Genotype evaluation,muscle histopathology and ultrastructure in a Duchenne muscular dystrophy animal model

AIM: To evaluate the genotype , muscle histopathology and ultrastructure in dko mice. METHODS: Dystrophin/Utrophin-deficient double knockouts (dko) mice were obtained from university of Oxford, UK. Genotype of filial generation of heterozygote was evaluated by PCR-SSP. HE staining and fluorescent immunohistochemistry by SABC-Cy3 were used to detect striated muscle of dko mouse, and the muscle ultrastructure was observed by transmission electron microscope(TEM). RESULTS: In 112 filial generation mice, there were 28 mdx (25.0%), 26 dko (23.2%) and 58 heterozygote (51.8%), which coincided with the law of Mendelian inheritance. HE staining showed that the myocytes were not very uniform, there were phenomenon of round outline, centrally nucleated fibers, widening interspace, inflammatory cell infiltration and connective tissue proliferation in dko mice. There were no any immunofluorescent expression of dystrophin and utrophin in sarcolemma in dko mice. TEM showed sarcolemma breakage, separation and edema, and loose myofibril texture, inflammatory cell infiltration and connective tissue proliferation in dko mice. CONCLUSION: PCR-SSP is a very quick and accurate way for genotype evaluation of filial generation. The pathophysiology of dko mouse was very similar to Duchenne muscular dystrophy (DMD), and dko mouse is an ideal animal model for study of DMD clinical therapy.     

Investigations on the Occurrence of Hereditary Diseases in the Danish Cattle Population 1989-1991

Agerholm, J.S., A. Basse and K. Christensen: Investigations on the occurrence of hereditary diseases in the Danish cattle population 1989-1991. Acta vet. scand. 1993, 34, 245-253.– The methods of the Danish Bovine Genetic Disease Programme are outlined, and the results obtained during the first 3 years in function are described. The most common disease reported was spinal muscular atrophy in calves of the Red Danish Dairy breed with 312 reports. Necropsy was performed on 162 cases, and spinal muscular atrophy was diagnosed in 82 of these. Bovine progressive degenerative mye-loencephalopathy, rectovaginal constriction, syndrome of arthrogryposis and palatoschisis, hereditary chondrodysplasia (2 different types), syndactylism, epitheliogenesis imperfecta, and osteogenesis imperfecta was diagnosed with 1 case each. Lethal trait A46 was diagnosed in 4 calves. Some of these diseases have not previously been described in Denmark, and epitheliogenesis imperfecta was for the first time diagnosed in the Hereford breed. Chromosome translocation 1/29 was detected in the Blonde d’Aquitaine (BAQ), Limousine, and Red Danish Dairy breed. The aberration occurred frequently in BAQ. Furthermore, a complex chromosome translocation t(l;8;9)(q45;ql3;q26) was detected in the Red Danish Dairy breed.     

ULTRASONOGRAPHIC FEATURES OF DIFFUSE HEPATOCELLULAR DISORDERS IN DAIRY CATTLE

Ultrasonography of the liver was performed in 200 Holstein-Friesian dairy cattle using a 3.5 MHz transducer with a linear array electronic scanner. Liver samples were taken, processed and examined microscopically and the fat occupying rate was calculated. The hepatic ultrasonograms were evaluated according to the presence of bright pattern, dark pattern, deep attenuation, vascular blurring and blurring of edges. Of the 200 animals, 96 had a normal liver, 63 had hydropic degeneration of the liver, 37 had fatty infiltration of the liver, 3 had liver dystrophy and I had hepatic amyloidosis, diagnosed through histopathological examination. Amyloidosis was characterized by bright pattern and blurring of edges. Liver dystrophy had higher percentages of bright pattern and blurring of edges than normal liver. Hydropic degeneration had higher percentages of dark pattern and blurring of edges than normal liver. Fatty infiltration had higher percentages of bright pattern, deep attenuation, vascular blurring and blurring of edges than normal liver. The present results suggest that different ultrasonographic patterns can be observed in various diffuse hepatocellular disorders in dairy cattle     

Radiographic,MRI, and CT findings in a young dog with Becker-like muscular dystrophy

An 8-month-old, male, crossbreed dog was presented for macroglossia, reduced mandibular extension, ptyalism, dysphagia, and regurgitation. Serum creatine kinase and aspartate aminotransferase activity were markedly increased. Thoracic radiographs showed an axial gastro-esophageal hiatal hernia, diaphragmatic thickening, and asymmetry. Magnetic resonance imaging of the head showed a severely enlarged tongue, symmetric increase in size of the geniohyoid and mylohyoid muscles, and diffuse masticatory hypomyotrophy. Whole-body CT ruled out other musculoskeletal abnormalities and further characterized the radiographic and MRI findings. Muscular histopathology was consistent with Becker muscular dystrophy.     

The electroretinographic phenotype of dogs with Golden Retriever muscular dystrophy

Objective To characterize the flash electroretinogram (ERG) in the Golden Retriever muscular dystrophy (GRMD) dog and to compare the results with those from a control group of Golden Retrievers. To investigate whether similar abnormalities of the ERG as those found in a majority of human patients with Duchenne muscular dystrophy (DMD) are also observed in the GRMD dog, the canine model for DMD. Animals Five GRMD dogs and five age‐matched clinically normal Golden Retrievers. Procedure An ophthalmic examination was carried out prior to performing electroretinography under general anesthesia. Rod, combined rod–cone and oscillatory potentials responses were recorded after dark adaptation. Responses to 30‐Hz‐flicker were recorded after light adaptation. The ERG responses of the GRMD dogs were compared with those of the control dogs by use of a Wilcoxon signed rank test. Results GRMD dogs had significantly reduced a and b‐wave amplitudes after dim white flash stimuli (rod response) and reduced a‐wave amplitude after bright white flash stimuli (rod–cone response). Conclusion and clinical relevance The ERG abnormalities observed in the GRMD dog suggest a dysfunction in the rod signaling pathway. These ERG alterations are different from those observed in human patients with DMD.     

外源性硫化氢对骨骼肌疲劳的影响简

试验旨在探讨硫化氢供体硫氢化钠改善疲劳状态下肌力减退的作用。采用脉冲式电流直接刺激腓肠肌作为疲劳模型,对比硫氢化钠和任氏液分别对蟾蜍腓肠肌疲劳的影响。结果表明,硫氢化钠在浓度为5×10^-4、1×10^-3、2×10^-3 mol/L时,离体腓肠肌达到最大收缩幅度90%、50%和10%的时间极显著长于在任氏液作用下的离体腓肠肌(P<0.01);5.6 mg/kg硫氢化钠也极显著延长了在体腓肠肌达到最大收缩幅度90%、50%和10%的时间(P <0.01)。表明外源性硫化氢具有延缓肌肉疲劳的作用。     

氧化鱼油对鲤鱼生产性能和肌肉组织结构的影响

将新鲜鱼油(过氧化物值 ,POV ,1.28meqO2/kg)和POV分别为59.28、118.79、159.85、189.37、331.82和367.86meqO2/kg 的6个氧化程度不同的鱼油按3 %加入半纯化饲料中 ,投喂60g左右二龄鲤鱼鱼种9周。结果表明 :氧化鱼油损害鲤鱼生产性能 ,即降低鲤鱼增重率(P<0.05)、增加饲料系数(P<0.05) ;氧化鱼油导致肝体比(HSI)、肾体比(KSI)和脾体比(SSI)增加 ,以POV为59.28meqO2/kg 的低氧化程度鱼油增加幅度最高 ,超过此氧化程度 ,增加态势相对减弱 ,这预示低量氧化产物刺激肝胰脏、肾脏和脾脏增生 ,高量氧化产物破坏性增强 ,抑制增生作用 ;氧化鱼油破坏肌肉组织 ,使肌纤维间隙急剧扩大、肌原纤维降解、肌原纤维模式紊乱。     

Glutathione Peroxidase Activity and Erythrocyte Lipid Peroxidation as Indices of Selenium and Vitamin E Status in Young Pigs

A randomized, blocked 2³ factorial experiment was conducted with 48 pigs from sows fed a diet low in selenium and vitamin E. From 3 to 12 weeks of age the piglets were kept in single pens and fed a basic diet consisting mostly of barley, dried skim milk, soybean meal and dried yeast, and containing 55 µg selenium and 3 mg vitamin E per kg. The treatment factors — i.e. feed supplements — were 2 levels of Se (nil, 60 µg/kg), 2 levels of vitamin E (nil, 50 mg/kg), and 2 levels of the feed antioxidant ethoxyquin (nil, 150 mg/kg). Blood samples, collected at termination of the experiment, were examined for glutathione peroxidase activity (GSH-Px) and resistance against erythrocyte lipid peroxidation (ELP) to evaluate Se and vitamin E status, respectively. Analysis of variance showed the GSH-Px activity to be litter-dependent (P < 0.001) and influenced by selenium supplementation (P < 0.001) but not by the other supplements or by interactions between supplements. Resistance against ELP was influenced only by vitamin E supplementation (P < 0.001). GSH-Px and ELP thus seem to be valuable and simple methods for evaluating, respectively, Se status and vitamin E status in growing pigs.     

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background

The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.

Methods

A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.

Results

Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.

Conclusion

We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.