Changes of biochemical indicators of blood serum and the skeletal muscle homogenate of the hind limbs of the rats in acute ischemia-reperfusion and in the condition of using of the carbacetam

Annotation. Simulation of ischemia-reperfusion causes the biochemical disorders and changes in the prooxidant-antioxidant system, both in the serum and skeletal muscles of the affected limb. The severity of local and systemic postischemic imbalance decreases in with correction with medicine from the group of nootropics – carabacetam. The aim of the study was determining the degree of metabolic disorders based on the analysis of changes in serum biochemical parameters and muscle homogenate in ischemic-reperfusion disorders caused by the imposition of an arterial tourniquet and under conditions of correction with carbacetam. Materials and methods. A biochemical study of blood serum and muscle homogenate of 60 rats was performed. Ischemia was simulated by applying SWAT rubber tourniquets on the hind limbs of the rats, on the inguinal fold, for 2 hours, and reperfusion was modeled by removing the tourniquet and restoring of blood circulation through 2 hours after its application. Observations of the animals were carried out during for 14 days. 30 rats in the reperfusion period were intraperitoneally administered the carbacetam at a dose of 5 mg per kilogram of body weight 1 time a day for 14 days of the reperfusion period. The Blood serum was separated and the soft tissue samples were taken below the turniquet site for the biochemical studies. Statistical processing of the material was performed using the package of programs “Microsoft Excel”. The largest deviations in serum were observed after 1 day of the reperfusion: an increase in creatinine by 28.16 % (p<0.005), total bilirubin by 20.75 % (p<0,005), ALT by 96.14 % (p<0,005), AST by 147.27 % (p<0.005) and alkaline phosphatase by 70.84 % (p<0.005), as well as an increase in prooxidant activity of CD by 77.27 % (p<0.005), CT 147.47 % (p<0,005) and TBC-AL by 82.52 % (p<0,005) against the background of a decrease in SOD activity by 9.77 % (p<0.005) and an increase in catalase levels by 15.37 % (p<0.005) relative to the value of the control group. The development of ischemia and reperfusion was accompanied by the largest increase in the level of prooxidants (CD – by 27.55 % (p<0.005), CT – by 47.34 % (p<0.05), TBC-AL – by 62.09 %) (p<0.005)) in the homogenate of the skeletal muscles after 2 hours after restoration of the blood supply. Antioxidant activity, on the other hand, decreased by 15.06 % (p<0.005) SOD and by 8.96 % (p<0.005) catalase after 1 day. After 14 days, a gradual increase and return of most of the studied values to the level of the control group. Under the influence of the carbacetam, the biochemical parameters of blood serum most significantly decreased in relative to the value of the groups without correction after 7 days of the experiment. The level of total bilirubin was lower by 29.65 % (p˂0.05), ALT by 14.95 % (p˂0.05), AST by 19.84 % (p˂0.05), alkaline phosphatase by 25.53 % (p˂0.05), and the difference in the content of diene, triene conjugates and TBC-LP was the largest after 1 day – by 17.09 % (p<0.005), 20,41 % (p<0.05) and 11.24 % (p<0.05), respectively. Under the conditions of correction, the indicators of SOD and catalase activity increased, being significantly higher than in the groups of the animals without correction, especially after 7 days of reperfusion – by 29.41 % (p<0.005) and by 15.71 % (p<0.05) in accordance. The content of the diene, triene conjugates and TBC-LP muscle homogenate was statistically significantly lower in the groups with the introduction of the carbacetam than in the groups of the animals without correction. The largest difference between the groups was found after 1 day with a difference of 9.16 % (p<0.05), 7.35 % (p<0.005) and 16.05 % (p<0.05), respectively. Antioxidant activity of SOD and catalase after 1 day was higher by 27.41 % (p<0.005) and by 25.13 % (p<0.005), compared with the group of the animals without introduction of the carbacetam. Thus, simulated ischemia-reperfusion is accompanied by the greatest biochemical disorders in the serum – after 1 day, and in the muscle homogenate – after 2 hours of the reperfusion. After 14 days of the experiment, most indicators still do not return to the level of the control group. The application of the correction helps to reduce the severity of biochemical disorders at the systemic and local levels, especially during periods of their greatest deviations.

Consistent expression pattern of myogenic regulatory factors in whole muscle and isolated human muscle satellite cells after eccentric contractions in humans

Skeletal muscle satellite cells (SC) play an important role in muscle repair following injury. The regulation of SC activity is governed by myogenic regulatory factors (MRF), including MyoD, Myf5, myogenin, and MRF4. The mRNA expression of these MRF in humans following muscle damage has been predominately measured in whole muscle homogenates. Whether the temporal expression of MRF in a whole muscle homogenate reflects SC-specific expression of MRF remains largely unknown. Sixteen young men (23.1 ± 1.0 yr) performed 300 unilateral eccentric contractions (180°/s) of the knee extensors. Percutaneous muscle biopsies from the vastus lateralis were taken before (Pre) and 48 h postexercise. Fluorescence-activated cell sorting analysis was utilized to purify NCAM+ muscle SC from the whole muscle homogenate. Forty-eight hours post-eccentric exercise, MyoD, Myf5, and myogenin mRNA expression were increased in the whole muscle homogenate (~1.4-, ~4.0-, ~1.7-fold, respectively, P < 0.05) and in isolated SC (~19.3-, ~17.5-, ~58.9-fold, respectively, P < 0.05). MRF4 mRNA expression was not increased 48 h postexercise in the whole muscle homogenate ( P > 0.05) or in isolated SC ( P > 0.05). In conclusion, our results suggest that the directional changes in mRNA expression of the MRF in a whole muscle homogenate in response to acute eccentric exercise reflects that observed in isolated muscle SC. NEW & NOTEWORTHY The myogenic program is controlled via transcription factors referred to as myogenic regulatory factors (MRF). Previous studies have derived MRF expression from whole muscle homogenates, but little work has examined whether the mRNA expression of these transcripts reflects the pattern of expression in the actual population of satellite cells (SC). We report that MRF expression from an enriched SC population reflects the directional pattern of expression from skeletal muscle biopsy samples following eccentric contractions.

TNFα Mechanically Sensitizes Masseter Muscle Nociceptors by Increasing Prostaglandin E2 Levels

TNFα induces mechanical sensitization of rat masseter muscle nociceptors, which takes 2–3 h to manifest and is mediated through activation of P55 and P75 receptors. This study was undertaken to determine whether TNFα induces nociceptor mechanical sensitization through the release of other algogenic substances such as glutamate, prostaglandin E2 (PGE2), and/or nerve growth factor (NGF), which have been shown to induce mechanical sensitization of muscle nociceptors. Masseter muscle homogenate levels of PGE2 and NGF were measured 3 h after injection of TNFα (1 μg) or vehicle control using commercially available kits. Interstitial glutamate concentration was measured after injection of TNFα or vehicle control using a glutamate-selective biosensor probe. Diclofenac, a cycloxygenase inhibitor that blocks the synthesis of PGE2, d-2-amino-5-phophonovaleric acid (APV), a competitive N-methyl-d-aspartate (NMDA) receptor antagonist, and a tyrosine kinase A (TrkA) receptor antibody, which blocks NGF-induced masseter muscle nociceptor sensitization, were used to assess the contribution of PGE2, glutamate, and NGF to TNFα-induced nociceptor sensitization. PGE2 and glutamate concentrations were significantly elevated 3 h after TNFα injection into the masseter muscle. Injection of diclofenac partially reversed the TNFα-induced decreases in the mechanical threshold (MT) of masseter muscle nociceptors, whereas vehicle control, APV, and TrkA antibody did not significantly alter nociceptor MT. These results suggest that TNFα-induced mechanical sensitization of masseter muscle nociceptors is mediated in part by increased PGE2 levels. The findings of this study support the hypothesis that TNFα induces a delayed mechanical sensitization of masseter muscle nociceptors indirectly by the release of PGE2.

Biochemical and Immunohistochemical Evidence for a Non-muscle Myosin at the Neuromuscular Junction in Bovine Skeletal Muscle

We identified 220-kD protein in bovine skeletal muscle homogenate by affinity chromatography on an agarose column and subsequent SDS-PAGE. Peptide mass fingerprinting (MALDI mass spectrometry) and internal sequence analysis revealed that this protein has homology with several members of the myosin superfamily, particularly with human cardiac β-myosin heavy chain (β-MHC). A rabbit polyclonal antibody against the 220-kD protein specifically stained a 220-kD band in Western blots of skeletal muscle homogenate. Immunohistochemical experiments on cryostat sections demonstrated that in skeletal muscle this protein is exclusively localized at the neuromuscular junctions, no immunoreactivity being present at the myofibril level. Because of its relative homology with cardiac β-MHC, we also investigated the distribution of the 220-kD protein in bovine heart. In cardiac fibers, 220-kD protein-related immunoreactivity was restricted to the intercalated disks, whereas myofibrils were completely devoid of specific immunoreactivity. This distribution pattern was completely different from that of cardiac β-MHC, which involved myofibrils. Because of the above biochemical and immunohistochemical features, the 220-kD protein we have identified is suggested to be a novel member of the non-muscle (non-sarcomeric) myosin family.

Effects of tissue fractionation on exercise-induced alterations in SR function in rat gastrocnemius muscle

Because studies into exercise-induced alterations in sarcoplasmic reticulum (SR) Ca2+ sequestration have produced conflicting reports, we have hypothesized that the differences in SR Ca(2+)-adenosinetriphosphatase (ATPase) activity and Ca2+ uptake in SR fractions observed in different studies are due to different SR isolation techniques. To investigate this possibility, rat white and red gastrocnemius muscles from control and run animals were studied by using two conventional isolation techniques to obtain a crude microsomal fraction and an isolated SR vesicle (SRV) fraction. Indexes of CM and SRV function were compared with measurements from whole muscle homogenate. Treadmill running to exhaustion did not alter SR protein yields, percent SR extraction, or basal or Ca(2+)-ATPase purification in either fraction. Ca(2+)-activated ATPase activity was not altered by exercise in any of the fractions examined, but Ca2+ uptake was reduced in the homogenates (9.48 +/- 1.4 to 6.90 +/- 0.8 nmol . mg-1.min-1) and SRV fractions (84.0 +/- 11.5 to 50.7 +/- 14.0 nmol . mg-1.min-1) from the red gastrocnemius at free Ca2+ concentrations of 600-700 nM. These data indicate that reductions in SR Ca2+ uptake are dissociated from changes in Ca(2+)-ATPase in vitro and occur only in a specific population of vesicles. The mechanisms underlying these alterations are not known but may involve a reduction in the number of Ca(2+)-ATPase enzymes or a selective sedimentation of damaged vesicles in the SRV fraction.