Low Dose Carbon Black Nanoparticle Exposure Does Not Aggravate Allergic Airway Inflammation in Mice Irrespective of the Presence of Surface Polycyclic Aromatic Hydrocarbons.

SUBJECT:lindner-mouse-ova-cbnp

OVA-sensitised female Balb/c mouse exposed to CBNP

Female 7-9 week old Balb/c mice, OVA-free diet, water ad libitum; OVA i.p. on days 1, 14, 21 and OVA aerosol challenge on days 26-28

SUBJECT:lindner-mouse-intrapulmonary-airways

Microdissected intrapulmonary airways of OVA-sensitised Balb/c mouse

Female 7-9 week old Balb/c mice; n = 4-8 per group

SUBJECT:lindner-mouse-tracheal-epithelium

Isolated tracheal epithelial cells of OVA-sensitised Balb/c mouse

Female 7-9 week old Balb/c mice; n = 4-8 per group

SUBJECT:lindner-mouse-lung-histology

Paraffin-embedded lung of OVA-sensitised Balb/c mouse

Female 7-9 week old Balb/c mice; n = 7-8 per group

SUBJECT:lindner-mouse-trachea-histology

Paraffin-embedded trachea of OVA-sensitised Balb/c mouse

Female 7-9 week old Balb/c mice; n = 2-3 per group

SUBJECT:lindner-mouse-explanted-trachea

Freshly explanted trachea of OVA-sensitised Balb/c mouse

Female 7-9 week old Balb/c mice; trachea held at 30 C in HEPES-buffered Ringer solution, epithelium facing upwards

BALF:lindner-cytokines-cbnp

BAL fluid cytokine and chemokine levels - CBNP during established allergic airway inflammation

Table 2, cytometric bead array, concentrations in pg/mL, mean +/- SEM, n = 11-16, OVA/CBNP groups compared with the OVA/H2O group by Mann-Whitney U test. NULL RESULT for aggravation: no CBNP increased any cytokine or chemokine relative to OVA/H2O. Values without a named measurement slot, in the order PBS/H2O, OVA/H2O, OVA/P90, OVA/P90-BaP, OVA/P90-9NA, OVA/AS-PAH - Mcp-1: 0.01+/-0.01, 27.0+/-5.7, 12.7+/-3.9*, 7.4+/-5.5*, 11.7+/-5.7*, 0.6+/-0.3*; IL-4: 0.8+/-0.6, 1.2+/-0.2, 0.7+/-0.1, 0.7+/-0.1*, 0.9+/-0.2, 0.6+/-0.2*; IL-5: 0.8+/-0.3, 12.7+/-2.1, 10.4+/-1.8, 13.4+/-1.8, 11.1+/-1.8, 11.7+/-2.2; IL-13: 0.03+/-0.01, 7.3+/-1.1, 4.4+/-0.9*, 5.7+/-1.1, 4.4+/-0.6*, 5.4+/-1.2; IL-17A: 0.2+/-0.1, 0.4+/-0.1, 0.3+/-0.1, 0.3+/-0.1, 0.4+/-0.1, 0.3+/-0.1 (* p < 0.05 vs OVA/H2O). Every significant CBNP effect was a DECREASE, i.e. an attenuation rather than an aggravation.

Exposure conditions

Outputs

Evidence

BALF:lindner-immune-cells-cbnp

BAL fluid differential immune cell count - CBNP during established allergic airway inflammation

Figure 1b, differential count of macrophages, lymphocytes, eosinophil and neutrophil granulocytes per mL BAL fluid, n = 18-25, mean +/- SEM, Mann-Whitney U test versus OVA/H2O. Macrophage, lymphocyte and granulocyte numbers were increased in the OVA control relative to the PBS control. NULL RESULT: CBNP exposure did not increase immune cell numbers. The only significant CBNP effects were decreases in eosinophil number after P90-BaP and P90-9NA. Values are plotted, not tabulated, so no numeric measurements are recorded here.

Exposure conditions

Outputs

Evidence

LF:lindner-ahr-methacholine

Total lung resistance in response to methacholine - CBNP during established allergic airway inflammation

Figure 1c, invasive measurement of total lung resistance in response to a 100 mg/mL methacholine dose, n = 7-8, mean +/- SEM, Mann-Whitney U test versus OVA/H2O. Airway hyperresponsiveness was increased in the OVA control compared with the PBS control, confirming the asthma model. NULL RESULT: airway hyperresponsiveness in the CBNP groups was not different from the OVA/H2O group. Reported as a plot only, so no numeric values are transcribed.

Exposure conditions

Outputs

Evidence

GE:lindner-cyp1a1-intrapulmonary

Cyp1a1 mRNA in intrapulmonary airways - CBNP during established allergic airway inflammation

Table 3, Cyp1a1 mRNA in microdissected intrapulmonary airways, n-fold expression normalised to RPL32, mean +/- SEM, n = 4-8, Mann-Whitney U test versus OVA/H2O. NULL RESULT: no CBNP increased Cyp1a1. Cyp1a1 was below the limit of detection in tracheal epithelial cells in every group, so that airway section is not recorded here.

Exposure conditions

Outputs

Evidence

GE:lindner-cyp1b1-trachea

Cyp1b1 mRNA in tracheal epithelial cells - CBNP during established allergic airway inflammation

Table 3, Cyp1b1 mRNA in isolated tracheal epithelial cells, n-fold expression normalised to RPL32, mean +/- SEM, n = 4-8, Mann-Whitney U test versus OVA/H2O. NULL RESULT: no CBNP significantly increased Cyp1b1, including the PAH-coated particles. For comparison, Cyp1b1 in intrapulmonary airways was 1.0+/-0.1 (PBS/H2O), 0.6+/-0.1 (OVA/H2O), 0.6+/-0.1 (P90), 0.8+/-0.1 (P90-BaP), 0.5+/-0.1 (P90-9NA) and 0.9+/-0.1 (AS-PAH).

Exposure conditions

Outputs

Evidence

GE:lindner-ho1-intrapulmonary

HO-1 mRNA in intrapulmonary airways - CBNP during established allergic airway inflammation

Table 3, heme oxygenase-1 (HO-1) mRNA in microdissected intrapulmonary airways, n-fold expression normalised to RPL32, mean +/- SEM, n = 4-8, Mann-Whitney U test versus OVA/H2O. NULL RESULT for the oxidative stress MIE: HO-1 did not increase after any CBNP. HO-1 in tracheal epithelial cells was likewise flat: 1.2+/-0.3 (PBS/H2O), 1.0+/-0.2 (OVA/H2O), 1.1+/-0.2 (P90), 1.1+/-0.2 (P90-BaP), 1.1+/-0.4 (P90-9NA), 1.1+/-0.3 (AS-PAH). Glutathione reductase (Gr) in intrapulmonary airways was 1.0+/-0.1, 1.3+/-0.1, 1.1+/-0.1, 1.5+/-0.1, 1.0+/-0.1 and 1.5+/-0.1 for the same group order.

Exposure conditions

Outputs

Evidence

GE:lindner-gpx3-trachea

Gpx3 mRNA in tracheal epithelial cells - CBNP during established allergic airway inflammation

Table 3, glutathione peroxidase 3 (Gpx3) mRNA in isolated tracheal epithelial cells, n-fold expression normalised to RPL32, mean +/- SEM, n = 4-8, Mann-Whitney U test versus OVA/H2O. Gpx3 fell after the OVA challenge itself (1.0+/-0.1 in PBS/H2O to 0.3+/-0.1 in OVA/H2O) and no CBNP raised it back or lowered it further significantly. Gpx3 in intrapulmonary airways was essentially unchanged across all six groups (1.0+/-0.1, 0.9+/-0.1, 0.8+/-0.1, 1.0+/-0.1, 1.0+/-0.1, 1.0+/-0.1). The authors note that the lowered antioxidant defence in the OVA model did not translate into CBNP-induced cytokine or chemokine induction.

Exposure conditions

Outputs

Evidence

GE:lindner-il6-intrapulmonary

IL-6 mRNA in intrapulmonary airways - CBNP during established allergic airway inflammation

Table 3, IL-6 mRNA in microdissected intrapulmonary airways, n-fold expression normalised to RPL32, mean +/- SEM, n = 4-8, Mann-Whitney U test versus OVA/H2O. NULL RESULT: no CBNP increased IL-6. P90-9NA significantly DECREASED IL-6 (4.3+/-0.5 vs 7.9+/-1.2, p < 0.05). IL-6 in tracheal epithelial cells was 1.2+/-0.3, 1.5+/-0.6, 2.4+/-1.3, 3.1+/-1.7, 0.6+/-0.3 and 3.5+/-1.8 for the same group order, with no significant differences.

Exposure conditions

Outputs

Evidence

GE:lindner-il13-intrapulmonary

IL-13 mRNA in intrapulmonary airways - CBNP during established allergic airway inflammation

Table 3, IL-13 mRNA in microdissected intrapulmonary airways, n-fold expression normalised to RPL32, mean +/- SEM, n = 4-8, Mann-Whitney U test versus OVA/H2O. NULL RESULT: no CBNP increased IL-13. P90-9NA significantly DECREASED IL-13 (34+/-4 vs 52+/-8, p < 0.05), consistent with the decreased IL-13 protein in its BAL fluid. IL-13 in tracheal epithelial cells was 1.1+/-0.3, 41+/-14, 27+/-7, 20+/-9, 26+/-10 and 31+/-10 for the same group order, with no significant differences. Other inflammatory mRNAs in intrapulmonary airways for the same group order - Mcp-1: 1.0+/-0.6, 12+/-5, 11+/-4, 10+/-4, 8+/-3, 11+/-4; KC: 1.0+/-0.3, 2.1+/-0.6, 1.7+/-0.3, 1.9+/-0.3, 1.1+/-0.2, 1.5+/-0.3; IL-17a: 1.0+/-2.9, 13+/-3, 13+/-3, 11+/-1, 8+/-2, 19+/-4.

Exposure conditions

Outputs

Evidence

GCM:lindner-mucins-trachea

Muc5ac and Muc5b mRNA in tracheal epithelial cells - CBNP during established allergic airway inflammation

Table 3, Muc5ac and Muc5b mRNA in isolated tracheal epithelial cells, n-fold expression normalised to RPL32, mean +/- SEM, n = 4-8, Mann-Whitney U test versus OVA/H2O. The unmodified P90 particles significantly INCREASED Muc5ac (101+/-15 vs 35+/-10, p < 0.01); none of the PAH-coated particles or AS-PAH changed either mucin. Muc5b was flat in the trachea in every group.

Exposure conditions

Outputs

Evidence

GCM:lindner-mucins-intrapulmonary

Muc5ac and Muc5b mRNA in intrapulmonary airways - CBNP during established allergic airway inflammation

Table 3, Muc5ac and Muc5b mRNA in microdissected intrapulmonary airways, n-fold expression normalised to RPL32, mean +/- SEM, n = 4-8, Mann-Whitney U test versus OVA/H2O. The unmodified P90 particles significantly DECREASED Muc5b (18+/-3 vs 31+/-5, p < 0.05) while Muc5ac was unchanged, the mirror image of the tracheal response. No PAH-containing CBNP changed either mucin here.

Exposure conditions

Outputs

Evidence

GCM:lindner-pas-intrapulmonary

PAS goblet cell surface area and stored mucus volume in intrapulmonary airways - CBNP

Figure 2a-c, stereological quantification of the epithelial basement membrane surface area covered by goblet cells (Sgc/Sep) and the stored mucus volume per basement membrane area (Vmucin/Sep) on PAS-stained lung sections, n = 7-8. NULL RESULT: no CBNP increased either measure relative to OVA/H2O. Reported as plots only, so no numeric values are transcribed.

Exposure conditions

Outputs

Evidence

GCM:lindner-pas-trachea

PAS-positive cells in tracheal epithelium after P90 exposure

Figure 2d,e, PAS-positive cells as a percentage of non-ciliated cells in the tracheal epithelium, n = 2-3. Stained only for the OVA/H2O and OVA/P90 groups, as a follow-up to the P90-induced Muc5ac mRNA increase. NULL RESULT: the number of PAS-positive cells was not increased after P90 exposure, so the Muc5ac mRNA rise was not accompanied by more mucus-storing goblet cells. Reported as a plot only and with a very small n, so no numeric values are transcribed.

Exposure conditions

Outputs

Evidence

CILIA:lindner-cbf-trachea

Tracheal ciliary beat frequency - CBNP during established allergic airway inflammation

Figure 3c, mean ciliary beat frequency of explanted tracheae, each data point one animal, mean +/- SEM, calculated from at least 40 ciliated cells at four tracheal regions per animal. The OVA challenge alone did NOT affect ciliary beat frequency. The only CBNP effect was a DECREASE after the unmodified P90; the PAH-coated particles and AS-PAH left beat frequency unchanged. Reported as a scatter plot only, so no numeric values are transcribed. The authors attribute the P90 effect to the more adhesive, viscous Muc5ac-rich mucus agglutinating the cilia rather than to cilia damage, since ATP stimulation restored the beat frequency equally in all groups.

Exposure conditions

Outputs

Evidence

CILIA:lindner-cbf-trachea-atp

Tracheal ciliary beat frequency after ATP stimulation - CBNP

Figure 3d, mean ciliary beat frequency after adding ATP (10 uM) to the explanted trachea at the end of each experiment, calculated from at least 30 ciliated cells at three tracheal regions per animal. Beat frequency was equally increased in all test groups, which the authors read as evidence that P90 did not damage the cilia themselves - the P90-associated beat frequency decrease is attributed to the mucus layer, not to cilia injury. Reported as a plot only, so no numeric values are transcribed.

Exposure conditions

Outputs

Evidence

MCC:lindner-transport-trachea

Tracheal particle transport speed - CBNP during established allergic airway inflammation

Figure 3b, mean transport speed of 4.5 um polystyrene particles on the explanted tracheal epithelium, each data point one animal, mean +/- SEM. The OVA challenge alone decreased transport speed relative to the PBS control, which the authors attribute to the mucus on the epithelium decelerating transport rather than to a ciliary defect. NULL RESULT: CBNP exposure did not further decrease particle transport. Reported as a scatter plot only, so no numeric values are transcribed.

Exposure conditions

Outputs

Evidence

COMP:lindner-il6-balf-p90

BAL fluid IL-6, OVA/P90 vs OVA/H2O - no change

Table 2. NULL RESULT reported by the paper: P90 did not increase BAL fluid IL-6 (0.8+/-0.2 vs 1.1+/-0.3 pg/mL, not significant). No change magnitude is stated by the paper, so none is recorded.

Statistical test: Mann-Whitney U test

COMP:lindner-il8-balf-p90

BAL fluid KC (murine IL-8 homologue), OVA/P90 vs OVA/H2O - no change

Table 2. NULL RESULT: P90 did not increase BAL fluid KC (16.7+/-4.3 vs 21.6+/-5.7 pg/mL, not significant). The paper describes KC as the murine homolog of human IL-8, which is why it is carried in il8_concentration.

Statistical test: Mann-Whitney U test

COMP:lindner-lung-resistance-p90

Lung resistance to methacholine, OVA/P90 vs OVA/H2O - no change

Figure 1c. NULL RESULT: airway hyperresponsiveness to methacholine in the CBNP groups was not different from OVA/H2O. Values are plotted only, so no magnitude is available.

Statistical test: Mann-Whitney U test

COMP:lindner-cyp1a1-intrapulmonary-p90

Cyp1a1 mRNA in intrapulmonary airways, OVA/P90 vs OVA/H2O - no change

Table 3. NULL RESULT: the exposure to CBNP did not increase Cyp1a1 mRNA (1.3+/-0.4 vs 1.0+/-0.2 n-fold, not significant), i.e. no AhR pathway activation.

Statistical test: Mann-Whitney U test

COMP:lindner-cyp1b1-trachea-as-pah

Cyp1b1 mRNA in tracheal epithelial cells, OVA/AS-PAH vs OVA/H2O - no change

Table 3. AS-PAH carries a mixture of at least 12 surface PAH species and gave the numerically highest Cyp1b1 value (2.8+/-0.9 vs 1.2+/-0.3 n-fold), but the paper states that no CBNP increased the Cyp enzymes, i.e. the difference was not significant.

Statistical test: Mann-Whitney U test

COMP:lindner-ho1-intrapulmonary-p90

HO-1 mRNA in intrapulmonary airways, OVA/P90 vs OVA/H2O - no change

Table 3. NULL RESULT for the oxidative stress MIE: HO-1 mRNA did not increase after CBNP exposure (1.1+/-0.1 vs 1.3+/-0.1 n-fold, not significant).

Statistical test: Mann-Whitney U test

COMP:lindner-muc5ac-intrapulmonary-p90

Muc5ac mRNA in intrapulmonary airways, OVA/P90 vs OVA/H2O - no change

Table 3. Airway-region specificity: while P90 raised Muc5ac in the trachea, Muc5ac mRNA in the intrapulmonary airways remained constant (29+/-4 vs 44+/-11 n-fold, not significant).

Statistical test: Mann-Whitney U test

COMP:lindner-muc5b-trachea-p90

Muc5b mRNA in tracheal epithelial cells, OVA/P90 vs OVA/H2O - no change

Table 3. The mirror of the intrapulmonary Muc5b decrease: P90 had no effect on Muc5b mRNA in the tracheal epithelial cells (2.2+/-0.2 vs 2.2+/-0.6 n-fold).

Statistical test: Mann-Whitney U test

COMP:lindner-muc5ac-trachea-as-pah

Muc5ac mRNA in tracheal epithelial cells, OVA/AS-PAH vs OVA/H2O - no change

Table 3. The PAH-containing CBNP did not change mucin mRNA expression; AS-PAH gave 39+/-20 vs 35+/-10 n-fold. Read with the P90 result, this is the paper's basis for suggesting that the higher specific surface area of uncoated P90, not the surface PAH, drives the Muc5ac response.

Statistical test: Mann-Whitney U test

COMP:lindner-pas-trachea-p90

PAS-positive cells in tracheal epithelium, OVA/P90 vs OVA/H2O - no change

Figure 2d,e. NULL RESULT: the number of PAS-positive cells was not increased after P90 exposure, so the P90-induced Muc5ac mRNA rise did not translate into more mucus-storing goblet cells. Plotted only (n = 2-3), so no magnitude is available.

Statistical test: Mann-Whitney U test

COMP:lindner-transport-rate-p90

Tracheal particle transport speed, OVA/P90 vs OVA/H2O - no change

Figure 3b. NULL RESULT: the exposure to the CBNP did not further decrease the particle transport, even though the OVA challenge itself had already slowed it. Plotted only, so no magnitude is available.

Statistical test: Mann-Whitney U test

COMP:lindner-il1beta-balf-as-pah

BAL fluid IL-1 beta, OVA/AS-PAH vs OVA/H2O - decreased

Table 2. AS-PAH reduced IL-1 beta protein in the BAL fluid (0.5+/-0.1 vs 1.3+/-0.2 pg/mL). The paper marks significance as p < 0.05 without an exact p-value and states no change magnitude, so neither is recorded. This is an attenuation of the allergic inflammation, not an aggravation.

Statistical test: Mann-Whitney U test

COMP:lindner-il1beta-balf-p90-9na

BAL fluid IL-1 beta, OVA/P90-9NA vs OVA/H2O - decreased

Table 2. P90-9NA reduced IL-1 beta protein in the BAL fluid (0.7+/-0.2 vs 1.3+/-0.2 pg/mL), marked p < 0.05. Another attenuating effect of a PAH-coated particle.

Statistical test: Mann-Whitney U test

COMP:lindner-il6-mrna-intrapulmonary-p90-9na

IL-6 mRNA in intrapulmonary airways, OVA/P90-9NA vs OVA/H2O - decreased

Table 3. P90-9NA significantly decreased IL-6 mRNA in intrapulmonary airways (4.3+/-0.5 vs 7.9+/-1.2 n-fold), marked p < 0.05.

Statistical test: Mann-Whitney U test

COMP:lindner-il13-mrna-intrapulmonary-p90-9na

IL-13 mRNA in intrapulmonary airways, OVA/P90-9NA vs OVA/H2O - decreased

Table 3. P90-9NA significantly decreased IL-13 mRNA in intrapulmonary airways (34+/-4 vs 52+/-8 n-fold), marked p < 0.05, consistent with the decreased IL-13 protein in its BAL fluid.

Statistical test: Mann-Whitney U test

COMP:lindner-muc5ac-trachea-p90

Muc5ac mRNA in tracheal epithelial cells, OVA/P90 vs OVA/H2O - increased

Table 3. The clearest positive finding of the paper: unmodified P90 increased Muc5ac mRNA in the tracheal epithelial cells (101+/-15 vs 35+/-10 n-fold), marked p < 0.01. The paper reports a direction and a significance threshold rather than an exact p-value or a change magnitude, so neither is recorded.

Statistical test: Mann-Whitney U test

COMP:lindner-muc5b-intrapulmonary-p90

Muc5b mRNA in intrapulmonary airways, OVA/P90 vs OVA/H2O - decreased

Table 3. Unmodified P90 decreased Muc5b mRNA in the intrapulmonary airways (18+/-3 vs 31+/-5 n-fold), marked p < 0.05. Because Muc5b is required for effective mucociliary clearance, the authors read this as a potential worsening of the allergic airway inflammation despite the overall null result.

Statistical test: Mann-Whitney U test

COMP:lindner-beat-frequency-p90

Tracheal ciliary beat frequency, OVA/P90 vs OVA/H2O - decreased

Figure 3c. The unmodified P90 decreased the tracheal ciliary beat frequency; the PAH-coated particles and AS-PAH did not. Plotted only, so no magnitude is available. The authors attribute the effect to the Muc5ac-rich mucus agglutinating the cilia, not to cilia damage, because ATP stimulation raised beat frequency equally in all groups.

Statistical test: Mann-Whitney U test

COMP:lindner-beat-frequency-p90-bap

Tracheal ciliary beat frequency, OVA/P90-BaP vs OVA/H2O - no change

Figure 3c. The PAH-coated P90 particles did not affect the ciliary beat frequency, which together with their unchanged Muc5ac mRNA is the paper's basis for pointing at the higher specific surface area of unmodified P90 as the critical particle characteristic.

Statistical test: Mann-Whitney U test

KER:lindner-muc5ac-to-cbf

Increased Muc5ac-rich mucus leads to decreased ciliary beat frequency

The mechanism the authors propose for the single P90-specific effect on ciliary function: the P90-induced Muc5ac increase makes the released mucus more adhesive and viscous, which agglutinates the cilia. The inference is explicitly hypothetical in the paper ("we suggest", "might have"), and rests on a plotted beat frequency difference plus the observation that ATP stimulation restored beat frequency equally in all groups, so evidence support is recorded as weak.

KER:lindner-muc5b-loss-to-mcc

Decreased Muc5b in intrapulmonary airways leads to impaired mucociliary clearance

Muc5b is required for airway defence against pathogens and for effective mucociliary clearance, so the authors read the P90-induced decrease of Muc5b mRNA in the intrapulmonary airways as probably affecting mucociliary clearance. No clearance measurement was made in the intrapulmonary airways in this study - the measured particle transport speed is tracheal and was unchanged by CBNP - so this link is an inference rather than a measured relationship and is recorded as indirect with weak support.

PROTOCOL:lindner-cba-001

Cytometric bead array for BAL fluid cytokines and chemokines

BAL fluid centrifuged; cytokines and chemokines relevant for lung inflammation determined in the supernatant with CBA Flex Set Kits (BD Bioscience). Fluorescent intensity read on a BD Accuri C6 flow cytometer, analysed with FCAP Array v3.0. Results reported as absolute protein concentrations (pg/mL).

Equipment: CBA Flex Set Kits (BD Bioscience, San Jose, CA, USA); BD Accuri C6 flow cytometer (BD Bioscience); FCAP Array v3.0 Software (BD Bioscience)

PROTOCOL:lindner-bal-diffcount-001

Bronchoalveolar lavage and differential immune cell count

Trachea cannulated and lungs rinsed with 1 mL D-PBS containing protease inhibitor. BAL volume recorded and total immune cell number counted in a Neubauer chamber. For the differential count, 50 uL of BAL fluid was cytospinned (500 x g, 5 min) and macrophages, lymphocytes, eosinophil and neutrophil granulocytes stained by the Pappenheim method or with Diffquick solutions. At least 100 immune cells counted per sample; results given as total cells per mL BAL fluid.

Equipment: Neubauer counting chamber; Cytospin (Thermo Scientific); Diffquick solutions (Medion Grifols Diagnostics AG)

PROTOCOL:lindner-lungfx-001

Invasive measurement of total lung resistance with methacholine challenge

Airway responsiveness to methacholine assessed invasively as total lung resistance using Finepoint RC Units (Data Science International). Mice anaesthetised, tracheotomised with a cannula and mechanically ventilated. Data represent the airway resistance in response to a 100 mg/mL methacholine dose.

Equipment: Finepoint RC Units (Data Science International, New Brighton, MN, USA); Tracheal cannula and mechanical ventilator

PROTOCOL:lindner-qpcr-trachea-001

TaqMan real-time RT-PCR on isolated tracheal epithelial cells

Total RNA isolated from tracheal epithelial cells. Real-time RT-PCR in TaqMan Universal PCR Master Mix on an ABI PRISM 7900HT or StepOnePlus Sequence Detector System; probes tagged with reporter 6-FAM and quencher Tamra. Expression changes calculated as the difference in cycle threshold to the housekeeping gene RPL32 with the delta-delta-CT method and reported as n-fold expression (2^-ddCT).

Equipment: ABI PRISM 7900HT / StepOnePlus Sequence Detector System (Applied Biosystems); TaqMan Universal PCR Master Mix (Life Technologies)

PROTOCOL:lindner-qpcr-airways-001

SYBR Green real-time RT-PCR on microdissected intrapulmonary airways

Distal airways microdissected from surrounding parenchyma under a stereomicroscope and pooled. Real-time RT-PCR on a Roche Light Cycler 480 II using Light Cycler 480 SYBR green I Master. Cycling: 95 C 10 min, then 45 cycles touch-down 63-58 C for 8 s at 0.5 C/s and 72 C for 10 s. Expression changes calculated as n-fold difference in cycle threshold to the housekeeping gene RPL32, normalised by the rule of proportion.

Equipment: Roche Light Cycler 480 II Instrument (Roche Diagnostics); Light Cycler 480 SYBR green I Master (Roche Diagnostics)

PROTOCOL:lindner-pas-lung-001

PAS staining and stereological quantification of goblet cells in lung sections

Lungs fixed ex situ by intra-tracheal instillation of 4% phosphate buffered paraformaldehyde, stored overnight in 4% paraformaldehyde and paraffin embedded with randomised orientation (orientator technique). 2 um sections stained with periodic acid-Schiff reagent, imaged at 20-fold magnification. Surface area of mucin-containing goblet cells per epithelial basal membrane surface area (Sgc/Sep) and volume of PAS-stained epithelial mucin per Sep (Vmucin/Sep) determined with the newCAST computer-assisted stereology toolbox.

Equipment: Olympus BX-51 microscope with DP-25 digital camera; newCAST stereology toolbox (Visiopharm, Hoersholm, Denmark); Periodic acid-Schiff reagent

PROTOCOL:lindner-pas-trachea-001

PAS staining of tracheal epithelium and counting of PAS-positive cells

Tracheae pinned on a cork plate, fixed in 4% phosphate-buffered paraformaldehyde, paraffin embedded and longitudinally sliced in fractionated series. 7 um sections stained via PAS reaction and haematoxylin. PAS-positive cells counted in at least 2 areas 490 um apart, each at least 350 um wide, one slice every 70 um, with at least 520 total epithelial cells counted. Reported as the percentage of the total number of non-ciliated cells.

Equipment: Microtome; Periodic acid-Schiff reagent and haematoxylin

PROTOCOL:lindner-cbf-001

Video microscopy of ciliary beat frequency in explanted trachea

Trachea samples pinned epithelium-up in a Sylgard-coated Delta T4 culture dish under 2 mL HEPES-Ringer solution, held at 30 C. Cilia motion recorded with 1000 images at 100 Hz in at least four areas per trachea; ciliary beat frequency of 10 ciliated cells measured per area and determined by Fourier transformation of grey level changes over time with Matlab-based software. Mean frequency calculated per trachea. ATP (10 uM) added at the end of each experiment to activate cilia beating.

Equipment: Zeiss Examiner.D1 microscope, Achroplan 40x/0.80 W objective; SMX-150M camera (EHD Imaging GmbH); Bioptechs Delta T4 Culture Dish Controller

PROTOCOL:lindner-transport-001

Particle transport speed on explanted tracheal epithelium

Transport of 4.5 um polystyrene particles (2 x 10^6 particles/mL) recorded on the explanted tracheal epithelium at 30 C with 200 images at 12 Hz from at least four different areas per trachea. Particle transport speed determined with Image-Pro Plus 6.0 and the mean speed calculated for each trachea.

Equipment: Zeiss Examiner.D1 microscope, 20x/1.0 DIC (UV) VIS-R objective; 4.5 um polystyrene particles; Image-Pro Plus 6.0 (Medium Cybernetics)