This study describes a technique to establish a silicosis rat model with the inhalation of silica through the whole body in an inhalation chamber. The rats with silicosis could closely mimic the pathological process of human silicosis in an easy, cost-effective manner with good repeatability.
The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent models continue to be an essential tool for studying human silicosis. For silicosis, the classic pathological process needs to be inducible via the inhalation of freshly generated quartz particles, which means specifically inducing human occupational disease. This study described a technique to establish and effectively mimic the pathological dynamic evolution process of silicosis. Further, the technique had good repeatability with no surgery involved. The inhalation exposure system was fabricated, validated, and used for toxicology studies on respirable particle inhalation. The critical components were as follows: (1) bulk dry SiO2 powder generator adjusted with an air-flow controller; (2) 0.3 m3 whole-body inhalation exposure chamber accommodating up to 3 adult rats; (3) a monitoring and control system for regulating oxygen concentration, temperature, humidity, and pressure in real-time; and (4) a barrier and waste disposal system for protecting laboratory technicians and the environment. In summary, the present protocol reports the inhalation via the whole body, and the inhalation chamber created a reliable, reasonable, and repeatable rat silicotic model with low mortality, less injury, and more protection.
Silicosis, which is caused by the inhalation of silica, is the most serious occupational disease in China, accounting for more than 80% of the total number of occupational disease reports every year1. The etiology of silicosis is clear, and it can be prevented and controlled, but no effective treatment method is available2. Many drugs have been proven to be effective in basic studies, but they have imprecise clinical effects3,4. Therefore, the pathological and physiological mechanisms of silicosis still need to be explored.
Many studies have used a one-time infusion of silica into the trachea of rats or mice to investigate the pathogenesis of silicosis5,6. Although these rodent silicotic models could be obtained in a short time7, these methods still had challenges, such as animal trauma and high mortality. Some studies have involved instilling stored silica into the lungs to induce a nonspecific lung reaction, but did not mention silicotic nodules in mice8. Furthermore, away from acute silicosis, chronic exposure to silica in occupational environments induced significantly lower pulmonary inflammation and elevated the levels of anti-apoptotic markers, rather than pro-apoptotic markers, in the lungs9. Therefore, a reliable, reasonable, and repeatable animal model is needed to explore the pathogenesis of silicosis further.
The present study describes a method to mimic the disease process of patients with silicosis through silica inhalation via the whole body, air-delivered particles in an inhalation chamber, which comprised an air-delivered silica generator, a whole-body chamber, and a waste disposal system. This method is simple, easy to operate, and effectively mimics the pathological dynamic evolution process of silicosis. Also, many possible mechanisms and the pathogenesis of silicosis are identified using this method10,11,12. The proposed protocol is anticipated to help further investigations in the related field of silicosis research.
All animal experiments were conducted according to the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Committee on the Ethics of North China University of Science and Technology (protocol code LX2019033 and 2019-3-3 of approval). Male Wistar rats, 3 weeks of age, were used for the present study. All rats were kept in static cages with wood shavings. The animals were maintained in a 12 h/12 h light/dark cycle, and were provided with food and water ad libitum. Follow-up experiments were conducted after 1 week of adaptive feeding.
1. Animal preparation
2. Silica preparation
CAUTION: Silica dust inhaled by the human body can damage the lungs. Therefore, individuals must wear overalls, medical gloves, and protective masks (N95) during operations.
3. Silica exposure to the rats
4. Acquisition and fixation of lung tissues
5. Hematoxylin and eosin (H&E) staining
6. Immunohistochemical staining
Using the proposed method, some potential mechanisms and the pathogenesis of silicosis were explored in rats. The schematic of the inhalation chamber is shown in Figure 1. The chamber consisted of an air-delivered silica generator, a whole-body chamber, and a waste disposal system, as previously described17. The pulmonary functions, levels of inflammatory factors in the serum and lung, collagen deposition, and myofibroblast differentiation were reported in the previous studies10,18,19. The differential expression of miRNA, lncRNA, and mRNA was reported in our previous reports20,21,22. No rats died after silica exposure in the aforementioned multiple-batch studies.
The classic pathological characteristics of rats with silicosis were summarized previously23. The silicotic nodules consisted of silica-contained macrophages. Figure 2 presents the collagen deposition in rats with silicosis. The polarized lens revealed silica in macrophages. Figure 3 presents the dynamic evolution of silicotic nodules by immunohistochemical staining of CD68; other alternative markers included inducible nitric oxide synthase or arginase-124. As mentioned earlier23, the rats exposed to silica for 24 weeks showed visible and observable lesions, including collagen deposition in silicotic nodules, periodic acid-Schiff positive staining, and impaired pulmonary functions. On the other hand, the other organs (heart, spleen, and liver) did not show morphological differences between control rats and rats with silicosis (Figure 4). The kidney of rats exposed to silica for 24 weeks had mild degenerative changes in the proximal convoluted tubules. The abnormal bone metabolism was well documented in our previous studies10,17. Overall, these studies highlighted that the proposed protocol could mimic the progression of silicosis in humans well.
Figure 1: Schematic of the exposure control apparatus. (A) Air-delivered silica generator. (B) Whole-body chamber. (C) Instrument panels. (D) Exposure control apparatus. (E) All components are assembled to form a working instrument; the chamber comprises an air-delivered silica generator, a whole-body chamber, and a waste disposal system. (F,G) Air detector. Please click here to view a larger version of this figure.
Figure 2: H&E staining and collagen deposition in rats with silicosis. H&E staining of rats exposed to silica for 2 and 24 weeks. The alveolar structure of rats was still intact, and the alveolar wall was thickened after 2 weeks of silica inhalation. The alveolar structure of rats disappeared, and large areas of fibrosis were formed after 24 weeks of silica inhalation. The silica particles were trapped in the lung lobes of rats (2 and 24 weeks), and the collagen fibers of rats (24 weeks) were observed under a polarized light microscope. Scale bar: 50 µm. Please click here to view a larger version of this figure.
Figure 3: Dynamic evolution of silicotic nodules detected by immunohistochemical staining of CD68. (A) As the exposure time increased (from 2 to 24 weeks), the area of silicotic nodules gradually increased, and the adjacent silicotic nodules gradually fused into large nodules. (B) The pattern of silicon nodules. Scale bar: 50 µm. Please click here to view a larger version of this figure.
Figure 4: H&E staining of the lungs, kidney, liver, spleen, and bone of control rats and rats with silicosis. (A) H&E staining of the lungs, kidney, liver, spleen, and bone of control rats. Scale bar: 1 mm. (B) H&E staining of the lungs, kidney, liver, spleen, and bone of control rats. Scale bar: 50 µm. Multiple fibrotic lesions of varying sizes were formed in rats exposed to silica compared with the control rats. No significant differences in the kidney, liver, and spleen were found between control rats and rats with silicosis, but the bone loss was observed in rats with silicosis. (C) H&E staining of the lungs, kidney, liver, spleen, and bone of rats with silicosis. Scale bar: 1 mm. (D) H&E staining of the lungs, kidney, liver, spleen, and bone of rats with silicosis. Scale bar: 50 µm. Please click here to view a larger version of this figure.
Measuring time (min) | Volume (L) | W1 (g) | W2 (g) | Concentrations (mg/m3) |
10 | 460 | 0.40 | 0.43 | 65.22 |
20 | 923 | 0.40 | 0.46 | 65.01 |
30 | 1404 | 0.40 | 0.49 | 64.1 |
Table 1: Concentrations of silica in the whole-body chamber.
As the leading cause of silicosis, silica plays a decisive role in molding. The silica particles inhaled by patients with pneumoconiosis are fresh, free silica particles produced by mechanical cutting. Silica can generate reactive oxygen species either directly on freshly cleaved particle surfaces or indirectly through its effect on the macrophages25. Therefore, the grinding of silica particles is of high importance. In the proposed protocol, silica was ground with agate mortar for more than 90 min to make it finer, more irregular, and increase the surface area. As reported, the airborne concentrations of crystalline silica26 must not be lower than 0.05 mg/m3. However, this protocol might have an issue with inaccurate dust concentrations; the uncertainty of dust concentration was mainly associated with the absence of a built-in dust concentration monitoring system. The actual silica concentration was calculated using the mass of SiO2 entering the dust cabinet and the gas flow rate. The volume of SiO2 was based on the speed of the rotary plate, rather than the mass of SiO2 actually entering the cabinet. Hence, possible solutions to the problem were checking the volume of silica in the chamber twice a week to ensure that the rats were exposed to the same volume of silica each time or placing a concentration-measuring device in the dust chamber, the latter being the best solution.
The limitations of this model were also apparent: (1) the relationship between the exposure dose and its biological effect is only approximate because the respiratory tract of rats is different to that of humans; (2) the uncertainty of dust concentration existed; (3) the method required the purchase of special equipment; (4) the volume of the dust chamber and the number of dust-infected rats was limited; (5) the mouse silicosis model could not be constructed because the respiratory tract of mice was narrow and silica dust could not be deposited in the lungs; also, the mouse model was cheaper, and it was easy to generate transgenic or KO mice.
The conventional construction of the silicosis animal model mainly included two methods: bronchial injection and inhalation of SiO2. In bronchial injection, the mortality was closely related to the perfusion dose, and the invasive surgery inevitably caused additional collateral damage27. To replace the intratracheal injection model, some scholars established a silicosis model using an ultrasonic atomized silica suspension for inhalation28. However, ultrasonic atomization could not control the concentration of silica in the air after atomization, the repeatability was poor, and typical fibrotic lesions could not be formed using this modeling method. Another economical, practical, and effective model was the mouse nasal drip model29, but this method injected liquid silica into the trachea and was not as good as inhaling it. The exposure control apparatus has a multiple air intake system so that the silica in the inhalation chamber is evenly distributed, the data are accurate, and the dust distribution in the dust chamber is uniform. Hence, the test environment was stable for a long time, and relevant parameters were observed and recorded at any time.
The significance of establishing animal disease or injury models is to mimic the pathological process of disease or injury caused by pathogenic factors to the greatest extent possible. Therefore, a good animal model is as close to human disease as possible. By inhalation exposure to silica, the rats could freely inhale pathogenic silica particles in the dust chamber. The weekly and daily exposure sessions also fully mimicked the working hours of pneumoconiosis workers. Using this modeling method, we identified pathological changes such as epithelial-mesenchymal transition, activation of transfer growth factor signals, activation of macrophages, and activation of senescence-related signals during silicosis in rats. Some of the results were confirmed in human samples18. Recently, we have also begun to study the dynamic pathological changes in the evolution of silicosis by this method23.
This simple, low-cost, and easily-repeatable protocol is also of great importance at a time when the incidence of silicosis is making a comeback in the world30. After the 8-week inhalation exposure to 100 mg quartz/m3, 20% of silica remained in the rat lungs after 6 and 12 months31. Also, the researchers investigated the extent to which an animal in a similar device could inhale and exhale air; the concentration of the gas inhaled by the animals slightly changed32. The protocol still holds great promise, for example, by combining it with microcomputed tomography to observe the dynamic evolution of silicosis and combining it with the transcriptome database to verify the pathological process of silicosis and validate new anti-inflammatory and anti-fibrotic systemic therapies.
The authors have nothing to disclose.
This work was funded by the National Natural Science Foundation of China (82003406), the Natural Science Foundation of Hebei Province (H2022209073), and the Science and Technology Project of Hebei Education Department (ZD2022127).
Air detector (compressive atmospheric sampler) | Qingdao Xuyu Environmental Protection Technology Co. LTD | ||
Anatomical table | No specific brand is recommended. | ||
Antibody of CD68 | Abcam | ab201340 | |
DAB | ZSGB-BIO | ZLI-9018 | |
Electric heating air-blowing drier | Shanghai Yiheng Scientific Instrument Co., LTD | ||
Electronic balance | OHRUS | ||
Embedding machine | leica | ||
Exhaust gas discharge device | HOPE Industry and Trade Co. Ltd. | ||
Generator systems | HOPE Industry and Trade Co. Ltd. | ||
Gloves (thin laboratory gloves) | The secco medical | ||
Hematoxylin and eosin | BaSO Diagnostics Inc. | BA4025 | |
HOPE MED 8050 exposure control apparatus | HOPE Industry and Trade Co. Ltd. | ||
Inhalation chamber | HOPE Industry and Trade Co. Ltd. | ||
Injection syringe | No specific brand is recommended. | ||
Light microscope | olympus | ||
Object slide | shitai | ||
PV-6000 (HRP-conjugated goat anti-mouse IgG polymer) | Beijing Zhongshan Jinqiao Biotechnology Co. Ltd | s5631 | |
Silicon dioxide | Sigma-Aldrich | ||
Slicing machine | leica | RM2255 | |
Waste gas treatment device | HOPE Industry and Trade Co. Ltd. | ||
Wet box | Cooperative plastic Products Factory | ||
Xylol | Tianjin Yongda Chemical Reagent Co., LTD |