Neutrophil extracellular traps in CSF and serum of dogs with steroid-responsive meningitis-arteritis.

In steroid-responsive meningitis-arteritis (SRMA), inflammatory dysregulation is driven by neutrophilic granulocytes resulting in purulent leptomeningitis. Neutrophils can generate neutrophil extracellular traps (NET). Uncontrolled NET-formation or impaired NET-clearance evidently cause tissue and organ damage resulting in immune-mediated diseases. The aim of the study was to verify that NET-formation is detectable in ex vivo samples of acute diseased dogs with SRMA by visualizing and measuring NET-markers in serum and cerebrospinal fluid (CSF) samples. CSF-samples of dogs with acute SRMA (n = 5) and in remission (n = 4) were examined using immunofluorescence (IF)-staining of DNA-histone-1-complexes, myeloperoxidase and citrullinated Histone H3 (H3Cit). Immunogold-labeling of H3Cit and neutrophil elastase followed by transmission electron microscopy (TEM) were used to determine ultrastructural NET-formation in the CSF of one exemplary dog. H3Cit-levels and DNase-activity were measured in CSF and serum samples using an H3Cit-ELISA and a DNase-activity-assay, respectively in patients with the following diseases: acute SRMA (n = 34), SRMA in remission (n = 4), bacterial encephalitis (n = 3), meningioma with neutrophilic inflammation (n = 4), healthy dogs (n = 6). NET-formation was detectable with IF-staining in n = 3/5 CSF samples of dogs with acute SRMA but were not detectable during remission. Vesicular NET-formation was detectable in one exemplary dog using TEM. DNase-activity was significantly reduced in dogs suffering from acute SRMA compared to healthy control group (p < 0.0001). There were no statistical differences of H3Cit levels in CSF or serum samples of acute diseased dogs compared to dogs under treatment, dogs suffering from meningioma or bacterial encephalitis or the healthy control group. Our findings demonstrate that NET-formation and insufficient NET-clearance possibly drive the immunologic dysregulation and complement the pathogenesis of SRMA. The detection of NETs in SRMA offers many possibilities to explore the aetiopathogenetic influence of this defence mechanism of the innate immune system in infectious and non-infectious canine neuropathies.

Clinical signs, causes, and outcome of central cord syndrome in 22 cats.

OBJECTIVE: To describe the signalment, clinical findings, presumptive or definitive diagnosis, and outcome in cats with central cord syndrome (CCS). ANIMALS: 22 cats. CLINICAL PRESENTATION: Cats evaluated for CCS at 7 referral hospitals between 2017 and 2021 were included. Information retrieved from medical records included signalment, physical and neurological examination findings, diagnostic investigations, definitive or presumptive diagnosis, treatment, and follow-up. RESULTS: Median age at presentation was 9 years. Two neuroanatomical localizations were associated with CCS: C1-C5 spinal cord segments in 17 (77.3%) cats and C6-T2 spinal cord segments in 5 (22.7%) cats. Neuroanatomical localization did not correlate with lesion location on MRI in 8 (36.3%) cats. The most common lesion location within the vertebral column was over the C2 and C4 vertebral bodies in 6 (27.2%) and 5 (22.7%) cats, respectively. Peracute clinical signs were observed in 11 (50%) cats, acute in 1 (4.5%), subacute in 4 (18%), and chronic and progressive signs were seen in 6 (40.9%) cats. The most common peracute condition was ischemic myelopathy in 8 (36.3%) cats, whereas neoplasia was the most frequently identified chronic etiology occurring in 5 (22.7%) cats. Outcome was poor in 13 (59%) cats, consisting of 4 of 11 (36.6%) of the peracute cases, 3 of 4 (75%) of the subacute cases, and 6 of 6 of the chronic cases. CLINICAL RELEVANCE: Central cord syndrome can occur in cats with lesions in the C1-C5 and C6-T2 spinal cord segments. Multiple etiologies can cause CCS, most commonly, ischemic myelopathy and neoplasia. Prognosis depends on the etiology and onset of clinical signs.

Investigation of the presence of specific neural antibodies in dogs with epilepsy or dyskinesia using murine and human assays.

BACKGROUND: Autoimmune mechanisms represent a novel category for causes of seizures and epilepsies in humans, and LGI1-antibody associated limbic encephalitis occurs in cats. HYPOTHESIS/OBJECTIVES: To investigate the presence of neural antibodies in dogs with epilepsy or dyskinesia of unknown cause using human and murine assays modified for use in dogs. ANIMALS: Fifty-eight dogs with epilepsy of unknown cause or suspected dyskinesia and 57 control dogs. METHODS: Serum and CSF samples were collected prospectively as part of the diagnostic work-up. Clinical data including onset and seizure/episode type were retrieved from the medical records. Screening for neural antibodies was done with cell-based assays transfected with human genes for typical autoimmune encephalitis antigens and tissue-based immunofluorescence assays on mouse hippocampus slices in serum and CSF samples from affected dogs and controls. The commercial human und murine assays were modified with canine-specific secondary antibody. Positive controls were from human samples. RESULTS: The commercial assays used in this study did not provide unequivocal evidence for presence of neural antibodies in dogs including one dog with histopathologically proven limbic encephalitis. Low titer IgLON5 antibodies were present in serum from one dog from the epilepsy/dyskinesia group and in one dog from the control group. CONCLUSION AND CLINICAL IMPORTANCE: Specific neural antibodies were not detected using mouse and human target antigens in dogs with epilepsy and dyskinesia of unknown origin. These findings emphasize the need for canine-specific assays and the importance of control groups.

Immunoglobulin profiling with large high-density peptide microarrays as screening method to detect candidate proteins for future biomarker detection in dogs with steroid-responsive meningitis-arteritis.

Steroid responsive meningitis arteritis (SRMA) is an aberrant Th2-mediated systemic inflammatory disease in dogs. The etiopathogenesis still remains unclear as no triggering pathogen or autoantigen could be found so far. HYPOTHESIS: Large high-density peptide microarrays are a suitable screening method to detect possible autoantigens which might be involved in the pathogenesis of SRMA. METHODS: The IgA and IgG profile of pooled serum samples of 5 dogs with SRMA and 5 dogs with neck pain due to intervertebral disc herniation (IVDH) without ataxia or paresis were compared via commercially available high-density peptide microarrays (Discovery Microarray) containing 29,240 random linear peptides. Canine distemper virus nucleoprotein (CDVN) served as positive control as all dogs were vaccinated. Common motifs were compared to amino acid sequences of known proteins via databank search. One suitable protein was manually selected for further analysis with a smaller customized high-density peptide microarray. RESULTS: Pooled serum of dogs with SRMA and IVDH showed different IgA and IgG responses on Discovery Microarray. Only top IgG responses of dogs with SRMA showed a common motif not related to the control protein CDVN. This common motif is part of the interleukin 1 receptor antagonist protein (IL1Ra). On IL1Ra, dogs with SRMA displayed IgA binding to an additional epitope, which dogs with IVDH did not show. DISCUSSION: IL1Ra is an anti-inflammatory acute phase protein. Different immunoglobulin binding patterns on IL1Ra could be involved in the pathogenesis of SRMA and IL1Ra might be developed as future biomarker for SRMA.

Case report: Lympho-histiocytic meningoencephalitis with central nervous system vasculitis of unknown origin in three dogs.

Meningoencephalitis of unknown origin (MUO) is an umbrella term for a variety of subtypes of meningoencephalitis of dogs and cats with no identifiable infectious agent. In dogs, granulomatous meningoencephalitis (GME), necrotizing meningoencephalitis (NME), and necrotizing leukoencephalitis (NLE) are the most commonly reported subtypes. However, sporadically there are reports about other subtypes such as greyhound encephalitis or eosinophilic meningoencephalitis. The following case series presents three dogs with peracute to acute progressive signs of encephalopathy. The magnetic resonance imaging (MRI) of two dogs (post mortem n = 1/2) showed severe, diffuse swelling of the cortical gray matter with increased signal intensity in T2weighted (w) and fluid-attenuated inversion recovery (FLAIR) and decreased signal intensity in T1w. Additionally, focal to multifocal areas with signal void in both dogs and caudal transforaminal herniation of the cerebellum in one dog was observed. Post mortem histopathological examination revealed lympho-histiocytic encephalitis and central nervous system (CNS) vasculitis in all dogs. No infectious agents were detectable by histopathology (hematoxylin and eosin stain), periodic acid-Schiff reaction (PAS), Ziehl-Neelsen stain and immunohistochemistry for Canine adenovirus-1, Parvovirus, Listeria monocytogenes, Parainfluenzavirus, Toxoplasma gondii, Herpes-suis virus, Pan-Morbillivirus, Tick born encephalitis virus, Severe acute respiratory syndrome coronavirus (SARS-CoV) 2. Furthermore, two dogs were tested negative for rabies virus. To the best of the authors' knowledge, this is the first report of a lympho-histiocytic encephalitis with CNS vasculitis with no identifiable infectious agent. It is suggested to consider this as an additional subtype of MUO with severe clinical signs.

Concomitant necrotizing encephalitis and granulomatous meningoencephalitis in four toy breed dogs.

The term "meningoencephalitis of unknown origin" (MUO) describes a group of different encephalitides in dogs in which no infectious agent can be identified and a multifactorial etiology is suspected. Among others, genetic factors and unknown triggers seem to be involved. Included are necrotizing leukoencephalitis (NLE), necrotizing meningoencephalitis (NME), and granulomatous meningoencephalitis (GME). In this case series, we describe the histopathological findings of four toy breed dogs with focal or multifocal necrotizing encephalitis and mainly lymphocytic perivascular infiltrates on histopathological examination. At the same time, however, in all dogs, focal or multifocal high-grade angiocentric granulomatous inflammatory lesions were evident with focal histiocytic perivascular infiltrates in the brain. The former changes are typical for NLE and NME. In contrast, the latter changes are indicative of GME. This case series shows that the boundaries between the necrotizing and granulomatous variants of MUO might be smooth and suggests that NLE, NME, and GME are not as distinct as previously described. This finding could be a crucial piece of the puzzle in the study of the pathogenesis of MUO as individual susceptibility and specific triggers could be responsible for the manifestation of the different MUO subtypes.

Biopsy Characteristics, Subtypes, and Prognostic Features in 107 Cases of Feline Presumed Immune-Mediated Polyneuropathy.

Inflammatory polyradiculoneuropathy (IMPN) is one of the causes of sudden onset of neuromuscular signs such as para-/tetraparesis in young cats. Even though most cases have a favorable outcome, persistent deficits, relapses, and progressive courses are occasionally seen. As clinical presentation does not always appear to predict outcome and risk of recurrence, this study was initiated to screen for prognostic biopsy findings in a large cohort of histologically confirmed IMPN cases with clinical follow-up. In total, nerve and muscle specimens of 107 cats with biopsy diagnosis of presumed autoreactive inflammatory polyneuropathy and 22 control cases were reviewed by two blinded raters for a set of 36 histological parameters. To identify patterns and subtypes of IMPN, hierarchical k-means clustering of 33 histologic variables was performed. Then, the impact of histological parameters on IMPN outcome was evaluated via an univariate analysis to identify variables for the final multivariate model. The data on immediate outcome and follow-up were collected from submitting neurologists using a purpose-designed questionnaire. Hierarchical k-means clustering sorted the tissues into 4 main categories: cluster 1 (44/129) represents a purely inflammatory IMPN picture, whereas cluster 2 (47/129) was accompanied by demyelinating features and cluster 3 (16/129) by Wallerian degeneration. Cluster 4 (22/129) reflects normal tissues from non-neuropathic control cats. Returned questionnaires provided detailed information on outcome in 63 animals. They were categorized into recovered and non-recovered. Thereby, fiber-invasive infiltrates by mononuclear cells and mild fiber loss in intramuscular nerve branches correlated with higher probabilities of recovery. Remyelination in semithin sections, on the other hand, is correlated with a less favorable outcome. Animals grouping in cluster 1 had a tendency to a higher probability of recovery compared to other clusters. In conclusion, diagnosis of feline IMPN from nerve and muscle biopsies allowed for the identification of histologic features that were positively or negatively correlated with outcome.

Case Report: Unable to Jump Like a Kangaroo Due to Myositis Ossificans Circumscripta.

A male 10-year-old captive red kangaroo (Macropus rufus) was presented with a chronic progressive pelvic limb lameness and reluctance to jump. The general examination revealed a palpable induration of the lumbar epaxial muscles. Magnetic resonance imaging performed under general anesthesia revealed bilateral almost symmetric, well-circumscribed mass lesions in superficial erector spinae muscles. The lesions had irregular to multilobulated appearance with hyper-, hypo-, and isointense areas in T2- and T1-weighted (w) sequences without contrast enhancement. On computed tomography, a peripheral rim of mineralization was apparent. Histopathological analysis of a muscle biopsy showed osseous trabeculae with rare clusters of chondrocytes indicating metaplasia of muscle tissue to bone. No indications of inflammation or malignancy were visible. The clinical, histopathological, and imaging workup of this case was consistent with myositis ossificans circumscripta. This disorder is particularly well-known among human professional athletes such as basketball players, where excessive, chronic-repetitive force or blunt trauma causes microtrauma to the musculature. Metaplasia of muscle tissue due to abnormal regeneration processes causes heterotopic ossification. The kangaroo's clinical signs improved with cyto-reductive surgery, cage rest, weight reduction, and meloxicam without further relapse.

Clinical Course and Diagnostic Findings of Biopsy Controlled Presumed Immune-Mediated Polyneuropathy in 70 European Cats.

There is a paucity of information on the clinical course and outcome of young cats with polyneuropathy. The aim of the study was to describe the clinical features, diagnostic investigations, and outcome of a large cohort of cats with inflammatory polyneuropathy from several European countries. Seventy cats with inflammatory infiltrates in intramuscular nerves and/or peripheral nerve biopsies were retrospectively included. Information from medical records and follow up were acquired via questionnaires filled by veterinary neurologists who had submitted muscle and nerve biopsies (2011-2019). Median age at onset was 10 months (range: 4-120 months). The most common breed was British short hair (25.7%), followed by Domestic short hair (24.3%), Bengal cat (11.4%), Maine Coon (8.6%) and Persian cat (5.7%), and 14 other breeds. Male cats were predominantly affected (64.3%). Clinical signs were weakness (98.6%) and tetraparesis (75.7%) in association with decreased withdrawal reflexes (83.6%) and, less commonly, cranial nerve signs (17.1%), spinal pain/hyperesthesia (12.9%), and micturition/defecation problems (14.3%). Onset was sudden (30.1%) or insidious (69.1%), and an initial progressive phase was reported in 74.3%. Characteristic findings on electrodiagnostic examination were presence of generalized spontaneous electric muscle activity (89.6%), decreased motor nerve conduction velocity (52.3%), abnormal F-wave studies (72.4%), pattern of temporal dispersion (26.1%) and unremarkable sensory tests. The clinical course was mainly described as remittent (49.2%) or remittent-relapsing (34.9%), while stagnation, progressive course or waxing and waning were less frequently reported. Relapses were common and occurred in 35.7% of the cats' population. An overall favorable outcome was reported in 79.4% of patients. In conclusion, young age at the time of diagnosis and sudden onset of clinical signs were significantly associated with recovery (p < 0.05). Clinical and electrodiagnostic features and the remittent-relapsing clinical course resembles juvenile chronic inflammatory demyelinating polyneuropathy (CIDP), as seen in human (children/adolescents), in many aspects.

Evaluation of the effect of phenobarbital administration on the biochemistry profile, with a focus on serum liver values, in epileptic cats.

OBJECTIVES: Phenobarbital (PB) is the most common antiseizure drug (ASD) used for the management of feline epilepsy. In dogs, PB is known to cause serum liver enzyme induction and hepatotoxicity, especially after administration long term or in high concentrations. In cats, insufficient evidence is available to draw similar conclusions. The aim of this study was to evaluate the effect of PB administration on the serum biochemistry profile of epileptic cats. As an additional objective, other adverse effects arising, related to PB treatment, were recorded. METHODS: Medical records of four veterinary centres were retrospectively reviewed for epileptic cats receiving PB treatment. Cats were included if they had a diagnosis of idiopathic epilepsy or structural epilepsy; a normal baseline serum biochemistry profile; at least one follow-up serum biochemistry profile; no concurrent disease or had not received medication that could possibly influence liver function or lead to serum liver enzyme induction. Alkaline phosphatase, alanine aminotransferase (ALT), aspartate transaminase and gamma-glutamyl transferase activities, and total bilirubin, bile acids, glucose, albumin, total protein, urea and creatinine concentrations before and during PB administration were recorded. PB serum concentration was also recorded, when available. RESULTS: Thirty-three cats (24 males, nine females) with a median age of 3 years (range 2 months to 12 years) met the inclusion criteria. Idiopathic or structural epilepsy was diagnosed in 25 (76%) and eight (24%) cats, respectively. The follow-up period ranged from 9 to 62 months. This study found an increase in ALT in three cats, possibly related to a PB serum concentration >30 µg/ml. No statistically significant increase in serum liver enzymes or other evaluated biochemistry parameters was found by comparing pre- and post-treatment parameters. CONCLUSIONS AND RELEVANCE: PB administration did not result in hepatic enzyme induction or other biochemical abnormalities in cats. This strengthens the safety profile of PB as an ASD in cats.

Mitochondrial PCK2 Missense Variant in Shetland Sheepdogs with Paroxysmal Exercise-Induced Dyskinesia (PED).

Four female Shetland Sheepdogs with hypertonic paroxysmal dyskinesia, mainly triggered by exercise and stress, were investigated in a retrospective multi-center investigation aiming to characterize the clinical phenotype and its underlying molecular etiology. Three dogs were closely related and their pedigree suggested autosomal dominant inheritance. Laboratory diagnostic findings included mild lactic acidosis and lactaturia, mild intermittent serum creatine kinase (CK) elevation and hypoglycemia. Electrophysiological tests and magnetic resonance imaging of the brain were unremarkable. A muscle/nerve biopsy revealed a mild type II fiber predominant muscle atrophy. While treatment with phenobarbital, diazepam or levetiracetam did not alter the clinical course, treatment with a gluten-free, home-made fresh meat diet in three dogs or a tryptophan-rich, gluten-free, seafood-based diet, stress-reduction, and acetazolamide or zonisamide in the fourth dog correlated with a partial reduction in, or even a complete absence of, dystonic episodes. The genomes of two cases were sequenced and compared to 654 control genomes. The analysis revealed a case-specific missense variant, c.1658G>A or p.Arg553Gln, in the PCK2 gene encoding the mitochondrial phosphoenolpyruvate carboxykinase 2. Sanger sequencing confirmed that all four cases carried the mutant allele in a heterozygous state. The mutant allele was not found in 117 Shetland Sheepdog controls and more than 500 additionally genotyped dogs from various other breeds. The p.Arg553Gln substitution affects a highly conserved residue in close proximity to the GTP-binding site of PCK2. Taken together, we describe a new form of paroxysmal exercise-induced dyskinesia (PED) in dogs. The genetic findings suggest that PCK2:p.Arg553Gln should be further investigated as putative candidate causal variant.

Meningioma and associated cerebral infarction in three dogs.

BACKGROUND: In dogs, meningiomas mostly cause chronic progressive clinical signs due to slow tumor growth. CASE PRESENTATION: In contrast, three dogs were presented with the history of chronic generalized tonic-clonic seizures and peracute deterioration with sudden onset of neurological deficits in accordance with an extensive unilateral forebrain lesion. Magnetic resonance imaging examinations of the dogs revealed a well-delineated extraaxial T2W hyperintense mass in the rostral forebrain with homogeneous contrast enhancement. Additionally, an intraaxial, well-demarcated, unilateral lesion was apparent in the parenchyma supplied by the middle cerebral artery. In two cases, necropsy revealed meningothelial meningioma in the rostral fossa and marked eosinophilic neuronal necrosis, a sign of ischemia, focal malacia, edema and gliosis in the temporal lobe and hippocampus because of a focal thrombosis of the middle cerebral artery. In the third case symptomatic treatment resulted in improvement of clinical signs enabling a good quality of life for the patient. CONCLUSIONS: In dogs with structural epilepsy caused by meningioma, acute deterioration of clinical signs can be associated with ischemic infarctions as a potential complication.

Intervertebral disc herniation in two coatis (Nasua nasua) and postoperative laminectomy membrane formation.

Magnetic resonance imaging revealed spinal cord compression due to intervertebral disc herniation of Hansen type I and II in the thoracolumbar vertebral column in two middle-aged coatis (Nasua nasua) with chronic progressive paraparesis. Surgical treatment included hemilaminectomy and partial corpectomy in one and dorsal laminectomy in the other coati. Both coatis recovered well after surgery. One showed unremarkable gait 6 and 15 months post surgery, while the other one suffered from recurrence of paraparesis leading to euthanasia because of deterioration of neurological signs 20 months after the first surgery. Necropsy revealed formation of a laminectomy membrane compressing the spinal cord. Histopathological signs of spinal cord injury and findings of degenerative processes in the intervertebral disc were comparable to those described in dogs. In conclusion, this case report shows for the first time that surgical intervention seems to be a useful and safe treatment in chronic intervertebral disc herniation in coatis, but relapses are possible.

Comparison of surgical and conservative treatment of hydrated nucleus pulposus extrusion in dogs.

BACKGROUND: Whether compressive cervical myelopathy caused by hydrated nucleus pulposus extrusion (HNPE) in dogs should be treated surgically or conservatively has been debated. Only 1 recent study has contradicted the former predominant reports of surgical treatment for HNPE. HYPOTHESIS AND METHOD: Single center retrospective study to compare the outcome of client-owned dogs with HNPE after decompressive surgery or conservative treatment. ANIMALS: Thirty-six dogs diagnosed with HNPE confirmed by magnetic resonance imaging (MRI). RESULTS: Eighteen of 36 dogs underwent surgery whereas 18 dogs were managed conservatively including cage rest and physiotherapy. The most common affected intervertebral disc space was C4-5. In 3 dogs, HNPE was diagnosed at the level of T13-L1. Median time to regain ambulation was 6.6 days (range, 0-28 days) after surgery and 5.9 days (range, 0-15 days) with conservative management (P = .37). Only the length of a potential intramedullary lesion in cervical HNPE detected by MRI had an influence on the prognosis to gain ambulatory status in a time period of ≤9 days (P = .0035) and on short-term survival (P = .0011). CONCLUSIONS AND CLINICAL IMPORTANCE: Conservative management of HNPE in the cervical as well as in the thoracolumbar region represents a reasonable alternative to surgery, showing similar favorable outcome.

Effects of murine and human bone marrow-derived mesenchymal stem cells on cuprizone induced demyelination.

For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC) have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS). In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.