-
Oral gavage at a volume of 400 μL normal saline [200 mg/(kg∙body weight)] was given to the mice in the control group. The IVIS Spectrum imaging showed that the FI of different tissues varied greatly (Table 1). Of all the organs collected, the heart had the lowest intensity of autofluorescence. In contrast, the FI of excrement and the large intestine was high, more than 18 and 15 times greater than that of the heart, respectively. In the quality control step, we tested the food and water consumed by the animals in advance and found that the food contained a strong fluorescence signal. Although we previously fasted the animals for at least 12 h, the data for the digestive system may have been elevated due to the food residues. Except for the small intestine (P = 0.043), there was no statistically significant difference in the other tissues at the different time points. This difference could have been caused by food residues.
Organs/tissues Different time points (Mean ± SD) 0.5 h (n = 3) 1 h (n = 3) 2 h (n = 3) 4 h (n = 3) Total Excrement 277.90 ± 114.01 216.66 ± 37.82 289.96 ± 108.78 155.04 ± 42.63 234.89 ± 90.92 Large intestine 235.95 ± 97.01 213.92 ± 64.14 176.70 ± 35.30 149.55 ± 67.29 194.03 ± 68.68 Stomach 136.24 ± 73.77 167.07 ± 60.01 202.77 ± 62.81 135.92 ± 19.57 160.50 ± 57.06 Bladder 109.20 ± 21.35 182.18 ± 143.60 155.57 ± 7.12 127.20 ± 23.96 143.54 ± 69.19 Small intestine 78.67 ± 12.57 180.44 ± 58.70 69.18 ± 1.01 78.38 ± 10.44 101.67 ± 54.29 Lung 34.26 ± 4.08 31.34 ± 4.06 34.45 ± 6.50 42.14 ± 9.84 35.55 ± 6.98 Skeletal muscle 31.19 ± 4.51 29.68 ± 4.46 30.89 ± 5.96 28.55 ± 8.88 30.08 ± 5.41 Liver 26.63 ± 4.68 20.77 ± 2.97 24.38 ± 0.55 29.47 ± 5.50 25.31 ± 4.71 Kidney 23.56 ± 2.01 23.94 ± 5.39 22.99 ± 2.51 24.92 ± 5.81 23.85 ± 3.72 Spleen 22.86 ± 11.13 16.53 ± 1.87 13.34 ± 0.85 9.59 ± 3.23 15.58 ± 7.15 Heart 11.88 ± 0.39 13.38 ± 5.01 12.63 ± 1.52 13.08 ± 0.97 12.74 ± 2.35 Table 1. Tissue fluorescence intensity in the control group at different time points (unit: [(p/sec)/(uW/cm2)]/g, 1.00 × 108)
-
In our previous study, the highest FI in the 100 nm PS group was observed at 0.5 h when the observation intervals were set to 0.5, 1, 2, and 4 h. To explore the exact absorption peak times, additional experiments were carried out using 100 nm fluorescent microspheres at higher doses [250 mg/(kg∙body weight)] and shorter time intervals (t = 5, 15, 30, and 45 min). The results are shown in Figure 1. Even when the exposure dose was increased, the highest FI in blood was still observed at t = 0.5 h (P < 0.05).
-
Figure 2 shows the time course of FI in the digestive system. At 0.5 h after the administration of 100 nm NPs, the FI of the stomach, small intestine, and large intestine were 19.24, 7.67, and 3.70 times higher than in the control group, respectively (P < 0.05). For the stomach and small intestine, the highest FI occurred at 0.5 h, and then began to decrease, eventually reaching the same level as the control group. In contrast, there was an increasing trend in excrement. The FI began to increase significantly from 1 h, and the highest level occurred at 4 h after exposure (12.79 times, P < 0.05). Similar trends were detected in the 3 μm group. The FI of the stomach and small intestine peaked at 0.5 h (stomach 2.32 times higher, small intestine 2.03 times higher, P < 0.05), then gradually decreased over time, and declined to the same level as the control group at 4 h. The FI of the large intestine increased at 1 h (1.91 times higher, P < 0.05), and then followed a decreasing trend, and the changes in excrement were not significant. In the 10 μm group, the FI in the stomach increased by approximately 3 times at 0.5, 1, and 2 h (P < 0.05), and displayed a downward trend. No significant change was found in the small intestine. From 2 h, the levels in the large intestine and excrement increased significantly (large intestine 6.43 times, stool 21.46 times, P < 0.05), and still maintained a high level compared with the control group at 4 h (P < 0.05).
Figure 2. Time course changes of fluorescence intensity in stomach, small intestine, large intestine, and excrement after administering NMPs by gavage at a dose of 200 mg/(kg∙body weight). a: compared with control, P < 0.05; b: compared with 0.5 h, P < 0.05; c: compared with 1 h, P < 0.05; d: compared with 2 h, P < 0.05. NMPs, nano-/microplastics.
-
There was no significant difference between the three treatment groups compared with the control group at different observation time points in the heart (P > 0.05, Figure 3A). The FI in the lung peaked at 2 h (1.31 times higher, P < 0.05), and decreased to the control level (0.97 times) at 4 h after exposure in the 3 μm group. There was no significant increase in the 100 nm and 10 μm groups at any time points after the oral gavage (Figure 3B).
Figure 3. Time course changes of fluorescence intensity in heart (A) and lung (B) after administering NMPs by gavage at a dose of 200 mg/(kg∙body weight). a: compared with control, P < 0.05; b: compared with 0.5 h, P < 0.05; c: compared with 1 h, P < 0.05; d: compared with 2 h, P < 0.05. NMPs, nano-microplastics.
-
The liver, spleen, kidney, and bladder are abdominal organs, and their anatomical position prevented direct contact with NMPs from oral administration. The only significant increase in FI was found in the liver at 4 h (1.34 times higher, P < 0.05) in the 100 nm group. No significant difference was found in the 3 and 10 μm groups compared with the control group at each observation time point (Figure 4A). In the kidney, the FI began to increase slightly after 1 h, and reached a peak at 4 h in the 100 nm group (1.39 times higher, P < 0.05) compared with the control group. The FI curve of the 10 μm group showed the same fluctuations, and the highest level reached 1.24 times at 4 h (P < 0.05). There was no significant change in the 3 μm group (Figure 4C), and there was no significant change observed in the spleen and bladder (Figure 4B and 4D).
Figure 4. Time course changes of fluorescence intensity in liver (A); spleen (B); kidney (C); bladder (D) after administering NMPs by gavage at a dose of 200 mg/(kg∙body weight). n = 3; a: compared with control, P < 0.05; b: compared with 0.5 h, P < 0.05; c: compared with 1 h, P < 0.05. NMPs, nano-/microplastics.
-
Skeletal muscle accounts for about 30%−40% of mammalian body weight[23], and it receives a large blood supply, especially when the animal undertakes physical activity. In our previous study, the FI increased significantly in the blood at 0.5 h after the oral gavage, and was maintained at a relatively high level at 4 h. It was therefore assumed that NMPs would accumulate in the skeletal muscle through blood circulation. Therefore, the skeletal muscle of the animals was collected for observation in this experiment. The IVIS Spectrum results showed that an increased FI was only observed at 4 h after exposure in the 100 nm group, which was statistically significant compared to the control group (1.77 times, P < 0.05). However, no change in FI was observed in the 3 and 10 μm groups (Figure 5).
-
To confirm the results obtained from the IVIS Spectrum, we further performed H&E staining to verify the changes in fluorescence signals. The mice with the strongest FI at any observational timepoint compared with its control group were selected to perform H&E staining. Samples of stomach at 0.5 h in all NMPs treated groups; small intestine at 0.5 h in the 100 nm and 3 μm treated group; large intestine at 0.5 h in the 100 nm treated group, 1 h in the 3 μm treated group and 2 h in the 10 μm treated group; lung at 2 h in the 3 μm treated group, liver at 4 h in the 100 nm treated group, kidney at 4 h in the 100 nm and 10 μm treated groups, and skeletal muscle at 4 h in the 100 nm treated group, were therefore examined. The H&E staining results are shown in Figures 6−7.
Figure 6. Histological examination of the digestive system (200×). The first column is the image of H&E staining. The second is the merge of both fluorescence microspheres and DAPI dye. The third is the fluorescence image of DAPI dye. The fourth is the fluorescence image of fluorescence microspheres (green). DAPI, 4',6-diaminidine-2-phenylindole.
Figure 7. Histological examination of lung, liver, kidney and skeletal muscle (200×). The first column is the image of H&E staining. The second is the merge of both fluorescence microspheres and DAPI dye. The third is the fluorescence image of DAPI dye. The fourth is the fluorescence image of fluorescence microspheres (green). DAPI, 4',6-diaminidine-2-phenylindole. Black arrows indicate inflammation.
In the digestive system, the largest FI was found in the stomach and small intestine in the 100 nm group. By contrast, in the large intestine, a stronger FI was detected in the 10 μm group than in the 100 nm and 3 μm groups (Figure 6). Most fluorescence signals were found in the mucosal layer in the large intestine, and the magnitude of the fluorescence signals gradually decreased from the muscular layer to the serosa membrane (Figure 6H–J). The histological analysis of small intestine showed that mucosal injury, epithelial damage, disintegration of the lamina propria, hemorrhage, accompanied by slight infiltration of inflammatory cells were detected in the 100 nm group, whereas, swelling and congestion of the intestinal villi and exfoliation of epithelial cells were shown in the 3 μm group (Figure 6E2–J2). By contrast, there was no hyperemia or significant infiltration of inflammatory cells in the 10 μm group.
In observed organ/tissues, no evidence of changes in cellular structure was shown in 3 and 10 μm groups. In 100 nm group, only mild congestion and small amounts of inflammatory cells were shown in lung. Slight congestion and vacuolation in the cytoplasm of the hepatocytes were found in liver. Stronger fluorescence signals were also found in the central vein of the liver and lung (Figure 7 and Supplementary Figure S1, available in www.besjournal.com). In the kidney, the glomerulus was swollen and neutrophilia was infiltrated slightly. In the skeletal muscle, the inward migration appeared in muscle fiber cell nucleus and the distance between muscle cells increased, accompanied by an uneven size and shape (Figure 7).
Figure S1. Histological examination of lung and kidney in other sizes of NMPs (200×). The first column is the image of H&E staining. The second is the merge of both fluorescence microspheres and DAPI dye. The third is the fluorescence image of DAPI dye. The fourth is the fluorescence image of fluorescence microspheres (green). DAPI, 4',6-diaminidine-2-phenylindole. Black arrows indicate inflammation.
HTML
Tissue Autofluorescence Intensity for the Control Group at Different Time Points
The Absorption Peak of 100 nm Fluorescent Microspheres in Blood by IVIS Spectrum
The Absorption of 100 nm, 3 μm, and 10 μm Fluorescent Microspheres in the Digestive System by IVIS Spectrum
The Absorption of 100 nm, 3 μm, and 10 μm Fluorescent Microspheres in Thoracic Organs by IVIS Spectrum
The Absorption of 100 nm, 3 μm, and 10 μm Fluorescent Microspheres in Abdominal Organs by IVIS Spectrum
The Absorption of 100 nm, 3 μm, and 10 μm Fluorescent Microspheres in Skeletal Muscles by IVIS Spectrum
Histological Confirmation
23219+Supplementary Materials.pdf |