Noninvasive Electromagnetic Phrenic Nerve Stimulation in Critically Ill Patients: A Feasibility Study.

BACKGROUND: Electromagnetic stimulation of the phrenic nerve induces diaphragm contractions, but no coils for clinical use have been available. We recently demonstrated the feasibility of ventilation using bilateral transcutaneous noninvasive electromagnetic phrenic nerve stimulation (NEPNS) before surgery in lung-healthy, normal-weight patients in a dose-dependent manner. RESEARCH QUESTION: Is NEPNS feasible in critically ill patients in an ICU setting? STUDY DESIGN AND METHODS: This feasibility nonrandomized controlled study aimed to enroll patients within 36 h of intubation who were expected to remain ventilated for ≥ 72 h. The intervention group received 15-min bilateral transcutaneous NEPNS bid, whereas the control group received standard care. If sufficient, NEPNS was used without pressure support to ventilate the patient; pressure support was added if necessary to ventilate the patient adequately. The primary outcome was feasibility, measured as time to find the optimal stimulation position. Further end points were sessions performed according to the protocol or allowing a next-day catch-up session and tidal volume achieved with stimulation reaching only 3 to 6 mL/kg ideal body weight (IBW). A secondary end point was expiratory diaphragm thickness measured with ultrasound from days 1 to 10 (or extubation). RESULTS: The revised European Union regulation mandated reapproval of medical devices, prematurely halting the study. Eleven patients (five in the intervention group, six in the control group) were enrolled. The median time to find an adequate stimulation position was 23 s (interquartile range, 12-62 s). The intervention bid was executed in 87% of patients, and 92% of patients including a next-day catch-up session. Ventilation with 3 to 6 mL/kg IBW was achieved in 732 of 1,701 stimulations (43.0%) with stimulation only and in 2,511 of 4,036 stimulations (62.2%) with additional pressure support. A decrease in diaphragm thickness was prevented by bilateral NEPNS (P = .034) until day 10. INTERPRETATION: Bilateral transcutaneous NEPNS was feasible in the ICU setting with the potential benefit of preventing diaphragm atrophy during mechanical ventilation. NEPNS ventilation effectiveness needs further assessment. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT05238753; URL: www. CLINICALTRIALS: gov.

First non-invasive magnetic phrenic nerve and diaphragm stimulation in anaesthetized patients: a proof-of-concept study.

BACKGROUND: Mechanical ventilation has side effects such as ventilator-induced diaphragm dysfunction, resulting in prolonged intensive care unit length of stays. Artificially evoked diaphragmatic muscle contraction may potentially maintain diaphragmatic muscle function and thereby ameliorate or counteract ventilator-induced diaphragm dysfunction. We hypothesized that bilateral non-invasive electromagnetic phrenic nerve stimulation (NEPNS) results in adequate diaphragm contractions and consecutively in effective tidal volumes. RESULTS: This single-centre proof-of-concept study was performed in five patients who were 30 [IQR 21-33] years old, 60% (n = 3) females and undergoing elective surgery with general anaesthesia. Following anaesthesia and reversal of muscle relaxation, patients received bilateral NEPNS with different magnetic field intensities (10%, 20%, 30%, 40%); the stimulation was performed bilaterally with dual coils (connected to one standard clinical magnetic stimulator), specifically designed for bilateral non-invasive electromagnetic nerve stimulation. The stimulator with a maximal output of 2400 Volt, 160 Joule, pulse length 160 µs at 100% intensity was limited to 50% intensity, i.e. each single coil had a maximal output of 0.55 Tesla and 1200 Volt. There was a linear relationship between dosage (magnetic field intensity) and effect (tidal volume, primary endpoint, p < 0.001). Mean tidal volume was 0.00, 1.81 ± 0.99, 4.55 ± 2.23 and 7.43 ± 3.06 ml/kg ideal body weight applying 10%, 20%, 30% and 40% stimulation intensity, respectively. Mean time to find an initial adequate stimulation point was 89 (range 15-441) seconds. CONCLUSIONS: Bilateral non-invasive electromagnetic phrenic nerve stimulation generated a tidal volume of 3-6 ml/kg ideal body weight due to diaphragmatic contraction in lung-healthy anaesthetized patients. Further perspectives in critically ill patients should include assessment of clinical outcomes to confirm whether diaphragm contraction through non-invasive electromagnetic phrenic nerve stimulation potentially ameliorates or prevents diaphragm atrophy.

Safety and Feasibility of Noninvasive Electromagnetic Stimulation of the Phrenic Nerves.

BACKGROUND: Mechanical ventilation is widely used in ICU patients as a lifesaving intervention. Diaphragmatic atrophy and thinning occur from lack of contractions of the diaphragm during mechanical ventilation. It may prolong weaning and increase the risk of respiratory complications. Noninvasive electromagnetic stimulation of the phrenic nerves may ameliorate the atrophy seen with ventilation. The objective of this study was to show that noninvasive repetitive electromagnetic stimulation is safe, feasible, and effective to stimulate the phrenic nerves in both awake individuals and anesthetized patients. METHODS: A single-center study with 10 subjects overall, 5 awake volunteers and 5 anesthetized subjects. We used a prototype electromagnetic, noninvasive, simultaneous bilateral phrenic nerve stimulation device in both groups. In the awake volunteers, we assessed time-to-first capture of the phrenic nerves and safety measures, such as pain, discomfort, dental paresthesia, and skin irritation. In the anesthetized subjects, time-to-first capture as well as tidal volumes and airway pressures at 20%, 30%, and 40% stimulation intensity were assessed. RESULTS: Diaphragmatic capture was achieved in all the subjects within a median (range) of 1 min (1 min to 9 min 21 s) for the awake subjects and 30 s (20 s to 1 min 15 s) for the anesthetized subjects. There were no adverse or severe adverse events in either group, nor any dental paresthesia, skin irritation, or subjective pain in the stimulated area. Tidal volumes increased in all the subjects in response to simultaneous bilateral phrenic nerve stimulation and increased gradually with increasing stimulation intensity. Airway pressures corresponded to spontaneous breathing of ∼2 cm H2O. CONCLUSIONS: Noninvasive phrenic nerve stimulation can be safely performed in awake and anesthetized individuals. It was feasible and effective in stimulating the diaphragm by induction of physiologic and scalable tidal volumes with minimum positive airway pressures.

Feasibility of transesophageal phrenic nerve stimulation.

BACKGROUND: Every year, more than 2.5 million critically ill patients in the ICU are dependent on mechanical ventilation. The positive pressure in the lungs generated by the ventilator keeps the diaphragm passive, which can lead to a loss of myofibers within a short time. To prevent ventilator-induced diaphragmatic dysfunction (VIDD), phrenic nerve stimulation may be used. OBJECTIVE: The goal of this study is to show the feasibility of transesophageal phrenic nerve stimulation (TEPNS). We hypothesize that selective phrenic nerve stimulation can efficiently activate the diaphragm with reduced co-stimulations. METHODS: An in vitro study in saline solution combined with anatomical findings was performed to investigate relevant stimulation parameters such as inter-electrode spacing, range to target site, or omnidirectional vs. sectioned electrodes. Subsequently, dedicated esophageal electrodes were inserted into a pig and single stimulation pulses were delivered simultaneously with mechanical ventilation. Various stimulation sites and response parameters such as transdiaphragmatic pressure or airway flow were analyzed to establish an appropriate stimulation setting. RESULTS: Phrenic nerve stimulation with esophageal electrodes has been demonstrated. With a current amplitude of 40 mA, similar response figures of the diaphragm activation as compared to conventional stimulation with needle electrodes at 10mA were observed. Directed electrodes best aligned with the phrenic nerve resulted in up to 16.9 % higher amplitude at the target site in vitro and up to 6 cmH20 higher transdiaphragmatic pressure in vivo as compared to omnidirectional electrodes. The activation efficiency was more sensitive to the stimulation level inside the esophagus than to the inter-electrode spacing. Most effective and selective stimulation was achieved at the level of rib 1 using sectioned electrodes 40 mm apart. CONCLUSION: Directed transesophageal phrenic nerve stimulation with single stimuli enabled diaphragm activation. In the future, this method might keep the diaphragm active during, and even support, artificial ventilation. Meanwhile, dedicated sectioned electrodes could be integrated into gastric feeding tubes.

Visual Attention and Saccadic Oculomotor Control in Parkinson's Disease.

BACKGROUND: In patients with Parkinson's disease (PD) we aimed at differentiating the relation between selective visual attention, deficits of programming and dynamics of saccadic eye movements while searching for a target and hand-reaction time as well as hand-movement time. Visual attention is crucial for concentrating selectively on one aspect of the visual field while ignoring other aspects. Eye movements are anatomically and functionally related to mechanisms of visual attention. Saccadic dysfunction might confound selective visual attention in PD. METHODS: We studied visual selective attention in 22 medicated PD patients (clinical ON status, mild to moderate disease severity) and 22 age matched controls. We looked for possible interferences through oculomotor deficits. Two tasks were compared: free viewing of photographs and time optimal visual search of a hidden target. Visual search times (VST), task related dynamics of saccades, and hand-reaction and hand-movement times were analyzed. RESULTS: In the free viewing task mild to moderately affected PD patients did not differ statistically from healthy subjects with respect to saccade dynamics. However, patients differed significantly from healthy subjects in the time optimal visual search task with 25% lower rates of successful searches. Hand movement reaction time did not differ in both groups, whereas hand movement execution time was significantly prolonged in PD patients. CONCLUSION: Saccadic oculomotor control and hand movement reaction times were intact, whereas in our less severely affected treated PD patients, visual selective attention was not. The highly reduced successful search rate might be related to disturbed programming and delayed execution of saccades during time optimal visual search due to decreased execution of serial-order sequential generation of saccades.

Protein phosphatase 2A (PP2A)-specific ubiquitin ligase MID1 is a sequence-dependent regulator of translation efficiency controlling 3-phosphoinositide-dependent protein kinase-1 (PDPK-1).

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.

Small Rho GTPases are key regulators of peripheral nerve biology in health and disease.

A thorough knowledge of the cellular and molecular basis of the structure and function of peripheral nerves is of paramount importance not only for a better understanding of the fascinating biology of the peripheral nervous system but also for providing critical insights into the various diseases affecting peripheral nerves as the firm foundation of potential treatments. Genetic approaches in model organisms, in combination with research on hereditary forms of neuropathies, have contributed significantly to our progress in this field. In this review, we will focus on recent advances using these synergistic approaches that led to the identification of small Rho GTPases and their regulators as crucial functional players in proper development and function of myelinated peripheral nerves, with a particular emphasis on the cell biology of Schwann cells in health and disease.

Comparative 3'UTR analysis allows identification of regulatory clusters that drive Eph/ephrin expression in cancer cell lines.

Eph receptors are the largest family of receptor tyrosine kinases. Together with their ligands, the ephrins, they fulfill multiple biological functions. Aberrant expression of Ephs/ephrins leading to increased Eph receptor to ephrin ligand ratios is a critical factor in tumorigenesis, indicating that tight regulation of Eph and ephrin expression is essential for normal cell behavior. The 3'-untranslated regions (3'UTRs) of transcripts play an important yet widely underappreciated role in the control of protein expression. Based on the assumption that paralogues of large gene families might exhibit a conserved organization of regulatory elements in their 3'UTRs we applied a novel bioinformatics/molecular biology approach to the 3'UTR sequences of Eph/ephrin transcripts. We identified clusters of motifs consisting of cytoplasmic polyadenylation elements (CPEs), AU-rich elements (AREs) and HuR binding sites. These clusters bind multiple RNA-stabilizing and destabilizing factors, including HuR. Surprisingly, despite its widely accepted role as an mRNA-stabilizing protein, we further show that binding of HuR to these clusters actually destabilizes Eph/ephrin transcripts in tumor cell lines. Consequently, knockdown of HuR greatly modulates expression of multiple Ephs/ephrins at both the mRNA and protein levels. Together our studies suggest that overexpression of HuR as found in many progressive tumors could be causative for disarranged Eph receptor to ephrin ligand ratios leading to a higher degree of tissue invasiveness.

Alternative polyadenylation signals and promoters act in concert to control tissue-specific expression of the Opitz Syndrome gene MID1.

BACKGROUND: Mutations in the X-linked MID1 gene are responsible for Opitz G/BBB syndrome, a malformation disorder of developing midline structures. Previous Northern blot analyses revealed the existence of at least three MID1 transcripts of differing lengths. RESULTS: Here we show that alternative polyadenylation generates the size differences observed in the Northern blot analyses. Analysis of EST data together with additional Northern blot analyses proved tissue-specific usage of the alternative polyadenylation sites. Bioinformatic characterization of the different 3'UTRs of MID1 revealed numerous RNA-protein interaction motifs, several of which turned out to be conserved between different species. Furthermore, our data suggest that mRNA termination at different polyadenylation sites is predetermined by the choice of alternative 5'UTRs and promoters of the MID1 gene, a mechanism that efficiently allows synergistic function of 5' and 3'UTRs. CONCLUSION: MID1 expression is tightly regulated through concerted action of alternative promoters and alternative polyadenylation signals both during embryonic development and in the adult.

Essential and distinct roles for cdc42 and rac1 in the regulation of Schwann cell biology during peripheral nervous system development.

During peripheral nervous system (PNS) myelination, Schwann cells must interpret extracellular cues to sense their environment and regulate their intrinsic developmental program accordingly. The pathways and mechanisms involved in this process are only partially understood. We use tissue-specific conditional gene targeting to show that members of the Rho GTPases, cdc42 and rac1, have different and essential roles in axon sorting by Schwann cells. Our results indicate that although cdc42 is required for normal Schwann cell proliferation, rac1 regulates Schwann cell process extension and stabilization, allowing efficient radial sorting of axon bundles.

Cdc42 and Rac1 signaling are both required for and act synergistically in the correct formation of myelin sheaths in the CNS.

The formation of myelin sheaths in the CNS is the result of a complex series of events involving oligodendrocyte progenitor cell (OPC) proliferation, directed migration, and the morphological changes associated with axon ensheathment and myelination. To examine the role of Rho GTPases in oligodendrocyte biology, we have used a conditional tissue-specific gene-targeting approach. Ablation of Cdc42 in cells of the oligodendrocyte lineage did not affect OPC proliferation, directed migration, or in vitro differentiation, but it led to the formation of a unique and stage-specific myelination phenotype. This was characterized by the extraordinary enlargement of the inner tongue of the oligodendrocyte process and concomitant formation of a myelin outfolding as a result of abnormal accumulation of cytoplasm in this region. Ablation of Rac1 also resulted in the abnormal accumulation of cytoplasm in the inner tongue of the oligodendrocyte process, and we provide genetic evidence that rac1 synergizes with cdc42 in a gene dosage-dependent way to regulate myelination.

Neuronal migration in the murine rostral migratory stream requires serum response factor.

The central nervous system is fundamentally dependent on guided cell migration, both during development and in adulthood. We report an absolute requirement of the transcription factor serum response factor (SRF) for neuronal migration in the mouse forebrain. Conditional, late-prenatal deletion of Srf causes neurons to accumulate ectopically at the subventricular zone (SVZ), a prime neurogenic region in the brain. SRF-deficient cells of the SVZ exhibit impaired tangential chain migration along the rostral migratory stream into the olfactory bulb. SVZ explants display retarded chain migration in vitro. Regarding target genes, SRF deficiency impairs expression of the beta-actin and gelsolin genes, accompanied by reduced cytoskeletal actin fiber density. At the posttranslational level, cofilin, a key regulator of actin dynamics, displays dramatically elevated inhibitory phosphorylation at Ser-3. Our studies indicate that SRF-controlled gene expression directs both the structure and dynamics of the actin microfilament, thereby determining cell-autonomous neuronal migration.