Our experiment was conducted on 30 male Wistar rats of average body weight 187,82±12,56 g at the beginning of the study. The animals were divided into three groups: A, B, C. The animals in group A were exposed to scent free e-cig vapour. During the 10 min exposition, the rats were placed in a 0.1 m³ PCV cage and were exposed to the vapour with the use of the pump (0.18 kW; 1.4/1.6 A; 230 V; 50/60 Hz). It was installed on one side of the box, while e-cigarette aerosol was puffed on the other. This mechanism generates airflow into the cage. The cage containing 5 animals at a time was hermetically sealed with the two holes (e-cig and pump connection points) that were left open. Animals were exposed in order to consume 0.6 ml/day of e-liquid containing 12 mg/ml of nicotine, propylene glycol and water produced by Inawera Dot Com Sp. Z o.o. Sp. K. One cycle of treatment consisted of 5 min puff followed by 20 min stop. During the experiment the e-cig voltage was set at 5.5 V. At the end of the cycle the animals were transferred to a clean cage. Animals were subjected to 1 cycle/day for 5 consecutive days/week, and for 6 consecutive weeks (27) The rats in group B were exposed to the smoke from 10 traditional cigarettes with the same total nicotine dose as in group A. In total, one group of animals was exposed to 210 mg of nicotine. The concentration of nicotine in serum of each rat was not tested. Therefore, it is not possible to precisely define the dose of nicotine received by single rat. Our study is based on the official list of the substances in the liquid as reported by the manufacturer. No additional tests were performed to assess the liquid composition.

The rats in group C constituted the control group and they were exposed to the same inhalation-related stress that other rats but without the nicotine element. The animals were decapitated without anaesthesia 24 h after last exposition and their lungs were dissected. Body weight of rats at the time of sacrifice was: 290.73 SD 15,69 g (group A), 287.67 SD 21.34 g (group B), 324.38 SD 19, 16 g (group C).

Our study involves histological analysis of multiple organs as well as some biochemical and molecular markers measurement by ELISA. Therefore, we had to carefully choose the right euthanasia method, so that it would not affect our results. Due to multiple concerns, we have chosen the decapitation without prior anesthaesia. It was performed by an experienced worker of the Experimental Medicine Facility of the Medical University of Lublin. The CO₂ euthanasia is known to negatively affect the lungs, the study of which are important part of our project (28,29). Pentobarbital on the other hand significantly changes the biochemistry of brain, including the acetylcholinesterase activity (30). Moreover, similarly to CO₂ it also alters the blood biochemical markers (31). Enflurane, halothane, isoflurane and sevoflurane negatively affect rat sperm (32). Moreover, isoflurane euthanasia significantly impact the metabolism of liver, possibly changing the glycogen levels (33). The cervical dislocation was rejected as it requires prior anesthesia. Concussion on the other hand could significantly damage the brain, therefore, it was also not considered.

To conclude, decaptation was chosen because contrary to other methods accepted by EU Directive 2010/63/EU it does not induce significant changes to any organs except for the neck and does not alter the biochemistry. Bearing the responsibility, we have obtained the necessary approval of Bioethical Committee for the whole experiment, including the decapitation.

After fixing the material in 10% buffered formalin, the organs were embedded in paraffin blocks. The material was then tailored into 5-µm-thick sections. The experiment was conducted with the formal approval of the local animal care committees: ‘Local Ethics Committee for Animal Experiments’ by University of Life Sciences in Lublin (30/2015). The study was carried out in accordance with Directive 2010/63/Eu of The European Parliament and of The Council of 22 September 2010 on The Protection of Animals Used For Scientific Purposes.

The histomorphological evaluation of the tissues stained with H&E was performed with the use of a light microscope using lenses x10, x40 and x100. Fifty fields of view from each animal were analysed. Samples were stained by Masson's Trichrome method to assess collagen deposition and fibrosis. PAS staining was done to visualise the blood-air barrier. We used a standard procedure of described stainings. The thickness of the membrane forming blood-air barrier was measured using a microscope with digital camera Olympus BX41, lens x100 and CellSense software in the PAS stained sections. Optical density of the picture of the trichrome-stained area was outlined and quantified using ImageJ software and its associated colour deconvolution plugin as described previously (34,35).

This immunohistochemical staining was performed with the use of antibodies directed against α-Smooth Muscle Actin (α-SMA, Elabscience, polyclonal rabbit anti-human, -mouse and -rat antibody, E-AB-33323, dilution 1:200, previously used by Li et al (36) to assess myofibroblasts that are responsible for collagen fiber production and form blood vessel walls. The exposure of the antigenic sites was performed thermally by incubation in citrate buffer solution with pH=6, in a microwave oven at 800 W, for 3 cycles lasting 5 min each. In order to inhibit endogenous peroxidase activity slides were incubated in 0.3% perhydrol (H₂O₂) in methanol for 30 min. The samples were incubated in normal serum for 1 h to block the non-specific bindings. The material was incubated at 4˚C overnight (17 h) in diluted primary antibody. The diaminobenzidine solution (DAB) and hematoxylin colouration were used to visualize the reaction (5 min). Negative controls were prepared in a similar manner, only the specific primary antibody was omitted. The material was evaluated with a light microscope using lenses x10 and x40. Quantification of the vessel number was determined by the visualization of the α-SMA muscle actin in the vessel wall cells. Blood vessels were counted in 50 fields of view under 100x lens. Elastic fibers were evaluated in orcein staining.

The obtained test results were statistically analysed with Statistica 13.0 (StatSoft). The Shapiro-Wilk test was used to assess the data distribution and the Kruskal-Wallis test was used to calculate the P-values. In this regard, a probability (P-value) <0.05 was considered statistically significant.

A collapse of parenchyma, hyperhagia, hyperplasia of type II of pneumocytes, and an increased number of macrophages was observed in both experimental groups (Fig. 1). Furthermore, in the e-cig group eosinophils and mononuclear cells infiltration was noted, as well as thickened alveolar septa, hyperaemia, intrabronchiolar erythrocytes and the increase of mucus production. In the conventional cigarette group irregularity of alveolar lumen was observed, as well as features of emphysema, vacuolization of cells in the alveolar septa, mucus intrabronchioles and hemorrhage into bronchiole and alveolar lumen.

In the Masson's Trichrome staining, sections of increased collagen deposition within thickened alveolar septa and initial fibrosis was observed in both experimental groups (Fig. 1). The highest optical density score was noted in the conventional cigarette group (Table I). In the control group, normal appearance of the lung tissue and collagen fibres was noted, the latter only surrounded the bronchioles.

In the PAS staining, an increase of the blood-air barrier-forming membrane thickness was observed in both experimental groups (Table I and Fig. 2). In the orcein staining delicate elastin fibres were noted in the control group. An initial elastolysis was observed in both experimental groups-4 elastic fibres were disrupted, sparse, irregular and thickened (Fig. 2).

The highest expression of α-SMA was seen in the conventional cigarette group B (Fig. 2). The most numerous myofibroblasts were within parenchyma of that same group (20±5 in one field of view), fewer in e-cig group A (17±6 in one field of view) and just single ones in the control group. Numbers of α-SMA positive blood vessels were the highest in the conventional cigarette group (Table I).