Tissue classification in a canine model of Duchenne Muscular Dystrophy using quantitative MRI parameters.

Duchenne Muscular Dystrophy (DMD) is a genetic disorder caused by dystrophin protein deficiency. Muscle biopsy is the gold standard to determine the disease severity and progression. MRI has shown potential for monitoring disease progression or assessing the treatment effectiveness. In this study, multiple quantitative MRI parameters were used to classify the tissue components in a canine model of DMD disease using histoimmunochemistry analysis as a "ground truth". Results show that multiple MRI parameters may be used to reliably classify the muscular tissue and generate a high-resolution tissue type maps, which can be used as potential non-invasive imaging biomarkers for the DMD.

Localized MRI and histological image correlation in a canine model of duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked disorder. Therapeutic assessments currently require muscle biopsy to ascertain information about the status of disease progression. MRI shows potential to be used in place of muscle biopsy for therapeutic assessments. In this work, localized histological data and various MRI parameters were correlated in a canine model of DMD. The results indicate several MRI parameters may be useful as biomarkers of disease progression.

Diaphragm remodeling and compensatory respiratory mechanics in a canine model of Duchenne muscular dystrophy.

Ventilatory insufficiency remains the leading cause of death and late stage morbidity in Duchenne muscular dystrophy (DMD). To address critical gaps in our knowledge of the pathobiology of respiratory functional decline, we used an integrative approach to study respiratory mechanics in a translational model of DMD. In studies of individual dogs with the Golden Retriever muscular dystrophy (GRMD) mutation, we found evidence of rapidly progressive loss of ventilatory capacity in association with dramatic morphometric remodeling of the diaphragm. Within the first year of life, the mechanics of breathing at rest, and especially during pharmacological stimulation of respiratory control pathways in the carotid bodies, shift such that the primary role of the diaphragm becomes the passive elastic storage of energy transferred from abdominal wall muscles, thereby permitting the expiratory musculature to share in the generation of inspiratory pressure and flow. In the diaphragm, this physiological shift is associated with the loss of sarcomeres in series (∼ 60%) and an increase in muscle stiffness (∼ 900%) compared with those of the nondystrophic diaphragm, as studied during perfusion ex vivo. In addition to providing much needed endpoint measures for assessing the efficacy of therapeutics, we expect these findings to be a starting point for a more precise understanding of respiratory failure in DMD.

MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were administered with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with magnetic resonance imaging (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a 3-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.

Cardiac structure and function in female carriers of a canine model of Duchenne muscular dystrophy.

This investigation tested the hypothesis that carriers of golden retriever muscular dystrophy (GRMD), a genetically homologous condition of Duchenne muscular dystrophy (DMD), have quantifiable abnormalities in myocardial function, structure, or cardiac rhythm. Eleven GRMD carriers and four matched controls had cardiac evaluations and postmortem examinations. 24-h ECG Holter monitoring disclosed ventricular ectopy in 10 of 11 carriers and 2 of 4 controls. Conventional echocardiography failed to demonstrate significant differences between carriers and controls in systolic function. All carriers had multifocal, minimal to marked myofiber necrosis, fibrosis, mineralization, inflammation, and/or fatty change in their hearts. Immunohistochemistry revealed a mosaic dystrophin deficiency in scattered cardiac myofibers in all carriers. No controls had cardiac histologic lesions; all had uniform dystrophin staining. Despite cardiac mosaic dystrophin expression and degenerative cardiac lesions, GRMD carriers at up to 3 years of age could not be distinguished statistically from normal controls by echocardiography or 24-h Holter monitoring.

Muscular dystrophy in dogs: does the crossing of breeds influence disease phenotype?

Golden Retriever (GR) muscular dystrophy is an inherited degenerative muscle disease that provides an excellent model for Duchenne muscular dystrophy in humans. This study defined the histopathologic lesions, including the distribution of type I and II muscle fibers (FTI and FTII), in 12 dystrophic and 3 nondystrophic dogs between 7 and 15 months of age. The authors were interested in studying the influence on disease phenotype from crossing the base GR breed with Yellow Labrador Retrievers. The dystrophic dogs were divided according to breed: GRs and Golden Labrador Retrievers (GLRs). On hematoxylin and eosin staining, histopathologic lesions were more severe in GRs than GLRs. Six of eight GR muscles (75%) had a severe lesion grade (grade 3). In contrast, seven GLR muscles (87.5%) had mild lesions (grade 2), and only one had severe lesions (grade 3). Changes in fiber-type distribution were more pronounced in GRs versus GLRs. FTI:FTII ratio inversion was observed in three dystrophic GRs but only one GLR. The mean diameter of FTI and FTII was smaller in GRs and GLRs than in nondystrophic dogs (P < .01). The FTI of five GR muscles (62.5%) were larger than those of GLRs, whereas only one GLR muscle was larger (P < .05). The differential was less pronounced for FTII, with four GR muscles being larger and three GLR being larger. Observations indicate that crossing the base GR breed with Labrador Retrievers lessened the severity of the GR muscular dystrophy phenotype.

Myofiber injury and regeneration in a canine homologue of Duchenne muscular dystrophy.

OBJECTIVE: To test the hypothesis that differential skeletal muscle involvement, previously observed in dogs with a homologue of Duchenne muscular dystrophy, correlates with the histochemical markers of myofiber injury and regeneration. DESIGN: Evidence of injury (cellular penetration by Evans blue dye, immunoglobulin G expression, hematoxylin and eosin staining of necrotic figures), myofiber regeneration (fetal myosin heavy chain isoform expression), and morphologic indices in the cranial sartorius (CS), long digital extensor, and vastus lateralis muscles were examined in five dogs with dystrophy and five normal dogs. RESULTS: Only the CS muscle, at 1 mo, demonstrated significant differences in injury when compared with age-matched controls. By 6 mo, the long digital extensor and vastus lateralis also suffered greater than normal injury. Only the dystrophic CS tissue expressed a notable increase in mean myofiber diameter when compared with other muscles at 6 mo. Normal CS muscles revealed a distinct population of small myofibers at this age. CONCLUSION: The CS seems unique in its selective pathologic involvement. These differences may contribute to the marked regenerative response of this muscle in the dystrophic state. An improved understanding of mechanisms by which some dystrophin-deficient canine muscles remain spared from injury may provide clues to investigate and prevent the degenerative processes in humans.

In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide.

In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric oligonucleotide to induce host cell mismatch repair mechanisms and correct the chromosomal mutation to wild type. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated in vivo repair of the GRMD mutation that was sustained for 48 weeks. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of exons 5-10 demonstrated increasing levels of exon 7 inclusion with time. An isolated exon 7-specific dystrophin antibody confirmed synthesis of normal-sized dystrophin product and positive localization to the sarcolemma. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism (RFLP)-PCR and sequencing the PCR product. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscle of a live animal using a chimeric oligonucleotide.

Use of whole body hyperthermia as a method to heat inaccessible tumours uniformly: a phase III trial in canine brain masses.

In this study, whole body hyperthermia (WBH) was assessed as a means of heating intracranial tumours uniformly. Twenty-five dogs received radiation therapy and 20 the combination of radiation and WBH. Total radiation dose was randomly assigned and was either 44, 48, 52, 56 or 60 Gy. Because of WBH toxicity, intercurrent disease or tumour progression, seven of the 45 dogs received less than the prescribed radiation dose. For WBH, the target rectal temperature was 42 degrees C for 2h and three treatments were planned. In five of the 20 dogs randomized to receive WBH, only one WBH treatment was given because of toxicity. WBH toxicity was severe in six dogs, and resulted in death or interruption in treatment. Most tumours did not undergo a complete response, making it impossible to differentiate tumour recurrence from brain necrosis as a cause of progressive neuropathy. Therefore, survival was the major study endpoint. There was no survival difference between groups. One-year survival probability (95% CI) for dogs receiving radiation therapy alone was 0.44 (0.25, 0.63) versus 0.40 (0.19, 0.63) for dogs receiving radiation and WBH. There was no difference in the incidence of brain necrosis in the two treatment groups. Results suggest that use of WBH alone to increase the temperature of intracranial tumours as a means to improve radiation therapy outcome is not a successful strategy.

Contraction force generated by tarsal joint flexion and extension in dogs with golden retriever muscular dystrophy.

Force generated due to torque caused by tarsal joint flexion and extension was measured noninvasively at 3, 4.5, 6, and 12 months of age in dogs with the Duchenne homologue, golden retriever muscular dystrophy (GRMD). Absolute and body-weight-corrected GRMD twitch and tetanic force values were lower than normal at all ages (P<0.01 for most). Tarsal flexion and extension were differentially affected. Flexion values were especially low at 3 months, whereas extension was affected more at later ages. Several other GRMD findings differed from normal. The twitch/tetany ratio was generally lower; post-tetanic potentiation for flexion values was less marked; and extension relaxation and contraction times were longer. The consistency of GRMD values was studied to determine which measurements will be most useful in evaluating treatment outcome. Standard deviation was proportionally greater for GRMD versus normal recordings. More consistent values were seen for tetany versus twitch and for flexion versus extension. Left and right limb tetanic flexion values did not differ in GRMD; extension values were more variable. These results suggest that measurement of tarsal tetanic force should be most useful to document therapeutic benefit in GRMD dogs.

Molecular analysis of a spontaneous dystrophin 'knockout' dog.

We have determined the molecular basis for skeletal myopathy and dilated cardiomyopathy in two male German short-haired pointer (GSHP) littermates. Analysis of skeletal muscle demonstrated a complete absence of dystrophin on Western blot analysis. PCR analysis of genomic DNA revealed a deletion encompassing the entire dystrophin gene. Molecular cytogenetic analysis of lymphocytes from the dam and both dystrophic pups confirmed a visible deletion in the p21 region of the affected canine X chromosome. Utrophin is up-regulated in the skeletal muscle, but does not appear to ameliorate the dystrophic canine phenotype. This new canine model should further our understanding of the physiological and biochemical processes in Duchenne muscular dystrophy.

Hydranencephaly and cerebellar hypoplasia in two kittens attributed to intrauterine parvovirus infection.

Six weeks after vaccination with modified live feline parvovirus vaccine, a cat gave birth to five kittens, three of which died soon afterwards. The remaining two kittens (A and B) survived, but at 8 weeks of age were unable to walk and showed abnormal behaviour, with lack of menace and oculovestibular responses, and severe dysmetria. These signs suggested multifocal disease associated with the cerebrum and cerebellum. Magnetic resonance imaging demonstrated severe bilateral (kitten A) or unilateral (kitten B) hydrocephalus or hydranencephaly, combined with cerebellar agenesis (kitten A) or severe hypoplasia (kitten B). Hydranencephaly was confirmed histopathologically in both kittens. Parvovirus was isolated from the kidney of one kitten. Parvoviral DNA was amplified by the polymerase chain reaction (PCR) from paraffin wax-embedded brain of both kittens. The severe malformations observed in these kittens presumably resulted from an in-utero parvovirus infection, possibly due to vaccination, that occurred late in the first, or early in the second, trimester of pregnancy.

Abnormalities of the thoracic bellows: stress fractures of the ribs and hiatal hernia.

The thoracic bellows mechanism consists of the rib cage and the diaphragm. The purpose of this study was to determine if nontraumatically acquired lesions of the bellows were secondary to underlying disease. Abnormalities of the bellows, specifically stress fractures of the ribs and hiatal hernia, were found in 21 dogs and cats with underlying cardiopulmonary disease, neuromuscular disease, or metabolic disease. A case-control study of Bulldogs demonstrated that hiatal hernia was associated with the more severe manifestations of brachycephalic syndrome. Stress fractures occurred mostly in females and in cats, and involved multiple ribs. Fractures were usually related to severe respiratory effort, but also occurred in association with metabolic disease. Hiatal hernia was also associated with severe respiratory effort, but may be exacerbated if a neuromuscular disorder affecting the diaphragm is present. Abnormalities of the thoracic bellows, such as rib stress fractures and hiatal hernia, may be signs of underlying disease, rather than being primary causes of disease.

Late-onset progressive spinocerebellar degeneration in Brittany Spaniel dogs.

Eight Brittany Spaniel dogs, seven females and one male, between 7 and 14 years old presented with clinical neurological signs of spinocerebellar disease of about 6 months to 4 years duration. Clinically the dogs had a dramatic forward "saluting" movement of the thoracic limbs, hypermetria of the pelvic limbs, cerebellar ataxia and intention tremors. Terminally, dogs crawled in a crouched thoracic posture with neck extension. Lesions were confined to cerebellum, medulla oblongata and spinal cord. The most severe lesion was diffuse Purkinje cell loss with massive neurofilament accumulation in degenerating cells. There was some bilateral neuronal degeneration in the dorsal horns of the spinal cord and in the gracilis and cuneate nuclei. There was bilateral sporadic axonal degeneration in the dorsal columns and lateral and ventromedial areas of the spinal cord. The etiology of this syndrome was not determined.

Alternative dystrophin gene transcripts in golden retriever muscular dystrophy.

Golden retriever muscular dystrophy (GRMD), the canine model of Duchenne muscular dystrophy (DMD), is caused by a splice site mutation in the dystrophin gene. This mutation predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. Western blot analysis of skeletal muscle from GRMD dogs reveals a slightly truncated 390-kD protein that is approximately 91% the size of normal dystrophin. This 390-kD dystrophin suggests that GRMD dogs, like some DMD patients, employ a mechanism to overcome their predicted frameshift. Reverse-transcriptase polymerase chain reaction on GRMD muscle has revealed two in-frame dystrophin transcripts which lack either exons 3-9 or exons 5-12. Both transcripts could be translated into a dystrophin protein of approximately 390 kD. An understanding of how truncated dystrophin is produced in GRMD may allow this mechanism to be manipulated toward a potential therapy for DMD.

Evaluating motor end-plate-targeted injections of botulinum toxin type A in a canine model.

Tarsal joint forces were measured in dogs over 70 days following botulinum toxin type A (BTX-A) injections. Three dogs were injected at motor end-plates located by electromyography (EMG), while 3 dogs were similarly injected, but without EMG guidance. Extension forces were significantly (P < 0.05) smaller in limbs injected at motor end-plates than in corresponding limbs on days 14 and 35. There were no significant differences at other times. Using these techniques, EMG end-plate targeting potentiates effects of BTX-A.

Atraumatic rupture of the gastrocnemius muscle after corticosteroid administration in a dog.

A 6-year-old spayed female Shetland Sheepdog was referred for evaluation of lameness, muscle atrophy, and a partial plantigrade stance of the right hind limb of 5 weeks' duration. Without history of trauma, atraumatic rupture of the right gastrocnemius muscle was diagnosed. Surgical repair was unsuccessful. The dog then developed signs of hyperadrenocorticism. Results of ACTH stimulation and low-dose dexamethasone suppression tests were consistent with iatrogenic adrenal suppression. One deleterious effect of excessive use of corticosteroids on muscle and connective tissue is degenerative myopathy. Steroid-induced myopathy with subsequent rupture of the gastrocnemius muscle was suspected in this dog. Clinical signs of myopathy most often develop with use of triamcinolone acetonide; therefore, care should be taken when administering this and other corticosteroids.

Fibrocartilaginous embolism of the spinal cord in dogs: review of 36 histologically confirmed cases and retrospective study of 26 suspected cases.

Clinical features of 36 dogs with histologically confirmed fibrocartilaginous embolism (FCE) were contrasted with those of 26 dogs in which FCE was suspected based on characteristic clinical findings and the absence of compressive spinal cord disease on myelography. Dogs with confirmed and suspected FCE were of similar signalment, and had acute, nonprogressive dysfunction, often associated with trauma or exercise. The "suspected" group included fewer giant breeds and more often had asymmetric lesions, intact nociception, and upper motor neuron involvement. Dog size and severity of clinical signs probably contributed to owners choosing euthanasia in dogs with confirmed lesions. Accordingly, data from such patients may be skewed relative to these clinical features.

Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs.

OBJECTIVES: To use exon 7-specific genomic polymerase chain reaction (PCR) products to identify the genotypes of normal, affected, and carrier female dogs in pedigrees segregating Golden Retriever muscular dystrophy (GRMD), and to confirm the concordant segregation of the mutation in all carrier and affected dogs presently available. DESIGN: The GRMD mutation is found in the consensus splice acceptor site in intron 6 of the canine dystrophin gene. PCR cycle-sequencing and restriction fragment length polymorphism/PCR were used for determination of the pattern of segregation of the point mutation which causes GRMD. ANIMALS: Normal, clinically affected, and obligate carrier dogs in pedigrees of GRMD. PROCEDURE: DNA from blood was amplified, using PCR and primers that bracket all of exon 7 of the canine dystrophin gene as well as 100 base pairs of intron on either side. PCR products were either cycle-sequenced directly or submitted to a second round of PCR, using 1 of the original primers coupled with a mutagenic restriction fragment length polymorphism-primer, which thus creates an artificial restriction site. Digestion with Stu I detected the normal allele. To detect the affected allele, Sau96 I was used to digest the 310-base pair exon 7 genomic fragment directly. CONCLUSIONS: Simple, clear diagnosis of carrier status was possible using these methods. This mutation is passed through all carrier and affected dogs in both United States GRMD colonies and the colony in Australia. CLINICAL RELEVANCE: Rapid, accurate diagnosis of carrier and affected dogs will enhance study of this homologue of Duchenne muscular dystrophy.

Cystic meningiomas in 2 dogs.

Two dogs with signs of forebrain disease had hypodense lesions on computed tomography evaluation. Magnetic resonance imaging of the first dog showed a hypointense lesion on the T1-weighted scan and a hyperintense lesion on T2-weighted scanning. At surgery, both dogs had a primary cystic intracranial lesion, and the abnormal tissue adjacent to the cyst had histological features of meningioma. Each dog underwent whole brain irradiation after surgery, and 1 dog lived for 3 years after treatment. While uncommon, meningioma should be considered as a differential diagnosis in dogs with cystic intracranial lesions.