Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

<p>Abstract</p> <p>Background</p> <p>The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression.</p> <p>Methods</p> <p>Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes.</p> <p>Results and Discussion</p> <p>We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent <it>in situ </it>hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age.</p> <p>Conclusion</p> <p>Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.</p

Kinematic gait analyses in healthy Golden Retrievers

Kinematic analysis relates to the relative movement between rigid bodies and finds application in gait analysis and other body movements, interpretation of their data when there is change, determines the choice of treatment to be instituted. The objective of this study was to standardize the march of Dog Golden Retriever Healthy to assist in the diagnosis and treatment of musculoskeletal disorders. We used a kinematic analysis system to analyse the gait of seven dogs Golden Retriever, female, aged between 2 and 4 years, weighing 21.5 to 28 kg, clinically normal. Flexion and extension were described for shoulder, elbow, carpal, hip, femorotibialis and tarsal joints. The gait was characterized lateral and had accepted hypothesis of normality for all variables, except for the stance of hip and elbow, considering a confidence level of 95%, significance level α = 0.05. Variations have been attributed to displacement of the stripes during movement and the duplicated number of reviews. The kinematic analysis proved to be a consistent method of evaluation of the movement during canine gait and the data can be used in the diagnosis and evaluation of canine gait in comparison to other studies and treatment of dogs with musculoskeletal disorders

Bortezomib (PS-341) Treatment Decreases Inflammation and Partially Rescues the Expression of the Dystrophin-Glycoprotein Complex in GRMD Dogs

Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin-associated proteins in the muscle fiber membrane

Immunohistochemistry of TGF-β and dystrophin in skeletal muscle.

<p><b>A and E.</b> Muscle from healthy dogs. TGF-β is detected around the vessels in muscles (A), α-dystroglycan (E) and dystrophin (I) patterns in the sarcoplasmic membrane. <b>B:</b> Muscle from CD at T1 shows greater TGF-β expression in the endomysium of the fibers and more deposition of connective tissue at T1. <b>C and D:</b> TD after treatment with bortezomib (T1) showed lower deposition of connective tissue and lower expression of TGF-β in the endomysium of the fibers. Original magnification: 40×; bar: 50 µm. <b>F, G and H:</b> Neither untreated (CD) (J) nor treated GRMD dogs (TD) (K and L) showed expression of dystrophin in the sarcoplasmic membrane, indicating that bortezomib did not rescue this protein during the treatment. Original magnification: 20×; bar: 100 µm.</p

Ultrastructural analysis of connective tissue in muscles from GRMD dogs.

<p><b>A:</b> Muscle from a healthy dog. There was a narrow endomysium space and a lower deposition of connective tissue (→). <b>B:</b> An untreated GRMD dog (CD). The endomysium of this dog exhibits a higher deposition of connective tissue and hypercontracted fibers (HF). <b>C and D:</b> Treated GRMD dogs (TD) showed a lower deposition of connective tissue and endomysium, and few fibers were hypercontracted (HF). Original magnification: 3,500×. <b>E:</b> In muscles from healthy dogs, the mitochondria were preserved and had the same electron density as the fibers, and the mitochondrial cristae were visible. <b>F:</b> GRMD dogs demonstrated abnormal mitochondria, had a higher electron density, and were smaller, and the cristae were not visible. <b>G and H:</b> Abnormal fiber (<b>NF</b>) with macrophage invasion (<b>M</b>), complete loss of membrane integrity and myofibrillar structure showing a finely granular cytoplasm. Activated fibroblasts (<b>F</b>) with a prominent rough endoplasmic reticulum (→) were present in the endomysium. Original magnification: A and B, 8,900×; C, 3,500×; D, 5,600×.</p

Serum and hematological parameters in GRMD dogs.

<p>Mean values for altered serum and hematological parameters from GRMD dogs during the nine weeks of the study. The times represent the bortezomib treatment period in the treated dogs (K1 and B7). The parameter with the greatest change was serum CK concentration, with a high magnitude range of 40 to 70 times the reference value from Kaneko et al. (1997). CK: creatine kinase</p

Clinical signs in GRMD dogs.

<p>The main clinical signs observed in GRMD dogs during treatment with bortezomib (three, four and five months of age). There were different phenotypes, but the main clinical signs related to muscular dystrophy progression were common to all of the dogs in the study. For physiological studies of GRMD, please see the reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061367#pone.0061367-Yang1" target="_blank">[63]</a> showing an <i>in situ</i> protocol to measure the force generated by a single muscle in dogs.</p

Immunohistochemistry and western blot for β-dystroglycan.

<p><b>A:</b> Muscle from a healthy dog shows the β-dystroglycan pattern in sarcoplasmic membranes. <b>B:</b> Untreated GRMD dog. <b>C and D</b>: Western blot and immunohistochemical analysis from a TD show higher expression of β-dystroglycan in muscle fibers than CD after treatment with bortezomib. Using ImageJ software and measuring the blot band intensity, we found that TD-K1 had a 2-fold increase in the expression of β-dystroglycan. Original magnification: 40×; bar: 50 µm.</p

Immunohistochemistry of phospho-NFκB in skeletal muscle.

<p><b>A and C:</b> Muscle from a CD shows higher expression of the (active) phospho-NFκB in the nuclei of muscle fibers. <b>B and D:</b> GRMD dogs (TD), after treatment with bortezomib, showed a lower expression of phospho-NFκB in the nuclei of fibers, indicating proteasome inhibition and preservation of inactive NFκB in the cytoplasm. Original magnification: A and B, 10×; bar, 200 µm; C and D, 20×; bar, 100 µm. <b>E:</b> The phospho-NFκB positive nuclei were counted in 10 random fields, with images captured at 40×. Student's <i>t</i>-test was used to evaluate these results (* p<0.05). The CD showed more phospho-NFκB-positive nuclei, indicating more activation of proteasomal activity inducing pro-apoptotic factors and inflammatory molecules.</p

Histological analysis of H&E-stained skeletal muscle fibers after treatment with bortezomib and muscle collagen morphometry.

<p>A and B: CD showed a greater deposition of connective tissue in the endomysium and perimysium (▸), and the inflammatory cells formed groups or massive lesions (*) with a poor histopathological appearance. C and D: TD showed lower deposition of connective tissue in endomysium and perimysium (▸) and a lower presence of inflammatory cells (*); Original magnification: 10×; bar: 200 µm. E: Mean and SD of muscle collagen morphometry of slides stained with picrosirius red followed by quantitative analysis. Muscle from healthy dogs and TD and CD before (T0) and after (T1) treatment with bortezomib. The p-value was <0.0001 for comparing the collagen at T0 and T1 for CD. At T1 there was a statistically significant difference between the TD and CD, with higher collagen levels in CD (p = 0.0028).</p