primary motor, mixed or primary sensory functions (recurrent laryngeal, phrenic and plantar digital) within formalin-fixed paraffin-embedded (FFPE) and formalin-fixed frozen (FFF) tissues subjected to different antigen retrieval protocols. RESULTS: Immunohistochemistry of FFPE-derived nerve samples with selected antibodies and specific antigen retrieval methods enabled identification of myelinated and unmyelinated axons, cholinergic, sympathetic and peptidergic axons. The recurrent laryngeal and phrenic nerves are composed of myelinated cholinergic (motor), myelinated sensory fibres, unmyelinated adrenergic (sympathetic) axons and unmyelinated peptidergic (sensory) axons. In contrast, as expected, the plantar digital nerve had no myelinated motor fibres being mainly composed of myelinated sensory fibres, unmyelinated sympathetic and unmyelinated peptidergic sensory axons. MAIN LIMITATION: Attempts specifically to label parasympathetic fibres were unsuccessful in any nerve examined in both FFPE and FFF tissues. CONCLUSIONS: A panel of antibody markers can be used to reveal morphological and functional properties of equine nerves. Future work should enable better characterisation of morphological changes in equine neuropathies at various stages of disease development.
Subject(s)
Axons , Nerve Fibers, Myelinated , Animals , Horses , Immunohistochemistry , Peripheral NervesABSTRACT
CASE SUMMARY: A 15-year-old neutered male domestic shorthair cat presented with a 4-week history of dysphonia and reluctance to chew hard food. Oral examination revealed a mass lesion on the caudal soft palate. Biopsy was performed and histopathology with immunohistochemistry was most consistent with histiocytic sarcoma. CT of the head identified a discrete, left-sided, soft tissue mass lesion cranial to the tonsil with bilaterally symmetrical regional lymph nodes. CT of the thorax was unremarkable. Surgical removal achieved cytoreduction but not complete removal. Adjuvant chemotherapy with lomustine 10 mg (30 mg/m2) was initiated. The patient developed suspected pancreatitis 2-3 weeks postoperatively, so further chemotherapy treatment was discontinued, but supportive treatment with pain relief and appetite stimulants was provided. Three months postoperatively, the patient developed recurrent dysphonia, and oral examination revealed a suspicion of local recurrence. Confirmation of diagnosis (cytopathology or histopathology) was not obtained. Supportive treatment to maintain a good quality of life was continued and the patient was euthanased 6 months after diagnosis owing to progressive disease. RELEVANCE AND NOVEL INFORMATION: Only a few reports have been published describing histiocytic diseases of cats. To our knowledge, this is the first report of a feline histiocytic sarcoma of the oral cavity. Therefore, histiocytic sarcoma should be considered as a differential diagnosis in feline patients with an oral mass, especially if histopathology suggests a pleomorphic and poorly differentiated sarcoma. Immunohistochemistry for the confirmation of cell line origin would be strongly recommended.
ABSTRACT
INTRODUCTION: Equine recurrent laryngeal neuropathy (RLN) is a naturally occurring model of length-dependent axonopathy characterized by asymmetrical degeneration of recurrent laryngeal nerve axons (RLn). Distal RLn degeneration is marked, but it is unclear whether degeneration extends to include cell bodies (consistent with a neuronopathy). METHODS: With examiners blinded to RLN severity, brainstem location, and side, we examined correlations between RLN severity (assessed using left distal RLn myelinated axon count) and histopathological features (including chromatolysis and glial responses) in the nucleus ambiguus cell bodies, and myelinated axon count of the right distal RLn of 16 horses. RESULTS: RLN severity was not associated with RLn cell body number (P > .05), or degeneration. A positive correlation between the left and right distal RLn myelinated axon counts was identified (R2 = 0.57, P < .05). DISCUSSION: We confirm that RLN, a length-dependent distal axonopathy, occurs in the absence of detectable neuronopathy.
Subject(s)
Cell Body/pathology , Medulla Oblongata/pathology , Nerve Fibers, Myelinated/pathology , Neurons/pathology , Recurrent Laryngeal Nerve/pathology , Vocal Cord Paralysis/pathology , Animals , Atrophy , Cell Count , Horses , Recurrent Laryngeal Nerve/physiopathology , Vocal Cord Paralysis/physiopathologyABSTRACT
A female spayed domestic longhair cat aged 3 years and 9 months was referred for investigation of regurgitation and weight loss of 2 months' duration. Thoracic radiographs revealed a soft tissue mass within the cranial mediastinum causing focal oesophageal dilation. Computed tomography confirmed a contrast-enhancing mass located cranial to the heart base, possibly originating from the oesophagus. Exploratory thoracotomy revealed an intramural soft tissue mass within the ventral oesophageal wall. Surgical excision of the mass and reconstruction of the oesophagus around an oesophageal tube was performed successfully. Histopathological examination of the mass was compatible with a spindle cell tumour with a prominent vasoformative component. Immunohistochemistry was positive for α-smooth muscle actin and von Willebrand factor protein, and negative for CD117/c-kit protein. Both histopathological and immunohistochemical findings confirmed the diagnosis of an angioleiomyosarcoma. The cat was clinically well 6 months postoperatively. To our knowledge, this is the first report of an oesophageal angioleiomyosarcoma in a cat.
Subject(s)
Angiomyoma/veterinary , Cat Diseases/diagnostic imaging , Esophageal Neoplasms/veterinary , Leiomyosarcoma/veterinary , Angiomyoma/diagnostic imaging , Angiomyoma/surgery , Animals , Cat Diseases/pathology , Cat Diseases/surgery , Cats , Esophageal Neoplasms/diagnostic imaging , Esophageal Neoplasms/surgery , Esophagus/diagnostic imaging , Esophagus/pathology , Esophagus/surgery , Female , Immunohistochemistry/veterinary , Leiomyosarcoma/diagnostic imaging , Leiomyosarcoma/surgery , Radiography , Thoracotomy/veterinaryABSTRACT
A 15 year-old grey Thoroughbred gelding presented for investigation of chronic weight loss and recent onset of respiratory difficulty. Clinical examination confirmed tachypnoea with increased respiratory effort. Thoracic ultrasound examination detected pleural effusion. The dyspnoea was related to the large volume of pleural effusion and, following post-mortem examination, to the presence of a large mediastinal mass. Multiple pigmented masses, likely melanomas, were detected peri-anally. Thoracic radiography, cytological examination of the pleural fluid and a fine needle aspirate of a thoracic mass led to a presumptive diagnosis of malignant melanoma and this was confirmed at post mortem examination. Further metastatic spread to the central nervous system and right guttural pouch was also identified. In conclusion this case manifests the potential malignant behaviour of equine melanomas, and a review of proposed therapies for melanoma treatment highlights the therapeutic options and current areas of research.
ABSTRACT
Traumatic brain injury is the leading cause of death in children and young adults globally. Malignant cerebral oedema has a major role in the pathophysiology that evolves after severe traumatic brain injury. Added to this is the significant morbidity and mortality from cerebral oedema associated with acute stroke, hypoxic ischemic coma, neurological cancers and brain infection. Therapeutic strategies to prevent cerebral oedema are limited and, if brain swelling persists, the risks of permanent brain damage or mortality are greatly exacerbated. Here we show that a temporary and size-selective modulation of the blood-brain barrier allows enhanced movement of water from the brain to the blood and significantly impacts on brain swelling. We also show cognitive improvement in mice with focal cerebral oedema following administration in these animals of short interfering RNA directed against claudin-5. These observations may have profound consequences for early intervention in cases of traumatic brain injury, or indeed any neurological condition where cerebral oedema is the hallmark pathology.
Subject(s)
Brain Edema/etiology , Brain Edema/therapy , Brain Injuries/complications , Claudins/metabolism , Cognition/physiology , Animals , Blood-Brain Barrier/metabolism , Brain Edema/diagnostic imaging , Brain Injuries/diagnostic imaging , Child , Claudin-5 , Claudins/genetics , Humans , Intracranial Pressure/physiology , Male , Mice , RNA Interference , Tomography, X-Ray ComputedABSTRACT
A seven-month-old Cocker Spaniel had a cough, acute lethargy, decreased responsiveness, and episodes of hyperexcitability. There were bilateral generalized increased lung sounds, bilateral episcleral hemorrhage, and systemic hypertension. Prolonged buccal mucosal bleeding time and elevated D-dimer concentrations were detected. Radiographically, there was a generalized moderate unstructured interstitial pattern. In thoracic CT images, there was a diffuse moderate hyperattenuating appearance of the bronchial walls and interstitium and diffuse areas of moderate bronchiectasis. The brain CT images were characterized by marked hyperattenuating well-defined masses. In addition, there were smaller hyperattenuating and hypoattenuating masses scattered throughout the cerebral and cerebellar parenchyma. A zinc sulphate flotation test confirmed large numbers of Angiostrongylus vasorum L1 larvae. Despite therapy the dog continued to deteriorate and underwent euthanasia. Postmortem examination confirmed the presence of multiple intracranial and extracranial hemorrhages. Angiostrongylosis should be considered as one of the differential diagnoses in dogs presenting with neurologic signs consistent with acute intracranial haemorrhage.