1%) and EDTA-2K (0.05 mM) dissolved in PBS, and the number of total cells, neutrophils, macrophages, lymphocytes, and eosinophils were counted with an automatic erythrocyte analyzer (XT-2000iV, Sysmex Corporation, Hyogo, Japan). Lactate dehydrogenase (LDH) and total protein (TP) concentrations in the supernatant obtained by centrifugation of the BALF were measured with an automatic biochemical analyzer (TBA-200FR, Toshiba Medical Systems Corporation, Tochigi, Japan). Interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, granulocyte monocyte colony stimulating factor (GM-CSF), interferon (IFN)-γ, and tumor

necrosis factor (TNF)-α concentrations were measured using Regorafenib purchase a Rat Cytokine 10-Plex A Panel kit and Bio-Plex Suspension Array System (Bio-Rad Laboratories, Inc., Tokyo, Japan). The trachea, left lung, liver, kidney, spleen, and cerebrum were fixed with 10% (v/v) neutral phosphate-buffered formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histopathological evaluation under the light microscope. Morphology of the MWCNTs in the lung was observed with the light microscope. Sections of the right lung after lavage were fixed with glutaraldehyde and were resin-embedded to give ultrathin sections. Morphology of the individual tubes of instilled MWCNTs in the lung of rats was observed with TEM (JEM-100CX II, JEOL

Ltd., Tokyo, Japan). Statistical analyses of the body and lung weights, as well as the cell numbers and biochemical parameters in the BALF were PARP inhibitor conducted. Statistical significance was determined using multiple comparison tests between the negative control

and MWCNT-exposed groups. First, the Bartlett’s test was conducted. One-way layout analysis of variance was conducted when the variances were equal. Further, Dunnett’s multiple comparison tests were conducted when the differences between the groups were significant. DAPT in vivo The Kruskal–Wallis test was used when the variances were not equal and Steel’s multiple comparison tests were conducted when the differences were significant. Statistical significance was determined between the positive and negative control groups using intergroup comparison tests. First, the F-test was conducted; the Student’s t-test was used when the variances were equal, and the Aspin–Welch t-test was used when the variances were not equal. Statistical significances were judged at the 0.05 probability level. SAS System version 6.12 (SAS Institute Japan Ltd., Tokyo, Japan) was used for all the statistical analyses. SEM and TEM images of the bulk and dispersed MWCNT samples are shown in Fig. 1. In the bulk MWCNT samples, MWCNTs were in the form of agglomerates with sizes ranging from 50 to 100 μm, which are formed from tangled individual tubes with lengths of more than 10 μm (Fig. 1a).