Summary
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.
Abstract
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.
Introduction
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.
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Protocol
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
- Upon arrival, house all the rats in a specific pathogen-free (SPF) room.
- Randomly divide the healthy rats into two groups (n = 10): control rats inhaling pure air and rats with silicosis inhaling silica.
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.
- Ground the silica particles (see Table of Materials) with an agate mortar for 1.5 h before each exposure. This is because freshly fractured quartz produces larger quantities of active oxygen species than aged quartz13, and silica with a diameter of 1-5 µm is the most pathogenic.
- Weigh the silica (30 g) using an electronic balance after grinding, place it in a glass container, and bake it at 180 °C for 6 h in an electric heating air-blowing drier (see Table of Materials) to eliminate pathogens from the surface of the silica particles.
3. Silica exposure to the rats
- Connect the injection and the commercially available generator systems (see Table of Materials) and place the silica (30 g) in the generator. Check whether the connection pipeline is normal, the power cord is connected, and the power supply is normal.
- Check the water level of the spray tower and the humidifier of the waste gas treatment device (see Table of Materials) manually, and add water if it is insufficient (not up to the standard line).
- Add tap water to the spray tower of the waste gas treatment device and distilled water to the humidifier (Figure 1).
- Turn on the exhaust gas discharge device (see Table of Materials) and the air source switch to confirm whether the inside of the shielding cabinet is in a negative-pressure state.
- Confirm that the liquid mixing, powder mixing, pure gas flow control valves, and wastewater discharge valve under the inhalation chamber are closed.
- Place a total of 10 rats in the inhalation chamber (see Table of Materials), and close the inhalation compartment and the screened cabinet doors.
- Set the following experimental parameters on the instrument panel or in the computer: cabinet pressure: -50 to -30 Pa; oxygen concentration: 21%; cabinet temperature: 26-30 °C; humidity: 30%-70%; dust entry rate: 2.0-2.5 mL/min; and cabinet dust concentration: 60 ± 5 mg/m3.
NOTE: Observe the experimental data and equipment status continuously during the experiment. The equipment failure alarm prompted timely processing.- Expose each animal to silica continuously for 3 h per day, 5 days per week, and allow the animals in the control group to inhale pure air.
- On completion of the experiment, close the mixed gas flow control valve, and open the pure gas flow valve. Inject the pure gas continuously into the inhalation chamber.
NOTE: In the present study, the pure gas flow (7.0-7.5 m3/h) was injected for at least 20 min until the poisonous gas in the inhalation chamber was completely replaced.- Close the pure air-flow valve, open the door, take the rats out, and send them back to the pathogen-free room.
- Remove the rat rack and the branch pipe components in sequence and place them in the sink for cleaning. After rinsing, close the automatic cleaning valve and open the hatch.
- Wipe the inner wall with a clean cloth, or turn on the pure gas to dry the tank. Finally, carry out the disinfection. After cleaning and disinfecting with 75% ethanol, close the exhaust gate and, as soon as possible, slightly open the door of the inhalation cabin to evaporate the moisture, so that the inside of the inhalation cabin remains dry.
- Check the silica concentration in the cabinet with a comprehensive atmospheric sampler following the manufacturer's instructions (see Table of Materials) twice a week to ensure the stability of the silica concentration during the experiment. Calibrate the atmospheric sampler before sampling.
- Use a digital single-pan analytical balance for gravimetric determination. The calculated silica concentration was 65 mg/m3 (Figure 1 and Table 1).
NOTE: Weigh the filter paper before and after the absorption of silica. The concentration of silica was calculated using the following formula12:
where W2 = weight of the filter paper after sampling, W1 = weight of the filter paper before sampling, and V = volume of the air.
- Use a digital single-pan analytical balance for gravimetric determination. The calculated silica concentration was 65 mg/m3 (Figure 1 and Table 1).
4. Acquisition and fixation of lung tissues
- Euthanize the rats by intraperitoneal injection of pentobarbital (100 mg/kg body weight) and Lidocaine (4 mg/kg body weight). Assess the death by the loss of heartbeat14.
- At the end of the experiment, fix the right lower lung, kidney, liver, spleen, and bone with 4% paraformaldehyde for at least 24 h, embed in paraffin, and cut into 5 µm sections7,15.
5. Hematoxylin and eosin (H&E) staining
- Deparaffinize the paraffin sections in xylol (see Table of Materials) twice for 10 min each16, and rehydrate in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and distilled water for 3 min each.
- Stain the sections with hematoxylin (see Table of Materials) for 5 min, and then wash the sections with water10.
- Place the sections in 2% hydrochloric alcohol and then in distilled water until the color changes to blue.
- Stain the sections with eosin for 1 min, dehydrate them with 95% ethanol, make them transparent with xylene, seal them with neutral gum, and observe under a light microscope12.
6. Immunohistochemical staining
- Routinely wash the paraffin sections with water.
- Expose the antigens at high pressure (60 kPa) and high temperature (100 °C) for 80 s and then block with an endogenous peroxidase blocker (3%) for 15 min to eliminate the endogenous peroxidases7.
- Incubate the samples with antibodies directed against CD68 (1:200 dilution-add 4 µL of CD68 to 396 µL of antibody diluent; see Table of Materials) at 4 °C overnight.
- Incubate the samples with a secondary antibody (HRP-conjugated goat anti-mouse IgG polymer; see Table of Materials) at 37 °C for 30 min, and then wash the samples with 1x PBS.
- Visualize the immunoreactivity with 3,3-diaminobenzidine (DAB; see Table of Materials). After applying DAB to the tissue, observe the staining of the tissue under a light microscope10.
NOTE: The staining time varied from a few seconds to a few minutes according to the staining time of the tissue. The staining procedure was aborted by placing the sections in water. In this study, the brown staining of the tissue represented the positive expression of CD68. All antibodies were diluted in 1x PBS.
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Representative Results
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.
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Discussion
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.
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Disclosures
The authors declare no conflict of interest.
Acknowledgments
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).
Materials
| Name | Company | Catalog Number | Comments |
| 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 |
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