Roberto Poma

Peptidylarginine deiminase 2 (PAD2) overexpression in transgenic mice leads to myelin loss in the central nervous system

SUMMARY Demyelination in the central nervous system is the hallmark feature in multiple sclerosis (MS). The mechanism resulting in destabilization of myelin is a complex multi-faceted process, part of which involves deimination of myelin basic protein (MBP). Deimination, the conversion of protein-bound arginine to citrulline, is mediated by the peptidylarginine deiminase (PAD) family of enzymes, of which the PAD2 and PAD4 isoforms are present in myelin. To test the hypothesis that PAD contributes to destabilization of myelin in MS, we developed a transgenic mouse line (PD2) containing multiple copies of the cDNA encoding PAD2, under the control of the MBP promoter. Using previously established criteria, clinical signs were more severe in PD2 mice than in their normal littermates. The increase in PAD2 expression and activity in white matter was demonstrated by immunohistochemistry, reverse transcriptase-PCR, enzyme activity assays, and increased deimination of MBP. Light and electron microscopy revealed more severe focal demyelination and thinner myelin in the PD2 homozygous mice compared with heterozygous PD2 mice. Quantitation of the disease-associated molecules GFAP and CD68, as measured by immunoslot blots, were indicative of astrocytosis and macrophage activation. Concurrently, elevated levels of the pro-inflammatory cytokine TNF-α and nuclear histone deimination support initiation of demyelination by increased PAD activity. These data support the hypothesis that elevated PAD levels in white matter represents an early change that precedes demyelination.

Accuracy, intermethod agreement, and inter-reviewer agreement for use of magnetic resonance imaging and myelography in small-breed dogs with naturally occurring first-time intervertebral disk extrusion

OBJECTIVE To determine accuracy, intermethod agreement, and inter-reviewer agreement for multisequence magnetic resonance imaging (MRI) and 2-view orthogonal myelography in small-breed dogs with first-time intervertebral disk (IVD) extrusion. DESIGN Prospective evaluation study. ANIMALS 24 dogs with thoracolumbar IVD extrusion. PROCEDURES Each dog underwent MRI and myelography. Images obtained with each modality were independently evaluated and assigned standardized scores in a blinded manner by 3 reviewers. Results were compared with surgical findings. Inter-reviewer and intermethod agreements were assessed via κ statistics. Accuracy was assessed as the percentage of dogs for which ≥ 2 of 3 reviewers recorded findings identical to those determined surgically. RESULTS Inter-reviewer agreement was substantial for site (κ = 0.70) and side of IVD extrusion (κ = 0.62) in T2-weighted magnetic resonance images and was substantial for site (κ = 0.72) and fair for side of extrusion (κ = 0.37) in myelographic images. Agreement for site between each modality and surgical findings was near perfect (κ = 0.94 and 0.88 for MRI and myelography, respectively). Intermethod agreement was substantial for site (κ = 0.71) and moderate for side of extrusion (κ = 0.40). Accuracy of MRI for site and side was 100% when results for T1-weighted, T2-weighted, and contrast-enhanced T1-weighted sequences were combined. Accuracy of myelography was 90.9% and 54.5% for site and side, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Agreement between imaging results and surgical findings for identification of IVD extrusion sites in small-breed dogs was similar for MRI and myelography. However, MRI appeared to be more accurate than myelography and allowed evaluation of extradural compressive mass composition.

Investigation of the use of three electroencephalographic electrodes for long-term electroencephalographic recording in awake and sedated dogs

OBJECTIVE To compare electroencephalography (EEG) artifact associated with use of the subdermal wire electrode (SWE), gold cup electrode (GCE), and subdermal needle electrode (SNE) over an 8-hour period in sedated and awake dogs. ANIMALS 6 healthy dogs. PROCEDURES 8 EEG channels were recorded during 20-minute video-EEG recording sessions (intermittently at 0.5, 2, 4, 6, and 8 hours) with and without chlorpromazine sedation. Nonphysiologic artifacts were identified. Duration of artifact was summed for each channel. Number of unaffected channels (NUC) was determined. RESULTS NUC was significantly affected by electrode type and sedation over time; median for SWE (2.80 channels; 95% confidence interval [CI], 0.84 to 5.70 channels) was significantly different from medians for GCE (7.87 channels; 95% CI, 7.44 to 7.94 channels) and SNE (7.60 channels; 95% CI, 6.61 to 7.89 channels). After 4 hours, NUC decreased in awake dogs, regardless of electrode type. In awake dogs, duration of artifact differed significantly between SWE and GCE or SNE; medians at 8 hours were 61.55 seconds (95% CI, 21.81 to 173.65 seconds), 1.33 seconds (95% CI, 0.47 to 3.75 seconds), and 21.01 seconds (95% CI, 6.85 to 64.42 seconds), respectively. CONCLUSIONS AND CLINICAL RELEVANCE The SWE had a significant duration of artifact during recording periods > 2 hours, compared with results for the GCE and SNE, in awake dogs. The GCE, SNE, and sedation resulted in significantly more channels unaffected by artifact. For longer recordings, caution should be exercised in selecting EEG electrodes and sedation state, although differences among electrodes may not be clinically relevant.

Multisystem Axonopathy and Neuronopathy in Golden Retriever Dogs

Central axonopathies are uncommon in dogs. Several terms have been used to describe these diseases, such as axonopathy, leukomyelopathy, axonal degeneration, and neuroaxonal degeneration. The majority of reported cases were breed-specific with peculiar clinical and pathological features seen in each breed. The association of central axonopathy and motor neuronal degeneration has not been reported. We report a progressive multisystem central axonopathy and motor neuronopathy in 3 Golden Retriever littermates with unique clinical and pathological features. Dog 1 was a 3-month-old female intact Golden Retriever dog referred to the Small Animal Clinic of the Ontario Veterinary College (OVC) with a 6-week history of pelvic limb weakness. The owners noticed that the dog appeared smaller than the other littermates and that her pelvic limb musculature was underdeveloped. She had difficulty in getting up, and when resting in lateral recumbency had fine muscle tremors in the limb muscles. The clinical signs progressed to involve the thoracic limbs with generalized weakness observed 2 weeks before presentation. On physical examination, no other abnormalities were detected except for generalized muscle atrophy. On neurological examination, short-strided tetraparesis, with no evidence of proprioceptive ataxia, was observed. The postural reactions (proprioceptive positioning and hopping) were adequate, as were the cranial nerve reflexes and responses. The flexor and patellar reflexes were mildly decreased. Resting, high-frequency, low-amplitude muscle tremors were seen in all limbs. These tremors could be elicited by palpation of the limb muscles. Based on the findings of lower motor neuron (LMN) tetraparesis, absence of proprioceptive ataxia, normal postural reactions, and mildly decreased reflexes, a neuromuscular disease was suspected. CBC, biochemistry profile, and urinalysis were performed. Abnormalities on the biochemistry profile were a creatine kinase (CK) activity of 517U/L (reference range, 40–255U/L) and a globulin concentration of 17 g/L (reference range, 21–42 g/L). No cause for the mild hypoglobulinemia was identified and it was assumed to be associated with the young age of the dog. No abnormalities were identified on the CBC or urinalysis. Electromyography, nerve conduction studies, and nerve and muscle biopsies were proposed, but declined by the owners. The dog continued to deteriorate and was re-evaluated at 6 months of age. At that time, the dog was markedly tetraparetic and the patellar reflexes were no longer present. Some extensor muscle tone was still present. Euthanasia and necropsy were performed. Two other littermates had a similar history and presentation. Dog 2, a male intact Golden Retriever, began to show signs of weakness and tremors at 15 weeks of age. By 5 months of age, severe generalized muscle atrophy and fatigue were obvious. CBC and biochemistry profile at that time were normal. The dog was referred and examined at 8 months of age. The neurological examination identified a kyphotic posture (Fig 1), generalized muscle atrophy, neuromuscular type (short-stride) tetraparesis, absence of ataxia, mildly decreased spinal reflexes, and head and body tremors. The nature of the tremors was suggestive of muscle weakness and not intentional tremors as seen with cerebellar disorders. Cranial nerve examination and postural reactions were adequate. CBC and biochemistry profile did not identify any clinically relevant abnormalities. Serum and whole blood were submitted for metabolic screening for amino acids, organic acids, and carbohydrates to investigate a possible metabolic or storage disease. Results of these tests were within reference limits. Euthanasia and necropsy were performed. Dog 3, a male intact Golden Retriever littermate, was presented to the OVC at 8 months of age. Weakness and body tremors were first observed when the dog was 3 months old. At presentation, the dog was markedly tetraAbbreviations:

Effects of hypercapnia, hypocapnia, and hyperoxemia on blood oxygenation level-dependent signal intensity determined by use of susceptibility-weighted magnetic resonance imaging in isoflurane-anesthetized dogs.

OBJECTIVE To assess the effects of alterations in PaCO(2) and PaO(2) on blood oxygenation level-dependent (BOLD) signal intensity determined by use of susceptibility-weighted magnetic resonance imaging in brains of isoflurane-anesthetized dogs. ANIMALS 6 healthy dogs. PROCEDURES In each dog, anesthesia was induced with propofol (6 to 8 mg/kg, IV) and maintained with isoflurane (1.7%) and atracurium (0.2 mg/kg, IV, q 30 min). During 1 magnetic resonance imaging session in each dog, targeted values of PaCO(2) (20, 40, or 80 mm Hg) and PaO(2) (100 or 500 mm Hg) were combined to establish 6 experimental conditions, including a control condition (PaCO(2), 40 mm Hg; PaO(2), 100 mm Hg). Dogs were randomly assigned to different sequences of conditions. Each condition was established for a period of >or= 5 minutes before susceptibility-weighted imaging was performed. Signal intensity was measured in 6 regions of interest in the brain, and data were analyzed by use of an ANCOVA and post hoc Tukey-Kramer adjustments. RESULTS Compared with control condition findings, BOLD signal intensity did not differ significantly in any region of interest. However, signal intensities in the thalamus and diencephalic gray matter decreased significantly during both hypocapnic conditions, compared with all other conditions except for the control condition. CONCLUSIONS AND CLINICAL RELEVANCE In isoflurane-anesthetized dogs, certain regions of gray matter appeared to have greater cerebrovascular responses to changes in PaCO(2) and PaO(2) than did others. Both PaO(2) and PaCO(2) should be controlled during magnetic resonance imaging procedures that involve BOLD signaling and taken into account when interpreting findings.

Evaluation of the ADVIA 120 for analysis of canine cerebrospinal fluid

BACKGROUND Conventional techniques for canine cerebrospinal fluid (CSF) analysis require large sample volumes and are labor intensive and subject to operator variability. OBJECTIVE The purpose of this study was to evaluate the ADVIA120 CSF assay for analysis of canine CSF samples. METHODS CSF samples collected from 36 healthy control dogs and 17 dogs with neurologic disease were processed in parallel using the automated assay and established manual methods using a hemocytometer and cytocentrifugation. Results for WBC (total nucleated cell) count, RBC count, and differential nucleated cell percentages were compared using Spearman rank correlation coefficients and Bland-Altman bias plots. RESULTS Correlation coefficients for WBC and RBC counts were 0.57 and 0.83 for controls, and 0.92 and 0.94 for ill dogs, respectively. Coefficients for the percentages of neutrophils, lymphocytes, and monocytes were 0.53, 0.26, and 0.12 for controls and 0.77, 0.92, and 0.70 for dogs with neurologic disease. When data were combined (n=53), correlation coefficients were 0.86 and 0.91 for WBC and RBC counts, and 0.63, 0.43, and 0.30 for neutrophil, lymphocyte, and monocyte percentages. A 9.5% positive bias and 7.0% negative bias were obtained for the ADVIA 120 CSF assay for lymphocytes and macrophages in dogs with neurologic disease with Bland-Altman analysis. A 12.2% positive bias was found for lymphocyte percentage in dogs with neurologic disease. CONCLUSIONS Manual and automated CSF assays had moderate to excellent correlation for WBC and RBC concentrations, but results were more variable for differential cell percentages. The ADVIA assay may be more useful for assessment of canine CSF with adjustment of cell differentiation algorithms.

Effects of hypercapnia, hypocapnia, and hyperoxemia on brain morphometrics determined by use of T1-weighted magnetic resonance imaging in isoflurane-anesthetized dogs

OBJECTIVE To evaluate the effects of various combinations of PaCO2 and PaO2 values on brain morphometrics. ANIMALS 6 healthy adult dogs. PROCEDURES A modified Latin square design for randomization was used. Dogs were anesthetized with propofol (6 to 8 mg/kg, IV), and anesthesia was maintained with isoflurane (1.7%) and atracurium (0.2 mg/kg, IV, q 30 min). Three targeted values of PaCO2 (20, 40, and 80 mm Hg) and 2 values of PaO2 (100 and 500 mm Hg) were achieved in each dog, yielding 6 combinations during a single magnetic resonance (MR) imaging session. When the endpoints were reached, dogs were given at least 5 minutes for physiologic variables to stabilize before T1-weighted MR images were obtained. Total brain volume (TBV) and lateral ventricular volume (LVV) were calculated from manually drawn contours of areas of interest by use of a software program, with each dog serving as its own control animal. Three blinded investigators subjectively evaluated the lateral ventricular size (LVS) and the cerebral sulci width (CSW). Brain morphometric values were compared among the target blood gas states. RESULTS No significant differences in TBV were found among target states. The LVV was significantly greater during hypocapnia, compared with hypercapnia at the same PaO2 value. With regard to the subjective evaluations, there were no significant differences among evaluators or among combinations of PaO2 and PaCO2 values. CONCLUSIONS AND CLINICAL RELEVANCE The changes observed in LVV during hypocapnia and hypercapnia may serve as a potential confounding factor when neuromorphometric evaluations are performed in anesthetized dogs.