Particulate air pollution: major research methods and applications in animal models

Abstract

Ambient air pollution is composed of a heterogeneous mixture of gaseous and solid particle compounds in which primary particles are emitted directly into the atmosphere, such as diesel soot, while secondary particles are created through physicochemical transformation. Particulate matter (PM), especially fine and ultrafine particles, can be inhaled and deposited in the alveolar cavities and penetrate into circulation. An association between high levels of air pollutants and human disease has been known for more than half a century and increasing evidences demonstrate a strong link between exposure on PM and the development of systemic diseases, such as cardiovascular and neurological disorders. Experimental animal models have been extensively used to study the underlying mechanism caused by environmental exposure to ambient PM. Due to their availability, quality, cost, and genetically modified strains, rodent models have been widely used. Some common exposure approaches include intranasal instillation, intratracheal instillation, nose-only inhalation, whole-body inhalation, and intravenous injection have been reviewed with brief summary of its performance, merit, limitation, and application. We hope this would provide useful reference in advancing experimental researches about air pollution human health and disease development.

INTRODUCTION

Air pollution is composed of a heterogeneous mixture of gaseous and solid particle compounds that primarily include ozone (O₃), carbon monoxide (CO), sulfur dioxide (SO₂), nitrogen oxides (NO_x), liquids, and particulate matter (PM). PM is a complex mixture of extremely small particles and liquid droplets in which primary particles are emitted directly into the atmosphere, such as diesel soot, while secondary particles are created through physicochemical transformation of gases, such as nitrate and sulfate formation from gaseous nitric acid and sulfur dioxide, respectively. PM can be divided into coarse (10 to 2.5 μm; PM10–2.5), fine (<2.5 μm; PM_2.5), and ultrafine (<0.1 μm; PM_0.1) particles.¹ Of particular interest in PM are PM_2.5 and PM_0.1 because they are the PM that ultimately enters the lungs, although the chemical components from bigger PM could also be deposited in the alveolar cavities and penetrate into circulation.² An association between high levels of air pollutants and human disease has been known for more than half a century that nose and throat disturbance was found in urban cities as early as 1936.³ In a landmark study conducted in six US cities discovered that PM_2.5, or a more complex pollution mixture associated with PM_2.5, contributed to excess mortality in some major cities in the US.⁴ Ambient air pollution impacts every one of us, which starts from prenatal time period and lasts lifetime. It is a global challenge especially to those emerging countries like China and India.⁵ In the United States, “the number of people exposed to unhealthy levels of air pollution increased to more than 133.9 million people, higher than the 125 million in the years covered by the 2017 report (2013–2015)” according to this year’s “State of the Air” report from American Lung Association.⁶ Although sky visibility has increased over Europe,⁵ most European Union countries “fail to meet the bloc’s air quality standards and more than 1,000 Europeans die prematurely each day, ten times more than in road accidents”, and “pollution’s toll on health in Bulgaria and other eastern European countries was even worse than in Asian giants China and India”.⁷

Exposure to ambient PM_2.5 particles has profound health impact, especially when exposure occurs early in human lives, which may have direct impact on respiratory system, and indirect impact on other systems, such as cardiovascular, digestive, and neurological.^{1, 2, 8–12} A reduction in exposure to ambient PM_2.5 had contributed to significant and measurable improvements in life expectancy in the United States,¹³ reflecting the importance of lifetime exposure on human life quality and human disease development, even when the ambient concentrations were perceived “acceptable” in most of North American and European cities. Although life expectancy has been improved significantly since air pollution levels have been reduced,^{14, 15} the mechanisms of the effects of air pollution on human diseases remain unclear.

Experimental animal models have been extensively used to study the underlying mechanism of cardiovascular and respiratory diseases caused by environmental exposure to particulate air pollutants. Due to their availability, quality, cost, and genetically modified strains, mice, guinea pigs, and other rodents have been used in investigations related to pulmonary uptake and disposition of aerosolized particles. Some common exposure approaches include intranasal instillation, intratracheal instillation, nose-only inhalation, whole-body inhalation, and intravenous injection. Each of these approaches has its own merits, limitations, targeted organs and systems, and requirements for study design.^16–18 In this review, we intend to summarize major methods with their pros and cons about PM exposure in rodents, especially in mice. In addition, PM exposure on major human diseases that were conducted in rodent models are also highlighted. We hope this would provide useful reference in advancing experimental research in the understanding of air pollution in human health and disease.

EXPOSURE METHODS

Instillation via nose or trachea

Aspiration or instillation of foreign material like fluid into the lungs is frequently seen as accident or intentionally applied for drug delivery or other interventional purposes.¹⁹ As an experimental tool, it has been widely applied in toxicity studies, especially the investigations on the airborne PM. The procedure seems simple but generally requires animals to be sedated to avoid coughing.^{20, 21} The particles, either collected on site or purchased from a commercially available companies, such as from the National Institute of Standards and Technology (NIST),²² should be in sterile saline or phosphate-buffered saline (PBS), and resuspended with a desired form and concentration before the delivery. The exposure is performed by placing a lightly anesthetized animal in a supine position, and the particle suspension is instilled or aspired into the nasal cavity drop-wise using a micropipette. The intervention, depending on the hypothesis and animal model, can be 1–2 times per week for up to a few months.²³ As to the intratracheal instillation, it needs some instruments, along with anesthesia, to facilitate the process in mice.^{24, 25} Very similar to the nasal instillation, particles need to be suspended in sterile saline or PBS, and syringe, needle, or an endotracheal tube or catheter may be needed for the delivery.^{25, 26}

There are a few aspects that need to be considered. First, some technique and experience in it may be needed to make sure the consistence in the procedure and substantial amount of the particle containing fluid is delivered (into the lung). It is recommended to run a few pilot experiments with Typan blue solution in some mice to perfect the procedure and make sure that substantial amount of the solution reaches the lung tissue (by sacrificing the mice and examining the lung organ). Second, the dose delivered via nasal or tracheal instillation does not represent exposure to ambient levels of PM, but may be relevant for occupational or even ambient exposures, if careful calculation about the dosage is made.²⁵ Third, the amount of fluid containing the particles needs to be limited for each delivery and the frequency of the delivery also needs to be cautious in order to prevent pulmonary edema in the animals. While instillation via either nose or trachea has long been used in toxicity testing as an alternative to inhalation exposure due to its simplicity and cost saving for complex atmospheric generation and exposure systems, the outcomes are quite different between instillation and inhalation, ranging from lung interlobular distribution to the evaluation of airway hyperreactivity, bronchoalveolar lavage fluid constituents, and histopathology.²⁷

Injection intravenously or intraperitoneally

PM particles, especially PM_2.5 and PM_0.1 particles, are believed that, once inhaled, the insoluble PM_2.5 or PM_0.1 particles can translocate into the circulation, with the potential for direct effects on homeostasis and cardiovascular integrity.²⁸ It is even highly possible that PM_0.1 to cross the lung-blood barrier due to its much smaller sizes and propensity to form aggregates.^{2, 29}

Although one could argues that systemic administration of PM particles is not a physiologic mode of exposure compared to inhalation, it is believed and accepted that PM particles be directly injected intravenously serve as a convenient and appropriate method to investigate the mechanisms of action of translocated particles.^{30, 31} To do so, particles that are collected or purchased are suspended in sterile saline or PBS. Particle suspensions need to be sonicated and vortexed prior to intravenous administration to mouse tail vein to avoid particle aggregation.³⁰

Intraperitoneal injection is widely used as a means of administering substances, particularly injectable anesthetics. Due to some side effects that may happen, which primarily include inadvertent injection into the gut, abdominal fat and subcutaneous tissues, caution is needed during the performance. The procedure seems simple, which is not usually necessary to sterilize the skin with antiseptics or use of anesthetics.³² The particles with targeted concentration may be suspended in saline or PBS,³³ which is injected intraperitoneally once, or 2–3 times per week. A new needle should be used for each animal and the injecting fluid with the suspended particles should be at body temperature. If repeated injections are needed, consider some alternatives, such as the use of minipumps, which could last a few weeks without the need for external connections or frequent handling.³⁴ In addition to its “artificial” delivery that bypasses respiratory system compared to human inhalation, another downside is the exposure dosage that is difficult to determine or to correlate the exposure dose with the reality what human subjects are exposed.

Nose-only inhalational exposure

Nose-only inhalational exposure is relatively versatile, which requires inhalation holding tubes and has broad application, such as suspended ambient particles that are collected or commercially purchased, ambient or concentrated particles, gases, diesel exhaust particles, even like tobacco smoke/particles.^35–39 The system is technically less demanding although a labor-intensive regimen if a long-term exposure is planned.⁴⁰ However, it is most likely “artificial” exposure that is not “natural” in terms of what and how much (concentration) the animals are exposed. It is good for a relatively shortterm exposure and is a good alternative for mechanistic study, especially about chemical components on the adverse effects of human health and disease development.

Whole body inhalation exposure

Ambient inhalational exposure:

Ambient inhalation exposure, either to specific size of the particles or to specific component of the air pollutants, does require exposure chambers with certain technical support and maintenance. They can be indoor or outdoor, depending on the study goals and technical availability. An indoor one, such as an ambient PM_2.5 exposure chamber,^{41, 42} mainly consists exposure chambers that house the animals and a cyclone that is used to remove the particles larger than 2.5 μm. As to the control, an identical protocol with the exception that a High Efficiency Particulate Air filter (HEPA) is positioned in the inlet valve to the exposure system to remove all of the particles from that air stream.⁴¹ It can run continuously with very limited negative impact on the animals, and the exposure concentrations may fluctuate along with the ambient levels.

An outdoor one, represented by open-top chamber^{43, 44} and shed exposure chamber,^{45, 46} reflects the exposure to “natural air”. The open-top chamber system mainly includes exposure chambers of cylindrical structures while the ambient air is forced into the chamber by large fans and exits at the top. Since it is exposed to ambient air, a few monitoring devices may have to be provided in order to determine which air pollutants and at what levels of those air pollutants those animals are exposed.⁴⁴ For the control, one or more filters may be added to the system to reduce both particles and gaseous components in the clean chamber. The shed system is similar to the open-top chamber except that utility sheds are used.^{45, 46} One of the advantages of the outdoor systems is that it is movable and relatively easy to set up, which enables the potential to study specific area and location, such as traffic crossroad⁴⁴ or air pollutant emission source.^{45, 46} However, the setup and its location also require careful consideration of site safety, environmental factors (such as ambient temperature, humidity, noise, and light), sanitation, and availability of water and electricity for both animals and researchers.

Concentrated ambient particle (CAPs) inhalational exposure:

Several CAPs acute exposure studies in humans have been performed in the last 1–2 decades,^47–49 although most of them have been done in animal models. The best representative exposure system that may have the most productive outcomes is the versatile aerosol concentration enrichment system (VACES).^{40, 50, 51} This exposure system uses the principle of the condensational growth of the ambient particles followed by virtual impaction to concentrate the aerosol, and allows the particles larger than 2.5 μm in aerodynamic diameter removed at the concentrator inlet, and the remaining aerosol to be concentrated by inertial separation techniques that dispose of most of the carrier air, which enables delivery of concentrated streams of real world particles to human subjects or laboratory animals via whole body exposure.^{40, 52} While several modifications to the VACES have been made to improve its performance and to facilitate its use for daily operation, the basic design was illustrated in which each rodent was housed individually with a total number up to 128 mice or 24 rats could be exposed simultaneously.^{40, 50, 53} In a latest design, exposure chambers are modified so that the mice can be housed in their original cages during the exposure and the mice do not need to be housed individually.⁵⁴ There are several advantages with this system. First, it mimics human whole body, inhalational exposure with minimum impact on animal’s food intake and drinking during the exposure.^{40, 52} Second, it allows both short and long term exposures (so far it has demonstrated a 10-month continuous exposure in mice).^{12, 55} Third, it allows the ambient particles to be concentrated up to 10 folds (concentration enrichment factor 10) while both the particle numbers and volume size distributions are reasonably well preserved during the concentration enrichment process.⁴ Fourth, a few additional devices for monitoring physiological parameters or instrumental intervention, such as telemetry for EKG or blood pressure, or a rodent treadmill, can be used simultaneously during the exposure.^{4, 54} Fifth, due to its concentration capability and continuous exposure potential, it has the flexibility and advantage to have a research design within a reasonable time period while a maximal positive outcomes may be achieved.^{56, 57} Last but not least, the mice do not need training before the exposure and no any drug is need to sedate them during the exposure. Therefore, with those advantages mentioned above, it is regarded one of the best inhalational exposure systems up to date.

In addition to a station exposure system either indoor or outdoor, as illustrated above, an exposure system that is mobile, such as in a (semi-)trailer, has some advantages over a station one in terms of “precision” exposure to specific site and chemical components without compromising the others, such as safety and sanity of the animals and researchers. One type of mobile trailer exposure systems, which was called “OASIS-1” located in Ohio of USA, is based on the VACES mechanism that can house both mice and rats for extended time period.^55–61 Another type of mobile trailer exposure systems, which was called “AirCARE 1” and was primarily located in Michigan of USA. CAPs are generated from ambient PM_2.5 using a Harvard-type PM_2.5 concentrator and whole body animal exposures are conducted in Hinners chambers.^62–65

CONCLUSIONS AND PERSPECTIVE

Air pollution exposure has caused significant human and public health burden, which is especially critical in some developing countries, such as in China, India, and Brazil.^66–68 While sustained reductions in air pollution exposure have demonstrated an association with increased life expectancy and substantial improvements in air quality have been made in the past decades,¹⁴ “clear sky visibility” over land has decreased in most of the regions globally over the past 30 years.⁵ Therefore, we still have a long way to go and more efforts are needed in reducing air pollution levels and associated disease initiation and development. Future investigations into air pollution exposure on human diseases should have systemic diseases, especially cardiovascular diseases, as one of the priorities, since cardiovascular diseases have led the mortality and morbidity globally.² Another emphasis should be in neurological diseases due to the skyrocketing incidence of dementia and Alzheimer’s disease.⁶⁹ In addition, cancer study has been drawn attention,⁷⁰ and will continue to be increased especially in the developing world.⁷¹ As to the exposure methods, although conditions and limitations vary, a method via inhalation through nose, either at the ambient level or at the concentrated one, should be pursued with every effort in clinically relevant animal models. This is critical due to the fact that bypassing the respiratory system without inhalational exposure seems “artificial” that hardly exists in human reality, which has profound different responses in the body comparing to natural, inhalational exposure.²⁷ As to the exposure, PM cumulative exposure still seems to dominate the field but more should be focused on its chemical components, especially heavy metals, on human health effects and specific disease development due to chemical complexity of PM particles and significant differences among the cities and regions. As to the susceptible populations, clinically relevant animal models that mimic exposure in children, seniors, and people who have underlying chronic diseases should be emphasized.