P 1500

AAV9 expressing GFP-Cre under the control of CamKII promoter (pENN.AAV.CamKII.HI.GFP-Cre.WPRE.SV40, Addgene #105551) was used to delete the expression of PRs in the EC of adult PRfl/fl males. Control mice were injected with AAV9 expressing GFP under the control of CamKII promoter (pENN.AAV.CamKII0.4.eGFP.WPRE.rBG, Addgene # 105541). The injections were performed bilaterally. The stereotaxic coordinates used for viral injections were anteroposterior [AP] − 4.6; mediolateral [ML], −/+ 3.0; dorsoventral [DV], − 3.7 and-3.0 from dura for central and medial EC and AP − 3.7, ML −/+ 4.2, and DV − 3.5 and − 3.0 from dura for lateral EC. A Hamilton Company (Reno, NV) syringe (Hamilton 7000 Glass, 1 μl, 0.3302 mm) was loaded with virus solution and mounted in the peristaltic pump holder (Harvard Apparatus, Holliston, MA; P-1500), 200 nl of the virus was injected at each site at a flow rate of 200 nl/min. The experiments were performed two weeks after viral injection. At the end of the experiment, viral transduction of the EC cortical neurons was confirmed by evaluating GFP expression in the brain slices.

Cigarette smoke extract solution was prepared using a modification of the method described earlier (41 (link)). CS of two 3R4F research grade cigarettes (Kentucky Tobacco Research and Development Center at the University of Kentucky, Lexington, KY, USA) were bubbled into 10 ml of RPMI without FBS (Lonza, Basel, Switzerland) using a Harvard peristaltic pump (Harvard Apparatus, P-1500, Holliston, USA) at a 550 ml/min flow rate for approximately 3 min. CSE solution was filtered through a 0.22 µm filter (TPP, Trasadingen, Switzerland). This solution was considered as 100% stock and diluted with culture media to 0.5% for further experiments. The pH of each CSE solution was measured as a mean pH 7.28. The prepared CSE solution was used within 30 min of preparation. The OD of the inner particles of the CSE was measured spectrophotometrically at the wavelength of 360 nm and compared to the control media.

Micro particle image velocimetry (μPIV) was performed using deionized water as working fluid, seeded with 3.55 μm fluorescent polystyrene particles (530/607 nm, microParticles GmbH). The experimental set-up consisted of an epi-fluorescence microscope system including a 3D stage system (LaVision FlowMaster Mitas) in combination with a 532 nm Nd:YAG laser (New Wave, Solo II-15) and a 2048 × 2048 pixel CCD camera (LaVision Imager ProX 4 M). For image acquisition a 5X, 0.16 NA microscope objective was employed (Zeiss EC “Plan-Neofluar”). For data post-processing the commercial software package Davis 7 (LaVision) was used. The volume flow of fluids was controlled with a peristaltic pump (P1500, Harvard Apparatus) and measured with a flow sensor (PVDF Chemical Flowmeter 0.025–2.5 l/min, B.I.O-TECH e.K., Germany). For each flow condition and measurement position 25 two-frame experimental images were acquired at a trigger rate of 4 Hz. The cross-correlation technique was used for analysis. The final interrogation window of 128 × 128 pixels overlapped by 50%, the corresponding vector spacing was 62.5 μm. The depth of correlation was determined to be about 124 μm, following ref. 61 . After processing, the 25 experimental images were time-averaged. Further details on the experimental set-up have been previously reported in ref. 62 (link).