Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are localized and secreted by human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A Human lung tissue from control subjects, idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF was immune-stained with IL-11, IL-11Rα and αSMA and with secondary fluorescence antibodies. Representative images are showed. White colour represents co-localization of both antibodies. Yellow arrows indicate endothelial cells. B HPAECs and C HPASMCs were isolated from pulmonary arteries of control subjects, IPF and PH associated to IPF patients and cultured until passage 1. Cell culture supernatants were collected to measure IL-11 by ELISA. Data are presented as scatter dot blot with median and interquartile range values of n = 6 patients in each group. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Staining, Fluorescence, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are increased in whole lung homogenates, isolated pulmonary arteries and serum of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF. The protein expression of IL-11 and IL-11Rα in A , B isolated pulmonary arteries (70–500 µm of internal diameter), C , D serum, and E , F lung tissue homogenates. Protein expression was measured using ELISA kits. H CD31 protein expression was measured in isolated pulmonary arteries as endothelial cells marker by ELISA. H , I Human lung tissue from control subjects, IPF and PH associated to IPF was immune-stained with IL-11, IL-11Rα and alpha smooth muscle actin (αSMA) antibodies. Representative images are showed from non-fibrotic lung areas and fibrotic areas. J Vascular wall thickening was quantified in a total of 20–30 pulmonary arteries per patient. K , L Immunohistochemical score quantification of IL-11 and IL-11Rα in a total of 20–30 pulmonary arteries per patient. Scale bar: 100 µm. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison. M Spearman ρ correlation of IL-11 expression in isolated pulmonary arteries from PH + IPF and mean pulmonary artery pressure (mPAP). N indicates the number of patients in each graph

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Marker, Staining, Immunohistochemical staining, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: SiRNA-IL-11 transiently transfection attenuates bleomycin-induced lung fibrosis and pulmonary hypertension in transgenic Tie2-GFP mice. Wild-type (WT) siRNA(−) Tie2-GFP mice and IL-11-KO siRNA-IL-11 Tie2-GFP mice received a single intratracheal dose of bleomycin (1.5 U/kg) on day 1 ( n = 11) during 14 days. siRNA-IL-11 was administered intravenously and intranasally three times a week from day 1 to day 14. At day 14 the following parameters were measured. A Masson’s trichrome histological images are showed. Scale bar: 100 µm. B Ashcroft score lung fibrotic index, C hydroxyproline amount in lung tissue D right ventricular systolic pressure (RVSP) mmHg, E right ventricular (RV) hypertrophy measured by the ratio of RV/left ventricular (LV) + septo in mg/mg, F pulmonary artery remodeling and G inflammatory cells in bronchoalveolar lavage fluid (BALF) were measured. H Immunohistochemical analysis of αSMA, IL-11 and IL-11Rα. Scale bar: 50 µm. Black arrows show pulmonary arteries. I Co-immunofluorescence of αSMA/Tie2-GFP. Scale bar: 25 µm. White arrows indicates co-localizations. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Transfection, Transgenic Assay, Immunohistochemical staining, Immunofluorescence, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and soluble IL-11Rα induce human pulmonary artery endothelial cell (HPAEC) to mesenchymal transition (EnMT) and human pulmonary artery smooth muscle cell (HPASMC) to myofibroblast-like transition. A HPAEC and B HPASMC were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or their combination during 48 h replacing culture medium and stimulus each 24 h. Experiments were done between passages 2–3. Gene mRNA transcripts of different genes measured by quantitative PCR (qPCR) as 2 −ΔCt . Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin for protein. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 4 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα activates intracellular signal. A Human pulmonary artery endothelial cells (HPAEC) and B human pulmonary artery smooth muscle cells (HPASMC) were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination during 30 min. Experiments were done between passages 2–3. Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin or non-phosphorylated protein as indicate. Representative blots are sowed. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα promotes time-dependent proliferation and senescence in human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A HPAECs or B HPASMCs were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination at indicated times. Experiments were done between passages 2–3. Cell proliferation was measured by the BrDU kit at 24 h, 48 h, 72 h and 96 h. Cell senescence was measured after 72 h of cell stimulation using β-galactosidase histology and P21 expression. Results were expressed as % senescence (β-galactosidase blue positive cells) relative to the total number of cells in each field. P21 expression was measured by quantitative PCR (qPCR) as 2 −ΔCt and western blot. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3–4 control subjects performed in triplicate). P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Cell Stimulation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Dot Blot

Journal: bioRxiv

Article Title: There is no Biophysical Distinction between Temporal Interference Stimulation and Direct kHz Stimulation for Actuation of Peripheral Nerves

doi: 10.1101/2024.09.06.611584

Figure Lengend Snippet: a) The basic principle of TIS involves two kHz carriers at two different frequencies, f 1 and f 2 , which interact in a given medium via interference, creating areas of amplitude modulation (AM). At midpoints between stimulation electrodes, maximum AM depth is achieved. The AM occurs at a frequency which will be equal to the frequency difference between the two carriers, Δ f . TIS can be used in vivo to deliver transcutaneous stimulation, where AM occurs at an area of interest to deliver phasic stimulation at a target region. The experiments in this paper explore this effect on peripheral nerve targets containing motor neurons which enervate muscles. b) Periodic symmetric stimulation signals like a sinusoid do lead to net depolarization of cells, because of membrane rectification. The depolarizing half-period leads to more net Na + influx than K + outflux. c) Sinusoidal stimulation is governed by a characteristic strength-frequency (s-f) curve, where the threshold minimum is around 50 Hz. Higher frequencies result in larger stimulation threshold currents. d) 1 kHz sinusoidal stimulation demonstrated on a mammalian myelinated axon model, demonstrating the effect of rectification leading to net depolarization. The model shows the calculated membrane potential during application of an extracellular stimulating current. Three test cases are shown: no stimulation, (orange line V rest ), subthreshold stimulation (blue line), and suprathreshold stimulation (green line).All dashed lines are low-pass filtered, solid lines are not filtered. Subthreshold stimulation gives a net membrane depolarization, however when the stimulation current becomes suprathreshold, one can see the summation effect at work as the membrane depolarizes more with each successive sinusoidal period, until threshold is reached and action potentials fire at the preferred resonance frequency of the neuron (5 Hz in this case). The net depolarization caused by sub- and supra-threshold stimuli is due to larger contribution of Na + inward currents versus outward K + currents, broken down in the inset on the right which shows the total transmembrane current density calculated for this model neuron. The application of a kHz stimulus can be conceptualized as analogous to a cathodic DC stimulation. e) In this work, we compare TIS, which is delivered by two electrode pairs, causing interference and AM in the tissue, with premodulated kHz stimulation, which is delivered using a single pair of electrodes. We find that biophysically, the two stimulation modalities are equivalent. f) The peripheral nerve stimulation models tested in this work. Motorneuron (insect) / motor fiber (human) recruitment and downstream evoked movement is the evaluated biomarker.

Article Snippet: Electrical stimulation and recording hardware: The kHz stimulation experiments were conducted using the Digitimer DS4 (for in vitro prototyping and locust experiments), and the Digitimer DS5 (for experiments on human participants).

Techniques: In Vivo, Muscles, Membrane, Biomarker Discovery

Journal: bioRxiv

Article Title: There is no Biophysical Distinction between Temporal Interference Stimulation and Direct kHz Stimulation for Actuation of Peripheral Nerves

doi: 10.1101/2024.09.06.611584

Figure Lengend Snippet: for N=3 locusts. a) Experimental setup for stimulation and quantifying response using video recording of evoked leg extension ( extensor tibiae muscle) caused by stimulation of the N5 nerve, which is the chosen biomarker for stimulation. The stimulation electrode configurations for AM-SW and SW burst are shown, together with the 4-electrode TIS configuration. b) Current threshold/frequency dependence for N5 nerve stimulation. Three stimulation waveforms were tested: AM-SW, SW burst, and TIS, with the modulation frequency set to 1 Hz. The current value applies to one current source only, i.e . one carrier in the case of TIS. The inset image shows the evaluated biomarker: leg extension, which occurs at one-half the modulation period. c) Stimulation of the median nerve, comparing TIS with AM-SW and SW. Stimulation of the median nerve in the forearm results in reproducible motor response in the form of finger flexion. As a biomarker for stimulation, we track flexion of the middle finger. For tests shown in a-c, we arbitrarily define threshold response as the middle finger flexing until it contacts the palm. Two electrode placement configurations were used for median nerve stimulation. For AM-SW, constant SW, and SW burst, two-electrode arrangement, 10 mm ∅ primary electrode gel-assisted AgCl, versus large ground 4×4 cm pad. For TIS, two-pairs of 10 mm ∅ gel-assisted AgCl electrodes. d) s-f dependence experiment: Current threshold value for obtaining finger flexion to the palm of the hand (evoked flexion motion shown in inset photograph) as a function of stimulation current frequency in the range 500-12500 Hz, N=3 participants. e) Stimulation current threshold at 500, 2500, and 5000 Hz for sustained tetanic flexion evoked by a constant SW stimulus. The current magnitude is ramped from 0 over 30 seconds. Here the current threshold values overlap with the s-f dependence found for the other kHz waveforms.

Article Snippet: Electrical stimulation and recording hardware: The kHz stimulation experiments were conducted using the Digitimer DS4 (for in vitro prototyping and locust experiments), and the Digitimer DS5 (for experiments on human participants).

Techniques: Biomarker Discovery

Journal: bioRxiv

Article Title: There is no Biophysical Distinction between Temporal Interference Stimulation and Direct kHz Stimulation for Actuation of Peripheral Nerves

doi: 10.1101/2024.09.06.611584

Figure Lengend Snippet: a ) Leg deflection angle, Δ θ , caused by extensor tibiae actuation, as a function of modulation frequency Δ f . Different Δ f give phasic contraction, while Δ f = 0 causes tetanic contraction. Experiment is performed in 2-electrode AM-SW mode with a fixed frequency of 2500 Hz and fixed max current amplitude of 98 μA, which is the minimal threshold for movement at Δ f = 1 Hz. b) This experiment involves applying an at-threshold stimulus via AM-kHz, and then turning off the modulation, while keeping the current setpoint constant. This test shows that threshold current for the phasic stimulation and the tonic stimulation are equal. c) Effect of amplitude modulation frequency Δ f on median nerve stimulation. The graph shows the dependence of current threshold of evoked muscle contraction on the envelope frequency, for a fixed stimulation frequency of 3000 Hz. The characteristic U-shaped response predicted by simulations is demonstrated.

Article Snippet: Electrical stimulation and recording hardware: The kHz stimulation experiments were conducted using the Digitimer DS4 (for in vitro prototyping and locust experiments), and the Digitimer DS5 (for experiments on human participants).

Techniques:

Journal: bioRxiv

Article Title: There is no Biophysical Distinction between Temporal Interference Stimulation and Direct kHz Stimulation for Actuation of Peripheral Nerves

doi: 10.1101/2024.09.06.611584

Figure Lengend Snippet: a) Geometry used for simulation, reflecting the experiment with the human median nerve. Electrodes placed on the forearm are depicted in gray, the nerve is orange. b) 3D image of the calculated activating function (second-order spatial derivative of the electric potential) in x direction. c) Horizontal cross section showing the average activating function at the nerve depth. Electrode positions are outlined in black. d) Stimulation mode index (SMI, ) in vertical cross section for three different stimulation current ratios. The tonic region (SMI = 1) is shown in red, unstimulated region in blue (SMI = 0), and phasic regions (0 < SMI < 1) are found at between electrode pairs. Corresponding time dependance of the activation function is plotted at the median nerve location depicted as an orange circle. The horizontal red line is the stimulation threshold of 2000 V/m . In the red region the activating function is never below the stimulation threshold. In the blue region the activating function is never above the threshold, while in the in-between the regions the activation function goes above or below the threshold at the TIS beat frequency Δ f .

Article Snippet: Electrical stimulation and recording hardware: The kHz stimulation experiments were conducted using the Digitimer DS4 (for in vitro prototyping and locust experiments), and the Digitimer DS5 (for experiments on human participants).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: There is no Biophysical Distinction between Temporal Interference Stimulation and Direct kHz Stimulation for Actuation of Peripheral Nerves

doi: 10.1101/2024.09.06.611584

Figure Lengend Snippet: in a circular medium of diameter d , conductivity σ, stimulation current I and electric field amplitude E . The value of the scaled electric field is only a function of the geometry and is independent on the chosen values of I, σ , or spatial scale d . This picture explains why TIS will always result in higher amplitudes of unmodulated carriers superficial to the region of maximum amplitude modulation where phasic stimulation is delivered. Both stimulation methods lead to the same amplitude of phasic stimulation in the middle of the medium.

Article Snippet: Electrical stimulation and recording hardware: The kHz stimulation experiments were conducted using the Digitimer DS4 (for in vitro prototyping and locust experiments), and the Digitimer DS5 (for experiments on human participants).

Techniques: