Magnetic resonance imaging of the proximal upper extremity musculature in boys with Duchenne muscular dystrophy.

There is a pressing need for biomarkers and outcomes that can be used across disease stages in Duchenne muscular dystrophy (DMD), to facilitate the inclusion of a wider range of participants in clinical trials and to improve our understanding of the natural history of DMD. Quantitative magnetic resonance imaging (qMRI) and spectroscopy (MRS) biomarkers show considerable promise in both the legs and forearms of individuals with DMD, but have not yet been examined in functionally important proximal upper extremity muscles such as the biceps brachii and deltoid. The primary objective of this study was to examine the feasibility of implementing qMRI and MRS biomarkers in the proximal upper extremity musculature, and the secondary objective was to examine the relationship between MR measures of arm muscle pathology and upper extremity functional endpoints. Biomarkers included MRS and MRI measures of fat fraction and transverse relaxation time (T 2). The MR exam was well tolerated in both ambulatory and non-ambulatory boys. qMR biomarkers differentiated affected and unaffected participants and correlated strongly with upper extremity function (r = 0.91 for biceps brachii T 2 versus performance of upper limb score). These qMR outcome measures could be highly beneficial to the neuromuscular disease community, allowing measurement of the quality of functionally important muscles across disease stages to understand the natural history of DMD and particularly to broaden the opportunity for clinical trial participation.

Gene transfer of arginine kinase to skeletal muscle using adeno-associated virus.

In this study, we tested the feasibility of non-invasively measuring phosphoarginine (PArg) after gene delivery of arginine kinase (AK) using an adeno-associated virus (AAV) to murine hindlimbs. This was achieved by evaluating the time course, regional distribution and metabolic flux of PArg using (31)phosphorus magnetic resonance spectroscopy ((31)P-MRS). AK gene was injected into the gastrocnemius of the left hindlimb of C57Bl10 mice (age 5 weeks, male) using self-complementary AAV, type 2/8 with desmin promoter. Non-localized (31)P-MRS data were acquired over 9 months after injection using 11.1-T and 17.6-T Bruker Avance spectrometers. In addition, (31)P two-dimensional chemical shift imaging and saturation transfer experiments were performed to examine the spatial distribution and metabolic flux of PArg, respectively. PArg was evident in each injected mouse hindlimb after gene delivery, increased until 28 weeks, and remained elevated for at least 9 months (P<0.05). Furthermore, PArg was primarily localized to the injected posterior hindimb region and the metabolite was in exchange with ATP. Overall, the results show the viability of AAV gene transfer of AK gene to skeletal muscle, and provide support of PArg as a reporter that can be used to non-invasively monitor the transduction of genes for therapeutic interventions.

Longitudinal measurements of MRI-T2 in boys with Duchenne muscular dystrophy: effects of age and disease progression.

Duchenne muscular dystrophy (DMD) is characterized by an increased muscle damage and progressive replacement of muscle by noncontractile tissue. Both of these pathological changes can lengthen the MRI transverse proton relaxation time (T2). The current study measured longitudinal changes in T2 and its distribution in the lower leg of 16 boys with DMD (5-13years, 15 ambulatory) and 15 healthy controls (5-13years). These muscles were chosen to allow extended longitudinal monitoring, due to their slow progression compared with proximal muscles in DMD. In the soleus muscle of boys with DMD, T2 and the percentage of pixels with an elevated T2 (⩾2SD above control mean T2) increased significantly over 1year and 2years, while the width of the T2 histogram increased over 2years. Changes in soleus T2 variables were significantly greater in 9-13years old compared with 5-8years old boys with DMD. Significant correlations between the change in all soleus T2 variables over 2years and the change in functional measures over 2years were found. MRI measurement of muscle T2 in boys with DMD is sensitive to disease progression and shows promise as a clinical outcome measure.

Ultra-high-field MRI real-time imaging of HSC engraftment of the bone marrow niche.

The bone marrow (BM) undergoes extensive remodeling following irradiation damage. A crucial part of restoring homeostasis following irradiation is the ability of hematopoietic stem cells (HSCs) to home to and engraft specialized niches within the BM through a remodeling BM vascular system. Here we show that a combination of ultra-high-field strength magnetic resonance imaging (17.6 T, MRI) coupled with fluorescent microscopy (FLM) serves as a powerful tool for the in vivo imaging of cell homing within the BM. Ultra-high-field MRI can achieve high-resolution three-dimensional (3D) images (28 × 28 × 60 µm(3)) of the BM in live mice, sufficient to resolve anatomical changes in BM microstructures attributed to radiation damage. Following intra-arterial infusion with dsRed-expressing BM cells, labeled with superparamagnetic iron oxides, both FLM and MRI could be used to follow initial homing and engraftment of donor HSC to a limited number of preferred sites within a few cell diameters of the calcified bone-the endosteal niche. Subsequent histology confirmed the fidelity and accuracy of MRI to create non-invasive, high-resolution 3D images of donor cell engraftment of the BM in living animals at the level of single-cell detection.

Non-invasive assessment of lower extremity muscle composition after incomplete spinal cord injury.

STUDY DESIGN: Cross-sectional study. OBJECTIVE: (1) To quantify intramyocellular lipid (IMCL) content of the soleus muscle. (2) To assess the T(2) relaxation rates in the lower extremity skeletal muscles in persons with incomplete spinal cord injury (SCI). SETTING: Academic Institution, Florida. METHODS: Eight subjects (42+/-10 years old; 70+/-12 kg; 176+/-10 cm) with chronic (17+/-9 months post injury) motor SCI (C4-T12; ASIA C or D) and eight matched healthy controls were tested. Localized unsuppressed proton spectroscopy (H-MRS) was performed to estimate total lipid content and individual lipid components; IMCL and extramyocellular lipid (EMCL) from the soleus muscle. T(2)-weighted imaging of lower extremity muscles yielded muscle T(2) rates. RESULTS: The IMCL content of the soleus muscle was 3.3 times higher in the patient group as compared to controls (P=0.002; 0.0401 (0.0234-0.0849) versus 0.0123 (0.0090-0.0175)). Similarly, EMCL measures were 4.5 times higher as compared to the controls (P=0.002). Significant differences were observed in the T(2) relaxation times of the soleus and gastrocnemius muscles (P<0.05). CONCLUSION: The increased levels of IMCL might interfere with the glucose uptake in skeletal muscle; potentially predisposing persons with incomplete SCI to the development of peripheral insulin resistance. Marked elevations in the T(2) relaxation times of the locomotor muscles are reflective of an altered muscle composition.

A longitudinal study of skeletal muscle following spinal cord injury and locomotor training.

STUDY DESIGN: Experimental rat model of spinal cord contusion injury (contusion SCI). OBJECTIVE: The objectives of this study were (1) to characterize the longitudinal changes in rat lower hindlimb muscle morphology following contusion SCI by using magnetic resonance imaging and (2) to determine the therapeutic potential of two types of locomotor training, treadmill and cycling. SETTING: University research setting. METHODS: After moderate midthoracic contusion SCI, Sprague-Dawley rats were assigned to either treadmill training, cycle training or an untrained group. Lower hindlimb muscle size was examined prior to SCI and at 1-, 2-, 4-, 8-, and 12-week post injury. RESULTS: Following contusion SCI, we observed significant atrophy in all rat hindlimb muscles with the posterior muscles (triceps surae and flexor digitorum) showing greater atrophy than the anterior muscles (tibialis anterior and extensor digitorum). The greatest amount of atrophy was measured at 2-week post injury (range from 11 to 26%), and spontaneous recovery in muscle size was observed by 4 weeks post-SCI. Both cycling and treadmill training halted the atrophic process and accelerated the rate of recovery. The therapeutic influence of both training interventions was observed within 1 week of training and no significant difference was noted between the two interventions, except in the tibialis anterior muscle. Finally, a positive correlation was found between locomotor functional scores and hindlimb muscle size following SCI. CONCLUSIONS: Both treadmill and cycle training diminish the extent of atrophy and facilitate muscle plasticity after contusion SCI.

A quantitative study of bioenergetics in skeletal muscle lacking carbonic anhydrase III using 31P magnetic resonance spectroscopy.

Oxidative slow skeletal muscle contains carbonic anhydrase III in high concentration, but its primary function remains unknown. To determine whether its lack handicaps energy metabolism and/or acid elimination, we measured the intracellular pH and energy phosphates by (31)P magnetic resonance spectroscopy in hind limb muscles of wild-type and CA III knockout mice during and after ischemia and intense exercise (electrical stimulation). Thirty minutes of ischemia caused phosphocreatine (PCr) to fall and P(i) to rise while pH and ATP remained constant in both strains of mice. PCr and P(i) kinetics during ischemia and recovery were not significantly different between the two genotypes. From this we conclude that under neutral pH conditions resting muscle anaerobic metabolism, the rate of the creatine kinase reaction, intracellular buffering of protons, and phosphorylation of creatine by mitochondrial oxygen metabolism are not influenced by the lack of CA III. Two minutes of intense stimulation of the mouse gastrocnemius caused PCr, ATP, and pH to fall and ADP and P(i) to rise, and these changes, with the exception of ATP, were all significantly larger in the CA III knockouts. The rate of return of pH and ADP to control values was the same in wild-type and mutant mice, but in the mutants PCr and P(i) recovery were delayed in the first minute after stimulation. Because the tension decrease during fatigue is known to be the same in the two genotypes, we conclude that a lack of CA III impairs mitochondrial ATP synthesis.

Resistance training improves strength and functional capacity in persons with multiple sclerosis.

The purpose of this study was to evaluate the effect of an eight-week progressive resistance training programme on lower extremity strength, ambulatory function, fatigue and self-reported disability in multiple sclerosis (MS) patients (mean disability score 3.7 +/- 0.8). Eight MS subjects volunteered for twice weekly training sessions. During the first two weeks, subjects completed one set of 8-10 reps at 50% of maximal voluntary contraction (MVC) of knee flexion, knee extension and plantarflexion exercises. In subsequent sessions, the subjects completed one set of 10-15 repetitions at 70% of MVC. The resistance was increased by 2-5% when subjects completed 15 repetitions in consecutive sessions. Isometric strength of the quadriceps, hamstring, plantarflexor and dorsiflexor muscle groups was assessed before and after the training programme using an isokinetic dynamometer. Magnetic resonance images of the thigh were acquired before and after the exercise programme as were walking speed (25-ft), number of steps in 3 min, and self-reported fatigue and disability. Knee extension (7.4%), plantarflexion (52%) and stepping performance (8.7%) increased significantly (P < 0.05). Self-reported fatigue decreased (P < 0.05) and disability tended to decrease (P = 0.07) following the training programme. MS patients are capable of making positive adaptations to resistance training that are associated with improved ambulation and decreased fatigue.

Spectral quantitation by principal component analysis using complex singular value decomposition.

Principal component analysis (PCA) is a powerful method for quantitative analysis of nuclear magnetic resonance spectral data sets. It has the advantage of being model independent, making it well suited for the analysis of spectra with complicated or unknown line shapes. Previous applications of PCA have required that all spectra in a data set be in phase or have implemented iterative methods to analyze spectra that are not perfectly phased. However, improper phasing or imperfect convergence of the iterative methods has resulted in systematic errors in the estimation of peak areas with PCA. Presented here is a modified method of PCA, which utilizes complex singular value decomposition (SVD) to analyze spectral data sets with any amount of variation in spectral phase. The new method is shown to be completely insensitive to spectral phase. In the presence of noise, PCA with complex SVD yields a lower variation in the estimation of peak area than conventional PCA by a factor of approximately 2. The performance of the method is demonstrated with simulated data and in vivo 31P spectra from human skeletal muscle.

Longitudinal study of skeletal muscle adaptations during immobilization and rehabilitation.

This study describes the metabolic, morphologic, neurologic, and functional adaptations observed in the plantar flexors during 8 weeks of lower leg immobilization and 10 weeks of physical therapy following ankle surgery. A combination of magnetic resonance imaging and spectroscopy, isokinetic and isometric muscle testing, and simple functional tests revealed many adaptive changes due to immobilization, including atrophy, loss of muscle strength, reduced central activation, increase in fatigue resistance, and an increase in inorganic phosphate content. After 10 weeks of physical therapy all alterations were reversed, with the exception of a remaining 5.5% deficit in total muscle cross-sectional area.

Volumetric measurement of human calf muscle from magnetic resonance imaging.

Muscle mass is a determining factor in skeletal muscle function and is affected by inactivity, immobilization, disease, and aging. The aim of this study was to develop an objective and time-efficient method to quantify the volume and cross-sectional area of human calf muscles using three-dimensional magnetic resonance images. We have estimated the errors incurred in muscle volume measurements arising from artifacts known to occur in magnetic resonance imaging (MRI). The largest source of error was due to partial volume effects, which resulted in overestimation of phantom volumes ranging from 145 to 900 cc by 6% to 13%. The magnitude of this effect has been shown to increase with decreasing object size and decreasing spatial resolution. We have presented a straightforward correction for this effect, which has reduced the volume measurement error to less than 4% for all cases. Through the use of computer simulations, the correction algorithm has been shown to be independent of object shape and orientation. To reduce user subjectivity, a semiautomated computer program has been developed to segment MRI data for particular muscle groups. Images from seven human subjects were analyzed by the program, yielding muscle volumes of 154.2 +/- 23.2, 281.2 +/- 35.8, and 432.2 +/- 83.7 for the lateral gastrocnemius, medial gastrocnemius, and soleus, respectively.

Diet after pancreas transplantation.

OBJECTIVE: To determine whether discontinuation of insulin therapy and glucose monitoring and instructions to increase dietary salt and water intake after pancreas transplantation (PTX) resulted in changes in food choices. RESEARCH DESIGN AND METHODS: All PTX recipients who had completed a preoperative diet record, had received their PTX > 6 months before, had stable pancreas and kidney function, and were on a stable diet were invited to submit a 3-day post-PTX diet record. Of the 14 eligible, 11 agreed to participate and completed the study (2 women and 9 men). Their pre- and post-PTX diet records were analyzed by computer program. Weight, glycohemoglobin, blood pressure, medications, and fasting lipids both before and after PTX were also analyzed. RESULTS: The recipients were studied 576 +/- 60 days post-PTX, on average. Total calories and BMI were unchanged after PTX. Before PTX, 34% of calories were in fats, 49% in carbohydrate, and 17% in protein with no change in distribution of calories after PTX, although there was a trend toward greater saturated fat intake. Total salt intake was increased after PTX (P < 0.01) because of sodium bicarbonate administration, although dietary salt intake did not change. The HDL cholesterol concentration increased and cholesterol-to-HDL ratio decreased after PTX (P < 0.05), while the remaining lipids were unchanged. CONCLUSION: Weight, total calories and distribution of calories, and dietary salt were unchanged after PTX, and diet did not explain the changes in HDL cholesterol or cholesterol-to-HDL ratio. These preliminary diet results suggest that greater emphasis on dietary instruction may be needed after PTX.

High-performance liquid chromatographic assay for CI-980, a novel 1-deaza-7,8-dihydropteridine anticancer agent, in human plasma and urine.

CI-980, a 1-deaza-7,8-dihydropteridine, is a novel anticancer agent that is a potent mitotic inhibitor acting as a tubulin binder similar to the vinca alkaloids. CI-980 has shown equivalent or superior anticancer activity in vitro compared to vincristine and retains full activity against vincristine resistant tumors in vitro. A high-performance liquid chromatographic (HPLC) assay was developed and validated for human plasma and urine to support Phase 1 clinical trials. CI-980 and PD 080658, internal standard, were isolated from 2-ml samples of human plasma and urine by solid-phase extraction with Bond-Elut C18 cartridges. Urine samples must be pretreated with bovine serum albumin (BSA) to minimize the binding of CI-980 to glass and some plastics. The eluate from the cartridges for both matrices was evaporated to dryness and taken up in mobile phase. Zorbax RX C18 columns, mobile phase buffer of 10 mM ammonium dihydrogen phosphate at pH 7.5 and a flow-rate of 0.75 ml/min were used for both matrices. Column dimensions, column temperature and mobile phase acetonitrile-buffer ratio were 300 mm x 4.6 mm I.D., 30 degrees C and 38:62 (v/v), respectively, for the plasma assay and 250 mm x 4.6 mm I.D., 35 degrees C and 40:60 (v/v), respectively, for the urine assay. Column effluent was monitored fluorometrically for the plasma method using excitation and emission wavelengths of 388 nm and 473 nm, respectively. Ultraviolet detection at 380 nm was used for the urine method. Peak-area ratios were proportional to CI-980 concentrations from 0.2 to 25 ng/ml and 1 to 100 ng/ml for plasma and urine, respectively. CI-980 in water will bind to glass and plastics but not PTFE or stainless steel. Urine calibration standards were frozen prior to use in order to compensate for loss of CI-980 due to freezing in this matrix. The accuracy of the assay was within 4.7%, with a precision of 5.6% for both matrices. Recoveries ranged from 93.8 to 102% and 90.7 to 92.3% for plasma and urine, respectively. CI-980 was stable in plasma and urine for at least 275 and 217 days, respectively, when stored at -70 degrees C. The assay is suitable for studying the clinical pharmacokinetics of CI-980.

Nutritional support of the child with cancer.

The purpose of this article is to outline how the disease, treatment, and psychological state of a child with cancer impact on the child's nutritional status. The methods of assessing nutritional status, including anthropometric measurements, laboratory indices, clinical observation, dietary assessment, and psychosocial evaluation, are summarized. After nutrition assessment, the pediatric oncology nurse and the dietitian, along with the oncologist, the family, and the child, develop a plan of care. The specific roles of the pediatric oncology nurse and dietitian in the nutrition intervention are described.

The effects of hypothermia on amino acid neurotransmitter release from the cerebral cortex.

The effects of systemic hypothermia (33.5 degrees C) on the ischemia-evoked release of the neurotransmitter amino acids, glutamate, aspartate, gamma-amino-butyric acid (GABA) and glycine into rat cerebral cortical superfusates were evaluated in the rat four vessel occlusion model. Glutamate, aspartate and GABA, but not glycine, levels were enhanced during and following a 20 min period of ischemia. However, when compared with normothermic ischemic animals, no reductions in glutamate, aspartate or GABA levels in the superfusates were apparent either prior to, during or following forebrain ischemic episodes. Indeed, the superfusate levels of aspartate and GABA were transiently increased immediately following ischemia. Glycine levels were significantly depressed, both pre- and post-ischemia, in cortical superfusates from hypothermic animals in comparison with normothermic rats.

Brain adenosine and transmitter amino acid release from the ischemic rat cerebral cortex: effects of the adenosine deaminase inhibitor deoxycoformycin.

The effects of a potent adenosine deaminase inhibitor, deoxycoformycin, on purine and amino acid neuro-transmitter release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four-vessel occlusion. Purine and amino acid releases were compared from control ischemic animals and deoxycoformycin-pretreated ischemic rats. Ischemia enhanced the release of glutamate, aspartate, and gamma-aminobutyric acid into cortical perfusates. The levels of adenosine, inosine, hypoxanthine, and xanthine in the same perfusates were also elevated during and following ischemia. Deoxycoformycin (500 micrograms/kg) enhanced ischemia-evoked release of adenosine, indicating a marked rise in the adenosine content of the interstitial fluid of the cerebral cortex. Inosine, hypoxanthine, and xanthine levels were depressed by deoxycoformycin. Deoxycoformycin pretreatment failed to alter the pattern of amino acid neurotransmitter release from the cerebral cortex in comparison with that observed in control ischemic animals. The failure of deoxycoformycin to attenuate amino acid neurotransmitter release, even though it markedly enhanced adenosine levels in the extracellular space, implies that the amino acid release during ischemia occurs via an adenosine-insensitive mechanism. Inhibition of excitotoxic amino acid release is unlikely to be responsible for the cerebroprotective actions of deoxycoformycin in the ischemic brain.

Purine concentrations in the cerebrospinal fluid of unanesthetized rats during and after hypoxia.

A new technique for achieving repeated sampling of fourth ventricular cerebrospinal fluid (CSF) from the cisterna magna of unanesthetized rats is described. The sampling cannula is positioned extracranially, in contrast to previously published techniques which require insertion through the skull. CSF samples, withdrawn from unanesthetized rats before, during and after a 25 min period of inhalation of 5% oxygen in nitrogen, were analyzed for their adenosine and inosine contents by high pressure liquid chromatography. Adenosine and inosine levels increased during the hypoxic episode and were even higher 1 h later. They had declined, but were still above basal levels, 2-3 h after the hypoxic episode. Elevated CSF adenosine concentrations may be responsible for the generation of such persistent effects of hypoxia as post-hypoxic respiratory depression.

The effect of hyperglycemia on extracellular levels of adenosine in the hypoxic rat cerebral cortex.

The levels of adenosine in rat cerebral cortical superfusates were studied in rats prior to, and after, the administration of saline or D-glucose (3 g/kg). Hypoxia-evoked increases in purine release were significantly attenuated after glucose administration. After glucose administration, the falls in arterial blood pressure, which normally accompany systemic hypoxia, were reduced. To ensure that this was not the reason for the decrease in adenosine release, blood was withdrawn from a second group of hyperglycemic rats so that the post-glucose hypoxia was equivalent to the original control. Adenosine release was still significantly attenuated. This decrease in the levels of adenosine, a cerebroprotective agent, in the cerebral cortical extracellular space, may be a contributing factor to the detrimental effects of hyperglycemia on recovery from cerebral ischemia.

Effect of a brief hypoxic/hypotensive episode on the in vivo release of cerebral cortical gamma-aminobutyric acid and glycine.

gamma-Aminobutyric acid (GABA) and glycine levels in rat cerebral cortical superfusates rose during a 10-min period to reach stable concentrations of approximately 0.55 microM and approximately 12.3 microM, respectively. In cerebrospinal fluid withdrawn from the fourth ventricle, the GABA concentration was 0.1 microM, and that of glycine, 10.55 microM. GABA, and to a lesser extent glycine, concentrations increased in the cortical superfusates during and immediately following exposure of the rats to a 5-min period of 5% oxygen in nitrogen inhalation.

Hypoxia/hypotension evoked release of glutamate and aspartate from the rat cerebral cortex.

Glutamate and aspartate levels in cerebral cortical superfusates rose rapidly after introduction of artificial cerebrospinal fluid into cortical cups to reach stable levels within 5-10 min. These equilibrated levels (glutamate 1.33 +/- 0.1 microM; aspartate 0.29 +/- 0.04 microM), which are very similar to those in fourth ventricular CSF (glutamate, 1.5 +/- 0.2 microM; aspartate, 0.27 +/- 0.03 microM), are likely to reflect basal interstitial amino acid levels in the superficial layers of the cortex. During and following brief (5 min) hypoxemic/hypotensive episodes, release of both excitatory amino acids into the cortical superfusates was enhanced. The finding of an hypoxia/hypotension-evoked release of excitatory amino acids is consistent with recent speculation that these agents may play an important role in the destruction of neurons in ischemic regions of the brain.