Phenytoin,methysergide, and penicillamine in hereditary muscular dystrophy of the chicken

Chickens with an inherited muscular dystrophy (line 413) were treated from days 1 through 90 ex ovo with either phenytoin, methysergide, or d-penicillamine. Under the conditions of the study, phenytoin and methysergide were equally effective in improving the righting ability of dystrophic birds, but penicillamine had little or no effect. Phenytoin and penicillamine decreased the abnormally high creatine kinase activity in dystrophic plasma, but only phenytoin reduced the high acetylcholinesterase activity in plasma and fast-twitch muscles of dystrophic birds. Beneficial effects of methysergide and phenytoin were also shown in trials as short as 25 days. None of the treatments, improved the abnormal histological appearance of dystrophic muscles.     

Water deprivation: Beneficial effect on muscular dystrophy in chickens

Chicks affected with hereditary muscular dystrophy were deprived of water for 1 to 4 days at ages to 37 days ex ovo. Water deprivation partially alleviated impaired righting ability and reduced the typically elevated plasma creatine kinase activity by as much as 90%. Muscles from water-deprived chicks showed several qualitative histologic improvements, including decreased sarcoplasmic staining for acetylcholinesterase activity, reduced fiber diameters, and a decreased incidence of abnormally large rounded fibers, but retained the high degree of fiber diameter variability characteristic of dystrophic muscles. Feed deprivation reduced body weight to a similar extent as water deprivation but had lesser effects on creatine kinase activity and did not improve righting ability or muscle histology. Although the mechanism of the improvements is unknown, the magnitude and scope of the effects suggest that water deprivation beneficially alters a major abnormality in dystrophic chickens.     

Possible manifestation of the dystrophic X chromosome in muscle cultures from carriers of Duchenne muscular dystrophy

Multilayer cell clusters have been observed before confluence and before myotube formation in muscle cell cultures derived from open biopsies of 7 of 14 (50%) female carriers of Duchenne muscular dystrophy, and in a high percentage of other dystrophic cultures. By contrast, this abnormality was seen in only 12 of 204 (6%) muscle biopsies from patients with other neuromuscular disorders. It appears that cluster formation is independent of the amount of connective tissue present in vivo, because histopathological analysis of the carrier biopsies showed increased endomysial connective tissue in only two cases. These results suggest that cluster formation is an expression of a myogenic defect and that it may be a manifestation of the genetic abnormality in X-linked muscular dystrophy.     

Effect of exercise on chickens with hereditary muscular dystrophy

To assess the effect of daily physical exercise alone on the onset and development of muscle weakness found in chickens with hereditary muscular dystrophy, two separate exercise trials composed of 20 or 30 successive opportunities to right when placed in the supine position were conducted. The effects of exercise on the physically ability/disability were measured by a flip-test procedure administered to each exercised and unexercised animal every 5 days and at a time separate from the exercise procedure. The flip test consisted of giving each animal five successive opportunities to right after having been placed on its back. Blood samples were drawn weekly and assayed for plasma creatine phosphokinase (CPK) activities. Unexercised dystrophic chickens were found to reach a peak of physical ability as quantitated by the flip test at approximately day 23 ex ovo. Subsequently, a progressive decrease in flipping ability was observed. The dystrophic chickens which were exercised daily exhibited a temporary, yet significant improvement in righting ability to Day 48 ex ovo. Also, exercise elicited small but consistent increases in plasma CPK activities compared to the unexercised control values. Regular physical exercise alone was found to alter some of the measurable symptoms of avian muscular dystrophy. To evaluate the singular effect of drug treatment in dystrophic animals, we recommend that the periodic testing for physical improvement be at a time separate from drug administration and devoid of the influence of exercise.     

自噬在Duchenne型肌营养不良中的研究

目的检测Duchenne型肌营养不良症(DMD)患者骨骼肌中LC3和p62的表达情况,分析自噬在DMD骨骼肌细胞坏死中的作用。方法收集2008年1月~2015年5月在我院就诊的病理确诊为DMD的患者(DMD组,81例),另以怀疑为肌病,但肌肉病理未见明显病变者为对照组(6例)。所有入选者均行心肌酶学、肌电图、骨骼肌活检常规组织学和酶学染色、抗dystrophin-N,-C,-R和抗dysferlin免疫组织化学染色。检测其中6例DMD患者及对照组骨骼肌中LC3和p62的表达。结果 81例DMD患者均为男性,起病年龄(4.60±2.35)岁,首发症状多以双下肢起病为主。血清肌酸激酶值的高峰出现在患者年龄的6~8岁,随着肌细胞明显坏死,肌酸激酶水平下降,但仍高于正常。在DMD患者骨骼肌中,组织病理均示典型肌营养不良改变。半定量Western blot提示DMD患者骨骼肌中LC3-II的表达降低,而p62表达显著升高。结论自噬功能障碍可能参与了DMD骨骼肌细胞坏死的病理生理过程。     

In vivo effects of three calcium blockers on chickens with inherited muscular dystrophy

Genetically homozygous Line 413 dystrophic chickens were given in separate trials daily i.p. injections of aqueous solutions of the calcium blocker drugs, diltiazem, verapamil, or nifedipine. At a dosage of 20 mg/kg/day, drug therapy in each case significantly prolonged the functional ability of the dystrophic chickens as quantitated regularly by a standardized test for righting ability. Enhanced functional ability, however, was not generally accompanied by a decrease in the usually high plasma creatine kinase activity. In addition, there was no change in the pectoralis muscle mass or protein with any of the drug treatments. Moreover, no significant reduction in the abnormally high total muscle calcium was found with calcium blocker treatment. Also, there was no marked change in the histopathology of muscle from the drugtreated dystrophic chickens. We concluded that drugs with calcium entry blocker activity offer only limited benefit in retarding dystrophic symptoms expressed in the chicken (viz., short-term enhancement in righting ability).     

Significance of serum myoglobin in neuromuscular diseases and in carrier detection of Duchenne muscular dystrophy

In a retrospective study, the serum myoglobin concentration (S-myoglobin) was determined in patients with neuromuscular diseases and in carriers of Duchenne muscular dystrophy (DMD). Myoglobin was quantified by a sensitive radioimmunoassay. Serum creatine kinase (CK, EC 2.7.3.2) activity (S-CK) and serum creatine kinase B-subunit activity (S-CKB) were determined for comparison. Sera from 70 patients with various neuromuscular diseases and from 17 female relatives of patients with DMD were analysed. Increased levels of S-myoglobin were found both in dystrophic and in spinal myopathies. Because of a marked overlap of the range of values between the different dystrophic myopathies and even between the dystrophic and the spinal myopathies, S-myoglobin is of little value in the final diagnosis of neuromuscular diseases. In the detection of carriers of DMD, simultaneous determination of S-myoglobin and S-CK gave a higher detection rate compared to the detection rate with S-CK. S-CKB was normal in all carriers and only elevated in some of the patients with DMD and limb girdle muscular dystorphy.     

Increased activities of MB and BB isozymes of creatine kinase in denervated neonatal and adult rat muscle

Creatine kinase (CK) activity and isozyme patterns were assessed in newborn and adult rat anterior tibial muscle in response to denervation. Total CK activity was low in the control neonatal muscle, gradually increasing to the adult level within 1 month. Denervation prevented this normal increase, and, therefore, CK activity was reduced to 25% of control at 2 months. In the denervated adult muscle, total CK activity decreased to 50% of control within 3 weeks and remained at that level. Denervation of neonatal muscle resulted in a greater conservation of MB isozyme compared with controls. The alteration in BB isozyme expression was even more dramatic with a 33-fold difference expressed at 2 months in terms of percent total CK in denervated vs. control muscle. In denervated adult muscle, MB and BB isozyme activities increased gradually, attaining levels 3-fold and 13-fold, respectively, above control muscle at the end of the experimental period.     

Serum pyruvate kinase in different neuromuscular diseases and in carriers of muscular dystrophy

Serum pyruvate kinase and creatine kinase activities were measured in a group of patients with various neuromuscular diseases and in carriers of muscular dystrophy. Elevated values of PK were usually but not invariably associated with elevated levels of CK. The data showed that PK activity was elevated in all patients with DMD, high values generally correlating inversely with age or disease duration. In definite carriers, the level of PK was raised simultaneously with CK, while in potential carriers, classified by their relationship with MD patients in mothers, sisters and other relatives, the PK levels were elevated in 23%, 44% and 10% respectively, indicating, especially for sisters, an increased genetic probability of being a definite carrier. In this way, we have confirmed that the serum PK assay is more sensitive in younger subjects and that combined CK and PK measurement will be of value in detecting a higher proportion of potential carriers.

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Sommario Abbiamo misurato l'attività sierica della piruvato kinasi e della creatinfosfato kinasi in un gruppo di pazienti affetti da diverse malattie neuromuscolari e nelle portatrici di distrofia muscolare. A valori elevati di piruvato kinasi comunemente ma non invariabilmente corrispondevano valori elevati di creatinfosfato kinasi. I dati raccolti hanno dimostrato che i valori sierici di piruvato kinasi erano elevati in tutti i soggetti affetti da DMD, generalmente inversamente correlabili con l'età e la durata della malattia. Nelle portatrici certe la piruvato kinasi era aumentata parallelamente alla creatinfosfato kinasi, mentre tra le portatrici possibili, suddivise secondo la relazione di parentela con i soggetti affetti da DM, in madri, sorelle e parenti, si sono riscontrati valori patologici della piruvato kinasi rispettivamente nel 23%; 44% e 10%: da questi dati, soprattutto per le sorelle, possiamo presumere un incremento della probabilità genetica di essere portatrici certe. Abbiamo così confermato che il dosaggio della piruvato kinasi sierica è un test più sensibile nei soggetti giovani e che questo, combinato con il dosaggio della creatinfosfato kinasi, è significativo per l'individuazione di un maggior numero di portatrici possibili.

     

Effect of dantrolene in Duchenne muscular dystrophy.

We have demonstrated that maneuvers capable of reducing Ca influx into cells have beneficial effects in dystrophic hamsters and Duchenne muscular dystrophy. Since dantrolene inhibits Ca release from the sarcoplasmic reticulum, its effects on DMD was studied in 7 patients of 6 to 13 years of age (mean 10.8 years). Patients were studied for 4 years with tri-monthly evaluations of manual muscle testing (MMT), functional activity, and serum CK and aldolase. During the first 2-year period, no medicines were given and served as control. In the second 2-year period, dantrolene 8 mg/kg/d was administered. No side effects were observed. In 1 patient, mild weakness occurred that disappeared when the dose was reduced to 6 mg/kg/d. The 95% confidence limit for the difference in slopes of regression lines from tri-monthly MMT was asymmetric in favor of dantrolene in 5 of 7 patients. Serum CK did not differ between the first and second year of the control and treatment periods, respectively. However, it fell significantly from the second year of control to the first year of treatment (P = 0.003). The fall during the first year of treatment was significantly greater (P less than 0.01) than in age-matched natural history controls during the same length of observation. There was a 3-fold reduction in CK when the pooled values of the first and second year control vs. treatment periods were analyzed. No changes were observed in functional activity and serum aldolase. The data suggest that dantrolene reduces serum CK in DMD associated with a lessening trend in MMT deterioration.     

Axonal transport of the molecular forms of acetylcholinesterase in developing and regenerating peripheral nerve

In chick sciatic nerve, acetylcholinesterase (AChE) occurs in four main molecular forms characterized by their sedimentation coefficients in sucrose gradients, referred to as G1 (5S), G2 (7.5S), G4 (11S), and A12 (20S). Under normal conditions, we previously showed by accumulation technique that the G4 and A12 forms are rapidly transported along the axons, whereas G1 and G2 are carried much more slowly. Here, we used to the same technique to study the anterograde axonal transport of these different AChE forms during normal axonal growth and experimental regeneration. During the first 2 months after hatching, G4 and A12 transport virtually doubled, whereas G1 + G2 transport increased only slightly. After nerve cutting, crushing, or freezing, the flow rates of G1 + G2 and G4 in the regenerating proximal stump decreased by 75% at 4 to 7 days compared with control values and that of A12, by 90 to 95%. In crushed and frozen nerves the transport of all four AChE forms slowly recovered thereafter, but failed to attain control values even after 7 weeks. In cut nerves, on the contrary, no significant recovery of G1 + G2, or G4 transport occurred, but A12 transport began to recover by day 7. Taken together, our results show that axonal transport of G1 + G2, G4, and A12 is selectively regulated in chick sciatic nerve, and suggest that the A12 form of AChE might have a special role and/or destination in regenerating axons.     

Drug evaluation in muscular dystrophy of the chicken

A high-capacity, step-wise system for drug evaluation in chickens with inherited muscular dystrophy has been developed. This report details the protocol and presents the first results from a rapid evaluation of the ameliorative effects of drugs on the impaired righting ability of dystrophic chicks. Of 31 compounds evaluated, only methysergide and corticosterone significantly increased righting ability.     

假肥大型肌营养不良患者血清酶学与肌电图改变的相关性研究

目的研究假肥大型肌营养不良（DMD）患者血清酶学、肌电图改变,进一步分析之间的相关性。方法选择DMD患者45例,平均年龄（7．14±2．92）岁,分别测定血清磷酸肌酸激酶（cK）、乳酸脱氢酶（LDH）、肌酸激酶同工酶（CK—Mb）、肌红蛋白（Mb）等。并进行肌电图检测。结果所有DMD患者血清酶学均增高,平均CK（13109．38±3023．99）U／L,LDH（1258．99±331．5）U／L,CK—Mb（494．22士121．75）U／L,Mb（1417．07±461．47）U／L。共检测肌肉169块,发现肌源性损害肌肉73块,其中1DO％股四头肌存在肌源性损害。33．3％（15／45）存在肱二头肌肌源性损害,26．7％（12／45）三角肌存在肌源性损害。股四头肌时限下降百分比平均为（42．31±8．74）％。CK与Mb、CK—Mb、LDH均呈正相关,差异有统计学意义（P〈0．01）。Mb与CK—Mb、LDH均呈正相关,差异有统计学意义（P〈0．01）、CK—Mb与LDH呈正相关,差异有统计学意义（P〈0．01）。股四头肌时限下降百分比与CK、Mb、CK—Mb呈正相关,差异有统计学意义（P〈0．05）。CK、CK—Mb、股四头肌时限下降百分比等与病程明显相关,差异有统计学意义（P〈0．05）。结论血清CK、Mb是早期诊断DMD的特异性指标。联合肌电图检查,可提高诊断的敏感性和特异性和评估病程的进展速度。     

Plasma pyruvate kinase and creatine kinase activity in Becker muscular dystrophy

Plasma creatine kinase (CK) and pyruvate kinase (PK) were measured in 31 obligate carriers of Becker muscular dystrophy (BMD), 36 BMD patients and appropriate controls. Mean plasma CK was 108 U/l in obligate carriers and 62 U/l in 43 age- and sex-matched controls (P less than 0.001 carriers vs controls). Control CK reference range was 31-125 U/l (mean +/- 2 SD of log transformed values). Mean plasma PK was 40 U/l in obligate carriers and 34 U/l in 56 controls (P less than 0.02 carriers vs controls). Control PK reference range was 18-61 U/l. Values of CK above the reference range upper limit were found in 13 of 31 BMD obligate carriers but only 2 showed elevated PK values. The sensitivity of CK in determining BMD carrier status, although only 42%, was markedly better than PK at 6.5%. Mean plasma CK in BMD patients was 2366 U/l, a 19-fold increase over the control value of 127 U/l (P less than 0.001 patients vs controls). Control CK reference range was 40-316 U/l. In contrast, mean plasma PK in BMD patients was 353 U/l, only 7-fold higher than the mean control value of 57 U/l (P less than 0.001 patients vs controls). Control PK reference range was 22-126 U/l. Clearly, the estimation of plasma PK as a means of determining BMD carrier status is markedly inferior to CK. Previous reports of increased sensitivity of PK compared with CK may have been due to artefactually elevated PK levels produced during sample preparation.     

Drugs in muscular dystrophy of the chicken: corticosterone-21-acetate

In a previous series of 22-day evaluations of 31 compounds, only corticosterone-21-acetate (C-21-A) increased righting ability of genetically dystrophic chickens to a greater extent than the standard of comparison, methysergide maleate. In the present study, C-21-A was subjected to longer-term trials of up to 48 days, and additional signs of the myopathy were examined. The highest doses of C-21-A increased righting ability for the duration of the trials, decreased the typically elevated plasma levels of creatine kinase (CK) activity by more than 80%, and improved morphology of the dystrophic pectoralis major muscle at the light microscopic level. The major adverse effect of C-21-A, reduction of body weight, was consistently observed at the relatively high doses needed to increase righting ability. That alone, however, could not account for increased righting ability, and plasma CK activity was decreased even at doses that did not reduce body weight. The results show that C-21-A is the most effective compound yet tested in this system and, perhaps more significantly, provides the first evidence that it is possible to identify compounds that improve muscle morphology in a hereditary myopathy using a short-term, step-wise system.     

Increase in the amount of elongation factor 2 in chicken muscular dystrophy

The amount of elongation factor 2 (EF2) in the cytoplasm and ribosome of breast muscle cell from normal and dystrophic strains of chicken was measured. Concentration in the cytoplasm of 20-day-old embryonic dystrophic muscle was higher than that in normal muscle, but no difference in content was found in muscles of 15-day-old embryos. The amount of EF2 bound to ribosomes was identical in normal and dystrophic muscles during all stages of development. Peptide mapping patterns of partial proteolytic fragments of EF2 from normal and dystrophic chicken breast muscles were similar. The increase in cytoplasmic protein synthetic activity of dystrophic breast muscles reported previously seems to be due to the corresponding increase in the number of EF2 molecules rather than to their modification in dystrophic muscle.