ABSTRACT
BACKGROUND: Intra-arterial treatment of aneurysms by redirecting blood flow is a newer method. The redirection is based on a significantly more densely braided wire stent. The stent wall keeps the blood in the lumen of the stent and slows down the turbulent flow in the aneurysms. Stagnation of blood in the aneurysm sac leads to the formation of thrombus and subsequent exclusion of the aneurysm from the circulation. The aim of the study was to evaluate flow diverter device Pipeline for broad neck and giant aneurysm treatment. METHODS: Fifteen patients with discovered aneurysm of the internal carotid artery were treated between November 2010 and February 2014. The majority of aneurysms of the internal carotid artery were located intradural at the ophthalmic part of the artery. The patients were treated using a flow diverter device Pipeline, which was placed over the aneurysm neck. Treatment success was assessed clinically and angiographically using O'Kelly Marotta scale. RESULTS: Control angiography immediately after the release of the stent showed stagnation of the blood flow in the aneurysm sac. In none of the patients procedural and periprocedural complications were observed. 6 months after the procedure, control CT or MR angiography showed in almost all cases exclusion of the aneurysm from the circulation and normal blood flow in the treated artery. Neurological status six months after the procedure was normal in all patients. CONCLUSIONS: Treatment of aneurysms with flow diverter Pipeline device is a safe and significantly less time consuming method in comparison with standard techniques. This new method is a promising approach in treatment of broad neck aneurysms.
ABSTRACT
BACKGROUND: Haemangiomas of tongue are rare type of malformations. They can be treated mostly conservatively but in some cases they need more aggressive treatment with preoperative intra arterial embolization and surgical resection. Lesions of tongue that are localized superficially can also be treated with direct puncture and injection of sclerosing agent (absolute ethanol). CASE REPORT: We present a case of a 48 years old female patient, where we performed embolization of cavernous haemangioma with mixture of absolute ethanol and oil contrast. After the procedure the patient received analgetics and antioedematous therapy. After the sclerotization the planed surgery was abandoned. Control MRI examinations 6 and 12 months after the procedure showed only a small remnant of haemangioma and no signs of a larger relapse. CONCLUSIONS: In our case the direct puncture of haemangioma and sclerotherapy with ethanol proved to be a safe and effective method to achieve preoperative devascularization of the lesion. Direct puncture of the lesion is not limited by the anatomy of the vessels or vasospasm, which can occur during the intra-arterial approach.
ABSTRACT
PURPOSE: To evaluate effect of platelet-rich plasma gel (PRPG), locally administered during the anterior cruciate ligament (ACL) reconstruction, with two MRI methods. The proximal tibial tunnel was assessed with diffusion weighted imaging (DWI) and with dynamic contrast-enhanced imaging (DCE-MRI). MATERIALS AND METHODS: In 50 patients, standard arthroscopic ACL reconstructions were performed. The patients in the PRPG group (n = 25) received a local application of PRPG. The proximal tibial tunnel was examined by DWI and DCE-MRI, which were used to calculate apparent diffusion coefficient (ADC) values, as well as the contrast enhancement gradient (G(enh)) and enhancement factor (F(enh)) values. RESULTS: At 1 month, the calculated average ADC value in the PRPG group was significantly lower than in the control group. At 2.5 and at 6 months, G(enh) was significantly higher in the PRPG group. There were no significant differences in F(enh) between the groups at any control examination. CONCLUSION: DWI and DCE-MRI measurements indicate a reduced extent of edema during the first postoperative month as well as an increased vascular density and microvessel permeability in the proximal tibial tunnel at 1 and 2.5 postoperative months as the effect of the application of PRPG.
Subject(s)
Anterior Cruciate Ligament Injuries , Anterior Cruciate Ligament Reconstruction/methods , Arthroscopy/methods , Contrast Media/administration & dosage , Diffusion Magnetic Resonance Imaging/methods , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Joint Instability/surgery , Knee Injuries/surgery , Magnetic Resonance Imaging/methods , Platelet-Rich Plasma , Tibia/pathology , Administration, Topical , Adult , Capillary Permeability/physiology , Edema/diagnosis , Edema/prevention & control , Female , Gels , Humans , Male , Microcirculation/physiology , Middle Aged , Postoperative Complications/diagnosis , Postoperative Complications/prevention & control , Wound Healing/physiology , Young AdultABSTRACT
While serotonin (5-HT) co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+)-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.
Subject(s)
Insulin-Secreting Cells/metabolism , Insulin/metabolism , Protein Processing, Post-Translational , Serotonin/metabolism , rab GTP-Binding Proteins/metabolism , rab3A GTP-Binding Protein/metabolism , Animals , Cell Line , Diabetes Mellitus/etiology , Diabetes Mellitus/metabolism , Humans , Insulin Secretion , Intracellular Space/metabolism , Mice , Patch-Clamp Techniques , Rats , Transglutaminases/metabolism , Tryptophan Hydroxylase/deficiency , rab27 GTP-Binding ProteinsABSTRACT
Synapse formation at the neuromuscular junction (NMJ) requires an alternatively spliced variant of agrin (Z(+) agrin) that is produced only by neurons. Here, we show that Nova1 and Nova2, neuron-specific splicing factors identified as targets in autoimmune motor disease, are essential regulators of Z(+) agrin. Nova1/Nova2 double knockout mice are paralyzed and fail to cluster AChRs at the NMJ, and breeding them with transgenic mice constitutively expressing Z(+) agrin in motor neurons rescued AChR clustering. Surprisingly, however, these rescued mice remained paralyzed, while electrophysiologic studies demonstrated that the motor axon and synapse were functional-spontaneous and evoked recordings revealed synaptic transmission and muscle contraction. These results point to a proximal defect in motor neuron firing in the absence of Nova and reveal a previously unsuspected role for RNA regulation in the physiologic activation of motor neurons.
Subject(s)
Agrin/metabolism , Alternative Splicing/genetics , Antigens, Neoplasm/metabolism , Motor Neuron Disease/metabolism , Motor Neuron Disease/physiopathology , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/metabolism , Synapses/metabolism , Agrin/chemistry , Agrin/genetics , Amino Acid Sequence , Animals , Antigens, Neoplasm/genetics , Electrophysiology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Gene Expression Regulation , Mice , Mice, Knockout , Molecular Sequence Data , Motor Neuron Disease/genetics , Nerve Tissue Proteins/genetics , Neuro-Oncological Ventral Antigen , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/geneticsABSTRACT
OBJECTIVE: The aim of the study was to evaluate the feasibility of two quantitative MRI methods: diffusion weighted imaging (DWI) and dynamic contrast enhanced imaging (DCEI), for follow-up assessment of the tibial tunnel after reconstruction of the anterior cruciate ligament (ACL). MATERIALS AND METHODS: Twenty-three patients were examined by MRI at 1 and 6 months following ACL reconstruction. DWI and DCEI were utilized for evaluating the region of interest (ROI) within the proximal part of the tibial tunnel. From the resulting apparent diffusion coefficient (ADC) maps, ADC values were calculated. DCEI data were used to extract the enhancement factor (f(enh)) and the enhancement gradient (g(enh)) for the same ROI. RESULTS: Calculated ADC as well as the f(enh) and g(enh) had diminished to a statistically significant extent by 6 months after ACL reconstruction. The average ADC value diminished from 1.48 (10(-3) mm(2)/s) at 1 month to 1.30 (10(-3) mm(2)/s) at 6 months after reconstruction. The average f(enh) value decreased from 1.21 at 1 month to 0.50 at 6 months and the average g(enh) value decreased from 2.01%/s to 1.15%/s at 6 months, respectively. CONCLUSION: The study proved feasibility of DWI and DCEI for quantitative assessment of the tibial tunnel at 1 and 6 months after ACL reconstruction. Both methods have the potential for use as an additional tool in the evaluation of new methods of ACL reconstruction. To our knowledge, this is the first time quantitative MRI has been used in the follow-up to the ACL graft healing process.
Subject(s)
Anterior Cruciate Ligament/surgery , Image Enhancement/methods , Magnetic Resonance Imaging/methods , Postoperative Complications/diagnosis , Tibia/pathology , Adult , Anterior Cruciate Ligament Injuries , Anterior Cruciate Ligament Reconstruction/adverse effects , Contrast Media , Edema/diagnosis , Edema/etiology , Feasibility Studies , Female , Follow-Up Studies , Gadolinium DTPA , Graft Survival , Humans , Male , Postoperative Complications/etiology , Postoperative Period , Rupture/diagnosis , Rupture/etiology , Tibia/blood supply , Wound HealingABSTRACT
Acetylcholinesterase (EC 3.1.1.7, AChE) is one of the components of the neuromuscular junction (NMJ). Its expression and targeting in the skeletal muscle fiber is therefore under the control of the mechanisms responsible for the formation of the highly complex structure of this synapse. Recently, it has been demonstrated that myotubes of the C2C12 mouse muscle cell line form highly differentiated pretzel-like postsynaptic accumulations of acetylcholine receptors (AChRs) in the complete absence of the nerve if they are cultured on the laminin coating. This finding questions previously stressed importance of the nerve-derived factors in NMJ synaptogenesis and therefore deserves additional testing. The aim of this paper was to test whether the reported nerve-independency can be demonstrated also in the cultured human muscle meaning that the findings on C2C12 cultures can be extrapolated also to the human muscle. In our experiments aneurally cultured human myotubes failed to form AChR clusters on its surface, no matter if they were grown on normal gelatine or laminin coating. However, when innervated by neurons extending from the rat embryonic spinal cord, human myotubes formed AChR clusters with elaborate topography but strictly on the areas contacted by the nerve. One can hypothesize that higher nerve dependency of the NMJ synaptogenesis in humans in comparison to other species reflects species-specific differences in the organization of movement. Humans have the highest "fractionation of movement" capacity which probably requests different, more nerve-controlled development of the motor system including nerve-restricted development of the neuromuscular contacts.
Subject(s)
Neuromuscular Junction/physiology , Synapses/physiology , Acetylcholinesterase/metabolism , Animals , Cell Line , Mice , Microscopy, Confocal , Neuromuscular Junction/enzymology , Receptors, Cholinergic/metabolism , Synapses/enzymologyABSTRACT
OBJECTIVE: To analyse the early effect of colloidal 90Y beta irradiation, as used for radiation synovectomy (RSV), on the viability of human chondrocytes in an ex-vivo model. METHODS: Twenty osteochondral plugs (6 mm in diameter) were procured from femoral condyles of an adult male donor and stored in normal saline at 4 degrees C. The cartilage surfaces of 10 plugs were contaminated with colloidal 90Y citrate corresponding to the standard knee RSV dose (185 MBq) matched for the sample size (430 kBq). The remaining 10 plugs served as controls. At days 1, 2, 3, 6 and 13, two osteochondral plugs from each group were stained for viability with live/dead probes and scanned under a confocal laser microscope. The ratios of viable (green channel) and non-viable (red channel) pixels were acquired in four cartilage depth regions and statistically analysed with a regression model. RESULTS: The irradiation did not significantly alter the viable/non-viable pixel ratio during the first 2 days, but longer exposures led to a significant and time progressive reduction from 8.7% (day 3) to 12.5% (day 13). The ratio was less affected deeper in the cartilage, where it increased about 1% for every 100 microm from the surface. CONCLUSIONS: Surface exposure of human cadaveric cartilage to a therapeutic dose of colloidal 90Y decreased chondrocyte viability, expressed as the viable/non-viable pixel ratio, in the early post-irradiation period. The findings established in the ex-vivo simulation may reflect the changes in knee cartilage occurring after RSV therapy.
Subject(s)
Chondrocytes/metabolism , Colloids/chemistry , Radiopharmaceuticals/pharmacology , Synovectomy , Yttrium Radioisotopes/pharmacology , Cadaver , Cartilage/metabolism , Cartilage/pathology , Chondrocytes/pathology , Humans , Image Processing, Computer-Assisted , Knee , Male , Microscopy, Confocal , Middle Aged , Regression Analysis , Synovial Membrane/metabolism , TemperatureABSTRACT
Unlike rodent or avian muscle, which forms clusters of acetylcholine receptors (AChRs) on its surface, exhibits cross striations, and contracts spontaneously even if cultured in the absence of the nerve, human muscle must be innervated to reach such differentiation level under in vitro conditions (Kobayashi and Askanas, 1985; Mars et al., 2001). Because it is known that AChR clustering and other aspects of neuromuscular junction (NMJ) formation necessitate the activation of muscle-specific kinase (MuSK), one explanation of this inability of human muscle is that it has no MuSK or that it cannot be activated in the absence of the nerve. To test this hypothesis we analyzed cultured human muscle for the expression of MuSK at two stages of differentiation: postfusion myotube and innervated, contracting myotube. Analyses were carried out at the mRNA level, as no reliable anti-MuSK antibodies are available for the immunocytochemical demonstration of MuSK in cultured human muscle. The presence of MuSK, however, can be tested indirectly, as it can be activated in the absence of the nerve simply by growing muscle culture on laminin coating (Kummer et al., 2004). In the second part of our study, we therefore tested human myotubes for the presence and activation of MuSK by exposing them to laminin coating and by analyzing them afterwards for the areas of postsynaptic differentiation typical for NMJ formation.
Subject(s)
Muscle, Skeletal/innervation , Muscle, Skeletal/physiology , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cholinergic/genetics , DNA Primers , Humans , Organ Culture Techniques , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and skeletal muscle fibers. Essential for the formation of neuromuscular junction (NMJ) is the activation of MuSK, a muscle-specific receptor tyrosine kinase (DeChiara et al., 1996). In mice lacking MuSK, motor axons fail to stop and differentiate, acetylcholine receptors (AChRs) fail to cluster, and AChR genes are expressed uniformly in muscle (DeChiara et al., 1996; Gautam et al., 1996). The retrograde signals for presynaptic differentiation are not known. Because synapse-specific transcription, like presynaptic differentiation, is MuSK-dependent, it is possible that retrograde signals for presynaptic differentiation might be encoded by genes that are expressed preferentially by synaptic nuclei. To identify such synapse-specific genes we screened Affymetrix microarrays with RNA from the dissected, synapse-enriched, and extrasynaptic regions of skeletal muscle and further studied those genes that encode for the secreted or cell-surface proteins.
Subject(s)
Neuromuscular Junction/physiology , Oligonucleotide Array Sequence Analysis , Synapses/physiology , Acetylcholinesterase/genetics , Animals , CD24 Antigen/genetics , DNA-Binding Proteins/genetics , Mice , Muscle, Skeletal/physiology , Receptors, Cholinergic/genetics , Transcription Factors/geneticsABSTRACT
In spite of several reports demonstrating that acetylcholinesterase (AChE [EC 3.1.1.7]) expression is importantly regulated at the level of its mRNA, we still know little about the relationship between AChE mRNA level and the level of mature, catalytically active enzyme in the cell. Better insight into this relationship is, however, essential for our understanding of the molecular pathways underlying AChE synthesis in living cells. We have approached this problem previously (Grubic et al., 1995; Brank et al., 1998; Mis et al., 2003; Jevsek et al., 2004); however, recently introduced small interfering RNA (siRNA) methodology, which allows blockade of gene expression at the mRNA level, opens new possibilities in approaching the AChE mRNA-AChE activity relationship. With this technique one can eliminate AChE mRNA in the cell, specifically and at selected times, and follow the effects of such treatment at the mature enzyme level. In this study we followed AChE activity in siRNA-treated cultured human myoblasts. Our aim was to find out how the temporal profile of the AChE mRNA decrease is reflected at the level of AChE activity under normal conditions and after inhibition of preexisting AChE by diisopropyl phosphorofluoridate (DFP).AChE activity was determined at selected time intervals after siRNA treatment in both myoblast homogenates and in culture medium to follow the effects of siRNA treatment at the level of intracellular AChE synthesis and at the level of AChE secreted from the cell.
Subject(s)
Acetylcholinesterase/genetics , Gene Silencing , Muscle, Skeletal/enzymology , Cells, Cultured , Humans , Muscle, Skeletal/cytology , RNA, Messenger/genetics , Transcription, GeneticABSTRACT
BACKGROUND: Subarachnoid haemorrhage is a debilitating disease. The treatment options include surgical clipping or endovascular embolisation. Still, many controversies exist about which method is more convenient. METHODS: In the retrospective study from January 2006 to December 2013, 129 patients with subarachnoid haemorrhage were analysed. They were classified according to the WFNS grade and Fisher scale. The diagnosis of intracranial aneurysms was based on computerised tomographic angiography and digital subtraction angiography. All patients received the standard therapy against vasospasm. The treatment outcome was evaluated with Glasgow Outcome Scale. RESULTS: Of 129 patients, surgery was employed in 40, endovascular obliteration in 86 patients and 3 patients received both forms of treatment. Four factors were statistically significant for worse results in the univariate analysis: the age, WFNS grade, Fisher grade and the presence of clinical vasospasm. In the multivariate analysis, only the age, WFNS grade and the presence of clinical vasospasm remained statistically significant for the outcome. There was a trend towards better outcome for the patients that had endovascular treatment compared with patients who were treated surgically, although the difference was not statistically significant. CONCLUSIONS: Although the endovascular embolisation in relation to the surgical clipping is becoming a more popular treatment method for ruptured cerebral aneurysms it cannot offer reliable endovascular exclusion in all types of aneurysms. Based on our experience, it is therefore necessary to look at these two methods as complementary that may both be used separately or in combination for the well-being of the patient.
Subject(s)
Embolization, Therapeutic/statistics & numerical data , Endovascular Procedures/statistics & numerical data , Intracranial Aneurysm/therapy , Neurosurgical Procedures/statistics & numerical data , Subarachnoid Hemorrhage/therapy , Adolescent , Adult , Aged , Comorbidity , Female , Humans , Intracranial Aneurysm/epidemiology , Male , Middle Aged , Prevalence , Risk Factors , Slovenia/epidemiology , Subarachnoid Hemorrhage/epidemiology , Treatment Outcome , Vasospasm, Intracranial/epidemiology , Vasospasm, Intracranial/prevention & control , Young AdultABSTRACT
The results of our recent investigations on the expression and distribution of acetylcholinesterase (EC. 3.1.1.7, AChE) in the experimental model of the in vitro innervated human muscle are summarized and discussed here. This is the only model allowing studies on AChE expression at all stages of the neuromuscular junction (NMJ) formation in the human muscle. Since it consists not only of the motor neurons and myotubes but also of glial cells, which are essential for the normal development of the motor neurons, NMJs become functional and differentiated in this system. We followed AChE expression at various stages of the NMJ formation and in the context of other events characteristic for this process. Neuronal and muscular part were analysed at both, mRNA and mature enzyme level. AChE is expressed in motor neurons and skeletal muscle at the earliest stages of their development, long before NMJ starts to form and AChE begins to act as a cholinergic component. Temporal pattern of AChE mRNA expression in motor neurons is similar to the pattern of mRNA encoding synaptogenetic variant of agrin. There are no AChE accummulations at the NMJ at the early stage of its formation, when immature clusters of nicotinic receptors are formed at the neuromuscular contacts and when occasional NMJ-mediated contractions are already observed. The transformation from immature, bouton-like neuromuscular contacts into differentiated NMJs with mature, compact receptor clusters, myonuclear accumulations and dense AChE patches begins at the time when basal lamina starts to form in the synaptic cleft. Our observations support the concept that basal lamina formation is the essential event in the transformation of immature neuromuscular contact into differentiated NMJ, with the accumulation of not only muscular but also neuronal AChE in the synaptic cleft.
Subject(s)
Acetylcholinesterase/metabolism , Gene Expression Regulation, Enzymologic , Muscle Fibers, Skeletal/enzymology , Neuromuscular Junction/enzymology , Acetylcholinesterase/genetics , Animals , Cell Differentiation , Coculture Techniques , Humans , Muscle Fibers, Skeletal/cytology , Neuromuscular Junction/cytology , Rats , Spinal Cord/cytology , Spinal Cord/embryology , Spinal Cord/enzymologyABSTRACT
In spite of intensive investigations, the roles of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) in the central nervous system (CNS) remain unclear. A role recently proposed for BuChE as an explanation for survival of AChE knockout mice is compensation for AChE activity if it becomes insufficient. Neuronal contribution of both enzymes to the cholinesterase pool in the neuromuscular junction has also been suggested. These proposals imply that BuChE expression follows that of AChE and that, in addition to AChE, BuChE is also expressed in alpha-motor neurons. However, these assumptions have not yet been properly tested. Histochemical approaches to these problems have been hampered by a number of problems that prevent unambiguous interpretation of results. In situ hybridization (ISH) of mRNAs encoding AChE and BuChE, which is the state-of-the-art approach, has not yet been done. Here we describe rapid nonradioactive ISH for the localization of mRNAs encoding AChE and BuChE. Various probes and experimental conditions had been tested to obtain reliable localization. In combination with RT-PCR, ISH revealed that, in rat spinal cord, cells expressing AChE mRNA also express BuChE mRNA but in smaller quantities. alpha-Motor neurons had the highest levels of both mRNAs. Virtual absence of transcripts encoding AChE and BuChE in glia might reflect a discrepancy between mRNA and enzyme levels previously reported for cholinesterases.
Subject(s)
Acetylcholinesterase/metabolism , Butyrylcholinesterase/metabolism , RNA, Messenger/metabolism , Spinal Cord/enzymology , Acetylcholinesterase/genetics , Animals , Butyrylcholinesterase/genetics , Cell Count , Choline O-Acetyltransferase/metabolism , Female , Fluorescent Dyes , Immunohistochemistry , In Situ Hybridization/methods , RNA Probes , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , ThoraxABSTRACT
The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.
Subject(s)
Agrin/metabolism , Calcium Signaling , Cell Differentiation , Muscle Development , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Paracrine Communication , Spinal Cord/metabolism , Animals , Caffeine/pharmacology , Calcium Channels, L-Type/metabolism , Calcium Signaling/drug effects , Cells, Cultured , Chickens , Child , Child, Preschool , Coculture Techniques , Culture Media, Conditioned/metabolism , Humans , Mice , Microscopy, Fluorescence , Microscopy, Video , Muscle Fibers, Skeletal/drug effects , Muscle, Skeletal/drug effects , Muscle, Skeletal/growth & development , Muscle, Skeletal/innervation , Patch-Clamp Techniques , Rats , Rats, Wistar , Recombinant Proteins/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Spinal Cord/embryology , Time Factors , Tissue Culture TechniquesABSTRACT
A frequent finding in patients surviving critical illness myopathy is chronic muscle dysfunction. Its pathogenesis is mostly unknown; one explanation could be that muscle regeneration, which normally follows myopathy, is insufficient in these patients because of a high glucocorticoid level in their blood. Glucocorticoids can prevent stimulatory effects of proinflammatory factors on the interleukin (IL)-6 secretion, diminishing in this way the autocrine and paracrine IL-6 actions known to stimulate proliferation at the earliest, myoblast stage of muscle formation. To test this hypothesis, we compared the effects of major proinflammatory agents [tumor necrosis factor (TNF)-alpha and endotoxin lipopolysaccharide (LPS)] on the IL-6 secretion from the muscle precursors and then studied the influence of dexamethasone (Dex) on these effects. Mononuclear myoblasts, which still proliferate, were compared with myotubes in which this capacity is already lost. For correct interpretation of results, cultures were examined for putative apoptosis and necrosis. We found that constitutive secretion of IL-6 did not differ significantly between myoblasts and myotubes; however, the TNF-alpha- and LPS-stimulated IL-6 release was more pronounced (P < 0.001) in myoblasts. Dex, applied at the 0.1-100 nM concentration range, prevented constitutive and TNF-alpha- and LPS-stimulated IL-6 release at both developmental stages but only at high concentration (P < 0.01). Although there are still missing links to it, our results support the concept that high concentrations of glucocorticoids, met in critically ill patients, prevent TNF-alpha- and LPS-stimulated IL-6 secretion. This results in reduced IL-6-mediated myoblast proliferation, leading to the reduced final mass of the regenerated muscle.
Subject(s)
Dexamethasone/administration & dosage , Interleukin-6/metabolism , Lipopolysaccharides/administration & dosage , Muscle, Skeletal/cytology , Muscle, Skeletal/physiology , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/metabolism , Regeneration/physiology , Tumor Necrosis Factor-alpha/administration & dosage , Cell Differentiation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Combinations , Granulocyte Precursor Cells/drug effects , Granulocyte Precursor Cells/metabolism , Humans , Muscle, Skeletal/drug effects , Myoblasts, Skeletal/drug effects , Regeneration/drug effectsABSTRACT
The genes encoding several synaptic proteins, including acetylcholine receptors, acetylcholinesterase, and the muscle-specific kinase, MuSK, are expressed selectively by a small number of myofiber nuclei positioned near the synaptic site. Genetic analysis of mutant mice suggests that additional genes, expressed selectively by synaptic nuclei, might encode muscle-derived retrograde signals that regulate the differentiation of motor axon terminals. To identify candidate retrograde signals, we used a microarray screen to identify genes that are preferentially expressed in the synaptic region of muscle, and we analyzed one such gene, CD24, further. We show that CD24, which encodes a small, variably and highly glycosylated, glycosylphosphatidylinositol (GPI)-linked protein, is expressed preferentially by myofiber synaptic nuclei in embryonic and adult muscle, and that CD24 expression is restricted to the central region of muscle independent of innervation. Moreover, we show that CD24 has a role in presynaptic differentiation, because synaptic transmission is depressed and fails entirely, in a cyclical manner, after repetitive stimulation of motor axons in CD24 mutant mice. These deficits in synaptic transmission, which are accompanied by aberrant stimulus-dependent uptake of AM1-43 from axons, indicate that CD24 is required for normal presynaptic maturation and function. Because CD24 is also expressed in some neurons, additional experiments will be required to determine whether pre- or postsynaptic CD24 mediates these effects on presynaptic development and function.
Subject(s)
CD24 Antigen/metabolism , Muscle Fibers, Skeletal/metabolism , Neuromuscular Junction/metabolism , Synaptic Transmission , Animals , CD24 Antigen/genetics , Cell Nucleus/metabolism , Male , Mice , Mice, Mutant Strains , Molecular Sequence Data , Muscle Fibers, Skeletal/chemistry , Muscle, Skeletal/chemistry , Muscle, Skeletal/innervation , Muscle, Skeletal/metabolism , Neural Cell Adhesion Molecules/metabolism , Neuromuscular Junction/chemistry , Neuromuscular Junction/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/analysis , RNA, Messenger/metabolism , Synaptic Transmission/geneticsABSTRACT
We describe a new approach for studying insulin-induced exocytosis in individual, well-differentiated, innervated human muscle fibres. We used an in vitro system in which motor axons extending from embryonic rat spinal cord explants functionally innervate co-cultured human muscle fibres. Under such conditions, the human muscle fibres reach a high degree of differentiation that is never observed in non-innervated, cultured human muscle fibres. To monitor insulin-induced membrane dynamics, we used confocal microscopy to measure the fluorescence intensity changes of the styryl dye FM1-43, a marker for membrane area. The fluorescence intensity increased after insulin stimulation. This increase, as well as the intensity of staining for the glucose transporter 4 (GLUT4), was significantly higher in the innervated and contracting fibres than in myoblasts and myotubes. This shows that in vitro innervation of human muscle cells not only enhances the differentiation stage but also improves the insulin response. Our approach allows continuous monitoring and quantitative assessment of insulin-induced increase in cumulative exocytosis in individual human muscle fibres at a differentiation level practically corresponding to that of adult muscle. It is therefore a suitable system for studying various parameters affecting the mechanisms underlying insulin-induced GLUT4 translocation in human skeletal muscle.
Subject(s)
Exocytosis/physiology , Insulin/metabolism , Microscopy, Fluorescence/methods , Muscle Denervation , Muscle Fibers, Skeletal/metabolism , Animals , Cells, Cultured , Female , Fluorescent Dyes , Glucose Transporter Type 4 , Humans , In Vitro Techniques , Monosaccharide Transport Proteins/metabolism , Muscle Fibers, Skeletal/cytology , Muscle Proteins/metabolism , Pregnancy , Pyridinium Compounds , Quaternary Ammonium Compounds , Rats , Rats, WistarABSTRACT
Synaptic basal lamina is interposed between the pre- and postsynaptic membrane of the neuromuscular junction (NMJ). This position permits deposition of basal lamina-bound NMJ components of both neuronal and muscle fibre origin. One such molecule is acetylcholinesterase (AChE). The origin of NMJ AChE has been investigated previously as the answer would elucidate the relative contributions of muscle fibers and motor neurons to NMJ formation. However, in the experimental models used in prior investigations either the neuronal or muscular components of the NMJs were removed, or the NMJs were poorly differentiated. Therefore, the question of AChE origin in the intact and functional NMJ remains open. Here, we have approached this question using an in vitro model in which motor neurons, growing from embryonic rat spinal cord explants, form well differentiated NMJs with cultured human myotubes. By immunocytochemical staining with species-specific anti-AChE antibodies, we are able to differentiate between human (muscular) and rat (neuronal) AChE at the NMJ. We observed strong signal at the NMJ after staining with human AChE antibodies, which suggests a significant muscular AChE contribution. However, a weaker, but still clearly recognizable signal is observed after staining with rat AChE antibodies, suggesting a smaller fraction of AChE was derived from motor neurons. This is the first report demonstrating that both motor neuron and myotube contribute synaptic AChE under conditions where they interact with each other in the formation of an intact and functional NMJ.