Reconstitution of the complete rupture in musculotendinous junction using skeletal muscle-derived multipotent stem cell sheet-pellets as a “bio-bond”

Background.Significant and/or complete rupture in the musculotendinous junction (MTJ) is a challenging lesion to treat because of the lack of reliable suture methods. Skeletal muscle-derived multipotent stem cell (Sk-MSC) sheet-pellets, which are able to reconstitute peripheral nerve and muscular/vascular tissues with robust connective tissue networks, have been applied as a “bio-bond”.Methods.Sk-MSC sheet-pellets, derived from GFP transgenic-mice after 7 days of expansion culture, were detached with EDTA to maintain cell–cell connections. A completely ruptured MTJ model was prepared in the right tibialis anterior (TA) of the recipient mice, and was covered with sheet-pellets. The left side was preserved as a contralateral control. The control group received the same amount of the cell-free medium. The sheet-pellet transplantation (SP) group was further divided into two groups; as the short term (4–8 weeks) and long term (14–18 weeks) recovery group. At each time point after transplantation, tetanic tension output was measured through the electrical stimulation of the sciatic nerve. The behavior of engrafted GFP+tissues and cells was analyzed by fluorescence immunohistochemistry.Results.The SP short term recovery group showed average 64% recovery of muscle mass, and 36% recovery of tetanic tension output relative to the contralateral side. Then, the SP long term recovery group showed increased recovery of average muscle mass (77%) and tetanic tension output (49%). However, the control group showed no recovery of continuity between muscle and tendon, and demonstrated increased muscle atrophy, with coalescence to the tibia during 4–8 weeks after operation. Histological evidence also supported the above functional recovery of SP group. Engrafted Sk-MSCs primarily formed the connective tissues and muscle fibers, including nerve-vascular networks, and bridged the ruptured tendon–muscle fiber units, with differentiation into skeletal muscle cells, Schwann cells, vascular smooth muscle, and endothelial cells.Discussion.This bridging capacity between tendon and muscle fibers of the Sk-MSC sheet-pellet, as a “bio-bond,” represents a possible treatment for various MTJ ruptures following surgery.

The Assessment of Reperfusion Inflammatory Injury in Ischemic Preconditioned Diabetic Rats

Objective: The assessment of systemic reperfusion injury and the contractile force of the peripheral muscles post-acute ischemia of the hind limbs in healthy versus diabetic ischemic preconditioned rats.Method: The study included 16 Wistar rats divided into two groups: the control group and the diabetic ischemic preconditioned group. Acute ischemia was induced, followed by reperfusion. The assessment of reperfusion injury used biochemical, histopathological and functional determinations (peak tetanic tension-PTT, specific tension-ST).Results: Ischemia-reperfusion injury was more severe in control group regarding creatine-kinase (CK) (CK1=470.13 IU/L versus CK2=230.88 IU/L, p=0.0001) and myoglobin (390.25 ng/mL versus 47.99 ng/mL, p=0.025). Cytolysis enzymes were significantly increased in diabetic preconditioned rats (Alanine aminotransferase ALAT1=46 IU/L, ALAT2=167.8 IU/L, p=0.02; Aspartate aminotransferase ASAT1=106 IU/L, ASAT2=237.5 IU/L, p=0.016). Functional assessment (PTT and ST) highlighted roughly equal values. A paradoxical response occurred in diabetic rats (the contractile force increased during the period of the stimulation). Histopathological findings showed that rhabdomyolysis was more severe in the control group, while inflammatory systemic response due to reperfusion injury was less expressed in diabetic ischemic preconditioned rats.Conclusions: Ischemic preconditioning reduces the severity of reperfusion injury and allows the preservation of contractile muscle function in diabetic rats.

Diaphragmatic Function Is Preserved during Severe Hemorrhagic Shock in the Rat

Background: Acute diaphragmatic dysfunction has been reported in septic and cardiogenic shock, but few data are available concerning the effect of hemorrhagic shock on diaphragmatic function. The authors examined the impact of a hemorrhagic shock on the diaphragm. Methods: Four parallel groups of adult rats were submitted to hemorrhagic shock induced by controlled exsanguination targeting a mean arterial blood pressure of 30 mmHg for 1 h, followed by a 1-h fluid resuscitation with either saline or shed blood targeting a mean arterial blood pressure of 80 mmHg. Diaphragm and soleus strip contractility was measured in vitro. Blood flow in the muscle microcirculation was measured in vivo using a Laser Doppler technique. Muscle proinflammatory cytokine concentrations were also measured. Results: Hemorrhagic shock was characterized by a decrease in mean arterial blood pressure to 34 ± 5 mmHg (−77 ± 4%; P< 0.05) and high plasma lactate levels (7.6 ± 0.9 mM; P < 0.05). Although tetanic tension of the diaphragm was not altered, hemorrhagic shock induced dramatic impairment of tetanic tension of the soleus (−40 ± 19%; P < 0.01), whereas proinflammatory cytokine levels were low and not different between the two muscles. Resuscitation with either blood or saline did not further modify either diaphragm or soleus performance and proinflammatory cytokine levels. The shock-induced decrease in blood flow was much more pronounced in the soleus than in the diaphragm (−75 ± 13% vs. −17 ± 10%; P = 0.02), and a significant interaction was observed between shock and muscle (P < 0.001). Conclusion: Diaphragm performance is preserved during hemorrhagic shock, whereas soleus performance is impaired, with no further impact of either blood or saline fluid resuscitation.

Effects of selenium supplementation on the muscle contractile properties, fatigue and antioxidant enzymes activities of gastrocnemius muscles in trained and untrained rats

Selenium (Se) is an important component of cellular seleno ‐compounds andan integral component of glutathione Peroxidase (GPx), which catalyzes thereduction of harmful radicals produced during muscular exercise. The currentstudy was carried out to evaluate the muscle contractile properties andfatigue resistance of gastrocnemius muscle under selenium supplementationschemes in sedentary and exercise protocols as well as measure the antioxidantenzymes activity and lipid peroxidation. Rats were divided into fourgroups; sedentary Selenium supplementation (S), exercise Selenium (SE)groups, sedentary control (SC) and exercise control (EC) groups. The ratswere fed with 80 μg/kg body weights selenium for six weeks. The exerciseprotocol consisted the 40 jumps up to the height of 40 cm for 6 ‐week. Themuscle fatigue protocol consisted the trains of pulses of 40 Hz at every secondfor at least 2 min. Significant (P<0.05) increase was observed in treatedgroups than control in the muscle contractile properties like twitch tension(Pt), contraction time (CT) and twitch/tetanic tension Ratio (Pt/Po Ratio),tetanic tension (Po) and EMG amplitude. Decreased EMG failure and increasedfatigue index were observed in ES group. Moreover, a significant(P<0.05) increase and decrease in the GPx activity and lipid peroxidation respectivelywas also reported than SC and EC group. While there were nochanges reported in the activity of CAT and SOD enzymes. This study revealedthat the Se with jumping exercise induces muscle contractile propertiesand decreases the muscular fatigue.

Mechanical Responses of the Human Triceps Surae after Passive "Stretching" Training of the Plantarflexors in Conditions Modulating Weightlessness

Mechanical Responses of the Human Triceps Surae after Passive "Stretching" Training of the Plantarflexors in Conditions Modulating WeightlessnessThe effect of a 60-day 6° head-down tilt of bed rest with and without prolonged passive muscle "stretching" training on the mechanical properties of the human triceps surae muscle was studied in 13 healthy male subjects. One group (n = 6; mean age 30.8 ± 3.1 years) underwent a 60-day head-down tilt, and a second group (n = 7; mean age 30.4 ± 1.2 years) underwent head-down tilt with prolonged passive muscle stretching. Head-down tilt without prolonged passive muscle "stretching" training showed maximal voluntary contraction declined by 34 % (p < 0.05) and the electrically evoked tetanic tension at 150 impulses·s-1and isometric twitch contraction reduced by 17 % (p < 0.02) and 18 % (p < 0.05), respectively. Time-to-peak tension, and half-relaxation time of the twitch slightly decreased by 3% (p > 0.05), and 7 %, respectively, but total contraction time slightly increased. The difference between electrically evoked tetanic tension and the maximal voluntary contraction expressed as a percentage of electrically evoked tetanic tension (referred to as force deficiency), has also been calculated. The force deficiency increased by 61 % (p < 0.001). After head-down tilt with prolonged passive muscle "stretching" training, the time-to-peak tension did not change, and half-relaxation time of the twitch decreased. In addition, there was a 14 % lengthening in the total duration of the twitch. The results of prolonged passive muscle "stretching" training demonstrated a clear deterioration of voluntarily and electrically induced muscle contractions. Passive "stretching" training caused a decrease by 29 % (p < 0.05) in the maximal voluntary contraction. The isometric twitch contraction, and electrically evoked tetanic tension both showed reductions by 17 %, and by 19 % (p < 0.05), respectively. The force deficiency decreased significantly by 21 % (p < 0.02). The rate of rise of electrically evoked tetanic tension and feature of voluntary contractions significantly reduced during head-down tilt with prolonged passive muscle "stretching" training. These basic experimental findings concluded that prolonged passive "stretching" training of a single muscle did not prevent a reserve of neuromuscular function.

Electromechanical stimulation ameliorates inactivity-induced adaptations in the medial gastrocnemius of adult rats

The efficacy of high-load, short-duration isometric contractions, delivered as one vs. two sessions per day, on blunting inactivity-induced adaptations in the medial gastrocnemius (MG) were compared. Adult rats were assigned to a control (Con) or spinal cord-isolated (SI) group where one limb was stimulated (SI-Stim) while the other served as a SI control (SI-C). One bout of stimulation (BION microstimulator) consisted of a 100-Hz, 1-s stimulus, delivered every 30 s for 5 min with a 5-min rest period. This bout was repeated six times consecutively (SI-Stim1) or with a 9-h rest interval after the third bout (SI-Stim2) for 30 consecutive days. MG weights (relative to body weight) were 63, 72, and 79% of Con in SI-C, SI-Stim1, and SI-Stim2, respectively. Mean fiber size was 56% smaller in SI-C than in Con, and it was 19 and 31% larger in SI-Stim1 and SI-Stim2, respectively, compared with SI-C. Maximum tetanic tension was 42, 60, and 73% of Con in SI-C, SI-Stim1, and SI-Stim2, respectively. Specific tension was 77% of Con in SI-C, and at Con levels in both SI-Stim groups. SI increased the percent IIb myosin heavy chain composition (from 49 to 77%) and IIb+ fibers (from 63 to 79%): these adaptations were prevented by both Stim paradigms. These results demonstrate that 1) brief periods of high-load isometric contractions are effective in reducing inactivity-induced atrophy, functional deficits, and phenotypic adaptations in a fast hindlimb extensor, and 2) the same amount of stimulation distributed in two compared with one session per day is more effective in ameliorating inactivity-related adaptations.

The role of ascorbic acid and exercise in chronic ischemia of skeletal muscle in rats

This study was designed to investigate the effects of peripheral arterial insufficiency, exercise, and vitamin C administration on muscle performance, cross-sectional area, and ultrastructural morphology in extensor digitorum longus (EDL) and soleus (Sol) muscles in rats. Adult Wistar rats were assigned to ischemia alone (isch), ischemia-exercised (exe), ischemia-vitamin C (vit C), and ischemia-exercise-vitamin C (vit C + exe) groups. Ischemia was achieved via unilateral ligation of the right common iliac artery. Contralateral muscles within the same animal served as controls. Exercise protocol consisted of 50-min intermittent level running performed every other day for 5 days. Vitamin C (100 mg/kg body wt) was administered intraperitoneally on a daily basis throughout the 14 days of the experiment. With regard to the EDL muscle, ischemia alone reduced muscle strength, which was not recovered after vitamin C administration. Exercise alone following ischemia induced the most severe structural damage and cross-sectional area decrease in the muscle, yet the reduction in tetanic tension was not significant. Exercise in conjunction with vitamin C administration preserved ischemia-induced EDL muscle tetanic tension. In the Sol muscle, a significant reduction in single twitch tension after vitamin C administration was found, whereas the tetanic force of the ischemic Sol was not significantly decreased compared with the contralateral muscles in any group. Ischemic Sol muscle cross-sectional area was reduced in all but the exe groups. In Sol, muscle strength was reduced in the vit C group, and mean cross-sectional area of ischemic Sol muscles was reduced in all groups except the exe group. These results illustrate that mild exercise, combined with a low dose of vitamin C supplementation, may have beneficial effects on ischemic EDL muscle with a smaller effect on the Sol muscle.

Sarcomere strain and heterogeneity correlate with injury to frog skeletal muscle fiber bundles

Sarcomere length and first-order diffraction line width were measured by laser diffraction during elongation of activated frog tibialis anterior muscle fiber bundles (i.e., eccentric contraction) at nominal fiber strains of 10, 25, or 35% ( n = 18) for 10 successive contractions. Tetanic tension, measured just before each eccentric contraction, differed significantly among strain groups and changed dramatically during the 10-contraction treatment ( P < 0.01). Average maximum tetanic tension for the three groups measured before any treatment was 203.7 ± 6.8 kN/m2, but after the 10-eccentric contraction sequence decreased to 180.3 ± 3.8, 125.1 ± 7.8, and 78.3 ± 5.1 kN/m2 for the 10, 25, and 35% strain groups, respectively ( P < 0.0001). Addition of 10 mM caffeine to the bathing medium decreased the loss of tetanic tension in the 10% strain group but had only a minimal effect on either the 25 or 35% strain groups. Diffraction pattern line width, a measure of sarcomere length heterogeneity, increased significantly with muscle activation and then continued to increase with successive stretches of the activated muscle. Line width increase after each stretch was significantly correlated with the lower yield tension of the successive contractile record. These data demonstrate a direct association and, perhaps, a causal relationship between sarcomere strain and fiber bundle injury. They also demonstrate that muscle injury is accompanied by a progressive increase in sarcomere length heterogeneity, yielding lower yield tension as injury progresses.

Progression of kyphosis in mdx mice

Spinal deformity in the form of kyphosis or kyphoscoliosis occurs in most patients with Duchenne muscular dystrophy (DMD), a fatal X-linked disorder caused by an absence of the subsarcolemmal protein dystrophin. Mdx mice, which also lack dystrophin, show thoracolumbar kyphosis that progresses with age. We hypothesize that paraspinal and respiratory muscle weakness and fibrosis are associated with the progression of spinal deformity in this mouse model, and similar to DMD patients there is evidence of altered thoracic conformation and area. We measured kyphosis in mdx and age-matched control mice by monthly radiographs and the application of a novel radiographic index, the kyphotic index, similar to that used in boys with DMD. Kyphotic index became significantly less in mdx at 9 mo of age (3.58 ± 0.12 compared with 4.27 ± 0.04 in the control strain; P ≤ 0.01), indicating more severe kyphosis, and remained less from 10 to 17 mo of age. Thoracic area in 17-mo-old mdx was reduced by 14% compared with control mice ( P ≤ 0.05). Peak tetanic tension was significantly lower in mdx and fell 47% in old mdx latissimus dorsi muscles, 44% in intercostal strips, and 73% in diaphragm strips ( P ≤ 0.05). Fibrosis of these muscles and the longissimus dorsi, measured by hydroxyproline analysis and histological grading of picrosirius red-stained sections, was greater in mdx ( P < 0.05). We conclude that kyphotic index is a useful measure in mdx and other kyphotic mouse strains, and assessment of paralumbar and accessory respiratory muscles enhance understanding of spinal deformity in muscular dystrophy.