Effect of zinc-carnosine chelate compound on muscle function in mdx mouse

The effect of antioxidant Z-103, catena-(S)-[mu-[N(alpha)-(3-aminopropinyl)histidinnato-(2-)N(1),N(2),O:N(tau)]-zinc], on muscle function in the muscular dystrophy (mdx) mouse was examined by repetitive intraperitoneal administration in subjects aged 4 to 12 weeks. Z-103 administration at a dose of 150 mg/kg increased the load resistant time (LRT), during which the animal with a load holds itself upright on a wire net. The Z-103 administration reduced hypertrophy, the ratio of centronucleated myofibers, and the rate of decay for magnitude of twitch force elicited by 0.5 Hz of electricity to the extensor digitorum longus (EDL) muscle of 12-week-old mdx mice, with little effect on the magnitude of twitch force. The administration of Z-103 (100 mg/kg) had a lesser effect on LRT and the other characteristics examined for EDL muscles. The constituent of Z-103, Zn(2+) applied in the form of ZnSO(4) (5 mg/kg), carnosine (100 mg/kg), and the combination of the two had no beneficial effect on mdx mice. Z-103 (150 mg/kg) administered to normal mice increased LRT with little effect on the contractile properties of EDL muscles. These results suggest that the administration of Z-103 ameliorates muscle function in the mdx mouse.     

Power output of fast and slow skeletal muscles of mdx (dystrophic) and control mice after clenbuterol treatment

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy. We tested the null hypothesis that 20 weeks of clenbuterol treatment ( approximately 2 mg kg-1 day-1) of mdx and control mice would have no effect on the absolute and specific force (Po, kN m-2) and absolute and normalised power output (W kg-1) of extensor digitorum longus (EDL) and soleus muscles. For mdx and control mice, clenbuterol treatment produced modest increases in the mass of the two muscles but did not increase absolute or specific force or normalised power output. For absolute power output, only the EDL muscles of mdx mice showed a difference following treatment, with the power output of treated mice being 118 % that of the untreated mice. The modest effects of clenbuterol treatment on the dynamic properties of skeletal muscle provide little support for any improvement in muscle function for the dystrophic condition.     

Effect of zinc-carnosine complex on muscular function in frail distrophin-deficient (mdx) mice

The effect of zinc-carnosine complex (Z-103) on muscle function in dystrophin-deficient (mdx) mice was examined using several different courses of repetitive administration. Z-103 at a dose of 100 mg/kg increased the load resistant time (LRT), during which an animal bearing a load holds himself upright on a wire net, in mdx mice when administered at an age of less than about 4 months. The effect of Z-103 on LRT was independent of sex when given by intraperitoneal (I.P.) administration between 4 and 8 weeks of age. Administration of Z-103 from the age of 4 to 9 weeks had no significant effect on wet weight, magnitude or rate of rise of twitch force, or rate of decay of twitch force over time with twitch elicited by 0.5 Hz of electricity in the extensor digitorum longus muscle or calcium content in the gastrocnemius muscle, while it increased the magnitude of twitch force in the soleus muscle. These results suggest that Z-103 reduces fatigability of the whole body in mdx mice, possibly by increasing the contractility of slow fibers.     

Expression of the dystrophin isoform Dp116 preserves functional muscle mass and extends lifespan without preventing dystrophy in severely dystrophic mice

Dp116 is a non-muscle isoform of dystrophin that assembles the dystrophin-glycoprotein complex (DGC), but lacks actin-binding domains. To examine the functional role of the DGC, we expressed the Dp116 transgene in mice lacking both dystrophin and utrophin (mdx:utrn(-/-)). Unexpectedly, expression of Dp116 prevented the most severe aspects of the mdx:utrn(-/-) phenotype. Dp116:mdx:utrn(-/-) transgenic mice had dramatic improvements in growth, mobility and lifespan compared with controls. This was associated with increased muscle mass and force generating capacity of limb muscles, although myofiber size and specific force were unchanged. Conversely, Dp116 had no effect on dystrophic injury as determined by muscle histopathology and serum creatine kinase levels. Dp116 also failed to restore normal fiber-type distribution or the post-synaptic architecture of the neuromuscular junction. These data demonstrate that the DGC is critical for growth and maintenance of muscle mass, a function that is independent of the ability to prevent dystrophic pathophysiology. Likewise, this is the first demonstration in skeletal muscle of a positive functional role for a dystrophin protein that lacks actin-binding domains. We conclude that both mechanical and non-mechanical functions of dystrophin are important for its role in skeletal muscle.     

Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle

A therapeutic approach for Duchenne muscular dystrophy (DMD) is to up-regulate utrophin in skeletal muscle in an effort to compensate for the lack of dystrophin. We previously hypothesized that promotion of the slow, oxidative myogenic program, which triggers utrophin up-regulation, can attenuate the dystrophic pathology in mdx animals. Since treatment of healthy mice with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) enhances oxidative capacity and elicits a fast-to-slow fiber-type transition, we evaluated the effects of chronic AMPK stimulation on skeletal muscle phenotype and utrophin expression in mdx mice. Daily AICAR administration (500 mg/kg/day, 30 days) of 5-7-week-old mdx animals induced an elevation in mitochondrial cytochrome c oxidase enzyme activity, an increase in myosin heavy-chain type IIa-positive fibers and slower twitch contraction kinetics in the fast, glycolytic extensor digitorum longus muscle. Utrophin expression was significantly enhanced in response to AICAR, which occurred coincident with an elevated β-dystroglycan expression along the sarcolemma. These adaptations were associated with an increase in sarcolemmal structural integrity under basal conditions, as well as during damaging eccentric contractions ex vivo. Notably, peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) and silent information regulator two ortholog 1 protein contents were significantly higher in muscle from mdx mice compared with wild-type littermates and AICAR further increased PGC-1α expression. Our data show that AICAR-evoked muscle plasticity results in beneficial phenotypic adaptations in mdx mice and suggest that the contextually novel application of this compound for muscular dystrophy warrants further study.     

Muscle-bone interactions in dystrophin-deficient and myostatin-deficient mice

We have investigated muscle-bone interactions using two mouse mutants that are known to differ from normal mice in skeletal muscle growth and development: mice lacking myostatin (GDF8) and mice lacking dystrophin (mdx). Myostatin-deficient mice show increased muscle size and strength compared to normal mice, whereas the mdx mouse is a well-established animal model for Duchenne muscular dystrophy. The mdx mice have significantly larger hindlimb muscles than controls, and histological sections of the quadriceps muscles show dystrophic changes with extensive fibrosis. Femoral bone mineral density (BMD) and fracture strength (Fu) are significantly greater in mdx mice than controls, and these variables are more strongly correlated with quadriceps muscle mass than with body mass. In contrast, mdx mice do not shower high bone mineral density in the spine relative to controls, whereas myostatin-deficient mice have significantly increased BMD in the lumbar spine compared to normal mice. Both mdx mice and myostatin-deficient mice have expanded femoral trochanters for attachment of large hindlimb muscles, and both mutant strains show increased cross-sectional area moments of inertia mediolaterally (Iyy) but not anteroposteriorly (Ixx) compared to normal mice. These data suggest that lean (muscle) mass is a significant determinant of bone mineral density and strength in the limb skeleton, even when accompanied by a dystrophic phenotype. Likewise, increased muscle mass produces a marked increase in the external dimensions of muscle attachment sites, even when increased muscle size is accompanied by extensive fibrosis and muscle weakness.     

Improved contractile function of the mdx dystrophic mouse diaphragm muscle after insulin-like growth factor-I administration

Limited knowledge exists regarding the efficacy of insulin-like growth factor I (IGF-I) administration as a therapeutic intervention for muscular dystrophies, although findings from other muscle pathology models suggest clinical potential. The diaphragm muscles of mdx mice (a model for Duchenne muscular dystrophy) were examined after 8 weeks of IGF-I administration (1 mg/kg s.c.) to test the hypothesis that IGF-I would improve the functional properties of dystrophic skeletal muscles. Force per cross-sectional area was approximately 49% greater in the muscles of treated mdx mice (149.6 +/- 9.6 kN/m(2)) compared with untreated mice (100.1 +/- 4.6 kN/m(2), P < 0.05), and maintenance of force over repeated maximal contraction was enhanced approximately 30% in muscles of treated mice (P < 0.05). Diaphragm muscles from treated mice comprised fibers with approximately 36% elevated activity of the oxidative enzyme succinate dehydrogenase, and approximately 23% reduction in the proportion of fast IId/x muscle fibers with concomitant increase in the proportion of type IIa fibers compared with untreated mice (P < 0.05). The data demonstrate that IGF-I administration can enhance the fatigue resistance of respiratory muscles in an animal model of dystrophin deficiency, in conjunction with enhancing energenic enzyme activity. As respiratory function is a mortality predictor in Duchenne muscular dystrophy patients, further evaluation of IGF-I intervention is recommended.     

Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy

Skeletal muscle myofibers constantly undergo degeneration and regeneration. Histopathological features of 6 skeletal muscles (cranial tibial [CT], gastrocnemius, quadriceps femoris, triceps brachii [TB], lumbar longissimus muscles, and costal part of the diaphragm [CPD]) were compared using C57BL/10ScSn-Dmd ^mdx (mdx) mice, a model for muscular dystrophy versus control, C57BL/10 mice. Body weight and skeletal muscle mass were lower in mdx mice than the control at 4 weeks of age; these results were similar at 6–30 weeks. Additionally, muscular lesions were observed in all examined skeletal muscles in mdx mice after 4 weeks, but none were noted in the controls. Immunohistochemical staining revealed numerous paired box 7-positive satellite cells surrounding the embryonic myosin heavy chain-positive regenerating myofibers, while the number of the former and staining intensity of the latter decreased as myofiber regeneration progressed. Persistent muscular lesions were observed in skeletal muscles of mdx mice between 4 and 14 weeks of age, and normal myofibers decreased with age. Number of muscular lesions was lowest in CPD at all ages examined, while the ratio of normal myofibers was lowest in TB at 6 weeks. In CT, TB, and CPD, Iba1-positive macrophages, the main inflammatory cells in skeletal muscle lesions, showed a significant positive correlation with the appearance of regenerating myofibers. Additionally, B220-positive B-cells showed positive and negative correlation with regenerating and regenerated myofibers, respectively. Our data suggest that degenerative and regenerative features of myofibers differ among skeletal muscles and that inflammatory cells are strongly associated with regenerative features of myofibers in mdx mice.     

Clinical effectiveness of usual care with or without antidepressant medication for primary care patients with minor or mild-major depression: a randomized equivalence trial

Background

Duchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy.

Methods

Using the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression.

Results

We have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression.

Conclusion

Together, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation.     

Cloning of cDNA encoding a regeneration-associated muscle protease whose expression is attenuated in cell lines derived from Duchenne muscular dystrophy patients

In the dystrophin-mutant mdx mouse, an animal model for Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and the mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation by using cDNA microarrays with newly established skeletal muscle cell lines from mdx and normal mice. We found that in the mdx muscle cell line, 12 genes, including L-arginine:glycine amidinotransferase and thymosin beta4, are up-regulated, whereas 7 genes, including selenoprotein P and a novel regeneration-associated muscle protease (RAMP), are down-regulated. Northern blot analysis and in situ hybridization revealed that RAMP mRNA is predominantly expressed in normal skeletal muscle and brain, and its production is enhanced in the regenerating area of injured skeletal muscle in mice. RAMP expression was much lower in individual muscle cell lines derived from biopsies of six DMD patients compared to a normal muscle cell line. These results suggest that RAMP may play a role in the regeneration of skeletal muscle and that its down-regulation could be involved in the progression of DMD in humans.     

Matrix metalloproteinase-2 ablation in dystrophin-deficient mdx muscles reduces angiogenesis resulting in impaired growth of regenerated muscle fibers

Matrix metalloproteases (MMPs) are a family of endopeptidases classified into subgroups based on substrate preference in normal physiological processes such as embryonic development and tissue remodeling, as well as in various disease processes via degradation of extracellular matrix components. Among the MMPs, MMP-9 and MMP-2 have been reported to be up-regulated in skeletal muscles in the lethal X-linked muscle disorder Duchenne muscular dystrophy (DMD), which is caused by loss of dystrophin. A recent study showed that deletion of the MMP9 gene in mdx, a mouse model for DMD, improved skeletal muscle pathology and function; however, the role of MMP-2 in the dystrophin-deficient muscle is not well known. In this study, we aimed at verifying the role of MMP-2 in the dystrophin-deficient muscle by using mdx mice with genetic ablation of MMP-2 (mdx/MMP-2(-/-)). We found impairment of regenerated muscle fiber growth with reduction of angiogenesis in mdx/MMP-2(-/-) mice at 3 months of age. Expression of vascular endothelial growth factor-A (VEGF-A), an important angiogenesis-related factor, decreased in mdx/MMP-2(-/-) mice at 3 months of age. MMP-2 had not a critical role in the degradation of dystrophin-glycoprotein complex (DGC) components such as β-dystroglycan and β-sarcoglycan in the regeneration process of the dystrophic muscle. Accordingly, MMP-2 may be essential for growth of regenerated muscle fibers through VEGF-associated angiogenesis in the dystrophin-deficient skeletal muscle.     

Enzyme replacement therapy in the mouse model of Pompe disease

Deficiency of acid alpha-glucosidase (GAA) results in widespread cellular deposition of lysosomal glycogen manifesting as myopathy and cardiomyopathy. When GAA-/- mice were treated with rhGAA (20 mg/kg/week for up to 5 months), skeletal muscle cells took up little enzyme compared to liver and heart. Glycogen reduction was less than 50%, and some fibers showed little or no glycogen clearance. A dose of 100 mg/kg/week resulted in approximately 75% glycogen clearance in skeletal muscle. The enzyme reduced cardiac glycogen to undetectable levels at either dose. Skeletal muscle fibers with residual glycogen showed immunoreactivity for LAMP-1/LAMP-2, indicating that undigested glycogen remained in proliferating lysosomes. Glycogen clearance was more pronounced in type 1 fibers, and histochemical analysis suggested an increased mannose-6-phosphate receptor immunoreactivity in these fibers. Differential transport of enzyme into lysosomes may explain the strikingly uneven pattern of glycogen removal. Autophagic vacuoles, a feature of both the mouse model and the human disease, persisted despite glycogen clearance. In some groups a modest glycogen reduction was accompanied by improved muscle strength. These studies suggest that enzyme replacement therapy, although at much higher doses than in other lysosomal diseases, has the potential to reverse cardiac pathology and to reduce the glycogen level in skeletal muscle.     

骨髓干细胞移植治疗不同年龄mdx鼠的疗效研究

目的：研究不同年龄的Duchenne型肌营养不良鼠（mdx鼠）与骨髓干细胞移植后缺失蛋白表达的关系。 方法： 获取4-5周C57BL/6小鼠的骨髓干细胞，体外培养3 d，静脉移植到7Gy γ射线预处理的6周龄、8周龄两组各6只mdx鼠。移植12周后，对移植鼠骨骼肌dystrophin蛋白表达情况进行检测。 结果： 6周龄、8周龄两组mdx鼠，静脉移植1.2×107骨髓干细胞，3个月后，分别有16%和7%的骨骼肌纤维表达了dystrophin蛋白。 结论： 静脉移植同种、同系鼠骨髓干细胞的mdx鼠，3个月之后, 不同年龄mdx鼠骨骼肌细胞dystrophin蛋白表达的阳性率不同，幼年鼠骨髓干细胞移植有较高比率的缺失蛋白表达。     

Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice

Duchenne muscular dystrophy (DMD) is caused by the absence of a functional dystrophin protein and is modeled by the mdx mouse. The mdx mouse suffers an early necrotic bout in the hind limb muscles lasting from approximately 4 to 7 weeks. The purpose of this investigation was to determine the extent to which dystrophin deficiency changed the proteome very early in the disease process. In order to accomplish this, proteins from gastrocnemius from 6-week-old C57 (n = 6) and mdx (n = 6) mice were labeled with fluorescent dye and subjected to two-dimensional differential in-gel electrophoresis (2D-DIGE). Resulting differentially expressed spots were excised and protein identity determined via MALDI-TOF followed by database searching using MASCOT. Proteins of the immediate energy system and glycolysis were generally down-regulated in mdx mice compared to C57 mice. Conversely, expression of proteins involved in the Kreb’s cycle and electron transport chain were increased in dystrophin-deficient muscle compared to control. Expression of cytoskeletal components, including tubulins, vimentin, and collagen, were increased in mdx mice compared to C57 mice. Importantly, these changes are occurring at only 6 weeks of age and are caused by acute dystrophin deficiency rather than more chronic injury. These data may provide insight regarding early pathologic changes occurring in dystrophin-deficient skeletal muscle.     

Preservation of muscle force in Mdx3cv mice correlates with low-level expression of a near full-length dystrophin protein

The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mouse muscle but was expressed in mdx3cv mice as a near full-length protein at approximately 5% of normal levels. Consistent with previous reports, we found dystrophic muscle pathology in both mouse strains. Surprisingly, mdx3cv extensor digitorium longus muscle showed significantly higher tetanic force and was also more resistant to eccentric contraction-induced injury than mdx4cv extensor digitorium longus muscle. Furthermore, mdx3cv mice had stronger forelimb grip strength than mdx4cv mice. Immunostaining revealed utrophin up-regulation in both mouse strains. The dystrophin-associated glycoprotein complex was also restored in the sarcolemma in both strains although at levels lower than those in normal mice. Our results suggest that subtherapeutic expression levels of near full-length, membrane-bound dystrophin, possibly in conjunction with up-regulated utrophin levels, may help maintain minimal muscle force but not arrest muscle degeneration or necrosis. Our findings provide valuable insight toward understanding delayed clinical onset and/or slow disease progression in certain Becker muscular dystrophy patients.     

Expression of Dp260 in muscle tethers the actin cytoskeleton to the dystrophin-glycoprotein complex and partially prevents dystrophy

Dystrophin forms a mechanical link between the actin cytoskeleton and the extracellular matrix in muscle that helps maintain sarcolemmal integrity. Two regions of dystrophin have been shown to bind actin: the N-terminal domain and rod domain repeats 11-17. To better understand the roles of these two domains and whether the rod domain actin-binding domain alone can support a mechanically functional link with actin, we constructed transgenic mice expressing Dp260 in skeletal muscle. Dp260, the retinal isoform of dystrophin, lacks the N-terminal domain and a significant portion of the rod domain, but retains the rod domain actin-binding domain. Our results indicate that Dp260 expression restores a stable association between costameric actin and the sarcolemma, assembles the dystrophin-glycoprotein complex, and significantly slows the progression of the dystrophy in the dystrophin-deficient mdx mouse. We assessed the functional integrity of the mechanical link in Dp260 transgenic mdx mice and found that Dp260 muscles showed normal resistance to contraction-induced injury, but dramatic reductions in force generation similar to those found with mdx muscles. Morphologically, Dp260 muscles displayed reduced amounts of inflammation and fibrosis, but still showed a significant, albeit reduced, amount of degeneration/regeneration. These data demonstrate that protection from contraction-induced injury can dramatically ameliorate, but not completely halt, the dystrophic process. We suggest that a non-mechanical defect, attributed to the loss of the N terminus of dystrophin, is likely responsible for the residual dystrophy observed.     

A sandwich ELISA kit reveals marked elevation of titin N-terminal fragment levels in the urine of mdx mice

The mdx mouse is a model of Duchenne muscular dystrophy (DMD), a fatal progres-sive muscle wasting disease caused by dystrophin deficiency, and is used most widely in preclinical studies. Mice with dystrophin deficiency, however, show milder muscle strength phenotypes than humans. In human, the introduction of a sandwich enzyme- linked immunosorbent assay (ELISA) kit revealed a more than 700- fold increase in titin N- terminal fragment levels in the urine of pediatric patients with DMD. Notably, the urinary titin level declines with aging, reflecting progression of muscle wast-ing. In mouse, development of a highly sensitive ELISA kit has been awaited. Here, a sandwich ELISA kit to measure titin N- terminal fragment levels in mouse urine was developed. The developed kit showed good linearity, recovery, and repeatability in measuring recombinant or natural mouse titin N- terminal fragment levels. The titin N- terminal fragment concentration in the urine of mdx mice was more than 500- fold higher than that of normal mice. Urinary titin was further analyzed by extending the collection of urine samples to both young (3– 11 weeks old) and aged (56– 58 weeks old) mdx mice. The concentration in the young group was significantly higher than that in the aged group. It was concluded that muscle protein breakdown is active and persistent in mdx mice even though the muscle phenotype is mild. Our results pro-vide an opportunity to develop DMD treatments that aim to alleviate muscle protein breakdown by monitoring urinary titin levels.     

Cilostazol attenuates oxidative stress and apoptosis in the quadriceps muscle of the dystrophic mouse experimental model

Duchenne muscular dystrophy (DMD) is the most severe and frequent form of muscular dystrophy. The mdx mouse is one of the most widely used experimental models to understand aspects of the biology of dystrophic skeletal muscles and the mechanisms of DMD. Oxidative stress and apoptosis are present in early stages of the disease in mdx mice. The high production of reactive oxygen species (ROS) causes activation of apoptotic death regulatory proteins due to DNA damage and breakdown of nuclear and mitochondrial membranes. The quadriceps (QUA) muscle of the mdx mouse is a good tool to study oxidative events. Previous studies have demonstrated that cilostazol exerts an anti-oxidant effect by decreasing the production of reactive oxygen species (ROS). The present study aimed to evaluate the ability of cilostazol to modulate oxidative stress and apoptosis in the QUA muscle of mdx mice. Fourteen-day-old mdx mice received cilostazol or saline for 14 days. C57BL/10 mice were used as a control. In the QUA muscle of mdx mice, cilostazol treatment decreased ROS production (−74%), the number of lipofuscin granules (−47%), lipid peroxidation (−11%), and the number of apoptotic cells (−66%). Thus cilostazol showed anti-oxidant and anti-apoptotic action in the QUA muscle of mdx mice.     

Profile of the selective dopamine D-2 receptor agonist N-0437: its effects on palatability- and deprivation-induced feeding, and operant responding for food

N-0437 is a potent and highly selective dopamine D-2 receptor agonist, which has been used in the present series of experiments to investigate its potential anorectic properties. In doses of 0.3-3.0 mg/kg (IP), N-0437 significantly reduced consumption of a sweetened palatable mash in nondeprived mice (minimal effective dose, 0.3 mg/kg) and rats (minimal effective dose, 0.56 mg/kg). Reduction in food intake were also produced in rats by the less potent, but selective, D-2 agonist RU 24213 (effective at 10.0 mg/kg), and by d-amphetamine (1.0 and 3.0 mg/kg). The anorectic effect of N-0437 (1.0 mg/kg) was completely antagonized by the selective D-2 antagonist, YM-09151-2 (0.01 mg/kg). Over a series of 10 injections, N-0437 (1.0 mg/kg) maintained its effect to reduce palatable food intake. In food-deprived rats, N-0437 (0.3-3.0 mg/kg, IP) also reduced consumption of standard laboratory food, and dose-dependently reduced operant responding for food under a FR8 schedule of reinforcement. The results of the experiments are discussed in terms of a possible direct effect to reduce feeding responses resulting from stimulation of postsynaptic dopamine D-2 receptors.     

Functional regeneration in the hindlimb skeletal muscle of the mdx mouse

Summary The pattern of spontaneous skeletal muscle degeneration and clinical recovery in hindlimb muscles of the mdx mutant mouse was examined for functional and metabolic confirmation of apparent structural regeneration. The contractile properties, histochemical staining and myosin light chain and parvalbumin contents of extensor digitorum longus (EDL) and soleus (Sol) muscles of mdx and age-matched control mice were studied at 3–4 and 32 weeks. Histochemical staining (myofibrillar ATPase and NADH-tetrazolium reductase) revealed no significant change in slow-twitch-oxidative (SO) or fast-twitch-oxidative-glycolytic (FOG) fibre type proportions in mdx Sol apart from the normal age-related increase in SO fibres. At 32 weeks mdx EDL, however, showed significantly smaller fast-twitch-glycolytic (FG) and larger FOG proportions than those in control EDL. These fibre type distributions were confirmed by differential staining with antibodies to myosin slow-twitch and fast-twitch heavy chain isozymes. Frequency distribution of cross-sectional area for each fibre type showed a wider than normal range of areas especially in FOG fibres of mdx Sol, and FG fibres of mdx EDL, supporting previous observations using autoradiography of myofibre regeneration. Isometric twitch and tetanic tensions in Sol were significantly less than in controls at 4 weeks, but by 32 weeks, values were not different from age-matched controls. In mdx EDL at 3 weeks, twitch and tetanus tensions were significantly less, and time-to-peak twitch tensions were significantly faster than in control EDL. By 32 weeks, mdx EDL twitch and tetanus tensions expressed relative to muscle weight continued to be significantly lower than in age-matched controls, despite normal absolute tensions. The maximum velocity of shortening in 32-week mdx EDL was significantly lower than in control EDL. Myosin light chain distribution in mdx Sol exhibited significantly less light chain 2-slow (LC2s) and more light chain 1b-slow(LC1bs) at 32 weeks than age-matched control Sol. Gels of EDL from 32-week-old mdx mice showed significantly less light chain 2-fast-phosphorylated (LC2f-P) and light chain 3-fast (LC3f) and significantly more light chain 1-fast (LC1f) and light chain 2-fast (LC2f), but normal parvalbumin content compared to age-matched controls. These observations suggest that mdx hindlimb muscles are differentially affected by the disease process as it occurs in murine models of dystrophy. However, the uniqueness of mdx Sol and to a lesser extent EDL is that they also undergo an important degree of functional regeneration which is able to compensate spontaneously for degenerative influences of genetic origin. The mdx mutant may therefore be an important model for the study of regeneration by skeletal muscle, and of the nerve-muscle interactions which enable or restrict that regeneration.