Ultrasonographic Diagnosis of Thoracic Outlet Syndrome Secondary to Brachial Plexus Piercing Variation

Structural variations of the thoracic outlet create a unique risk for neurogenic thoracic outlet syndrome (nTOS) that is difficult to diagnose clinically. Common anatomical variations in brachial plexus (BP) branching were recently discovered in which portions of the proximal plexus pierce the anterior scalene. This results in possible impingement of BP nerves within the muscle belly and, therefore, predisposition for nTOS. We hypothesized that some cases of disputed nTOS result from these BP branching variants. We tested the association between BP piercing and nTOS symptoms, and evaluated the capability of ultrasonographic identification of patients with clinically relevant variations. Eighty-two cadaveric necks were first dissected to assess BP variation frequency. In 62.1%, C5, superior trunk, or superior + middle trunks pierced the anterior scalene. Subsequently, 22 student subjects underwent screening with detailed questionnaires, provocative tests, and BP ultrasonography. Twenty-one percent demonstrated atypical BP branching anatomy on ultrasound; of these, 50% reported symptoms consistent with nTOS, significantly higher than subjects with classic BP anatomy (14%). This group, categorized as a typical TOS, would be missed by provocative testing alone. The addition of ultrasonography to nTOS diagnosis, especially for patients with BP branching variation, would allow clinicians to visualize and identify atypical patient anatomy

Time course and reversibility of epithelial stratification by the hydrostatic pressure.

<p>(A) A vertical section of MDCK I cell sheets. Hydrostatic pressure from basal to apical side was applied to the MDCK I cell sheets at two days after seeding on filters, and the vertical section of cell sheets was observed at Day 2 and 1–12 days after application of the hydrostatic pressure (Days 3–14). MDCK I cells showed gradual development of epithelial stratification with time. (B) The reversibility of epithelial stratification by the hydrostatic pressure. Hydrostatic pressure from basal to apical side was applied to the MDCK I cell sheets for four days, and the hydrostatic pressure gradient was then eliminated by the increase of the culture medium in the apical side. (C) Stratification index under the conditions in (A) and (B). The upper side is apical side and the lower side is basal side. Scale bars = 20 μm.</p

Scanning electron microscopy and light microscopic images in a vertical section of MDCK I cells under the ‘Apical’ and ‘Basal’ conditions.

<p>(A) Diagrams of culture conditions. MDCK I cells were seeded at a density of 2 × 10⁵ cells/cm² on Transwell permeable filters, and the amounts of the culture medium in the apical and basal sides were varied at two days after seeding on filters. (B and C) Scanning electron micrographs of MDCK I cell sheets at low magnification (B) and high magnification (C) at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. A rugged surface of the cell sheet was observed in the ‘Basal’ condition. (D and E) A vertical section of MDCK I cell sheets at low magnification (D) and high magnification (E). MDCK I cells were seeded on filters, and cultured under the ‘Apical’ and ‘Basal’ conditions for four days. A vertical section of cell sheets was observed by light microscopy staining with toluidine blue. A multi-layered cell sheet with a number of raised cell clumps was observed under the ‘Basal’ condition. Scale bars = 100 μm for (B), 10 μm for (C), 50 μm for (D) and 20 μm for (E). <i>A</i>, apical side; <i>B</i>, basal side.</p

Effects of hydrostatic pressure on transepithelial transport.

<p>(A) Effects of hydrostatic pressure on transepithelial electrical resistance (TER) in MDCK I cells. The TER was changed to decrease at two days after application of the hydrostatic pressure under the ‘Basal’ condition. (B) The ratio of <i>P</i>_Na to <i>P</i>_Cl (<i>P</i>_Na/<i>P</i>_Cl) under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. The <i>P</i>_Na/<i>P</i>_Cl was measured at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. The value of <i>P</i>_Na/<i>P</i>_Cl under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition. (C) <i>P</i>_Na and <i>P</i>_Cl under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. The value of <i>P</i>_Na under the ‘Basal’ condition was approximately four-fold higher than that under the ‘Apical’ condition. (D) Immunofluorescence microscopy for claudin-2 and ZO-1 in MDCK I cells at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. Claudin-2 staining was clearly detected at cell-cell contacts in a limited region under the ‘Basal’ condition. Scale bar = 10 μm. (E) Immunoblots for claudin-2 and E-cadherin in MDCK I cells at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. MDCK II cells cultured on filters for six days were used as a positive control. A faint band of claudin-2 was detected under the ‘Basal’ condition in MDCK I cells. (F) Flux of fluorescein and 4 kDa FITC-dextran under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. The flux was measured at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. The flux of fluorescein under the ‘Basal’ condition was significantly lower than that under the ‘Apical’ condition. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 compared with the ‘Apical’ condition.</p

Effects of signal inhibitors and activators on cell proliferation and TER.

<p>(A) Effects of signal inhibitors and activators on the cell number in MDCK I cells. MDCK I cells were seeded at a density of 5.0 × 10⁴ cells/well in a 12-well plate, cultured for 48 h in the presence of the inhibitor or activator, and the cell number was counted with counting chamber after the trypsinization of the cells. (B) Effects of signal inhibitors and activators on the doubling time of MDCK I cells. The doubling time was calculated from the results in (A) on the assumption that the rate of cell proliferation was constant during the 48 h of the culture. (C) Effects of signal inhibitors and activators on TER. MDCK I cells were cultured on filters in the presence of the signal inhibitor or activator, and the TER was measured at each time point.</p

Effects of signal inhibitors and activators on the epithelial stratification by the hydrostatic pressure.

<p>(A) Light microscopic images in a vertical section under the ‘Apical’ condition in the presence of the signal inhibitor or activator in MDCK I cells. The upper side is apical side and the lower side is basal side. (B) Stratification index of MDCK I cells under the ‘Apical’ condition in the presence of the signal inhibitor or activator. The stratification index in the presence of H89 and SQ22536 was significantly higher than that in the control experiment (DMSO). (C) Light microscopic images in a vertical section under the ‘Basal’ condition in the presence of the signal inhibitor or activator in MDCK I cells. The upper side is apical side and the lower side is basal side. (D) Stratification index of MDCK I cells under the ‘Basal’ condition in the presence of the signal inhibitor or activator. The administration of H89 significantly increased the degree of epithelial stratification by the hydrostatic pressure, and the administration of forskolin significantly decreased the degree of the epithelial stratification. ** <i>p</i> < 0.01 compared with control (DMSO).</p

Transmission and freeze-fracture electron microscopy of MDCK I cells under the ‘Apical’ and ‘Basal’ conditions.

<p>(A) Transmission electron micrographs in a vertical section of MDCK I cell sheets at low magnification under the ‘Apical’ and ‘Basal’ conditions. MDCK I cells were observed at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. Cavities were observed within the multi-layered MDCK I cells under the ‘Basal’ condition. Scale bar = 5 μm. (B) A transmission electron micrograph in a vertical section of MDCK I cell sheets at high magnification under the ‘Basal’ condition. The structure of microvilli was observed at the surfaces of the cavities and apical cell membranes of the outermost cell layer. Scale bar = 1 μm. (C) An enlarged image in the region enclosed by the white line in the transmission electron micrograph (B). Plasma membranes between the adjacent cells were fused immediately below the surface of the cavity (<i>arrows</i>). Scale bar = 1 μm. (D) Freeze-fracture electron micrographs in a lateral view of MDCK I cells under the ‘Apical’ condition at low magnification (left panel) and high magnification (right panel). TJ strands were observed immediately below the microvilli (<i>arrow</i>). Scale bars = 5 μm for the left panel and 200 nm for the right panel. (E) Freeze-fracture electron micrographs in a lateral view of MDCK I cells under the ‘Basal’ condition at low magnification (upper left panel), an enlarged image in the region enclosed by the white line in the upper left panel (upper right panel), and further enlarged images in the regions enclosed by the white lines in the upper right panel (lower panels). TJ strands were observed immediately below the surface of the cavity within the multi-layered MDCK I cells (<i>arrows</i>). Scale bars = 5 μm for the upper left panel, 1 μm for the upper right panel, and 200 nm for the lower panels. <i>m</i>, microvilli; <i>n</i>, nucleus; *, cavity.</p

Light microscopic images in vertical sections of MDCK I cells, MDCK II cells and Caco-2 cells under the various hydrostatic pressure conditions.

<p>(A) A vertical section of MDCK I cell sheets. The culture medium in both the apical and basal sides was increased under the ‘Increase’ condition and decreased under the ‘Decrease’ condition. A slight degree of epithelial stratification was observed under the ‘Decrease’ condition. (B) A vertical section of MDCK I cell sheets. The culture medium in the apical side was almost eliminated and the hydrostatic pressure from basal to apical side was applied (‘HP+’ condition) or not applied (‘HP−’ condition) to the MDCK I cell sheets. Epithelial stratification was observed under the ‘HP+’ condition, whereas there was hardly any sign of epithelial stratification under the ‘HP−’ condition. (C) Stratification index under the conditions in (A) and (B). The degree of epithelial stratification (stratification index) was quantified as described in <i>Materials and Methods</i>. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 compared with the ‘Apical’ condition. (D and E) A vertical section of MDCK II cell sheets at low magnification (D) and high magnification (E). MDCK II cells were seeded on filters, and cultured under the ‘Apical’ and ‘Basal’ conditions for four days. Epithelial stratification was not observed under the ‘Apical’ and ‘Basal’ conditions. (F and G) A vertical section of Caco-2 cell sheets at low magnification (F) and high magnification (G). Caco-2 cells were seeded on filters, and cultured under the ‘Apical’ and ‘Basal’ conditions for eight days. A multi-layered cell sheet was observed under the ‘Basal’ condition. (H) Stratification index in MDCK II and Caco-2 cells. ** <i>p</i> < 0.01 compared with the ‘Apical’ condition in corresponding cells. The upper side is apical side and the lower side is basal side. Scale bars = 20 μm for (A), (B), (D) and (F) and 10 μm for (E) and (G).</p

Localization of claudin-3, E-cadherin, occludin and Na⁺/K⁺ ATPase under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells and barrier function of TJs around the cavities within the multi-layered epithelia.

<p>(A and B) Immunofluorescence microscopy for claudin-3 and E-cadherin in z-axis plane under the ‘Apical’ (A) and ‘Basal’ (B) conditions in MDCK I cells. Signals of claudin-3 were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (<i>arrows</i>). (C and D) Immunofluorescence microscopy for occludin and Na⁺/K⁺ ATPase in z-axis plane under the ‘Apical’ (C) and ‘Basal’ (D) conditions in MDCK I cells. Signals of occludin were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (<i>arrows</i>). (E and F) A tracer experiment using a primary amine-reactive biotinylation reagent was performed in MDCK I cells cultured under the ‘Apical’ (E) and ‘Basal’ (F) conditions. The biotinylation reagent was administered in the basal side for 10 min, and the bound biotin was detected by streptavidin. The biotinylation reagent appeared to stop at TJs around the cavities within the multi-layered epithelia under the ‘Basal’ condition (<i>arrows</i>). Scale bars = 10 μm.</p

The mean values of CT density (HU) in the phantom materials mimicking lung parenchyma and the mean values of maximum CT density in the tube walls.

<p>Average of measured values (SD).</p