Brain cytokine and chemokine mRNA expression in mice induced by intranasal instillation with ultrafine carbon black
Introduction
A number of epidemiological studies have reported that exposures to elevated level of particulate matter (PM) in urban air are associated with adverse health effects as well as with an increase in the morbidity and mortality related to pulmonary and cardiovascular diseases in susceptible populations (Peters et al., 1997a, Peters et al., 1997b, Penttinen et al., 2001). Particles having a size less than 100 nm in aerodynamic diameter are designated as ultrafine particles (UFPs). PM is a complex mixture of airborne particles, including salts, metals, UFPs and biological materials. Exposures to respirable particulate matter with aerodynamic diameters smaller than 10 μm (PM10) and 2.5 μm (PM2.5) have been linked to the exacerbation of respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) (Pope and Kanner, 1993, Schwartz et al., 1993) and increased cardiopulmonary mortality (Schwartz and Dockery, 1992). It is now anticipated that the adverse health effects of ambient air PM (PM10/PM2.5) are very likely driven by the combustion-derived UFPs (Oberdörster et al., 1994, Seaton et al., 1995, Donaldson and Stone, 2003). Besides, concerns about the possible adverse health effects of engineering-derived nanoparticles are also growing seriously (Hoet et al., 2004). Stochastic modeling of UFP deposition and clearance in the human respiratory tract has shown that UFPs in the ambient air can be divided into two groups, of particle size about 1–10 nm and about 10–100 nm (Hofmann et al., 2003). Our interest lay in analyzing the size-specific effects of these nano-size particle groups.
Considerable progress in understanding the effects of UFPs in the lungs has been achieved largely through the use of surrogate particles such as ultrafine carbon black (ufCB) and titanium dioxide. Carbon represents a major constituent of ambient air UFPs, which is present in the form of organic carbon and elemental carbon (Campbell et al., 2005). Ultrafine CB (ufCB) particles were found to be more toxic and inflammogenic than fine particles of same chemical, when delivered at an equal mass dose (Donaldson et al., 2001a, Donaldson et al., 2001b). In a previous study, we have demonstrated that intratracheal instillation of ufCB in mice triggered the induction of proinflammatory cytokine and chemokine release and mRNA in lung and mediastinal lymph nodes, size-specifically (Tin-Tin-Win-Shwe et al., in press). Research efforts to study the effects of such very small particles on the lung have advanced considerably (Heinrich et al., 1995, Driscoll et al., 1996). While the adverse health effects of ambient air PM-exposure on lung and heart organ systems have been studied well, studies addressing the possibility of effects on central nervous system are very limited. Our understanding of how ufCB may affect immunogenic responses, particularly in brain is poor. Very recently, Campbell et al., 2005, Campbell et al., 2005 studied the possible proinflammatory effects of inhaled PM components in the whole brain using an asthmatic model in mice and clearly demonstrated that inhaled PM components trigger a proinflammatory response in brain.
More than three decades ago, De Lorenzo (1970) demonstrated in squirrel monkeys that intranasally instilled colloidal ultrafine gold particles (50 nm) were anterogradely translocated along the axons of olfactory nerves to the olfactory bulbs. It has been also shown that the materials such as, metals and organic xenobiotics that come to contact with the olfactory epithelium can be transported to the brain via the olfactory neurons (Tjalve and Henriksson, 1999, Larsson and Tjalve, 2000). A study in rats indicated that inhaled ultrafine elemental 13C particles may translocate along the axons of olfactory nerve to the brain (Oberdörster et al., 2004). Although lung is considered as the main portal of entry for PM air pollution (Utell and Frampton, 2000), it is also possible that inhaled UFPs, by virtue of their extremely small size may reach the olfactory mucosa and get translocated to the brain via the olfactory pathway, which in turn might cause neurotoxic effects. Therefore, we hypothesized that environmental and occupational exposures to airborne ufCB particles by nasal respiration may lead to their accumulation in the nasal mucosa at the early stage and subsequent translocation along the olfactory nerve axon to the olfactory bulb, and further to the hippocampus, whereby they can stimulate the residing microglia and astrocytes. Stimulation of the resident microglia and astrocytes by the ufCB particles may accelerate the transcription of specific proinflammatory cytokine and chemokine genes, leading to an enhanced expression of the mRNAs in brain, an initial event of the molecular process of inflammation.
In the present study, to test this hypothetical possibility, mice were instilled with 14 and 95 nm CB particles intranasally in a physiologically relevant condition (i.e., in mice without any prior sensitization) and we sought to investigate whether exposures to ufCB particles could accelerate the transcription of some major proinflammatory and chemokine genes in the brain, to investigate whether the acceleration was only in the olfactory bulb or in the hippocampus or in both the brain compartments, and to investigate whether the effect was size-dependent or size-specific, we examined the expression of IL-1β and TNF-α cytokine mRNAs as well as the expression of CCL2, CCL3 and CXCL9 chemokine mRNAs in the olfactory bulb and hippocampus by a quantitative real-time PCR method.
Section snippets
Animals
Male BALB/c mice (7-week-old) were purchased from Charles River Japan Inc. (Tokyo, Japan). 8-week-old mice were used in all experiments. Food and water were given ad libitum. The mice were housed in plastic cages under controlled environmental conditions (temperature 23 ± 0.5 °C, humidity 50 ± 5%, lights on 07:00–19:00 h). The experiments were conducted under the control of the Ethics Committee of Animal Care and Experimentation of the National Institute for Environmental Studies, Japan.
Particles
The CB
Results
To investigate the effects of intranasal ufCB instillation in mice on the possible inflammatory biomarkers in brain, we examined the mRNA expression of proinflammatory cytokines and chemokines in olfactory bulb and hippocampus by a quantitative real-time PCR method using ABI Prism 7000. Four hours after the last intranasal instillation, we collected olfactory bulb and hippocampus and determined the mRNA expression of proinflammatory cytokines such as IL-1β and TNF-α and chemokines such as CCL2
Discussion
Inflammation is considered to play a major role in the adverse health effects related to the exacerbation of cardiopulmonary morbidities caused by PM-exposures (MacNee and Donaldson, 1999, Donaldson and Stone, 2003). A hallmark of CNS inflammation is the infiltration of monocytes/macrophages and T cells to the sites of injury. Campbell et al., 2005, Campbell et al., 2005 demonstrated that the concentrations of cytokines (IL-1β and TNF-α) in the whole brain of mice were increased after exposure
Acknowledgments
This work was supported by a grant from the Ministry of the Environment, Japan. We thank Dr. Shinji Wakamatsu (National Institute for Environmental Studies) for his encouragement during this study.
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