Brain magnetic resonance imaging in the DE50-MD dog model of Duchenne muscular dystrophy reveals regional reductions in cerebral gray matter.

BACKGROUND: Duchenne muscular dystrophy is a X-linked disease characterized by severe and progressive muscle weakness, alongside cognitive impairment and a range of neurobehavioral disorders secondary to brain dystrophin deficiency. Duchenne muscular dystrophy patients have reduced cerebral gray matter and altered white matter ultrastructure (detected by magnetic resonance imaging) compared to age-matched controls. METHODS: We studied the DE50-MD canine model of Duchenne muscular dystrophy, which is deficient in full length brain dystrophin (Dp427) isoforms and has a neurocognitive phenotype. Eight DE50-MD and 6 age-matched littermate wild type male dogs underwent serial brain magnetic resonance imaging from 14 to 33 months of age. RESULTS: Reduced regional gray matter was detected in DE50-MD dogs compared with wildtype, including the piriform lobe, hippocampus and cingulate gyrus. Lateral ventricle volume was larger in DE50-MD dogs. Differences did not progress over time. White matter volume did not differ between DE50-MD and wildtype dogs. There was no difference in brain nor cranial vault volume between DE50-MD and wildtype dogs. CONCLUSION: Dystrophin deficiency in the canine brain results in structural changes that likely contribute to the neurocognitive phenotype.

Pre-Existing Mature Oligodendrocytes Do Not Contribute to Remyelination following Toxin-Induced Spinal Cord Demyelination.

Remyelination is the regenerative response to demyelination. Although the oligodendrocyte progenitor is established as the major source of remyelinating cells, there is no conclusive evidence on whether mature, differentiated oligodendrocytes can also contribute to remyelination. Using two different inducible myelin-CreER mouse strains in which mature oligodendrocytes were prelabeled by the expression of membrane-bound Green fluorescent protein, we found that after focal spinal cord demyelination, the surrounding surviving labeled oligodendrocytes did not proliferate but remained at a consistent density. Furthermore, existing (prelabeled) oligodendrocytes showed no evidence of incorporation or migration into the lesioned area, or of process extension from the peripheral margins into the lesion. Thus, mature oligodendrocytes do not normally contribute to remyelination and are therefore not a promising target for regenerative therapy.

Retinoid X receptor activation reverses age-related deficiencies in myelin debris phagocytosis and remyelination.

The efficiency of central nervous system remyelination declines with age. This is in part due to an age-associated decline in the phagocytic removal of myelin debris, which contains inhibitors of oligodendrocyte progenitor cell differentiation. In this study, we show that expression of genes involved in the retinoid X receptor pathway are decreased with ageing in both myelin-phagocytosing human monocytes and mouse macrophages using a combination of in vivo and in vitro approaches. Disruption of retinoid X receptor function in young macrophages, using the antagonist HX531, mimics ageing by reducing myelin debris uptake. Macrophage-specific RXRα (Rxra) knockout mice revealed that loss of function in young mice caused delayed myelin debris uptake and slowed remyelination after experimentally-induced demyelination. Alternatively, retinoid X receptor agonists partially restored myelin debris phagocytosis in aged macrophages. The agonist bexarotene, when used in concentrations achievable in human subjects, caused a reversion of the gene expression profile in multiple sclerosis patient monocytes to a more youthful profile and enhanced myelin debris phagocytosis by patient cells. These results reveal the retinoid X receptor pathway as a positive regulator of myelin debris clearance and a key player in the age-related decline in remyelination that may be targeted by available or newly-developed therapeutics.

Developmental origin of oligodendrocytes determines their function in the adult brain.

In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function.

Perceptions and attitudes towards companion animal brain banking in pet owners: A UK pilot study.

Background: Detailed analysis of archived brain tissue is fundamental to advancing the understanding of neurological disease. The development of the UK Brain Bank Network (UBBN) has provided an invaluable resource to facilitate such research in the human medical field. Similar resources are needed in veterinary medicine. However, collection and archiving of companion animal brain tissue is a potentially sensitive area for pet owners and veterinary professionals. Methods: Using an online survey, we aimed to study pet owners' perceptions of brain banking. The survey included information on respondents, their views on organ donation, the UBBN and the Royal Veterinary College's Companion Animal Brain Bank (RVC CABB). Results: In total 185 respondents were included. The use of brain tissue from pets for research was supported by 87% of respondents, and 66% of respondents felt that they were highly likely or likely to donate their pet's brain tissue to a CABB. Furthermore, 94% felt that more information on tissue banking in companion animals should be readily available. Conclusions: We found that the perceptions of companion animal brain banking were positive in our respondents. Open dialogue and clear information provision on the process and benefits of the CABB could enhance awareness and thus facilitate brain donation for translational research.

Parallel roles of neuroinflammation in feline and human epilepsies.

Autoimmune encephalitis refers to a group of disorders characterised by a non-infectious encephalitis, often with prominent seizures and surface neuronal autoantibodies. AE is an important cause of new-onset refractory status epilepticus in humans and is frequently responsive to immunotherapies including corticosteroids, plasma exchange, intravenous immunoglobulin G and rituximab. Recent research suggests that parallel autoantibodies can be detected in non-human mammalian species. The best documented example is leucine-rich glioma-inactivated 1 (LGI1)-antibodies in domestic cats with limbic encephalitis (LE). In this review, we discuss the role of neuroinflammation and autoantibodies in human and feline epilepsy and LE.

Validation of DE50-MD dogs as a model for the brain phenotype of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disease, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterisation of dystrophin expression and function in the canine brain is lacking. We studied the DE50-MD canine model of DMD that has a missense mutation in the donor splice site of exon 50. Using a battery of cognitive tests, we detected a neurocognitive phenotype in DE50-MD dogs, including reduced attention, problem solving and exploration of novel objects. Through a combination of capillary immunoelectrophoresis, immunolabelling, quantitative PCR and RNAScope in situ hybridisation, we show that regional dystrophin expression in the adult canine brain reflects that of humans, and that the DE50-MD dog lacks full-length dystrophin (Dp427) protein expression but retains expression of the two shorter brain-expressed isoforms, Dp140 and Dp71. Thus, the DE50-MD dog is a translationally relevant pre-clinical model to study the consequences of Dp427 deficiency in the brain and to develop therapeutic strategies for the neurological sequelae of DMD.

Identification of quantitative polymerase chain reaction reference genes suitable for normalising gene expression in the brain of normal and dystrophic mice and dogs.

Background: In addition to progressive, debilitating muscle degeneration, ~50% of patients with Duchenne muscular dystrophy (DMD) have associated cognitive and behavioural disorders secondary to deficiency of dystrophin protein in the brain. The brain expresses a variety of dystrophin isoforms (Dp427, Dp140 and Dp71) whose functions remain to be fully elucidated. Detailed comparative analysis of gene expression in healthy and dystrophin-deficient brain is fundamental to understanding the functions of each isoform, and the consequences of their deficiency, with animal models representing a key tool in this endeavour. Reverse transcription quantitative real-time PCR (RT-qPCR) is a widely used method to study gene expression. However, accurate quantitative assessment requires normalisation of expression data using validated reference genes. The aim of this study was to identify a panel of suitable reference genes that can be used to normalise gene expression in the brain of healthy and dystrophic dogs and mice. Methods: Using the DE50-MD dog and mdx mouse models of DMD we performed RT-qPCR from fresh frozen brain tissue and employed the geNorm, BestKeeper and Normfinder algorithms to determine the stability of expression of a panel of candidate reference genes across healthy and dystrophic animals, and across different brain regions. Results: We show that SDHA, UBC and YWHAZ are suitable reference genes for normalising gene expression in healthy and dystrophic canine brain, and GAPDH, RPL13A and CYC1 in healthy and dystrophic murine brain. Notably, there was no overlap in the highest performing reference genes between the two species. Conclusions: Our findings suggest that gene expression normalisation is possible across six regions of the canine brain, and three regions of the murine brain. Our results should facilitate future work to study gene expression in the brains of normal and dystrophic dogs and mice and thus decipher the transcriptional consequences of dystrophin deficiency in the brain.

Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo.

Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.

Comparison between Hemilaminectomy with either Anulectomy or Partial Discectomy for Treatment of Thoracolumbar Intervertebral Disc Protrusion in Dogs.

OBJECTIVES: The aim of this study was to compare the clinical outcome of dogs undergoing a hemilaminectomy with anulectomy (HA) or a hemilaminectomy with partial discectomy (HPD) for treatment of thoracolumbar intervertebral disc protrusion. METHODS: Medical records from 2006 to 2015 were retrospectively reviewed. Dogs were included if they had clinical signs and imaging findings consistent with thoracolumbar intervertebral disc protrusion and had undergone surgical treatment with a HA or HPD. Outcome data were obtained via veterinary records and owner questionnaires. Recorded variables included age, sex, body weight, neurological deficits, surgical time, perioperative complications, postoperative neurological deterioration and recurrence of clinical signs. RESULTS: The two treatment groups showed no significant difference in signalment, clinical presentation and imaging findings. However, significant differences were detected in outcome. Early postoperative neurological deterioration was recorded in 16/29 dogs in the HA group and 7/24 dogs in the HPD group (p = 0.037). Sustained clinical improvement for a minimum of 18 months postoperatively was reported in 9/22 dogs in the HA group compared with 17/23 dogs in the HPD group (p = 0.019). CLINICAL SIGNIFICANCE: Hemilaminectomy with partial discectomy for decompression of thoracolumbar intervertebral disc protrusion was associated with decreased postoperative neurological deterioration and increased sustained clinical improvement compared with hemilaminectomy with anulectomy.

Surgical treatment of a paraspinal abscess with osteomyelitis and spinal cord compression in a rabbit.

CASE DESCRIPTION A 16-month-old neutered male Continental Giant rabbit (Lepus curpaeums) was referred for evaluation of a 7-day history of acute-onset, progressive, symmetric paraparesis. CLINICAL FINDINGS On initial examination, the rabbit was nonambulatory, and results of neurologic examination were consistent with a lesion affecting the T3-L3 spinal cord segments. Thoracic radiography showed irregular widening of the left T11-12 articular process joint. Marked dorsolateral and lateral extradural spinal cord compression with contrast enhancement of the adjacent epaxial muscles was evident on MRI images of the spine. TREATMENT AND OUTCOME A left-sided T11-T12 hemilaminectomy was performed, which revealed an abnormal and hypertrophic T11-12 articular process joint and an osteolytic lesion communicating with the vertebral canal. Copious purulent material causing marked spinal cord compression was evident, and the surgical site was lavaged extensively with sterile (0.9% NaCl) saline solution. Results of aerobic, anaerobic, and enriched bacteriologic cultures of swab specimens obtained from the surgical site were negative. Histologic analysis of biopsy samples revealed chronic purulent osteomyelitis, myositis, and fasciitis with necrosis, fibrosis, and dystrophic mineralization. The rabbit was discharged 48 hours after surgery. Ten weeks after surgery, the rabbit was ambulatory with mild paraparesis. On telephone follow-up 21 months after surgery, the owners indicated that the rabbit was healthy and expressed satisfaction with the treatment and outcome. CLINICAL RELEVANCE Paraspinal abscess with vertebral canal involvement should be considered as a differential diagnosis for rabbits with clinical signs of progressive T3-L3 myelopathy. Outcome for the patient of the present report suggested that surgical treatment including decompression and debridement can result in a favorable long-term outcome.

Developmental Origin of Oligodendrocyte Lineage Cells Determines Response to Demyelination and Susceptibility to Age-Associated Functional Decline.

Oligodendrocyte progenitors (OPs) arise from distinct ventral and dorsal domains within the ventricular germinal zones of the embryonic CNS. The functional significance, if any, of these different populations is not known. Using dual-color reporter mice to distinguish ventrally and dorsally derived OPs, we show that, in response to focal demyelination of the young adult spinal cord or corpus callosum, dorsally derived OPs undergo enhanced proliferation, recruitment, and differentiation as compared with their ventral counterparts, making a proportionally larger contribution to remyelination. However, with increasing age (up to 13 months), the dorsally derived OPs become less able to differentiate into mature oligodendrocytes. Comparison of dorsally and ventrally derived OPs in culture revealed inherent differences in their migration and differentiation capacities. Therefore, the responsiveness of OPs to demyelination, their contribution to remyelination, and their susceptibility to age-associated functional decline are markedly dependent on their developmental site of origin in the developing neural tube.

Clinical outcome following pneumonectomy for management of chronic pyothorax in four cats.

Pneumonectomy is the resection of all lung lobes from one side of the thorax. The clinical findings, treatment and outcome of four cases of feline chronic pyothorax managed with exploratory thoracotomy and pneumonectomy are reported. All cases were initially medically managed with thoracic drain placement and antibiosis. However, resolution was not achieved with medical therapy and diagnostic imaging findings consistent with an area of abscessation or marked lung lobe consolidation were identified, supporting a decision for surgical management. Surgical exploration was performed via median sternotomy and, on the basis of gross inspection, non-functional lung was removed. A left-sided pneumonectomy was performed in three cats and a right-sided pneumonectomy in one. All cases survived to discharge and an excellent quality of life was reported on long-term follow-up. Pneumonectomy appears to be well tolerated in the cat.