A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Summary

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Abstract

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg∙mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg∙mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Introduction

Pleural effusion is a very common clinical manifestation of pulmonary diseases with a variety of causes. Having a useful animal pleural effusion model is very important to study these pulmonary diseases, the roles of the two pleural membrane layers, the mechanisms of pleural effusion, and its treatment. However, some reported pleural effusion models mainly focus on the malignant pleural effusion or biological factors rather than the nanoparticles in the environment^1,2. Here, we introduce a new model of pleural effusion that is simple, safe and effective.

With the development of nanotechnology and the extensive use of nanoproducts, there is a concern about the potential hazards of nanomaterials to the environment and the human health^3,4. Nanomaterials introduce risk factors and potentially lead to novel hazards within the workplace or through environmental contamination. In vitro and in vivo studies show that nanomaterials can result in multi-organ damage to the lungs, the heart, the liver, the kidney, and the nervous system, as well as the reproductive and immune systems^5,6. Additionally, some studies reported that the specific toxicity of nanomaterials was due to their unique physicochemical properties^3,4,7.

We have reported that a group of workers with occupational exposure to nanomaterials clinically presented with pleural and pericardial effusion, pulmonary fibrosis and granuloma^8,9. Silica nanoparticles were isolated in these patients' pleural effusion⁹. In order to reproduce and verify the pleural effusion induced by the inhaled nanoparticles in human, we conducted the experiment by instilling the polyacrylate/nanosilica (PA/NPSi) via the respiratory tract in rats, which mimicked human respiration in a real environment, and found that intratracheal instillation of PA/NPSi could result in pleural effusion in rats. Here, we introduce how to make pleural effusion in rats by intratracheal instillation of PA/NPSi, and how to isolate nanoparticles in the pleural effusion. This model may be useful for the further study of nanotoxicology and pleural effusion diseases.

Protocol

The study followed guidelines of Capital Medical University (Beijing, P.R China) for the care and use of experimental animals. All procedures were approved by the Animal Ethical Committee of Capital Medical University in China.

1. Experimental preparations

NOTE: Acclimate the female specific pathogen-free Wistar rats (weight: 200 ± 10 g) to the experimental environments for a week before administration (Environmental conditions: light/dark:12h/12h, temperature 22 ± 2 °C, humidity 50 ± 10 %).

1. Use a fresh 10 mL of PA/NPSi suspensions (nanosilica Ø:20 ± 5 nm by in situ emulsion polymerization) diluted in normal saline at concentrations of 3.125, 6.25, and 12.5 mg/mL, respectively¹⁰. Before administration, sonicate the suspensions for 20-30 min and vortex for 10 min in order to prevent nanoparticles aggregation.
2. Equally divide a total of 20 rats into four groups: one group for each concentration of PA/NPSi (0, 3.125, 6.25, and 12.5 mg/mL).
3. To anesthetize them, place the rats in a closed container with 1.5 mL of ether (99.5%) or any other IACUC approved protocols. After 60-90 s of anesthesia, check for the lack of response to pedal reflex. Ensure that the rats are breathing.
4. Put the anesthetized rat on the board and fix its front teeth with a sterilized line of nylon on the board too.
5. Open its mouth and expose its fissure of glottis with the help of a surgical forceps and frontal lens.
6. Instill the rats with 0.5 mL of PA/NPSi suspension to each rat's lung for a total of 1 mL using a fine tube with a sterilized blunt needle into the bilateral bronchus.
7. Place the rats on a plastic board in a supine position and let the rats recover slowly in 5-10 min.

2. Ultrasound examination for pleural effusion

1. Use an ultrasound system with a linear array transducer (frequency: 8 MHz) to examine rats on days 1, 3, 7 and 14¹⁰.
2. Give anesthesia (10% chloral hydrate, 0.35 mL/100 g, i.p.) to the rats and check for the lack of pedal reflexes.
3. Remove the hair from rats' chest and upper abdomen using an electric shaver. Then place the rat on a mounting plate in a supine position.
4. Cover the skin with the coated gel, and then place the transducer on the intercostal space and subcostal area to detect the pleural fluid.
   NOTE: In order to detect the effusion accurately, the left and the right lateral positions were selected to perform an ultrasound examination.
5. Put the rat on a plastic board in a supine position after ultrasound examination and let the rats recover slowly in 10 min.

3. Chest CT scanning for pleural effusion

1. On days 7 and 14 post-administration, anesthetize the rats with 10% chloral hydrate (i.p). Consider it sufficient depth of anesthesia when the rat does not react to pedal reflexes.
   NOTE: Day 7 post-administration is the most appropriate time to observe pleural effusion by CT scanning.
2. Place the rat on a plastic sheet in a prone position and then scan its chest to investigate pleural effusion using a 64-channel CT. Use the following settings: 64 mm x 0.625 mm detector configuration, 120 kV (peak), and 350 mAs.

4. Collection of pleural effusion and isolation of nanoparticles in the pleural effusion

1. After chest CT scanning of rats and under anesthesia of chloral hydrate, check the pedal reflex of the rats, shave the hair from abdomen to chest, and then disinfect the skin by iodine.
2. Bring the rats to the surgical area.
3. Under anesthesia, quickly cut 1-1.5 cm of the skin and abdominal muscles to the xiphoid along the midline with the intact diaphragm.
4. Carefully open the chest and inspect bilateral pleural cavities with the help of tweezers, especially the bilateral costal phrenic angles. Collect 1-2 mL of the light-yellow pleural effusion with a 2 mL sterile syringe.
5. Once done, sacrifice the rats with IACUC approved protocol.
6. Centrifuge the pleural effusion in a 2 mL tube for 15 min at 300 x g in order to isolate the nanoparticles.
7. Use a drop of the upper layer which is the bright liquid and observe under a transmission electron microscope(TEM,) at an accelerating voltage of 60-80 kV.

Results

Using a thoracic ultrasound, we found no pleural effusions on day 1 in all groups. However, on day 3, the pleural effusion appeared in the 6.25 and 12.5 mg/kg∙mL groups. The effusion was mainly in the right costal phrenic angle, while the pericardial effusion only presented in 12.5 mg/kg∙mL group. Furthermore, on day 7, both pleural effusion (Video 1) and pericardial effusion (Video 2) were detected in 6.25 mg/kg∙mL group (

Discussion

Sonography is the most convenient tool for determining pulmonary diseases, due to its excellent sensitivity to the free fluid in the pleural cavity¹¹. That is because sonography can immediately detect the contrast in acoustic impedance of air and fluids in the lung¹². Besides, sonography is more flexible in a small animal's model than CT. Nevertheless, the air in the lung reflected the sound wave and impeded from observing the intrapulmonary changes after nanoparticles ...

Materials

Name	Company	Catalog Number	Comments
Acuson S2000 Color Doppler ultrasound system	Siemens Medical Solutions, Mountain View ,CA
Polyacrylate/nanosilica	Fudan University,Shanghai, China		made by order with nanosilica(20±5)nm
10% chloral hydrate	Beijing Chemical Works	302-17-0
Transmission electron microscope	JEM-1400Plus，JEOL Ltd., Japan.
Light speed 16 spiral computed tomography	GE Healthcare, US
Specific pathogen-free Wistar	Animal Center of Lianhelihua (Beijing, China)		Wistar rats