Functional characterization of murine muscle spindles

Coordinated movements require proprioceptive information, such as information about muscle tone as well as position and movement of extremities in space. The primary proprioceptive sensory receptors are muscle spindles. Muscle spindles are complex stretch-sensitive mechanoreceptors. They detect how much and how fast a muscle is lengthened. Muscle spindles consist of specialised skeletal muscle fibers, so called intrafusal fibers. In their central part, these fibers are surrounded by a proprioceptive afferent sensory neuron in an annulospiral shape. Here the speed as well as the length of the stretch is translated into action potential frequencies, which are proportional to the length change and the speed thereof. Both polar endings are innervated by efferent γ-motoneurons. Previously it was shown that AChRs are concentrated in the polar region at the contact site between intrafusal fiber and sensory neuron. To investigate the function of these AChRs, extracellular recordings from single unit proprioceptive-afferents of wildtype murine extensor digitorum longus muscles in the absence of γ-motoneuron activity was performed. I investigated the response during ramp-and-hold stretches as well as during sinusoidal vibrations in the presence and absence of the AChR inhibitors d-tubocurarine, α-bungarotoxin or of the choline reuptake inhibitor hemicholinium-3. In the presence of either drug, the resting action potential discharge frequency was not altered but the stretch-evoked action potential frequencies were increased. Additionally, the firing rate during sinusoidal vibrations at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles. These results indicate that ACh modulates muscle spindle function during stretch in the central region of intrafusal fibers by possibly fine-tuning muscle spindle sensitivity. As a second project, I investigated the morphology and function of muscle spindles from murine models of muscular dystrophies. Muscular dystrophies comprise a heterogeneous group of hereditary diseases, which are all characterised by progressive degeneration and weakness of skeletal muscles. Murine model systems for two distinct types of muscular dystrophy with very different disease etiologies, i.e. dystrophin- and dysferlin-deficient mice, were analysed. The total number and the overall structure of muscle spindles in soleus muscles of these mice appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β-dystroglycan in intrafusal fibers outside the region of contact to the sensory neuron. Moreover, extracellular recordings from single units of sensory afferents from muscle spindles of the extensor digitorum longus muscle were performed during ramp-and-hold stretches, as well as during sinusoidal vibrations. I demonstrate that mouse models for muscular dystrophy have an increased resting discharge but no change during the dynamic or static phase of ramp-and-hold stretches. Mutant muscle spindles show a higher action potential firing rate during sinusoidal vibrations with small amplitudes and low frequencies. I observed no exacerbated phenotype in DMDmdx- dysf-/- double transgenic mice compared to either single transgenic animal. These results demonstrate that a lack of dystrophin and or dysferlin lead to a change in muscle spindle function and suggest that an impaired proprioceptive feedback might contribute to the instable gait and the frequent falls in patients with muscular dystrophy. To test the hypothesis that an increased intracellular calcium ion concentration [Ca2+] in dystrophic muscles could cause the impaired proprioceptive function, extracellular recordings from single units of sensory afferents from muscle spindles of the extensor digitorum longus muscle were performed during ramp-and-hold stretches, as well as during sinusoidal vibrations in the presence and absence of the AChE inhibitor neostigmine and the calcium channel blocker nifedipine. After nifedipine and neostigmine administration an increased resting discharge but no change during the dynamic or static phase of ramp-and-hold stretches as well as a higher action potential firing rate during sinusoidal vibrations after neostigmine administration with small amplitudes and low frequencies was observed. Overall, I show that murine models of muscular dystrophy have an impaired muscle spindle function, which could contribute to the instable gait and posture observed in patients with muscular dystrophy, that these changes could be due to an increased intracellular [Ca2+] in muscles and that the AChR in the central part of the muscle spindles negatively modulates muscle spindle responses during stretch

Functional characterization of murine muscle spindles

Coordinated movements require proprioceptive information, such as information about muscle tone as well as position and movement of extremities in space. The primary proprioceptive sensory receptors are muscle spindles. Muscle spindles are complex stretch-sensitive mechanoreceptors. They detect how much and how fast a muscle is lengthened. Muscle spindles consist of specialised skeletal muscle fibers, so called intrafusal fibers. In their central part, these fibers are surrounded by a proprioceptive afferent sensory neuron in an annulospiral shape. Here the speed as well as the length of the stretch is translated into action potential frequencies, which are proportional to the length change and the speed thereof. Both polar endings are innervated by efferent γ-motoneurons. Previously it was shown that AChRs are concentrated in the polar region at the contact site between intrafusal fiber and sensory neuron. To investigate the function of these AChRs, extracellular recordings from single unit proprioceptive-afferents of wildtype murine extensor digitorum longus muscles in the absence of γ-motoneuron activity was performed. I investigated the response during ramp-and-hold stretches as well as during sinusoidal vibrations in the presence and absence of the AChR inhibitors d-tubocurarine, α-bungarotoxin or of the choline reuptake inhibitor hemicholinium-3. In the presence of either drug, the resting action potential discharge frequency was not altered but the stretch-evoked action potential frequencies were increased. Additionally, the firing rate during sinusoidal vibrations at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles. These results indicate that ACh modulates muscle spindle function during stretch in the central region of intrafusal fibers by possibly fine-tuning muscle spindle sensitivity. As a second project, I investigated the morphology and function of muscle spindles from murine models of muscular dystrophies. Muscular dystrophies comprise a heterogeneous group of hereditary diseases, which are all characterised by progressive degeneration and weakness of skeletal muscles. Murine model systems for two distinct types of muscular dystrophy with very different disease etiologies, i.e. dystrophin- and dysferlin-deficient mice, were analysed. The total number and the overall structure of muscle spindles in soleus muscles of these mice appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β-dystroglycan in intrafusal fibers outside the region of contact to the sensory neuron. Moreover, extracellular recordings from single units of sensory afferents from muscle spindles of the extensor digitorum longus muscle were performed during ramp-and-hold stretches, as well as during sinusoidal vibrations. I demonstrate that mouse models for muscular dystrophy have an increased resting discharge but no change during the dynamic or static phase of ramp-and-hold stretches. Mutant muscle spindles show a higher action potential firing rate during sinusoidal vibrations with small amplitudes and low frequencies. I observed no exacerbated phenotype in DMDmdx- dysf-/- double transgenic mice compared to either single transgenic animal. These results demonstrate that a lack of dystrophin and or dysferlin lead to a change in muscle spindle function and suggest that an impaired proprioceptive feedback might contribute to the instable gait and the frequent falls in patients with muscular dystrophy. To test the hypothesis that an increased intracellular calcium ion concentration [Ca2+] in dystrophic muscles could cause the impaired proprioceptive function, extracellular recordings from single units of sensory afferents from muscle spindles of the extensor digitorum longus muscle were performed during ramp-and-hold stretches, as well as during sinusoidal vibrations in the presence and absence of the AChE inhibitor neostigmine and the calcium channel blocker nifedipine. After nifedipine and neostigmine administration an increased resting discharge but no change during the dynamic or static phase of ramp-and-hold stretches as well as a higher action potential firing rate during sinusoidal vibrations after neostigmine administration with small amplitudes and low frequencies was observed. Overall, I show that murine models of muscular dystrophy have an impaired muscle spindle function, which could contribute to the instable gait and posture observed in patients with muscular dystrophy, that these changes could be due to an increased intracellular [Ca2+] in muscles and that the AChR in the central part of the muscle spindles negatively modulates muscle spindle responses during stretch

Clonal hematopoiesis in patients with stem cell mobilization failure:a nested case-control study

Inadequate mobilization of peripheral blood progenitor cells (PBPCs) is a limiting factor to proceeding with autologous hematopoietic cell transplantation (auto-HCT). To assess the impact of clonal hematopoiesis (CH) on mobilization failure of PBPC for auto-HCT, we investigated the characteristics of poor mobilizers (with a total PBPC collection &lt;2 × 106 CD34+ cells per kg) in a consecutive single-center cohort of 776 patients. Targeted error-corrected next-generation sequencing of 28 genes was performed in a nested case-control cohort of 90 poor mobilizers and 89 matched controls. CH was detected in 48 out of 179 patients (27%), with most patients carrying a single mutation. The presence of CH (detected at variant allele frequency [VAF] ≥ 1%) did not associate with poor mobilization potential (31% vs 22% in controls, odds ratio, 1.55; 95% confidence interval, 0.76-3.23; P = .238). PPM1D mutations were detected more often in poor mobilizers (P = .005). In addition, TP53 mutations in this cohort were detected exclusively in patients with poor mobilization potential (P = .06). The incidence of therapy-related myeloid neoplasms (t-MN) was higher among patients with mobilization failure (P = .014). Although poor mobilizers experienced worse overall survival (P = .019), this was not affected by the presence of CH. We conclude that CH at low VAF (1%-10%) is common at the time of stem cell mobilization. TP53 mutations and PPM1D mutations are associated with poor mobilization potential and their role in subsequent development of t-MN in these individuals should be established.</p

Evaluation of the Academy of Finland

The objective of the evaluation was to produce a comprehensive view of the activities of the Academy of Finland based on international and national expertise and to find means for improving the Academy's impact, operation and structures. Overall conclusion is that the Academy is working well, given the budgetary and policy constrains within which it functions, but that these constraints need to be addressed if the research and innovation system in Finland is once more to be well governed and can therefore allow the Academy to make a fuller contribution. The government needs a way to regain systemic perspective, and to decide how and to what extent it will tackle the societal challenges by thinking in terms of systemic transitions in addition to stable innovation systems. The current activities in government towards achieving the 4% target and reforming research and innovation funding are encouraging signs that this perspective can be regained, and a new dynamism injected into Finnish research and innovation policy. Based on the analysis and conclusions the evaluation gives recommendations to the government, to the Ministry and the Academy, and of the future role of the Academy

Contributions of myofascial pain in diagnosis and treatment of shoulder pain. A randomized control trial

<p>Abstract</p> <p>Background</p> <p>Rotator cuff tendinopathy and subacromial impingement syndrome present complex patomechanical situations, frequent difficulties in clinical diagnosis and lack of effectiveness in treatment. Based on clinical experience, we have therefore considered the existence of another pathological entity as the possible origin of pain and dysfunction. The hypothesis of this study is to relate subacromial impingement syndrome (SIS) with myofascial pain syndrome (MPS), since myofascial trigger points (MTrPs) cause pain, functional limitation, lack of coordination and alterations in quality of movement, even prior to a tendinopathy. MTrPs can coexist with any degenerative subacromial condition. If they are not taken into consideration, they could perpetuate and aggravate the problem, hindering diagnosis and making the applied treatments ineffective.</p> <p>The aims and methods of this study are related with providing evidence of the relationship that may exist between this condition and MPS in the diagnosis and treatment of rotator cuff tendonitis and/or SIS.</p> <p>Method/design</p> <p>A descriptive transversal study will be made to find the correlation between the diagnosis of SIS and rotator cuff tendonitis, positive provocation test responses, the existence of active MTrPs and the results obtained with ultrasonography (US) and Magnetic Renonance Imaging (MRI). A randomized double blinded clinical trial will be carried out in experimental conditions: A Protocolized treatment based on active and passive joint repositioning, stabilization exercises, stretching of the periarticular shoulder muscles and postural reeducation. B. The previously described protocolized treatment, with the addition of dry needling applied to active MTrPs with the purpose of isolating the efficacy of dry needling in treatment.</p> <p>Discussion</p> <p>This study aims to provide a new vision of shoulder pain, from the perspective of MPS. This syndrome can, by itself, account for shoulder pain and dysfunction, although it can coexist with real conditions involving the tendons.</p> <p>Trail Registration</p> <p>ISRCTN Number: 30907460</p

The effect of methocarbamol and mexiletine on murine muscle spindle function

Introduction/Aims The muscle relaxant methocarbamol and the antimyotonic drug mexiletine are widely used for the treatment of muscle spasms, myotonia, and pain syndromes. To determine whether these drugs affect muscle spindle function, we studied their effect on the resting discharge and on stretch-induced action potential frequencies of proprioceptive afferent neurons. Methods Single unit action potential frequencies of proprioceptive afferents from muscle spindles in the murine extensor digitorum longus muscle of adult C57BL/6J mice were recorded under resting conditions and during ramp-and-hold stretches. Maximal tetanic force of the same muscle after direct stimulation was determined. High-resolution confocal microscopy analysis was performed to determine the distribution of Na(v)1.4 channels, a potential target for both drugs. Results Methocarbamol and mexiletine inhibited the muscle spindle resting discharge in a dose-dependent manner with IC50 values around 300 mu M and 6 mu M, respectively. With increasing concentrations of both drugs, the response to stretch was also affected, with the static sensitivity first followed by the dynamic sensitivity. At high concentrations, both drugs completely blocked muscle spindle afferent output. Both drugs also reversibly reduced the specific force of the extensor digitorum longus muscle after tetanic stimulation. Finally, we present evidence for the presence and specific localization of the voltage-gated sodium channel Na(v)1.4 in intrafusal fibers. Discussion In this study we demonstrate that both muscle relaxants affect muscle spindle function, suggesting impaired proprioception as a potential side effect of both drugs. Moreover, our results provide additional evidence of a peripheral activity of methocarbamol and mexiletine

Real-time detection of p-phenylenediamine penetration into human skin by in vivo Raman spectroscopy

Background. Penetration, autoxidation and N-acetylation of p-phenylenediamine (PPD) have been studied in vitro and ex vivo. However, a clear understanding of in vivo PPD penetration and the formation of PPD derivatives is lacking. Objectives. To obtain insights into the in vivo penetration, clearance and formation of PPD derivatives in human skin. Methods. Patch test chambers containing PPD 1% pet. were applied to the forearms of two human volunteers, with increasing application times. Non-invasive Raman microspectroscopy was used for detection of PPD (derivatives) in skin at several follow-up times. Results. Application of a PPD 1% pet. patch for 30 min resulted in substantial amounts of PPD in the stratum corneum of 90 mg PPD/g keratin. PPD contents were highest after three applications for 1 h each (330 mg PPD/g keratin), followed by single applications for 2 h 40 min, 2 h, and 23 h. The PPD half-time in the skin was 3 h. No spectral contributions of Bandrowski's base, monoacetyl-PPD and diacetyl-PPD were detected. Conclusions. We have gained insights into the in vivo penetration of PPD in human skin by using non-invasive Raman spectroscopy. Penetration into the skin was fast, and the PPD concentrations detected in the stratum corneum were high. PPD was detected in both the stratum corneum and the viable epidermis. Oxidized or acetylated PPD derivatives could not be detected