Christopher L. Mariani

Terminology and Classification of Seizures and Epilepsy in Veterinary Patients

The classification of epileptic seizures and epilepsy is a controversial and dynamic topic that has undergone many iterations in human medicine. The International League against Epilepsy is a multinational organization that has formed a number of task forces and subcommittees to study this issue, and has ratified several reports outlining recommended terminology and classification schemes for human patients. Veterinary publications on this issue have generally adapted these schemes to fit small animal patients, but a formally endorsed system to classify seizures and epilepsy has never been developed for veterinary patients. This review outlines the classification systems that have been published for human patients and summarizes previous efforts by veterinary authors to utilize these methods. Finally, a set of definitions and terminology for use in veterinary patients is proposed, which includes a glossary of descriptive terminology for ictal semiology and a diagnostic scheme for classification of individual patients. This document is intended as a starting point of discussion, which will hopefully eventually result in a formally ratified document that will be useful for communication between health professionals, the design of clinical trials and for guiding treatment decisions and prognostication for veterinary patients with seizures.

Cerebral phaeohyphomycosis caused by Cladosporium spp. in two domestic shorthair cats.

Two domestic shorthair cats presented for clinical signs related to multifocal central nervous system dysfunction. Both cats had signs of vestibular system involvement and anisocoria, and one had generalized seizure activity. Cerebrospinal fluid analysis revealed a neutrophilic pleocytosis with protein elevation in one cat and pyogranulomatous inflammation in the second. Electroencephalography and brain-stem auditory-evoked potentials in the first cat confirmed cerebral cortical and brain-stem involvement. Euthanasia was performed in both cats, and postmortem diagnoses of phaeohyphomycosis secondary to Cladosporium spp. were made based on histopathology and fungal culture in both cats.

A Placebo-Controlled, Prospective, Randomized Clinical Trial of Polyethylene Glycol and Methylprednisolone Sodium Succinate in Dogs with Intervertebral Disk Herniation

Background Acute intervertebral disk herniation (IVDH) is a common cause of spinal cord injury in dogs and currently there is no proven medical treatment to counter secondary injury effects. Use of methylprednisolone sodium succinate (MPSS) or polyethylene glycol (PEG) as neuroprotectants is advocated but controversial because neither treatment has been tested in placebo‐controlled, randomized, blinded trials in dogs. Hypothesis Polyethylene glycol will improve the outcome of severe spinal cord injury caused by IVDH compared to MPSS or placebo. Animals Client‐owned dogs with acute onset of thoracolumbar IVDH causing paralysis and loss of nociception for <24 hours. Methods Dogs were randomized to receive MPSS, PEG, or placebo; drugs appeared identical and group allocation was masked. Drug administration was initiated once the diagnosis of IVDH was confirmed and all dogs underwent hemilaminectomy. Neurologic function was assessed 2, 4, 8, and 12 weeks postoperatively using an open field gait score (OFS) as the primary outcome measure. Outcomes were compared by the Wilcoxon rank sum test. Results Sixty‐three dogs were recruited and 47.6% recovered ambulation. 17.5% developed progressive myelomalacia but there was no association with group. There was no difference in OFS among groups. Although full study power was not reached, conditional power analyses indicated the futility of continued case recruitment. Conclusions This clinical trial did not show a benefit of either MPSS or PEG in the treatment of acute, severe thoracolumbar IVDH when used as adjunctive medical treatment administered to dogs presenting within 24 hours of onset of paralysis.

Generation and Characterization of Neurospheres from Canine Adipose Tissue-Derived Stromal Cells

Adipose tissue-derived stromal cells (ADSCs) have been identified as a powerful stem cell source for cellular transplantation therapy. The dog is increasingly used as a model of human neurological disease; however, few studies have reported induction of canine ADSCs to neural lineages. We characterized canine ADSCs and investigated whether they could be induced to differentiate into neural lineages. Subcutaneous adipose tissue collected from the dorsal epaxial region of adult dogs aged from 1 to 6 years was cultured to produce ADSCs that were then induced to neural lineages. RT-PCR, flow cytometry, and immunocytochemistry were performed to characterize these cell populations. Morphologically fibroblast-like ADSCs were isolated and had similar characteristics to mesenchymal stem cells. Under neurogenic conditions containing basic fibroblast growth factor and epidermal growth factor, ADSCs formed spherical cellular aggregates that resembled neurospheres. RT-PCR confirmed expression of Sox2 and CD90 by these aggregates. Expression of neural stem/progenitor markers (Nestin, Sox2, Vimentin) and neural lineage markers (A2B5, GFAP, Tuj1) was shown on immunocytochemistry. After differentiation, 60% of the cells were Tuj1 positive. In conclusion, we isolated and generated neural progenitor cells from canine ADSCs. ADSCs have potential for future autologous cell transplantation therapy for neurological disorders.

Nonspecific immunotherapy with intratumoral lipopolysaccharide and zymosan A but not GM-CSF leads to an effective anti-tumor response in subcutaneous RG-2 gliomas

PurposeNonspecific stimulation of cells of the immune system may be useful in generating an anti-tumor response for a variety of cancers and may work synergistically with currently available cytotoxic therapies. In this study we examined the response of syngeneic rat gliomas to treatment with several nonspecific stimulators of dendritic cells and macrophages alone or in combination with radiation therapy.Experimental designRG-2 gliomas were implanted subcutaneously and treated with intratumoral (IT) injections of the toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and zymosan A (ZymA) and the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). Combination treatment with IT LPS and single-fraction external beam radiotherapy (EBRT) was also evaluated.ResultsTreatment with IT LPS and ZymA delayed tumor growth compared to saline controls. Multiple doses of both substances were superior to single doses, and led to complete tumor regression in 71% (LPS) and 50% (ZymA) of animals. GM-CSF showed no anti-tumor effects in this study. Combinations of IT LPS and EBRT appeared to have a synergistic effect in delaying tumor growth. Rechallenge studies and IT LPS treatment of RG-2 tumors in nude rats suggested the importance of T cells in this treatment paradigm.ConclusionsDirect IT treatment with the TLR ligands LPS and ZymA are effective in generating an anti-tumor response. These treatments may synergize with cytotoxic therapies such as EBRT, and appear to require T cells for a successful outcome.

IDH1 and IDH2 hotspot mutations are not found in canine glioma.

Human diffuse and anaplastic astrocytomas, well-differenti-ated and anaplastic oligodendrogliomas and secondary glio-blastomas frequently (>70%) contain somatic mutations ofthe R132 codon of the cytoplasmic NADPþ-dependent iso-citrate dehydrogenase (IDH1) or the corresponding R172codon in its homolog, IDH2.

Rejection of RG-2 gliomas is mediated by microglia and T lymphocytes

SummaryImmunotherapy holds great promise for the treatment of invasive brain tumors, and we are interested specifically in evaluating immune stimulation of microglial cells as one potential strategy. In order to better understand the tumor fighting capabilities of microglial cells, we have compared the responses of syngeneic (Fisher 344) and allogeneic (Wistar) rat strains after intracranial implantation of RG-2 gliomas. Animals were evaluated by clinical examination, magnetic resonance imaging (MRI) and immunohistochemistry for microglial and other immune cell antigens. While lethal RG-2 gliomas developed in all of the Fisher 344 rats, tumors grew variably in the Wistar strain, sometimes reaching considerable sizes, but eventually all of them regressed. Tumor regression was associated with greater numbers of T cells and CD8 positive cells and increases in MHC I and CD4 positive microglia. Our findings suggest that the combined mobilization of peripheral and CNS endogenous immune cells is required for eradicating large intracranial tumors.

Identification of Novel Genetic Risk Loci in Maltese Dogs with Necrotizing Meningoencephalitis and Evidence of a Shared Genetic Risk across Toy Dog Breeds

Necrotizing meningoencephalitis (NME) affects toy and small breed dogs causing progressive, often fatal, inflammation and necrosis in the brain. Genetic risk loci for NME previously were identified in pug dogs, particularly associated with the dog leukocyte antigen (DLA) class II complex on chromosome 12, but have not been investigated in other susceptible breeds. We sought to evaluate Maltese and Chihuahua dogs, in addition to pug dogs, to identify novel or shared genetic risk factors for NME development. Genome-wide association testing of single nucleotide polymorphisms (SNPs) in Maltese dogs with NME identified 2 regions of genome-wide significance on chromosomes 4 (chr4:74522353T>A, p = 8.1×10−7) and 15 (chr15:53338796A>G, p = 1.5×10−7). Haplotype analysis and fine-mapping suggests that ILR7 and FBXW7, respectively, both important for regulation of immune system function, could be the underlying associated genes. Further evaluation of these regions and the previously identified DLA II locus across all three breeds, revealed an enrichment of nominal significant SNPs associated with chromosome 15 in pug dogs and DLA II in Maltese and Chihuahua dogs. Meta-analysis confirmed effect sizes the same direction in all three breeds for both the chromosome 15 and DLA II loci (p = 8.6×10–11 and p = 2.5×10−7, respectively). This suggests a shared genetic background exists between all breeds and confers susceptibility to NME, but effect sizes might be different among breeds. In conclusion, we identified the first genetic risk factors for NME development in the Maltese, chromosome 4 and chromosome 15, and provide evidence for a shared genetic risk between breeds associated with chromosome 15 and DLA II. Last, DLA II and IL7R both have been implicated in human inflammatory diseases of the central nervous system such as multiple sclerosis, suggesting that similar pharmacotherapeutic targets across species should be investigated.

Evaluation of matrix metalloproteinase-2 and -9 in the cerebrospinal fluid of dogs with intracranial tumors.

OBJECTIVE To identify matrix metalloproteinase (MMP)-2 and -9 in CSF from dogs with intracranial tumors. SAMPLE CSF from 55 dogs with intracranial tumors and 37 control dogs. PROCEDURES Latent and active MMP-2 and -9 were identified by use of gelatin zymography. The presence of MMPs in the CSF of dogs with intracranial tumors was compared with control dogs that were clinically normal and with dogs that had idiopathic or cryptogenic epilepsy or peripheral vestibular disease. Relationships between MMP-9 and CSF cell counts and protein were also investigated. RESULTS Latent MMP-2 was found in CSF samples from all dogs, although active MMP-2 was not detected in any sample. Latent MMP-9 was detected in a subset of dogs with histologically documented intracranial tumors, including meningiomas (2/10), gliomas (3/10), pituitary tumors (1/2), choroid plexus tumors (5/6), and lymphoma (4/4), but was not detected in any control samples. Dogs with tumors were significantly more likely than those without to have detectable MMP-9 in the CSF, and the presence of MMP-9 was associated with higher CSF nucleated cell counts and protein concentration. CONCLUSIONS AND CLINICAL RELEVANCE Latent MMP-9 was detected in most dogs with choroid plexus tumors or lymphoma but in a smaller percentage of dogs with meningiomas, gliomas, or pituitary tumors. Detection of MMP in CSF may prove useful as a marker of intracranial neoplasia or possibly to monitor response of tumors to therapeutic intervention.

Perianesthetic morbidity and mortality in dogs undergoing cervical and thoracolumbar spinal surgery

OBJECTIVE To evaluate and compare perioperative morbidity and mortality in dogs undergoing cervical and thoracolumbar spinal surgery. STUDY DESIGN Prospective case series. ANIMALS 157 dogs undergoing cervical or thoracolumbar spinal surgery. METHODS Data were collected sequentially on canine cases presented from the Neurology Section of the North Carolina State University Veterinary Teaching Hospital for anesthesia and surgery for cervical spinal cord disease. Simultaneously, data were collected on all thoracolumbar spinal surgery cases during the same time period. Data included signalment, drugs administered, surgical approach, disease process, cardiac arrhythmias during anesthesia, and outcome. RESULTS Data were collected from 164 surgical events in 157 dogs. There were 52 cervical approaches; four dorsal and 48 ventral. All thoracolumbar surgeries were approached dorsolaterally. Four dogs 4/52 (7.6%) undergoing a cervical approach did not survive to discharge. Two dogs (2/8; 25%) underwent atlanto-axial (AA) stabilization and suffered cardiovascular arrest and two dogs (2/38; 5.2%) undergoing cervical ventral slot procedures were euthanized following anesthesia and surgery due to signs of aspiration pneumonia. All dogs undergoing thoracolumbar surgery survived until discharge (112/112). Mortality in dogs undergoing cervical spinal surgery was greater compared with dogs undergoing thoracolumbar spinal surgery (p = 0.009), however, in dogs undergoing decompressive disc surgery, intraoperative death rates were not different between dogs undergoing a cervical compared with thoracolumbar approaches (p = 0.32) nor was there a significant difference in overall mortality (p = 0.07). CONCLUSION AND CLINICAL RELEVANCE Overall, dogs undergoing cervical spinal surgery were less likely to survive until discharge compared with dogs undergoing thoracolumbar spinal surgery. Mortality in dogs undergoing cervical intervertebral disc decompression surgery was no different than for dogs undergoing thoracolumbar intervertebral disc decompression surgery. However, dogs undergoing cervical intervertebral disc decompression surgery should be considered at risk for aspiration pneumonia.