(2023) 20:6
Tanaka et al. Particle and Fibre Toxicology
https://doi.org/10.1186/s12989-023-00517-x

Particle and Fibre Toxicology

Open Access

RESEARCH

Polycyclic aromatic hydrocarbons in urban
particle matter exacerbate movement
disorder after ischemic stroke via potentiationof neuroinflammation
Miki Tanaka1,2, Tomoaki Okuda3, Kouichi Itoh2, Nami Ishihara1, Ami Oguro1, Yoshiaki Fujii‑Kuriyama4,
Yu Nabetani5, Megumi Yamamoto6, Christoph F. A. Vogel7,8 and Yasuhiro Ishihara1,8*

Abstract
Background A recent epidemiological study showed that air pollution is closely involved in the prognosis ofischemic stroke. We and others have reported that microglial activation in ischemic stroke plays an important role inneuronal damage. In this study, we investigated the effects of urban aerosol exposure on neuroinflammation and theprognosis of ischemic stroke using a mouse photothrombotic model.
Results When mice were intranasally exposed to CRM28, urban aerosols collected in Beijing, China, for 7 days, micro‑
glial activation was observed in the olfactory bulb and cerebral cortex. Mice exposed to CRM28 showed increased
microglial activity and exacerbation of movement disorder after ischemic stroke induction. Administration of core par‑ticles stripped of attached chemicals from CRM28 by washing showed less microglial activation and suppression of
movement disorder compared with CRM28-treated groups. CRM28 exposure did not affect the prognosis of ischemic
stroke in null mice for aryl hydrocarbon receptor, a polycyclic aromatic hydrocarbon (PAH) receptor. Exposure to PM2.5
collected at Yokohama, Japan also exacerbated movement disorder after ischemic stroke.
Conclusion Particle matter in the air is involved in neuroinflammation and aggravation of the prognosis of ischemic
stroke; furthermore, PAHs in the particle matter could be responsible for the prognosis exacerbation.
Keywords Particle matter, Polycyclic aromatic hydrocarbons, Ischemic stroke, Inflammation, Edema*Correspondence:
Yasuhiro Ishihara
ishiyasu@hiroshima-u.ac.jp
1
Program of Biomedical Science, Graduate School of Integrated Sciencesfor Life, Hiroshima University, 1‑7‑1, Kagamiyama, Higashi‑Hiroshima,
Hiroshima 739‑8521, Japan
2
Laboratory for Pharmacotherapy and Experimental Neurology, Kagawa
School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki,
Kagawa 769‑2101, Japan
3
Faculty of Science and Technology, Keio University, Yokohama,
Kanagawa 223‑8522, Japan
4
Medical Research Institute, Molecular Epidemiology, Tokyo Medical
and Dental University, Bunkyo, Tokyo 113‑8510, Japan
5
Department of Applied Chemistry, Faculty of Engineering, Universityof Miyazaki, Miyazaki 889‑2192, Japan

6
Department of Environment and Public Health, National Institutefor Minamata Disease, Minamata, Kumamoto 867‑0008, Japan
7
Department of Environmental Toxicology, University of California, Davis,
Davis, CA 95616, USA
8
Center for Health and the Environment, University of California, Davis,
Davis, CA 95616, USA© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

Background
Air pollution contributed to more than 3% of the annual
disability-adjusted life years lost in the 2010 Global Burden of Disease comparative risk assessment, a notable
increase since the previous estimate was made in 2000
[1]. A Harvard study of six cities showed that air pollution was positively associated with death from lung cancer and cardiopulmonary disease but not with death fromother causes considered together [2], suggesting that the
respiratory system is one of the major targets of air pollution. Among several air pollutants, PM2.5 is considered
to be one of the major risk factors threatening humanhealth. Inhaled PM2.5 can reach the deep lung and thenact on alveolar macrophages and lung epithelial cells toelicit inflammatory reactions and/or oxidative stress.
Inflammation is closely associated with oxidative stress:
reactive oxygen species inactivate several protein phosphatases, such as PTP1B, which activate a certain kinase
pathway to lead to the activation of NF-κB, a master transcription factor for inflammatory gene expression, followed by the upregulation of inflammatory cytokines,
chemokines and other molecules [3, 4]. This inflammation and oxidative stress are considered to be involved inasthma aggravation, susceptibility to infection and lungcancer progression [5–7].
Recent evidence has shown that PM2.5 can distributeto organs/tissues other than the respiratory tract. Oberdorster et al. examined the particle distribution in the
body by administration of 13C carbon nanoparticles torats and subsequent measurement of the carbon isotope
ratio [8]. The 13C concentration increased in the lung at
the early stage of administration and was also elevated inbrain regions such as the cerebral cortex and cerebellum.
Interestingly, although 13C particles in the lung decreased
in a time-dependent manner, those in the brain were notmarkedly reduced for a long time. Wang et al. revealed
that iron oxide nanoparticles reach not only the olfactory bulb but also the cerebral cortex and hippocampus,
as shown by the synchrotron radiation microbeam X-ray
fluorescence method [9]. Diesel exhaust particles werealso reported to move into the olfactory bulb after chamber exposure, as shown by the detection of metals with
X-ray fluorescence analysis [10]. In addition, submicrometer-sized polystyrene particles were suggested to passthrough the axon and reach the opposite site of neurons[11]. In humans, magnetite nanoparticles were detected
within cells in the frontal cortex [12]. Therefore, particle
matter introduced by inhalation reaches and accumulates
in the brain, although its mechanism is under debate.
Microglia are derived from yolk-sac macrophages,
which settle in the brain during development and are
the primary immune cells of the central nervous system(CNS). Microglia are activated by several endogenous

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and exogenous substrates to engulf them and induce
inflammatory reactions [13]. Microglia are reported toengulf particles to induce cyclooxygenase-2 (COX-2)
expression and release reactive oxygen species [14]. Bacteria and dead cells engulfed by phagocytes are digested
to amino acids and other constituents, while silica andcarbon, which frequently occupy a PM2.5 core, cannot
be digested, and thus many engulfed cells die with therelease of alarmins that induce inflammatory reactions[15].
Increasing evidence shows that air pollution is closely
involved in neurological disorders. Daily exposure toelevated carbon monoxide concentrations could be associated with an increased risk of epileptic seizures, especially subclinical seizures [16]. Long-term exposure toambient PM2.5 has increased the risks of death and hospital admissions for Alzheimer’s disease [17]. Exposure
to black carbon and sulfate in PM2.5 had the strongest
associations with dementia incidence [18]. In rats, trafficrelated air pollution-exposed animals had more amyloid
plaque deposition, higher hyperphosphorylated tau levels, more neuronal cell loss, and greater cognitive deficits[19]. In addition, ambient PM2.5 represents a potentiallyubiquitous and modifiable risk factor for the loss of senseof smell [20].
There are several epidemiologic reports showing therelationships between air pollution and the onset ofischemic stroke. It has been suggested that the incidence and hospitalization rates of ischemic stroke are
increased in air-polluted areas [21]. Hypertension andatherosclerosis are known risk factors for the onset of
cerebral infarction, and PM2.5 is reported to be involved
in increases in blood pressure and aggravation of atherosclerosis [22, 23]. Such risk factors affected by PM2.5
may contribute to the increased incidence of stroke due
to air pollution. Recently, the relationships between components of PM2.5 and ischemic stroke have been investigated. Zhang et al. reported that the association ofions were generally consistent for ischemic stroke andthat polycyclic aromatic hydrocarbons (PAHs), especially indeno(1,2,3-cd)pyrene and benzo(b)fluoranthene,
were predominantely associated with ischemic stroke[24]. PAHs upregulate CYP1A1 by an aryl hydrocarbon
receptor (AhR)-dependent manner. CYP1A1 is a phase I
metabolizing enzyme which plays a key role in metabolic
activation of PAHs. Interestingly, CYP1A1 CC genotype, elevated enzyme activity by a T → C mutation, isreported to be associated with 5 times increased risk ofischemic stroke [25]. Therefore, PAHs included in PM2.5
might be one of important determinants for the risk ofischemic stroke.
It has recently become epidemiologically clear that
exposure to PM2.5 exacerbates the prognosis of cerebral

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

infarction. PM2.5 exposure has been reported to increase
poststroke hospital stay by 2–5 days or increase mortality
within 1 year after ischemic stroke [26, 27]. In addition, it
was found that the severity of cerebral infarction (NIHSS)
was reduced in areas surrounded by green and became
more severe in areas with heavy traffic [28]. However, themechanism by which PM2.5 exacerbates the prognosis of
cerebral infarction remains unclear. As mentioned above,
PM2.5 can act on the central nervous system and induce
inflammatory reactions. Therefore, in this study, weinvestigated the mechanism by which PM2.5 aggravatesthe prognosis of cerebral infarction, focusing on microglial activity.

Results
Neuroinflammation induced by intranasal exposure
to CRM28

CRM28 was collected from filters in a central ventilating
system in a building in Beijing and contains several kindsof soluble ions, metals, PAHs and endotoxins (Additional file 1: Tables S3, S4, S5 and S6). The particle size

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distribution of CRM28 is shown in Additional file 1: Fig.
S6. CRM28 included many particles with diameters lessthan 1 µm.
First, we measured the microglial activity of mice
exposed to CRM28 by IHC of Iba1 and CD68 in theolfactory bulb and cerebral cortex because the olfactory
bulb is considered to be first target of PM nasally administrated [8] and because ischemia is induced in the cerebral cortex. CRM28 was suspended in the saline andthen intranasally administered into mice. Treatment withsaline (vehicle) did not affect the expressions of Iba1 andCD68 compared with untreated mice (data not shown).
Exposure to 10 µg CRM28 once a day for 7 days did notshow microglial morphology evaluated by Iba1 staining
or CD68 expression levels in the olfactory bulb and cerebral cortex (Fig. 1). When CRM28 was administered at adose of 100 µg/mouse once a day for 7 days, an enlarged
cell body of microglia and increased expression of CD68
were observed in the olfactory bulb and cerebral cortex(Fig. 1), indicating microglial activation by intranasal
exposure to CRM28. The expression of glial fibrillary

Fig. 1 Microglial activation induced by intranasal administration of CRM28. CRM28 was suspended in water and intranasally (i.n.) administeredat doses of 0, 10 or 100 µg/mouse once a day for 7 days. Brain slices were stained with Iba1 and CD68 and observed by confocal microscopy. A
Representative stained pictures of the olfactory bulb. B Amoeboid scores. C The CD68-stained area of the olfactory bulb. The values are presented
as the mean ± S.D. (n = 6 animals in each group). The data were analyzed using one-way ANOVA [B F(2, 15) = 4.524, p = 0.0290, C F(2, 15) = 6.770,
p = 0.0080] followed by Dunnett’s multiple comparisons test. D Representative stained pictures of the cerebral cortex. E Amoeboid scores. F The
CD68-stained area of the cerebral cortex. The values are presented as the mean ± S.D. (n = 6 animals in each group). The data were analyzed using
one-way ANOVA [E: F(2, 15) = 9.860, p = 0.0018, F: F(2, 15) = 21.83, p < 0.0001] followed by Dunnett’s multiple comparisons test

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

acidic protein in the cerebral cortex was not alteredby the nasal administration of 100 µg CRM28 (Additional file 1: Fig. S1), suggesting that astrocytes could
not respond to CRM28. When primary neurons, astrocytes or microglia prepared from mice were treated with
10 µg/mL CRM28, an inflammatory reaction, which ischaracterized by an increase in inflammatory molecules
such as IL-6, KC and COX-2, was strongly induced in
microglia, while neurons showed no response, and astrocytes showed less response than microglia (Additional
file 1: Fig. S2). In addition, serum cytokine levels were notchanged by CRM28 exposure (Additional file 1: Fig. S3),
indicating that systemic inflammation did not occur after
CRM28 exposure. Therefore, the major target of CRM28
in the brain is considered to be microglia.
Exacerbation of the ischemic stroke prognosis by CRM28

Ischemic stroke was induced by cortical photothrombosis with Rose Bengal injection. CRM28 exposure atdosages of 10 and 100 µg/mouse/day for 7 days did notaffect mortality by 1 week after ischemic stroke induction (data not shown). Coordinated movement after
ischemic stroke was evaluated by rotarod tests. Retention time on the rod 1 day after ischemic stroke induction largely decreased compared with the preischemic
period and then gently recovered in a time-dependent
manner in the vehicle group (Fig. 2). Preexposure to
10 µg CRM28 for 7 days showed no effect on coordinated movement after ischemic stroke. Administrationof 100 µg CRM enhanced the decreases in retention
1 day after induction of ischemic stroke, and notably,
the recovery after stroke was delayed by 100 µg CRM

Fig. 2 Impairment of movement disorder after ischemic stroke
elicited by CRM28 exposure. CRM28 was suspended in water andintranasally (i.n.) administered at doses of 0, 10 or 100 µg/mouse oncea day for 7 days. Coordinated movement was measured by a rotarod
test before (day 0) and 1, 3, 5 and 7 days after photothrombosis
induction. The reported values are the mean ± S.D. (n = 8 animals ineach group). The area under the curve (AUC) from 1 to 7 days wascalculated, and the AUC was analyzed using one-way ANOVA [F(2,21) = 8.363, p = 0.0021] followed by Dunnett’s multiple comparisonstest. The p values of the vehicle versus 10 µg CRM groups and vehicle
versus 100 µg CRM groups were 0.666 and 0.0018, respectively

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28 exposure (Fig. 2). Treatment with saline did notshow the changes in rotarod retention time comparedwith untreated group (data not shown). These results
indicate that CRM28 exposure can exacerbate the prognosis of ischemic stroke.
Edema appears to be critical in the pathogenesis ofischemic stroke, and severe edema can induce exencephaly, leading to a significant aftereffect or occasionally lethality. Recently, we reported that regulation of
the progression of ischemic edema by activated microglia is critical for infarct progression and neuronal
dysfunction [29]. Therefore, we next examined neuroinflammation, vasogenic edema and infarcts that occur
due to ischemia.
The cell body of microglia in the peripheral areaof the ischemic region enlarged a day after ischemic
stroke induction, as evaluated by Iba1 staining (Fig. 3A,
B). CD68 expression also increased 1 day after ischemic
stroke (Fig. 3A, C), clearly indicating ischemic activation of microglia. Although exposure to 10 µg CRM28
did not show any change in microglial morphology orCD68 expression, 100 µg CRM28 significantly potentiated the morphological change in microglia fromramified to ameboid and the increase in CD68 expression elicited by ischemia (Fig. 3). Therefore, additiveeffects of neuroinflammation by CRM28 exposure andischemia are considered to be observed in the peripheral area of the ischemic region. Magnetic resonanceimaging (MRI) T2WIs can be used to visualize both theinfarct and edema regions, and 2,3,5-triphenyltetrazolium chloride (TTC) staining can identify the infarct.
Thus, the size of the edema region can be determinedby calculating the difference in the size of the T2WI
hyperintense region from that of the TTC-unstained
area [30]. A high contrast area by T2WI appeared, andan infarct also occurred a day after ischemic stroke
induction, and the difference between both was considered to be the edema area (Fig. 4). Exposure to 10 µg
CRM did not affect the areas of T2-high contrast andTTC staining after ischemia, but 100 µg CRM28 significantly increased the T2 high contrast area and ischemic
edema (Fig. 4). Importantly, TTC-stained areas did notchange among the vehicle, 10 µg and 100 µg CRM28treated groups (Fig. 4), indicating that CRM28 exposure does not affect infarct size. Blood pressure andblood coagulation showed no change during CRM28
exposure (Additional file 1: Figs. S4 and S5). Thus,
hemodynamics is unlikely to be involved in edema formation. Overall, 100 µg CRM28 exposure for 7 days
can enhance ischemic neuroinflammation, which might
induce the potentiation of ischemic edema, followedby exacerbation of movement disorder after ischemic
stroke.

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

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Fig. 3 Enhancement of microglial activation during ischemia induced by CRM28 exposure. CRM28 was suspended in water and intranasally (i.n.)
administered at doses of 0, 10 or 100 µg/mouse once a day for 7 days. One day after induction of photothrombosis, brain slices were preparedand stained with Iba1 and CD68. The peripheral area of the ischemic area in the cerebral cortex was observed with a confocal microscope. A
Representative stained pictures of the cerebral cortex. B Amoeboid scores. C The CD68-stained area of the cerebral cortex. The values are presented
as the mean ± S.D. (n = 6 animals in each group). The data between vehicle and vehicle + PT1d were analyzed using Student’s t test. The data
among vehicle + PT1d, 10 µg + PT1d and 100 µg + PT1d were analyzed using one-way ANOVA [B: F(2, 15) = 12.57, p = 0.0006, C: F(2, 15) = 9.223,
p = 0.0024] followed by Dunnett’s multiple comparisons test

Contribution of PAHs in CRM28 to enhanced
neuroinflammation and aggravated prognosis
after ischemic stroke

We next investigated which component in CRM28 isinvolved in neuroinflammation and the potentiationof movement disorder after ischemic stroke. CRM28
was washed with water and organic solvents to produce
core particles. Particle size distribution was not affectedby washing procedure (Additional file 1: Fig. S6). The
contents of water-soluble ions in the core were muchless than those in CRM28 (Additional file 1: Table S3),
although the metal concentrations in the core were very
similar to those in CRM28 (Additional file 1: Table S4).
Of note, the PAH contents in the core decreased toapproximately one-tenth of the concentration in CRM28
after washing (Additional file 1: Table S5). Endotoxin
contents were also reduced by washing (Additional file 1:
Table S6). Bone marrow-derived macrophages were prepared from ICR mice and then exposed to CMR28, core
particles or dichloromethane extracts to examine thepotential for inducing inflammation. CYP1A1 expression
largely induced by CRM28 was significantly suppressedby treatment with core (Fig. 5A), reflecting the decreases
in PAH contents in core particles. This consideration
is supported by the results that the extracts induced
CYP1A1 expression (Fig. 5A). Although TNFα and
COX-2 expression was largely induced by treatment with
CRM28, their expression was significantly suppressed bycore administration (Fig. 5B, C). The extracts had higher
potency to increase TNFα and COX-2 expression thancore particles (Fig. 5B, C). Therefore, the core lost thepotency to induce inflammation by washing comparedwith CRM28. When mice were intranasally administered
100 µg core particles for 7 days, microglial morphology did not change, and CD68 expression remained low(Fig. 5D, E), indicating that microglia were not affectedby core particles. Therefore, chemical compounds wereattached to CRM28 and compounds detached from
CRM28 by the washing procedure can induce microglial activation by intranasal administration as CRM28.

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

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Fig. 4 Enhancement of brain edema after ischemic stroke by CRM28 exposure. CRM28 was suspended in water and intranasally (i.n.) administeredat doses of 0, 10 or 100 µg/mouse once a day for 7 days. One day after induction of photothrombosis, T1WI and T2WI were obtained for each mouseby MRI, and then the infarct area was determined by TTC staining. A Representative images of T1WI, T2WI and TTC staining. B T2 hyperintense lesion(T2WI), TTC-unstained area (Infarct) and edema region (T2 hyperintense lesion except infarct) were quantified by ImageJ software. The values arepresented as the mean ± S.D. (n = 5 animals in each group). The data were analyzed using one-way ANOVA [T2WI F(2, 12) = 9.124, p = 0.0039, Infarct
F(2, 12) = 1.708, p = 0.2225, Edema F(2, 12) = 4.893, p = 0.0279] followed by Dunnett’s multiple comparisons test

Then, the mice were treated with core particles for 7 days
and then subjected to ischemic stroke. Interestingly, theseverity of movement disorder in mice treated with thecore after ischemic stroke was similar to that in vehicletreated mice. Therefore, compounds attached to CRM28
could be involved in the aggravation of prognosis after
ischemic stroke.
We previously reported that AhR, which is a receptor
for PAHs to induce the upregulation of inflammatory
molecules, in microglia is involved in neuroinflammation and subsequent brain damage after middle cerebral

artery occlusion [30]. In addition, large parts of the PAHs
in CRM28 were left by the washing procedure (Additional file 1: Table S5). Thus, we examined the effects of
CRM28 on neuroinflammation and movement disorder
after ischemia, focusing on PAHs in CRM28. Bone marrow macrophages isolated from wild-type or Aryl hydrocarbon receptor knockout (AhR KO) mice were exposed
to 10 µg/mL CRM28 for 6 h. The mRNA expression of
CYP1A1, TNFα and COX-2 was increased by CRM28
administration in wild-type macrophages, while in AhR
KO macrophages, there was no increase in CYP1A1

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

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Fig. 5 Contribution of attached compounds around CRM28 to neuroinflammation and movement disorder after ischemic stroke. Bonemarrow-derived macrophages were treated with 10 µg/mL CRM28 or its core for 6 h, and then mRNA expression of A CYP1A1, B TNFα and C COX-2
was measured by real-time PCR. The values are presented as the mean ± S.D. (n = 5). The data were analyzed using Student’s t test (vehicle group vs.
CRM28-treated group) and one-way ANOVA [A F(2, 12) = 36.85, p < 0.0001, B F(2, 12) = 5.875, p = 0.0166, C F(2, 12) = 19.19, p = 0.0002] followed byDunnett’s multiple comparisons test. Multiple comparisons were corrected by Holm’s method. CRM28 or its core particle was suspended in water
and was intranasally (i.n.) administered at a dose of 100 µg/mouse once a day for 7 days. Brain slices were prepared and stained with Iba1 and CD68,
and then D amoeboid scores and E the CD68-stained area of the cerebral cortex were calculated. The values are presented as the mean ± S.D.
(n = 5). The data were analyzed using Student’s t test with Holm’s correction for multiple comparisons. F After 7 days of exposure to CRM28 or its
core, coordinated movement was measured by a rotarod test before (day 0) and 1, 3, 5 and 7 days after photothrombosis induction. The reported
values are the mean ± S.D. (n = 9 animals in each group). The area under the curve (AUC) from 1 to 7 days was calculated, and the AUC was
analyzed using Student’s t test with Holm’s correction for multiple comparisons. The p values of the vehicle versus 100 µg CRM groups and 100 µg
CRM versus 100 µg Core groups were 0.0016 and 0.0342, respectively

mRNA or suppression of TNFα and COX-2 expression
in the presence of CRM28 (Fig. 6A–C), suggesting that
PAHs in CEM28 contribute to the inflammatory reaction.
When wild-type mice or AhR KO mice were exposed to
100 µg CRM28 for 7 days, morphological changes andincreased CD68 expression were observed in wild-typemice (Fig. 6D–F). However, microglial cell body enlargement and CD68 expression induced by CRM28 weresignificantly attenuated in AhR KO mice (Fig. 6D–F).
Movement disorder after ischemic stroke induction was
also alleviated in AhR KO mice (Fig. 6G). Taken together,
these results suggest that PAHs in CRM28 are involvedin neuroinflammation and the aggravation of prognosis
after ischemic stroke.
Exacerbation of movement disorder after ischemic strokeby exposure to PM2.5

Finally, we confirmed the generality of the phenomenon:
CRM28-induced exacerbation of movement disorder
after ischemic stroke. PM2.5 was collected in spring 2020
at Yokohama, Japan, and was introduced to ICR miceat a dose of 100 µg for 7 days. Movement disorder after
ischemic stroke induction was alleviated compared withthat in vehicle-treated mice (Fig. 7). PM2.5 collected inTanaka et al. Particle and Fibre Toxicology(2023) 20:6

Page 8 of 14

Fig. 6 Significant role of PAHs included in CRM28 in neuroinflammation and movement disorder after ischemic stroke. Bone marrow-derived
macrophages prepared from wild-type and AhR KO mice were treated with 10 µg/mL CRM28 for 6 h, and then mRNA expression of A CYP1A1, B
TNFα and C COX-2 was measured by real-time PCR. The values are presented as the mean ± S.D. (n = 6). The data were analyzed using two-way
ANOVA [CYP1A1, F(1, 20) = 84.99, p < 0.0001; TNFα, F(1, 20) = 18.55, p = 0.0003; COX-2, F(1, 20) = 24.23, p < 0.0001] with Tukey’s corrected multiple
comparison tests. CRM28 was suspended in water and intranasally (i.n.) administered at a dose of 100 µg/mouse once a day for 7 days, followed bystaining of the cortex region with Iba1 and CD68. D Representative stained pictures of the cerebral cortex. E Amoeboid scores. F The CD68-stained
area of the cerebral cortex. The values are presented as the mean ± S.D. (n = 5). The data were analyzed using two-way ANOVA [E: F(1, 16) = 6.251,
p = 0.0237; F: F(1, 16) = 8.507, p = 0.0101] with Tukey’s corrected multiple comparison tests. G. Coordinated movement was measured by a rotarod
test before (day 0) and 1, 3, 5 and 7 days after photothrombosis induction. The reported values are the mean ± S.D. (n = 6 animals in each group).
The area under the curve (AUC) from 1 to 7 days was calculated, and the AUC was analyzed using two-way ANOVA [F(1, 20) = 25.97, p < 0.0001] with
Tukey’s corrected multiple comparison tests. The p values of the wild-vehicle versus wild-CRM groups, AhR KO-vehicle versus AhR KO-CRM groups,
and wild-CRM versus AhR KO-CRM groups were < 0.0001, 0.9943 and < 0.0001, respectively

winter 2018 at Yokohama, Japan showed the exacerbationof movement disorder after ischemia (data not shown).
Therefore, it is considered that PM2.5 exposure has thepotential to aggravate the prognosis after ischemic stroke.

Discussion
We have shown in this study that intranasal exposure to CRM28 caused neuroinflammation characterized by microglial activation. Induction of ischemia byphotothrombosis after CRM28 exposure exacerbated

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

Fig. 7 Aggravation of movement disorder after ischemic stroke
elicited by PM2.5 collected at Yokohama, Japan. PM2.5 collectedat Yokohama, Japan was suspended in water and was intranasally(i.n.) administered at a dose of 100 µg/mouse once a day for 7 days.
Coordinated movement was measured by a rotarod test before(day 0) and 1, 3, 5 and 7 days after photothrombosis induction. The
reported values are the mean ± S.D. (n = 7 animals in each group).
The area under the curve (AUC) from 1 to 7 days was calculated,
and the AUC was analyzed using Student’s t test. The p value of thevehicle versus 100 µg PM2.5 groups was 0.0013

postischemic neuroinflammation and motor dysfunction compared with the vehicle-treated group. Analysis using both the CRM28 core, in which chemical
substances attached to CRM28 were removed, and AhR
KO mice indicated that PAHs contained in CRM28
caused enhanced neuroinflammation and exacerbation of motor dysfunction after ischemia. We havereported that ischemia can induce inflammation [29]and that at least part of ischemic inflammation is AhRdependent [30]. Neuroinflammation has been reported
to exacerbate neuropathy due to cerebral infarction [31].
Therefore, CRM28-induced neuroinflammation is considered to act additively or synergistically with ischemiainduced inflammation to exacerbate postischemic motor
dysfunction.
The results we present here suggest that any inflammatory substance included in PM2.5 exacerbates the prognosis of ischemic stroke. In addition to PAHs, potent
AhR agonists such as polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated biphenyls (PCB) weredetected in the atmosphere, albeit in small quantities
in China and Japan [32, 33]. Therefore, these molecules
might contribute AhR-dependent neuroinflammation.
Ma et al. reported that binding of benzo[a]pyrene at environmentally relevant exposure concentrations alteredcytotoxicity to macrophages and they discussed the bioaccessibility of contaminants being carried [34]. In thisstudy, core did not affect neuroinflammation, suggesting that interaction of PAHs with core is important forneuronal damages. Furthermore, it is known that PM2.5
contains endotoxin as a component of bioaerosol. The
amount of endotoxin in CRM28 is small compared withthat in PM2.5 collected recently [35], probably because
it has been approximately 20 years since it was collected.

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Therefore, although endotoxin is a strong inflammatory
molecule, it is possible that PAHs have a strong effectin this study because of the few endotoxins in CRM28.
We showed that urban PM2.5 collected in Yokohama
also exacerbated the prognosis of cerebral infarction.
This PM2.5 contains many endotoxins compared with
CRM28, and we previously reported that not only PAHs
contained in urban PM2.5 but also endotoxins induce
inflammatory reactions [35]. In addition, we suggested
the crosstalk of AhR pathway and Toll-like receptor
pathway [36]. Therefore, the deterioration of prognosis
observed here may be due to endotoxins in addition to
PAHs in urban PM2.5.
The target of PAHs is AhR. AhR is known to be highly
expressed in the lungs and skin, which are barrier tissues
in the body, and it has also been reported to be expressedin several types of cells in the CNS, including microglia[37]. The activation of AhR plays either anti-inflammatory or proinflammatory roles in different environments.
Treatment with indole-3-carbinol, a natural ligand ofaryl hydrocarbon receptor, induced an increase in IL-1β
expression but suppressed LPS-elicited inflammatory
reactions in BV-2 microglia [38]. LPS markedly increases
inflammation by inducing AhR expression through activation of RelA and binding of RelA/p50 to an NF-κB
binding site identified in the human ahr gene promoter[39]. In an experimental autoimmune encephalomyelitis model, ­CX3CR1+ microglia-specific AhR deletion
upregulated the activation of proinflammatory astrocytes
and aggravated inflammation, indicating that microglial
AhR suppresses astrocytic inflammation [40]. On theother hand, in ischemic stroke models, AhR exacerbates
infarct formation [41], and downregulated IL-1β and IL-6
expression was detected in AhR KO mice [42]. We previously reported that AhR in microglia is involved in neuroinflammation and subsequent vasogenic edema after
middle cerebral artery occlusion [30]. The role of AhR inneuroinflammation is still controversial, but AhR might
be proinflammatory, at least in ischemia. Further study isneeded to uncover the mechanism of action of PAH-specific activation of AhR and its role in the brain. Because
astrocytic activity was not changed by CRM28 exposure,aquaporins in astrocytes are not likely to be involved in
edema exacerbation.
Increasing evidence has shown that PM2.5 via inhalation from the mouth and nose can target the CNS. In
fact, magnetite pollution nanoparticles have been foundin humans [12]. Multiple possibilities have been proposed
for the pathway by which PM2.5 enters the brain. Oberdorster et al. found that when rats were nasally exposed tocarbon (13C) particles with a particle diameter (more precisely, median diameter) of 36 nm, carbon microparticles
were detected in all brain regions, including the olfactory

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

bulb, cerebrum and cerebellum, a day after exposure[8]. This is considered to be olfactory nerve transport to
the CNS. Namely, it is hypothesized that PMs are taken
up into olfactory neurons, reach the olfactory bulb via
axonal transport, and then spread throughout the CNS
via intraneural spaces. In crab nerves, polystyrene particles with a particle size of 0.5 μm have been imaged moving in the axon at a speed of 20–40 μm per minute [11].
­TiO2 nanomaterials reportedly delivered through intranasal instillation, penetrated and impaired the brain in aparticle size-dependent manner [43]. ­Fe2O3 nanoparticles
also induced neuropathological changes including microglial activation [9]. Recently, it was reported that PM
exposure by breathing can be transported from the lungs
to the brain via the bloodstream [44]. The data suggestthat up to eight times the number of fine particles may
reach the brain by traveling, via the bloodstream, fromthe lungs than pass directly via the nose. CRM28 andurban PM2.5 were administered nasally in this study. The
particles are considered to be transported via the olfactory nerve system because inflammation at the olfactory bulb was detected after CRM28 exposure. However,
although systemic inflammation did not occur, lunginflammation was detected in this experimental protocol.
Therefore, particles inhaled into the lung might move to
the CNS. To address the dynamics of particles, we need
to observe PM2.5 in our body. However, there are limitations on observation methods for fine particle cores suchas carbon or silica, and it is currently extremely difficult
to observe small metals by electron microscopy becauseof artifacts derived from living animals. Another issue isthe determination of the pharmacokinetics of PM2.5 orother fine particles.
In this study, we administered CRM28 nasally at 10 and
100 µg/mouse/day and analyzed its neurological effects.
As a result, exposure to 10 µg/mouse/day for 7 days had
no effect on neuroinflammation or ischemic stroke prognosis, whereas exposure to 100 µg/mouse/day for 7 days
induced neuroinflammation and reduced poststroke
locomotion, exacerbating functional impairment compared with vehicle-treated mice. Converting the exposurein this study to the exposure per body weight for 7 days,
10 µg/mouse/day is 1.75 mg/kg. An approximate averagevalue of 40 g mouse weight was used for calculation. In
Japan, the environmental standard for PM2.5 stipulatedby the Ministry of the Environment is "the daily average value of PM2.5 is 35 μg/m3 or less". The ventilation
volume of a person with a body weight of 60 kg is calculated to be 0.5 L per breath, thus approximately 14.4
­m3 per day. Then, it became 8.4 µg/kg, as calculated by
the Japanese environmental standard. Regarding thiscalculation, if PM2.5 is inhaled for 208 days at the maximum value within the environmental standards in Japan,

Page 10 of 14

the exposure exceeds the no-observed-adverse-effectlevel estimated in this study. However, considering that
PM2.5 is slowly eliminated from the lung or brain [8,45], long-term exposure over years may be of concern.
On the other hand, although air pollution has regionaland seasonal characteristics, there are many areas wherePM2.5 in the atmosphere is relatively high throughout
the year; India’s average PM2.5 concentration level was
75 to over 85 μg/m3 in 2019 [46]. Another report showedthat the average concentration (± standard deviation) of
PM2.5 was 127 ± 77 μg/m3 from 2012 to 2021 in Delhi,
India, and the highest concentration of PM2.5 per week
was approximately 600 μg/m3 [47]. In China, it has beenreported that PM2.5 concentrations in Beijing, Jinan, andShanghai are 125.7 μg/m3 (18.6–355.5 μg/m3), 115.9 μg/
m3 (44.2–345.4 μg/m3), and 85.1 μg/m3 (24.3–232.8 μg/m3), respectively [48]. Therefore, the effect of PM2.5 on
the nervous system may not be negligible in such areas
with high PM2.5 concentrations. Recent progress inresearch on the effects of PM2.5 on the CNS has beenremarkable, and it is necessary to consider how to derive
the correlation between exposure and neurotoxicity.
Epidemiological studies have revealed that PM2.5
exacerbates many neurological diseases, including Alzheimer’s disease, Parkinson’s disease, epilepsy, autism,and dysosmia [20, 49–52]. Although the mechanismsof neurological effects of air pollution and/or PM2.5
are largely unknown, reports of animal experiments arebeing published recently. Patten et al. maintained Alzheimer’s disease model rats in a tunnel with heavy traffic for 14 months to investigate the effects on Alzheimer’s
disease pathology [19]. Inhaled nanosized particles weretranslocated into the hippocampus, where more amyloid
plaque deposition, higher hyperphosphorylated tau levels and more neuronal cell loss were detected comparedwith control animals. Notably, PM-exposed animals had
more microglial activation than control animals, indicating that PM exposure can enhance neuroinflammation
and Alzheimer’s disease pathology. To understand moredetails of the effects of PM on the brain, further mechanistic studies should be performed.

Conclusion
We investigated the effects of Beijing airborne particulate CRM28 and urban PM2.5 on cerebral infarction
prognosis. Nasal exposure to CRM28 induced neuroinflammation characterized by microglial activation andexacerbated motor dysfunction after ischemic stroke.
PAHs, including CRM28, can play a fundamental rolein enhanced neuroinflammation and exacerbated movement disorder. Therefore, in areas with severe air pollution, controlling the occurrence of PAHs may leadto the prevention of aggravation of cerebral infarction.

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

The significance of this study is that it identified components of PM associated with exacerbated prognosis for ischemic stroke, and this study could contributeto the creation of an environment in which people canlive safely and securely by the establishment of novel
environmental standards and/or regulation of emission
scientifically.

Methods
Mice

All animal procedures were performed in accordancewith the Fundamental Guidelines for Proper Conduct ofAnimal Experiments and Related Activities in Academic
Research Institutions under the Jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The Animal Care and Use Committee ofHiroshima University approved the experimental protocols (No. C18-16-4 and C20-33). Male C57BL/6J andICR mice were purchased from CREA Japan Inc. (Tokyo,
Japan). AhR-KO mice were generated as described previously [53]. The mice were maintained on a 12:12 h light/
dark cycle (light on from 8:00 a.m. to 8:00 p.m.) and had
free access to water and food. The mice were allowed toadapt to the facility for 1 week. We used a simple randomization method [54]. Four to five mice were housedin the same cage. No sample calculation was performed,
and the sample size was determined by our previous
studies [29, 30, 55]. Any animal was not excluded basedon the statistics. Two hundred forty mice were used inthis study. N values are included in the figure legends asappropriate.
Particle matter exposure

National Institute for Environmental Studies (NIES)
CRM28, urban aerosols collected on filters in a central
ventilating system in a building in the Beijing city centerfrom 1996 to 2005 [56], was purchased from the National
Institute for Environmental Studies. CRM28 was suspended in saline with sonication (10 W for 10 s) atconcentrations of 10 and 100 µg/10 µL. The resulting suspension of 10 µL was immediately applied into the nasal
cavity using a micropipette. Vehicle mice were intranasally administered the same amount of saline.
Immunohistochemistry (IHC)

IHC was performed according to our previous report[57]. The antibodies that were used are listed in Additional file 1: Table S1. Images were processed using the
Zen image acquisition software package (Carl Zeiss,
Oberkochen, Germany) and ImageJ software. The soma
area was evaluated using images of Iba1 staining, andthe amoeboid score was calculated by the following formula: (soma area/entire Iba1-stained area) × 100 (%).

Page 11 of 14

The Iba1- and CD68-costained areas were analyzed with
ImageJ software (National Institutes of Health, Bethesda,
MD, USA).
Total RNA extraction and qPCR

mRNA levels were determined according to the protocol described in our previous report [58]. The primer
sequences are presented in Additional file 1: Table S2.
mRNA levels were normalized to the level of the housekeeping gene β-actin, and the values of the treated samples were divided by those of the untreated samples tocalculate the relative mRNA levels.
Preparation of the photothrombosis model

Cortical photothrombosis was induced by Rose Bengal injection and subsequent radiation [59, 60]. Anesthesia was initially achieved with isoflurane at 3.5% andthen reduced to 2% during the surgical procedure. Mice
were placed in a stereotaxic frame, and then the scalp
was incised. Rose Bengal (10 mg/mL in sterile saline,
50 mg/kg) was injected intraperitoneally. The LED (170mW, 561 nm, light diameter 4 mm, M565L3, Thorlabs
Inc. Newton, NJ, USA) was illuminated for 20 min, positioned posterior 2 mm and left 2 mm relative to Bregma(motor and somatosensory area). Decreases in blood flow
were determined by laser Doppler flowmetry (Unique
Medical Co., Ltd., Tokyo, Japan), and mice with a 70 to
85% decrease in blood flow were used for subsequent
experiments. The scalp was sutured, and 1 mL of saline
was injected subcutaneously. The mice were allowed torecover in a 25 °C chamber for 1 h prior to returning totheir home cage. Mice were provided a dish of moist foodin their home cage for the duration of their poststroke
survival.
Rotarod test

The rotarod test was performed according to our previous report [55]. A rotarod apparatus (LE8200, Panlab,
Barcelona, Spain) was used to test the ability of mice tocoordinate movements. The rotarod test was performedby placing a mouse on a rotating drum and measuring
the time each animal was able to maintain its balancewalking on top of the rod. The speed of the rotarod
accelerated from 4 to 40 rpm over a 5-min period. Mice
were given 3 trials with a maximum time of 5 min and a
15 min intertrial rest interval. The average latency from 3
trials was calculated.
MRI

MRI was performed according to our previous paper[29]. Briefly, mice were anesthetized with isoflurane,
and body temperature was maintained at a constant
37 ± 0.2 °C. MRI data were acquired using an MRmini-SA

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

system (DS Pharma Biomedical, Osaka, Japan) consisting of a 1.5-Tesla permanent magnet, a compact computer-controlled console, and a solenoid MRI coil witha 30 mm inner diameter. T2-weighted images (T2WIs)
were obtained with the following parameters: repetition
time (ms)/echo time (ms) = 2500/69 and number of excitations = 4. To measure the signal intensity on T2WIs,
the mean signal intensity was determined using INTAGE
Realia Professional software (Cybernet Systems Co., Ltd.,Tokyo, Japan) and ImageJ software.
Staining of the infarct region

The infarct size was assessed by TTC staining [29]. Brains
were sliced into 1-mm-thick coronal sections 0.0 mm
from bregma and stained with 1% TTC solution in PBS at
37 °C for 10 min. TTC-stained brain sections were analyzed by ImageJ software (National Institutes of Health,Bethesda, MD, USA).
Washing procedure of CRM28

One milliliter of distilled water was added to 50 mg of
CRM28. The mixture was vortexed for 5 min and thencentrifuged at 30,000 × g for 5 min. This procedure wasrepeated twice. Then, one milliliter of acetone was added
to the resulting pellet. The mixture was vortexed for
5 min and then centrifuged at 30,000 × g for 5 min. This
procedure was repeated twice. Finally, one milliliter ofdichloromethane was added to the resulting pellet. The
mixture was vortexed for 5 min and then centrifuged at
30,000 × g for 5 min. This procedure was repeated twice.
The pellet was dried overnight in a desiccator. After theexact weight of the PM core was measured, 100 µg/10µL suspension was prepared with distilled water. Dichloromethane was evaporated and reconstituted in DMSO
to produce dichloromethane extracts, which were added
to cultured macrophages.
Preparation of bone marrow‑derived macrophages

Primary bone marrow progenitor cells were isolated anddifferentiated as described previously [61]. Briefly, thefemurs were isolated under sterile conditions, and bonemarrow cells were extracted with an RPMI mediumloaded syringe. The cells were passed through a 30 μm
cell strainer, and the supernatant was centrifuged for
5 min at 1000 × g to obtain cells. Bone marrow-derived
macrophage differentiation was performed in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF; 20 ng/mL; PeproTech, Cranbury, NJ, USA)for 7 days.
Collection of PM2.5

The PM2.5 used in this study was collected in Yokohama, Japan, in spring 2020, as previously described [62].

Page 12 of 14

Briefly, PM2.5 with an aerodynamic diameter of 2.5 μm
was separated using an impactor prior to entry into thecyclone device. Subsequently, the cyclone imparted acentrifugal force on the gas stream within a conicalshaped chamber and created a vortex inside the cyclone
body.
Statistics

All data are presented as the mean ± standard deviation(S.D.). All data were analyzed using GraphPad Prism 9(GraphPad Software, San Diego, CA, USA). Student’s ttest, one-way ANOVA with Dunnett’s corrected multiple comparison tests, and two-way ANOVA with Tukey’s
corrected multiple comparison tests were used to determine significant differences between the means of two or
more independent groups. The F value calculated fromANOVA is described in each figure legend, the p value
is indicated in each figure or legend, and significance wasconsidered when p values were less than 0.05.
Abbreviations
AhR	Aryl hydrocarbon receptor
CNS	Central nervous system
COX-2	Cyclooxygenase-2
IHC	Immunohistochemistry
MRI	Magnetic resonance imaging
PAH	Polycyclic aromatic hydrocarbon
T2WI	T2-weighted image
TTC​	2,3,5-Triphenyltetrazolium chloride

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12989-​023-​00517-x.
Additional file 1: Includes Fig. S1 to S6, Table S1 to S5 and related
methods.
Acknowledgements
The authors thank Masaho Fujikawa, Shinji Kawano and Kaede Namba fortechnical assistance during the course of this study.
Author contributions
Conceptualization, YI; methodology, MT, TO and YI; investigation, MT, TO, NI,
AO, YFK, YN, MY and YI; visualization, MT, KI and YI; funding acquisition, MT, TO,
and YI; project administration, KI and YI; Supervision, TO, CFAV; writing—origi‑
nal draft, MT and YI; writing—review and editing: all authors. All authors haveread and agreed to the published version of the manuscript.
Funding
This work was supported by Research Fellowships for Young Scientists (Grant
Number 20J10103 to MT), KAKENHI Grants from the Japan Society for the Pro‑
motion of Science (Grant Numbers 21K06702 to KI and 20H04341, 17H04714
and 15KK0024 to YI), the Environment Research and Technology Development
Funds of the Environmental Restoration and Conservation Agency of Japan(JPMEERF20165051 to TO and JPMEERF20205007 to YI) and the National
Institute of Environmental Health Sciences (R01ES029126 and R01ES032827
to CFAV).

Tanaka et al. Particle and Fibre Toxicology(2023) 20:6

Availability of data and materials
All data generated or analyzed during this study are included in this publishedarticle and its supplementary information file.

Declarations
Ethical approval and consent to participate
Laboratory animal applications in this study were approved by the Animal
Care and Use Committee of Hiroshima University approved the experimental
protocols (No. C18-16-4 and C20-33).
Competing interests
The authors declare they have nothing to disclose.
Received: 27 October 2022 Accepted: 7 February 2023

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