Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 effects^3,4. 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 silicosis^5,6. 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 method^10,11,12. The proposed protocol is anticipated to help further investigations in the related field of silicosis research.

Protokół

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

1. Upon arrival, house all the rats in a specific pathogen-free (SPF) room.
2. 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.

1. 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.
2. 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

1. 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.
   1. 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).
   2. Add tap water to the spray tower of the waste gas treatment device and distilled water to the humidifier (Figure 1).
2. 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.
   1. Confirm that the liquid mixing, powder mixing, pure gas flow control valves, and wastewater discharge valve under the inhalation chamber are closed.
3. Place a total of 10 rats in the inhalation chamber (see Table of Materials), and close the inhalation compartment and the screened cabinet doors.
4. 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.
   1. 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.
5. 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.
   1. Close the pure air-flow valve, open the door, take the rats out, and send them back to the pathogen-free room.
6. 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.
   1. 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.
7. 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.
   1. 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.

4. Acquisition and fixation of lung tissues

1. 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.
2. 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 sections^7,15.

5. Hematoxylin and eosin (H&E) staining

1. 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.
2. Stain the sections with hematoxylin (see Table of Materials) for 5 min, and then wash the sections with water¹⁰.
3. Place the sections in 2% hydrochloric alcohol and then in distilled water until the color changes to blue.
4. 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

1. Routinely wash the paraffin sections with water.
2. 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.
3. 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.
4. 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.
5. 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.

Wyniki

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 previou...

Dyskusje

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 mi...

Materiały

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