Excessive aggregation of fine particles may play a crucial role in adolescent spontaneous pneumothorax pathogenesis

Groups and specimens

The bullae (B group) and paravesicular (S group, normal adjacent tissue control) lung tissue specimens were obtained from 30 adolescent and young adult PSP patients around 15–29 years old who underwent a surgery of video-assisted thoracoscopic between November 2020 and October 2021 at Southern Yunnan Central Hospital (Honghe First People’s Hospital). PSP was characterized as the episode of spontaneous pneumothorax in the patient without any apparent pulmonary disease, inherited disease, or trauma disorder. The clinical characteristics of the patients, including body mass index, age, sex, height, weight, smoking habits, and pathological symptoms, were recorded and analyzed (Table 1). Another 30 normal lung tissues from patients aged younger than 30 years old with nonpneumothorax diseases were obtained as another control (N group). All tissue specimens were immediately collected after the surgery and stored in a freezer at −80 ° C. This study was authorized by the Medical Ethics Management Committee of Southern Yunnan Central Hospital (HH2020LLSC-2), and all people who participated in this study signed informed consent forms.

Materials

The 5250040 Varioskan Flash Microplate Reader used in this study was from Thermo Scientific (Waltham, MA, USA). The AK-RO-C2 Ultrapure Water Polishing System was from ELKAY (Chicago, IL, USA). The D3024R high-speed refrigerated centrifuge was from DLAB Scientific (Singapore, Singapore). The Nikon Eclipse E100 Orthostatic Optical Microscope and the Nikon DS-U3 Imaging System were from Nikon (Tokyo, Japan). The RM2016 Pathological Slicer was from Leica (Shanghai, China). The Image-Pro Plus 6.0 was from Media Cybernetics (Rockville, MD, USA). Absolute ethanol was purchased from Sinopharm Chemical (Beijing, China). The Victoria Blue Dye kit and neutral balsam were purchased from Servicebio (Wuhan, China). Normal rabbit serum was from Boster (Wuhan, China). The E0573 oven, pH 6.0 citric acid antigen-repairing solution, phosphate-buffered saline, bovine serum albumin, H&E stain, DAB horseradish peroxidase chromogenic kit, human MMP-9 enzyme-linked immunosorbent assay (ELISA) kit, human chemokine ligand 2 (CCL2)/chemoattractant protein-1 (MCP-1) ELISA kit, and MCP-1/CCL2 rabbit polyclonal antibody were purchased from Beyotime (Shanghai, China).

H & E staining

Routine sections of experimental lung tissue (with a thickness of approximately 4 µm) were placed in an oven for 4 h (60 ° C), treated with xylene, dehydrated with gradient alcohol (100%, 95%, and 70%, respectively), and rinsed with tap water. The slides were stained by hematoxylin dye for 6 min, rinsed with water for 1 min, differentiated with 0.5–1% hydrochloric acid alcohol for a moment (until the color changed from blue to red three times), and washed with water for more than 15 min. Next, the sections were immersed in 1% eosin alcohol for 1–2 min, rinsed with water for 1 min, and subjected to gradient alcohol (95%, 95%, and 100%, respectively) dehydration for 1 min each. Treatment with carbonate xylene (1:3) was performed for 10 s for dehydration, and treatment with xylene I for 1 min was carried out so that the sections became transparent. After drying, the sections were sealed with neutral gum. All standard H&E-stained samples were analyzed and recorded under a Nikon Eclipse E100 camera imaging capture system. Each stained sample with obvious histologic alteration was analyzed by the same pathological specialist in pulmonary pathology. Each test result was verified by another pathologcal expert. Pathological alterations, including lung histological features (such as bleb/bulla, fine particles, pathologic hyperplasia, granulation tissue, etc.) and cellular features (including macrophages, lymphocytes, giant cells, histiocytes, eosinophils, and neutrophils), were carefully recorded.

Fine particulate matter and alveolar macrophage (AM) examinations

The Swiss Giemsa dye kit (Pinofi Biotechnology, Wuhan, China) was applied for standard Wright-Giemsa (W-G) staining. The W-G-stained slides were observed under a light microscope. Three visual fields were randomly selected under the microscope and observed at 100×, 200×, and 400× magnifications, respectively. The distribution of fine particulate matter was observed using an image analysis system. The number of AMs was calculated from five randomly selected visual fields under a 400× high-power microscope.

Elastic fiber staining

The sample to be tested was fixed by 4% paraformaldehyde and stained by Victoria blue elastic fiber dye (Servicebio, Wuhan, China). The stained lung tissue was photographed by an Eclipse Ci-L microscope (Nikon, Tokyo, Japan). The target area of the image was magnified by 200×. The positive area of pulmonary elastic fibers was analysized by Image-Pro Plus 6.0. Three visual fields were analyzed in each slide. The percentage of the positive area (%) was calculated as follows: positive area/tissue area × 100.

Immunohistochemical (IHC) staining

After formalin fixation, the pulmonary tissue slice (4-µm-thick) was subjected to anti-human MCP-1 (1:100 dilution) polyclonal antibody or anti-human MMP-9 (1:200 dilution) monoclonal antibody IHC staining. The stained slices were analyzed by a Nikon Eclipse E100 Image System (Nikon, Tokyo, Japan). The obvious pathologically altered area was analyzed by two independent pathologists. The quality of the staining was semi-quantitatively evaluated as following: no staining (the positive cells were <10%); weak staining (the positive cells were approximately 10–50%); and strong staining (the positive cells were >50%). The H-score was calculated as follows: H = ∑(π × I) = (% of weakly stained area × 1) + (% of moderately stained area × 2) + (% of strongly stained area × 3), where π represents the percentage of the positive signal pixel area and I indicates the positive grade. The H-score value ranges between 0 and 300. A higher H-score indicates a stronger intensity of positive staining.

ELISA

Based on the manufacturer’s protocals provided with the ELISA kits (Beyotime, Shanghai, China), human MCP-1 and human MMP-9 were detected in the lysate of the pulmonary tissue specimens.

Statistical analysis

SPSS (version 25.0, IBM-SPSS, Chicago, IL, USA) was applied for statistical examination. Graphs (GraphPad Prism, San Diego, CA, USA) was version 8.02. Group comparisons of the histopathological data for the PSP and the non-PSP groups were examined with the Fisher’s exact test or chi-squared test. Differences of normally distributed continuous variable data between the PSP patients and the non-PSP controls were detected by the t-test. All statistical examines were two-tailed. P-value < 0.05 was defined as being significant.

PSP tissue had significant alveolar structural damage accompanied by fine particle aggregation and inflammatory reactions

The H&E staining experiments showed that the pulmonary alveolar structure was damaged and fused into bullae. In addition, a large number of dust particle-absorbed macrophages were observed in the alveolar cavity and alveolar interstitium in the B group (experimental group) (Figs. 1C–1D). The Victoria blue dye staining experiments also demonstrated that compared with the S and N groups (Figs. 1G–1H), the B group exhibited dilated alveoli, a widened septum, enlarged alveoli pores, a broken alveoli septum, a reduced pulmonary capillary bed, thickened intima of pulmonary arterioles, increased elastic fibers, and numerous deposited fine particulate matter in the stroma and alveolar cavity accompanied by a large number of AMs (Figs. 1E–1F). Furthermore, the W-G staining experiments showed that compared with the S and N groups, there was obvious aggregation of fine particulate matter and increased numbers of AMs in the alveolar wall and alveolar cavity in the B group (Fig. 2). These data suggest that there was fine particle matter aggregation and inflammation, accompanied with a broken pulmonary structure and pulmonary bullae formation in the PSP pulmonary tissue.

Significantly increased pulmonary elastic fiber content in the PSP patients by Victoria blue dye staining

After counting and calculation of the Victoria blue dye staining results, the pulmonary elastic fiber content in the B group was 10.67 ± 4.39 (95% confidence interval (CI) [9.026–12.31]. However, that in the S and N groups was 6.03 ± 2.45 (95% CI [5.114–6.943]) and 5.89 ± 2.92 (95% CI [4.801–6.98]), respectively, indicating that the pulmonary elastic fiber content of the B group was statistically higher than that of the S or N group. The difference between the B group and the S or N group was significant (P < 0.05). Nevertheless, no significant difference was found in the pulmonary elastic fiber content between the N and S groups (P = 0.842; Fig. 3A).

After staining, five fields of view were randomly obtained from the sections at 400 × magnification. The remarkable histopathological features of the PSP were fine particulate matter (87%), followed by AM accumulation (83%), pathologic hyperplasia (proliferation of fibrous tissue and capillary endothelial cells) (80%), eosinophilic infiltration (77%), hemorrhage (73%), and neutrophil infiltration (67%). The comparisons of the pathological findings between adolescent and young adult PSP (B and S groups) and non-PSP (N group) are presented in Table 2. Compared to the S and N groups, significant elevations of fine particulate matter and inflammation (Fig. 1) were found in the PSP patients (P < 0.05). Following, logistic detection also showed that the accumulation of AMs (odds ratio (OR): 0.116; 95% CI [0.034–0.39]; P < 0.05) and fine particulate matter (OR: 0.065; 95% CI [0.015–0.211]; P < 0.05) was obviously associated with the occurrence of PSP.

Significantly increased inflammation in the PSP patients

Using the W-G staining method, the number of AMs in each 100 cells was calculated in five randomly selected fields under 400 × magnification. The average number of AMs in the alveolar wall and the alveolar cavity of the B group was 9.95 ± 3.36 (95% CI [8.697–11.21]), which was statistically higher than that of the S group (7.17 ± 2.69; 95% CI [6.162–8.171]) and the N group (5.94 ± 2.32; 95% CI [5.072–6.809]; P < 0.05) (Fig. 3B). Combined with the findings above (eosinophilic infiltration (77%) and neutrophil infiltration (67%) in PSP patients), these results indicate that there was significant inflammation in the PSP patients.

Significantly increased MCP-1 and MMP-9 expression levels in the pulmonary tissue of the PSP patients

In order to further analyze the alterations of mesenchyme in lung tissue of the PSP patients, we detected the expression of MCP-1 and MMP-9. IHC examination indicated that the positive area (Fig. 4A; 30.63 ± 12.07%, CI [26.12–35.14]) and the H-score (Fig. 4B; 69.47 ± 26.83, CI [59.45–70.49]) of MCP-1 of the lung tissue in the B group were significantly increased comparing to those in the S group (15.67 ± 8.63%, CI [12.45–18.9]; 39.87 ± 22.25, CI [31.56–48.18], respectively) and the N group (16.72 ± 7.1%, CI [14.07–19.38]; 34.99 ± 8.26, CI [31.9–38.07], respectively) (P < 0.05). In addition, the positive area of MMP-9 (Fig. 4A; 15.24 ± 4.39%, CI [13.57–16.91]) and the H-score (Fig. 4B; 35.86 ± 4.26, CI [26.85–44.87]) in the B group were remarkably elevated than those in the S group (4.71 ± 3.60%, CI [3.34–6.08]; 18.98 ± 2.51, CI [13.68–24.28], respectively) and the N group (6.52 ± 5.53%, CI [4.42–8.62]; 14.08 ± 1.21, CI [11.52–16.64], respectively) (P < 0.05).

The ELISA results also showed that the MCP-1 (Fig. 3C; 1203.13 ± 84.98 pg/mL, 95% CI [1029–1377]) and MMP-9 (Fig. 4C; 1203.13 ± 84.98 pg/mL, 95% CI [26.5–44.3]) levels in the pulmonary tissue of the B group specimens were significantly increased comparing to those in the S (604.58 ± 57.86 pg/mL, 95% CI [486–723]; 9.43 ± 0.87 pg/mL, 95% CI [7.6–11.2], respectively) and N groups (564.79 ± 50.55 pg/mL, 95% CI [461–668]; 8.69 ± 0.68 pg/mL 95% CI: 7.3–10, separatively) (P < 0.05). These data reveal that there are chronic inflammatory reactions in the pulmonary tissue of PSP patients, with significant thickening of mesenchymal tissue, resulting in increased tissue fragility because of an imbalance in the proportion and distribution of extracellular matrix, elastic fibers, and collagen fibers, which could be the histological basis for the easy formation of PSP.