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.

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.

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.

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.

Temperature measurements in normal and tumor tissue of dogs undergoing whole body hyperthermia.

Temperature was measured in the left ventricle, aorta, liver, brain, lung, bone marrow, kidney, and spontaneous solid tumors in dogs undergoing whole body hyperthermia in a radiant heat device. Rectal temperature was found to be a satisfactory indicator of systemic arterial temperature during plateau temperature conditions but rectal temperature underestimated arterial temperature during heating and overestimated it during cooling. Lung temperature, based on small airway temperature, was the same as rectal temperature during plateau temperature conditions. Liver and brain temperatures were slightly higher (0.1-0.2 degree C) than rectal temperature during the plateau phase. During plateau temperature conditions, kidney temperature measurements were higher than rectal temperature when one site/kidney was measured but were lower than rectal temperature when two sites/kidney were measured suggesting invasive thermometry may have affected measured temperature values. Tibial marrow temperature was greater than rectal temperature during heating but fell below rectal temperature during plateau temperature conditions by as much as 1.3 degree C. Femoral marrow temperature was below rectal during heating but gradually exceeded it during steady state conditions, by 0.1-0.4 degree C. Temperature in solid tumors was variable, sometimes exceeding (0.6 degree C) and sometimes being less (1.8 degree C) than rectal temperature.

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.

Alpha-dystroglycan deficiency correlates with elevated serum creatine kinase and decreased muscle contraction tension in golden retriever muscular dystrophy.

The dystrophin-glycoprotein complex was examined in dystrophin-deficient dogs with golden retriever muscular dystrophy (GRMD) using immunoblot and immunofluorescence analysis. The dystrophin-associated proteins were substantially reduced in muscle from dogs with GRMD. Interestingly, regression analysis revealed a strong correlation between the amount of alpha-dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.

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.

Canine X-linked muscular dystrophy as an animal model of Duchenne muscular dystrophy: a review.

Canine X-linked muscular dystrophy is a spontaneously occurring, progressive, degenerative myopathy of dogs that is clinically and pathologically similar to Duchenne muscular dystrophy in man. The molecular basis for the disease has been shown to be a lack of dystrophin, the protein product of the Duchenne muscular dystrophy gene. Breeding colonies of dystrophic dogs have been established. This report reviews the findings of genetic, clinical, pathologic, molecular biologic, and immunocytochemical studies of the canine model, and compares the features of the canine disease to those of Duchenne dystrophy in man.

Notexin-induced muscle injury in the dog.

Notexin, a myotoxic phospholipase, was used to induce focal necrosis in the sartorius muscles of normal mixed-breed adult dogs and in 12-week-old beagles. Notexin injury caused pathologic changes similar to those of Duchenne muscular dystrophy (DMD) and its canine homologue, golden retriever muscular dystrophy (GRMD). All three conditions are characterized by increased serum creatine kinase (CK) levels, sarcolemmal defects, delta lesions, hyaline degeneration of myofibers, calcium-positive myofibers, and minimal effects on neurovascular structures. Four and 24 h after exposure to notexin, serum CK levels were elevated, and many myofibers were necrotic. In addition, by 24 h the necrotic areas were heavily invaded by mononuclear cells, and calcium-positive myofibers were prominent. Capillaries appeared intact even in areas of marked myonecrosis. Massive cellular infiltrate and myotube formation was evident at 3 days post injury. By 7 days, most affected fascicles were occupied by small immature myofibers. Regeneration was largely complete at 21 days. Our results suggest that notexin-induced muscle injury in dogs will be useful in the evaluation of potential therapies for DMD such as myoblast transplantation.

A role for mast cells in the progression of Duchenne muscular dystrophy? Correlations in dystrophin-deficient humans, dogs, and mice.

Dystrophin deficiency has been shown to be the underlying cause of Duchenne muscular dystrophy. Although dystrophin-deficient homologous animal models have been identified (dog, mouse, and cat), the clinical expression of the biochemical defect is species-specific. Thus, while the genetics and biochemistry of Duchenne dystrophy is understood, the pathophysiological cascade leading to muscle weakness in only humans and dogs remains obscure. To begin to dissect the pathophysiology at the histological level, we undertook a systematic study of mast cells in normal and dystrophin-deficient muscle. Mast cells have been implicated in the development of fibrosis in other disorders, and progressive fibrosis has been hypothesized to mediate the failure of muscle regeneration in human and dog dystrophin deficiency. Our results show a strong correlation between mast cell content and localization, and the clinico-histopathological progression in humans, dogs and mice. The mast cell increases were disease specific: other dystrophic myopathies with normal dystrophin generally did not show substantial increases in mast cell content or degranulation. Our data suggest that mast cell accumulation and degranulation may cause the grouped necrosis characteristic of dystrophin deficiency in all species.

Contraction tension and kinetics of the peroneus longus muscle in golden retriever muscular dystrophy.

Contraction tension and kinetics of the peroneus longus muscle were studied in dogs with the Duchenne homologue, golden retriever muscular dystrophy (GRMD), in advance of evaluating localized therapies such as myoblast transplantation. Absolute and both muscle- and body-weight-corrected twitch tension in GRMD dogs were low compared to normal litter mates at 3 months of age (p < 0.0005 for all). Tetanic tension was affected similarly. However, whereas absolute values were still reduced at 6 months (p < 0.0005 for twitch and 0.005 for tetany), twitch and tetanic tension corrected for either muscle or body weight was not statistically different, suggesting that the peroneus longus may be relatively spared in GRMD. Post-tetanic potentiation was more pronounced in GRMD versus normal dogs at both 3 (p < 0.0001) and 6 (p < 0.01) months. The degree of positive staircase at 3 months of age did not differ. Twitch contraction and relaxation times were dramatically prolonged, and there was concomitant sustained electrical activity, at, or before, 6 months of age in some severely affected dogs. Relatively few carriers were evaluated at these ages, but their values were similar to those of normal dogs. Apparent sparing of the peroneus longus muscle may limit application of this technique to evaluation of therapies administered early in life or in combination with toxins. Treatment to alter changes in contraction kinetics could also be assessed.

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.

Immunohistochemical detection of neural cell adhesion molecule and laminin in X-linked dystrophic dogs and mdx mice.

Although dystrophin deficiency is known to be the genetic and biochemical defect causing Duchenne muscular dystrophy (DMD), much remains unknown about the underlying factors affecting clinical and pathological expression of the disease. Two animal forms of muscular dystrophy resembling DMD have been described. Neural cell adhesion molecule (NCAM) and laminin expression were examined in the proliferation-competent mdx mouse and non-regenerative "golden retriever muscular dystrophy dog" (GRMD). The results showed that (1) NCAM expression was greater in dystrophic dogs and mice than in age-matched normal animals, (2) myoblast-specific NCAM was greater in mdx mice than in dystrophic dogs, and (3) laminin strongly labelled mdx and GRMD myofibre membranes but was also sometimes found in individual interstitial cells of mdx muscle. Expression of these proteins may partly determine the clinicopathological expression of dystrophin deficiency.

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.

Leucoencephalomalacia and cerebral white matter vacuolar degeneration in two related Labrador retriever puppies.

Two Labrador Retriever dogs from a common dam had similar neurological deficits consisting of cortical blindness, dullness, and loss of previously learned habits. Both were examined at 5 months of age, and histopathological examination revealed leucoencephalomalacia and vacuolar degeneration of the cerebral white matter. Histopathologic findings in these 2 dogs differed from those reported previously in Labrador Retrievers with spongy degeneration of central nervous system white matter. A nonlittermate full sibling to 1 of these dogs was examined at 1.5 years of age for similar clinical signs that did not progress for the next 25 months.

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.

Analysis of survival in a retrospective study of 86 dogs with brain tumors.

A retrospective study of 86 dogs with brain tumors was undertaken. Sixty-nine dogs had histologic confirmation of tumor type, whereas the remaining 17 dogs had CT evidence of a brain tumor. All dogs had neurologic abnormalities. Seven dogs received no treatment, 38 dogs received only symptomatic treatment, and 41 dogs received some form of definitive treatment, in addition to medical management. Types of definitive treatment included surgery, cobalt-60 radiation, whole-body hyperthermia, 125I implants, and chemotherapy, alone or in combination. The factor that was most associated with survival duration was mode of therapy. Those dogs who were treated with cobalt-60 radiation, with or without other combinations of therapy, lived significantly longer than dogs who received surgery (+/- 125I implants), or dogs who received symptomatic treatment (P = 0.01 and P less than 0.001, respectively). After statistic adjustment for treatment, multiplicity of brain involvement (solitary vs. multiple) provided prognostic information with respect to survival (P = 0.001), with dogs who had a solitary site of involvement having a better prognosis. After further adjustment, initial neurologic dysfunction (mild/moderate vs. severe) showed significance as prognostic variable (P = 0.005). Both the mild and moderate groups had a more favorable prognosis compared with dogs who had severe initial neurologic impairment. The median survival time for the 86 dogs was 1.0 month (range: 1 day-42.4 mo). Median survival times of dogs receiving: 1) no therapy or only symptomatic therapy, 2) surgery (+/- 125I), or 3) cobalt-60 radiation (+/- hyperthermia, +/- surgery) were 0.2, 0.9, and 4.9 months, respectively.

Feline spinal lymphosarcoma: a retrospective evaluation of 23 cats.

A retrospective study of pathologically confirmed cases of feline spinal lymphosarcoma (FSL) admitted to the Colleges of Veterinary Medicine at the University of Georgia and North Carolina State University from 1973 to 1988 was conducted. Two hundred fourteen cases of feline lymphosarcoma were diagnosed histopathologically; involvement of the central nervous system (CNS) was identified in 26 (12.1%). Twenty-three of these tumors involved the spinal cord, and 22 of the 23 were solitary. A predilection for the thoracic and lumbar vertebral canal was noted. Most cats with spinal disease were young, with mean and median ages of 43 and 24 months, respectively; 67 cats were 36 months of age or younger. In most cases, affected cats had acute neurological deterioration after an initial insidious course. Extraneural abnormalities were not consistently present. Neoplastic lymphocytes diagnostic of FSL were identified on cerebrospinal fluid (CSF) analysis in 6 of 17 cats evaluated. Sixteen of 17 cats evaluated had serologically positive test results for feline leukemia virus (FeLV) p27 antigen, and all cats tested for feline immunodeficiency virus (FIV) antibodies had negative test results.

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.