Establishing A Mouse Model for Respiratory Tract Extreme Low-temperature Air Inhalation

Summary

Cold environmental stimulation has been implicated in the development of various chronic diseases. Therefore, establishing animal models for preclinical research is crucial. This system addresses this need by offering a device that creates a stimulus model, meeting the requirements for basic research on pathogenic mechanisms.

Abstract

Currently, constructing a mouse model for cold environmental stimulation employs cold-heat plates and wearable cooling devices. These methods can partially fulfill the requirements for studying the responses and regulatory effects of mouse skin or neural circuits to cold stimulation. Numerous clinical studies have substantiated the correlation between exposure to low-temperature environments and the development of various diseases. Recently, there has been a growing emphasis on the continuous exchange of information between organs and tissues, providing a novel perspective on addressing longstanding issues within the human body. However, existing installations are unable to construct a model for mice inhaling cold air.

Although placing mice in a cold environment seems attractive, it has considerable limitations. While mice inhale cold air, their skin is also being stimulated by the cold environment, making it unclear whether resulting pathological changes are due to lung stimulation through the interaction of distant organs or due to the skin receptors and neural signal transmission. This creates considerable confusion in related research. This scheme presents a new approach for constructing a mouse model for extreme cold air inhalation stimulation. This device allows mice to inhale extremely low-temperature gases while their bodies remain at a normal temperature. It maximizes the simulation of the stimulating effects of extreme ambient temperatures on mice and meets the research needs for studying the relationship between extreme environmental temperatures and related diseases.

Introduction

This method primarily provides an extremely low-temperature air stimulation model in mice using a non-invasive, standardized, stable, and batch semiconductor refrigeration temperature feedback device. Clinical experiments related to low temperatures have confirmed a close relationship with the incidence and prognosis of various diseases. A time-series study involving 272 major cities in China obtained a total of 1,826,186 cases of non-accidental deaths. The relationship between temperature and mortality consistently indicates an inverted J-shaped curve, with the phase of high mortality rates due to cold being significantly longer than other temperatures. This suggests that the impact of low temperatures on stroke and cardiovascular diseases is unlimited to the cold phase; there is a continued influence during a period after the cold phase has subsided.

Among the non-accidental deaths, 14.33% can be attributed to environmental temperature factors, with moderate cold (-1.4 to 22.8 °C) and extreme cold (-6.4 to -1.4 °C) accounting for 10.49% and 1.14%, respectively. The causes of death include cardiovascular and cerebrovascular diseases at 17.48%, coronary heart disease at 18.76%, ischemic stroke at 14.09%, hemorrhagic stroke at 18.10%, respiratory system diseases at 10.57%, and chronic obstructive pulmonary disease at 12.57%¹. In China, epidemiological studies of stroke suggest a clear gradient from north to south². In the frigid climate of Northeastern China, the prevalence of stroke is 2.36 times higher compared to the southern region³. Substantial research has confirmed the direct impact of low-temperature environments on mortality rates and the incidence of stroke^4,5,6. Consequently, the significant climate temperature differences represent an environmental factor that cannot be ignored.

The lack of effective scientific reasoning explaining the correlation between low-temperature environments and increased rates of stroke and heart problems remains a topic of inquiry. While conventional wisdom suggests that cold temperatures may increase blood pressure through skin irritation and sympathetic excitation⁷, individuals typically take measures to insulate themselves and maintain body temperature equilibrium in response to cold conditions. When exposed to cold temperatures, modern humans rely on their respiratory system instead of the skin as the primary defense mechanism. While thick clothing can protect the skin from external cold, it cannot prevent inhaling cold air into the respiratory tract, exposing the trachea and alveoli to intense cold stimulation. Current methods for constructing animal models for low-temperature stimulation are primarily divided into two aspects. First, numerous studies have focused on exploring the response and regulatory mechanisms of mouse skin to low-temperature stimulation. One method involves placing mice on a plate that can control temperature changes (4-25°C) to investigate the specific regulatory mechanisms of body temperature regulation and avoidance behavior in response to cold stimuli^8,9. Other studies have placed cooling devices on the backs of mice to explore the role of neural circuits in body temperature regulation¹⁰.

Conversely, several studies have placed mice in small chambers with variable temperatures (4-30 °C). Research by Lal and colleagues and Qian et al. used this method to construct a mouse model of cold stimulation to explore the neural circuitry regulating the neuroendocrine control of cold-induced feeding behavior^11,12. However, the two methods mentioned have their limitations. First, the lowest temperature is 4 °C, which is insufficient to simulate extreme low-temperature air stimulation. This method cannot exclude the regulatory effects of the skin and neural circuits on the cold environment. As the primary site of air exchange, the lungs are also organs where cold-sensitive neurons are concentrated^13,14. The regulatory role of cold-sensitive neurons in various diseases has also been confirmed by several researchers^15,16,17. As a result, a method is urgently needed to stably, massively, and normatively construct a respiratory tract low-temperature animal model. Understanding the regulatory role of the lungs and cold-sensitive neurons in various chronic diseases under extreme low-temperature air stimulation is essential to provide a theoretical basis for preventing and treating stroke, coronary heart disease, and respiratory system diseases in cold regions. Our team addressed this critical gap by constructing a low-temperature device over the past two years. This device is characterized by repeatability, practicality, simple structure, and low cost, making it suitable for such studies.

Protocol

The Experimental Animal Ethics Committee has approved all procedures involving animal subjects at the First Affiliated Hospital of Harbin Medical University.

1. Assembly of device

NOTE: See Figure 1 for the device components.

1. Use metal connectors to secure two sets of brass shells to two sets of semiconductor cooling chips. Apply thermally conductive silicone grease between the brass shell and the semiconductor cooling chip.
2. Apply thermal grease between the brass shell and the semiconductor cooling chip.
3. Connect two groups of fans in every piece of semiconductor refrigeration piece below. Ensure that the brass shell, fan, semiconductor cooling chip, and thermally conductive silicone grease are securely connected as a complete unit.
4. Place the device as a whole in the gas collecting jar and two cover plates of the slots.
5. Install the plates above and below the gas collecting jar.
6. Connect the water inlet pipe and the water outlet pipes to the water inlet and outlet of the brass shell, respectively.
7. Connect four groups of brass shells to the top and bottom of the gas collection jar with pipes.
8. Connect the water pump to the water inlet pipe.
9. Place the water outlet pipe in the cistern.
10. Connect four sets of semiconductor coolers and water pumps to two sets of power supplies (12 V and 40 A).

2. Preparation of the animal for the experiment

NOTE: We used a C57Bl/6 male mouse aged 4 weeks for these experiments. It is recommended that the mouse be allowed to adapt to the fixator for 3-5 days before model preparation. The experimental environment should be at room temperature and kept quiet to avoid noise during the entire experiment.

1. Secure the mouse in the fixator: Place the mouse in the fixator, positioning the front part of the mouse's nose at the opening of the fixator. Use a matching sponge plug to fill and secure the rear part of the fixator, ensuring it has ventilation holes. Next, place the mouse fixator into the cylindrical slot of the gas collection jar (Figure 1D).

3. Experimental operation flow

1. Prepare an ice-water mixture and place it in the cistern.
   NOTE: For this step, ensure that the water level of the ice-water mixture is higher than the pump to ensure that the mixture is added in time throughout the experiment. Using water at room temperature is not recommended
2. Position the water pump within the ice-water mixture and ensure the outlet hose is submerged.
3. Place the temperature sensor probe inside the temperature-measuring hole of gas collection jar.
4. Connect the water pump to its power source and switch it on.
5. Connect the power supply to the temperature controller, plug the power adapter of the refrigeration unit into the temperature controller's power socket, and set the desired temperature range on the controller.
6. Set the temperature controller temperature range.
7. After the experiment, remove the mouse and place it back into its housing environment.
8. Switch off the power supply to the temperature controller.
9. Turn off the water pump.

4. Thermal imaging

NOTE: To demonstrate and verify that the mice can inhale extremely low-temperature air while maintaining normal body temperature within this apparatus, the temperature in the gas collection jar was measured using a thermal imaging camera (Figure 2).

1. Use a hand-held thermal imaging camera to ascertain the temperatures of both the air hole and the temperature-measuring hole of the gas collection jar from an optimal distance.
2. Position the red laser point, indicating the measurement location, precisely at the center of the designated temperature measurement area.
3. Employing the same methodology, remeasure the body temperature of the mice.

Results

We can observe the overall construction of this device, which includes a semiconductor refrigeration chip, thermally conductive silicone grease, a gas collection jar, a temperature controller, a fan, a water cooling circulation system, a mouse fixator, and a power adapter. A single unit can simultaneously accommodate the modeling needs of up to 16 mice (Figure 1A,B). The gas collection jar, water cooling circulation system, semiconductor refrigeration chip, fan, and mouse fixator constitute the main body of the device (Figure 1C). Inside the main body of the device, the front end of the mouse fixator can be inserted into the gas collection jar, ensuring that the nose of the mouse directly inhales the low-temperature gas (Figure 1D). Four sets of fans can be observed on the inner top side of the device's main body, with the back side of the fans connected to the semiconductor refrigeration chip through thermally conductive silicone grease (Figure 1E). The mouse fixator, used in conjunction with the device, is cylindrical, with the front end extendable into the gas collection jar. The fixator has ventilation holes in the middle and a plug at the rear for securing, meeting the fixation requirements for mice of different sizes (Figure 1F).

During the experiment, a temperature sensor probe is typically employed to continuously monitor the temperature within the gas collection jar in real time, as detailed in protocol step 3.3. To demonstrate and verify that the mice can inhale extremely low-temperature air while maintaining normal body temperature within this apparatus, the temperature in the gas collection jar was measured using a thermal imaging camera (Figure 2). The temperature within the gas collection jar was observed to be approximately -20 °C. The results clearly indicated that the body temperature of the mice within the restraining device remained unaffected. The results confirm that this device can enable mice to inhale extremely low-temperature gases while maintaining body temperature at a normal state. Currently, several researchers are focusing on the impact and pathological mechanisms of chronic diseases under extreme low-temperature conditions. This device can meet the needs of such research. By applying this device, a stable batch of animal models can be constructed, laying a solid foundation for subsequent studies.

Figure 1: The overall structure of the device and the decomposition diagram of each part. (A) The overall display of the device in the experimental state. (B) The upward view of the overall device indicates two sets of semiconductor refrigeration sheets and brass shells at the bottom of the gas collecting jar. (C) Partial enlarged image of the gas collecting jar. (D) After removing the locally enlarged image of the top device of the tank, it can be observed that the head end of the mouse fixator can be extended into the jar during the experiment, ensuring that only the nose of the mouse is exposed to the low-temperature environment. (E) Enlarged picture of the fan. (F) The image of the mouse fixation device indicates the head end of the device, the vent hole, and the glue plug at the tail. Please click here to view a larger version of this figure.

Figure 2: The monitoring diagram of the cooling effect during the operation of the device. (A) Under the monitoring of a far-infrared thermometer, the entire device can be observed in a low-temperature state. The insulation material attached to the device's exterior indicates a temperature of 13 °C. (B) Using a far-infrared thermometer to measure the temperature of the ventilation port where the mouse holder is placed during the experiment, it can be observed that the temperature inside the gas collection canister is -21.1 °C. The device has met our design requirements. (C) In the experiment, the mice were taken out of the device, and the far-infrared thermometer was used to measure the temperature of the nose and body of the mice. The results demonstrated that the temperature of the nose of the mice was significantly lower, while the temperature of the body remained normal. (D) A temperature-measuring hole is designed at the top of the experimental device. A far-infrared thermometer was used to measure the temperature inside the gas collecting jar through the temperature measuring hole, indicating that the internal temperature of the gas collecting jar is -21 °C, which proves that the device can be used for cold-temperature studies. Please click here to view a larger version of this figure.

Discussion

In constructing a low-temperature stimulation model, several key steps and precautions are necessary to ensure the accuracy of the experiment and the welfare of the animals. Use an ice-water mixture instead of room-temperature water to maintain a low-temperature state of the cooling water throughout the experiment, which helps simulate extremely low-temperature environments. Ensure that the recirculating cooling water system is unobstructed to ensure the efficient operation of the refrigeration system. When preparing an ice-water mixture, consider the following points: (1) An ice-water mixture means solid ice underneath and water above the solid ice; (2) Carefully observe the melting process of the solid ice during the experiment, as this may cause the ice to float. Such floating ice could potentially obstruct the pump surface, thereby hindering effective heat dissipation. At the end of the experiment, turn off the power supply to the refrigeration unit before the water pump. This ensures that the recirculating water system can continue dissipating heat from the refrigeration semiconductor after shutdown, preventing sudden temperature increases that could affect the results. For temperature monitoring, this device uses an intelligent temperature controller. The device's power supply is connected to the intelligent temperature controller, and the controller is used to set the temperature range. During the experiment, the temperature probe is placed in the gas collecting jar to monitor the temperature change continuously. Once the temperature exceeds the set range, the controller automatically disconnects the device's power supply to maintain the temperature within the set range.

If the refrigeration efficiency is significantly reduced or the refrigeration device fails during the experiment, consider the following possibilities: (1) Ensure the pump is in the ice-water mixture; (2) check if the water temperature is extremely high; (3) inspect the semiconductor refrigeration sheet for damaged; (4) verify that the power connection line of the semiconductor refrigeration sheet is broken.

This device has limitations. During the experiment, a large amount of ice-water mixture is needed to cool the semiconductor refrigeration sheet, and the cooling efficiency of the device is affected by the cooling efficiency of the semiconductor cooling chip. Future improvements may include designing the water-cycle refrigeration device as a closed-loop device and using refrigeration liquid or antifreeze as the cooling medium. Simultaneously, optimizing the appearance of the equipment and addressing the bare leakage of pipelines and power connection lines will enhance the convenience of carrying and popularizing the device. Furthermore, to maintain a consistent and stable inhalation of low-temperature gas by the mice, a mouse fixation device and rubber stopper were employed to immobilize the mice. Due to the inhalation of extremely cold gas, the stimulation process was limited in duration to safeguard the well-being of the mice and prevent frostbite and psychological issues. Future efforts will focus on enhancing the fixture to minimize additional harm during the stimulation phase in mice.

This technology is not only significant for basic scientific research on the effects of low-temperature environments on biological organisms but also has potential applications in studying the pathogenesis of chronic diseases in populations living in cold regions. The definition of extremely cold air varies across different regions and environments. However, by configuring the temperature control system, the device can be adjusted to meet research requirements for any temperature ranging from room temperature to -20 °C. This flexibility ensures that the device can accommodate the diverse temperature needs of researchers. Furthermore, it contributes to exploring the role of the lungs in inter-organ communication under low-temperature conditions, providing a solid foundation for preventing and treating stroke, coronary heart disease, and respiratory system diseases in cold regions. By following these steps and precautions, researchers can construct a stable and standardized animal model to study the effects of extreme low-temperature environments on biological organisms, providing a scientific basis for preventing and treating related diseases.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
brass shell	Shenzhen Haida high-tech electronic equipment company
cistern	Shenzhen Gongdecheng plastic industry
fan	ChaoJingYin electoronic.,LTD	4010	12 V
Gas collection jar	Shenzhen Gongdecheng plastic industry
intake/outlet pipe	Shanghai Shenchen rubber and plastic products factory
mouse fixator	Lanjieke technology
power	Shenzhen Haida high-tech electronic equipment company		12 V 40 A
Semiconductor cooling chip	Shenzhen Haida high-tech electronic equipment company	TEC2-19006	Double layer refrigeration
Sponge plug	Shenzhen Gongdecheng plastic industry
Stainless steel fastener	Shenzhen Haida high-tech electronic equipment company
thermally conductive silicone grease	Shenzhen Haida high-tech electronic equipment company	DRG102
water pump	Shenzhen Jutai pump Co.,LTD	DC005	12 V
Water pump power adapter	Chaoke power adapter factory	1210	100-240 V 50-60 HZ 12 V