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Inhalation Toxicology, 16:437–445, 2004
Copyright
c
Taylor & Francis Inc.
ISSN: 0895-8378 print / 1091-7691 online
DOI: 10.1080/08958370490439597
Translocation of Inhaled Ultrafine Particles to the Brain
G. Oberd¨orster
University of Rochester, Rochester, New York, USA
Z. Sharp, V. Atudorei
University of New Mexico, Albuquerque, New Mexico, USA
A. Elder, R. Gelein
University of Rochester, Rochester, New York, USA
W. Kreyling
National Research Center for Environment and Health (GSF), Neuherberg/Munich, Germany
C. Cox
National Institutes of Health, Bethesda, Maryland, USA
Ultrafine particles (UFP, particles <100 nm) are ubiquitous in ambient urban and indoor air
from multiple sources and may contribute to adverse respiratory and cardiovascular effects of
particulate matter (PM). Depending on their particle size, inhaled UFP are efficiently deposited
in nasal, tracheobronchial, and alveolar regions due to diffusion. Our previous rat studies have
shown that UFP can translocate to interstitial sites in the respiratory tract as well as to extrapul-
monary organs such as liver within 4 to 24 h postexposure. There were also indications that the
olfactory bulb of the brain was targeted. Our objective in this follow-up study, therefore, was
to determine whether translocation of inhaled ultrafine solid particles to regions of the brain
takes place, hypothesizing that UFP depositing on the olfactory mucosa of the nasal region will
translocate along the olfactory nerve into the olfactory bulb. This should result in significant in-
creases in that region on the days following the exposure as opposed to other areas of the central
nervous system (CNS). We generated ultrafine elemental
13
C particles (CMD = 36 nm; GSD =
1.66) from [
13
C] graphite rods by electric spark discharge in an argon atmosphere at a concen-
tration of 160 µg/m
3
. Rats were exposed for 6 h, and lungs, cerebrum, cerebellum and olfactory
bulbs were removed 1, 3, 5, and 7 days after exposure.
13
C concentrations were determined
by isotope ratio mass spectroscopy and compared to background
13
C levels of sham-exposed
controls (day 0). The background corrected pulmonary
13
C added as ultrafine
13
C particles on
day 1 postexposure was 1.34 µg/lung. Lung
13
C concentration decreased from 1.39 µg/g (day 1)
to 0.59 µg/g by 7 days postexposure. There was a significant and persistent increase in added
13
C
in the olfactory bulb of 0.35 µg/g on day 1, which increased to 0.43 µg/g by day 7. Day 1
13
C con-
centrations of cerebrum and cerebellum were also significantly increased but the increase was
inconsistent, significant only on one additional day of the postexposure period, possibly reflecting
translocation across the blood–brain barrier in certain brain regions. The increases in olfactory
bulbs are consistent with earlier studies in nonhuman primates and rodents that demonstrated
that intranasally instilled solid UFP translocate along axons of the olfactory nerve into the CNS.
We conclude from our study that the CNS can be targeted by airborne solid ultrafine particles
and that the most likely mechanism is from deposits on the olfactory mucosa of the nasopha-
ryngeal region of the respiratory tract and subsequent translocation via the olfactory nerve.
Received 21 July 2003; sent for revision 13 August 2003; accepted 22 September 2003.
This work was supported by the U.S. Environmental Protection Agency STAR Program, grant R827354, and by NIEHS grant ESO1247. We
acknowledge the excellent technical assistance of N. Corson, P. Mercer, and A. Lunts, and the valuable editorial assistance of J. Havalack. The
views expressed by the authors are their own and do not necessarily reflect those of the U.S. EPA.
Address correspondence to Dr. G¨unter Oberd¨orster, Department of Environmental Medicine, University of Rochester, 575 Elmwood Avenue,
Med. Ctr. Box 850, Rochester, NY 14642, USA. E-mail: gunter
oberdorster@urmc.rochester.edu
437
438 G. OBERD
¨
ORSTER ET AL.
Depending on particle size, >50% of inhaled UFP can be depositing in the nasopharyngeal
region during nasal breathing. Preliminary estimates from the present results show that ∼20%
of the UFP deposited on the olfactory mucosa of the rat can be translocated to the olfactory
bulb. Such neuronal translocation constitutes an additional not generally recognized clearance
pathway for inhaled solid UFP, whose significance for humans, however, still needs to be estab-
lished. It could provide a portal of entry into the CNS for solid UFP, circumventing the tight
blood–brain barrier. Whether this translocation of inhaled UFP can cause CNS effects needs to
be determined in future studies.
Ultrafine particles (UFP, particles <100 nm) are ubiquitous
in the ambient air, both indoor and outdoor, originating from
many anthropogenic and natural sources. Several epidemio-
logical studies have shown that ambient UFP are associated
with increased respiratory and cardiovascular morbidity and
mortality in susceptible people (Wichmann et al., 2000; Peters
et al., 1997a, 1997b; Penttinen et al., 2001; von Klot et al.,
2002; Pekkanen et al., 2002). The deposition of inhaled UFP
in the respiratory tract is governed by diffusional processes,
yet there are significant differences within the ultrafine particle
size range with respect to the efficiency of their maximal de-
position in different regions of the respiratory tract, as depicted
in Figure 1. About 90% of inhaled UFP around 1 nm in size
deposit in the nasopharyngeal region (Swift et al., 1992; Cheng
et al., 1996), whereas only about 10% of this size deposit in
the tracheobronchial and essentially none in the alveolar re-
gion; in contrast, 5- to 10-nm UFP deposit in all 3 regions with
about 20–30% efficiency, whereas 20-nm UFP are predicted to
be deposited in the alveolar region up to 50% and only about
10% each in the nasopharyngeal and tracheobronchial regions
FIG. 1. ICRP (1994) model of fractional depositions of in-
haled particles ranging from 0.6 nm to 20 µm in the na-
sopharyngeal/laryngeal (NPL), the tracheobronchial (TB), and
the alveolar (A) regions of the human respiratory tract dur-
ing nasal breathing. Note that within the ultrafine particle size
range (<100 nm) there are significant differences in each of the
3 regions with regard to their deposition probabilities.
(ICRP, 1994). Thus, each of the three regions of the respiratory
tract is targeted differently by a given size of UFP (Figure 1).
Their fate after deposition seems to differ from that of larger
particles, at least as far as solid or poorly soluble UFP are con-
cerned. We have found that within 4 h after whole-body inhala-
tion exposure of rats, ultrafine polytetrafluoroethylene (PTFE)
particles (CMD ∼ 20 nm) had translocated to submucosal sites
of the conducting airways and to the pulmonary interstitium of
the alveolar region (Oberd¨orster et al., 2000). Other rat inhala-
tion studies have shown that ultrafine elemental
13
C particles
(CMD ∼30 nm) had accumulated to a large degree in the liver
of rats by 24 h after exposure, indicating efficient translocation
into the blood circulation (Oberd¨orster et al., 2002). Suggested
pathways into the blood could be across the alveolar epithelium
as well as across intestinal epithelium from particles swallowed
into the gastrointestinal (GI) tract. On the other hand, using a
method of intratracheal inhalation of ultrafine
192
Ir particles in
rats, Kreyling et al. (2002) found only minimal translocation
(<1%) from the lung to extrapulmonary organs, although they
reported 10-fold higher translocation of smaller (15 nm) com-
pared to larger (80 nm) UFP. Nemmar et al. (2002) reported
findings in humans that inhalation of
99m
Tc-labeled ultrafine
carbon particles (Technegas) resulted in the rapid appearance
of the label in the blood circulation shortly after exposure and
also in the liver. They suggested that this at least partly indi-
cated translocation of inhaled UFP into the blood circulation.
In contrast, other studies in humans with
99m
Tc-labeled car-
bon particles (33 nm) by Brown et al. (2002) did not confirm
such uptake into the liver, and the authors cautioned that the
findings by Nemmar et al. (2002) were likely due to soluble
pertechnetate rather than labeled UFP.
It appears, therefore, that UFP size and chemistry (e.g., car-
bon vs. metal) are important determinants for extrapulmonary
translocation of UFP. We had observed in an earlier pilot study
with inhaled ultrafine
13
C particles a significant increase of
13
C in the olfactory bulb of rats, which led us to hypothesize
that routes for extrapulmonary translocation of solid UPF other
than via the blood circulation exist, that is, involving neuronal
pathways from deposits on the nasal olfactory mucosa via the
olfactory nerve. We decided, therefore, to conduct a more de-
tailed study in rats with a follow-up period of 7 days after UFP
inhalation for measuring their retention in lung and different
brain sections. We report here the significant accumulation of
13
C in the olfactory bulb over the 7-day postexposure period,
suggesting that a most likely pathway of translocation of solid
ULTRAFINE PARTICLE TRANSLOCATION TO THE BRAIN
439
UFP from the upper respiratory tract is indeed via the olfactory
nerve.
METHODS
Generation of Particles
Ultrafine
13
C particles were generated from pure [
13
C]
graphite electrodes placed in an electric spark discharge gen-
erator supplied with argon in the discharge chamber (Palas
soot generator, Karlsruhe, Germany). The [
13
C]graphite elec-
trodes were made by extruding a slurry of amorphous
13
Cpow-
der (Isotec, Inc., Miamisburg, OH) mixed with [
13
C
6
]glucose
(Isotec, Inc., Miamisburg, OH) through a syringe to produce
3.5-mm-diameter cylinders. These were baked in an argon at-
mosphere by slowly ramping up the temperature to 200
◦
C
within 1.5 h in order to degas the extrusion and decompose
the glucose. The electrodes were subsequently graphitized at
2400
◦
C in argon.
13
C Measurement
Isotopic measurements were performed by continuous-flow
isotope ratio mass spectrometry (Brand, 1996), using a Carlo
Erba elemental analyzer coupled to a Finnigan Mat Delta Plus
mass spectrometer. The results are reported using the conven-
tional δ notation, relative to the PDB standard (fossilized calcite
standard), expressed as per mille change:
δ-
13
C =
13
C/
12
C
sample
−
13
C/
12
C
standard
13
C/
12
C
standard
× 1000 [1]
where
13
C/
12
C
standard
is the ratio of the reference material (PDB)
and
13
C/
12
C
sample
is the ratio of the sample. Reproducibility
was better than 0.1‰ for both standards and samples, which
translates into a detection limit of about 1 ppm of added
13
C
(the
13
C/
12
C ratio for PDB is 0.0112372). Since mammalian
tissues have a lower
13
C/
12
C ratio than the reference carbonate,
the δ-
13
C values are negative.
Animal Exposure and Particle Characterization
Male Fischer-344 rats, 14 wk old, at a body weight of 284 ±
9 g, were used for the study. The exposure was for6hin
compartmentalized whole-body exposure chambers and was
performed in 2 sessions. Particle number concentration in the
inhalation chamber was measured by a condensation nuclei
counter (TSI model 3022A), and ultrafine particle size distri-
bution by a scanning mobility particle sizer (SMPS, TSI model
3080). Particle mass concentration was determined by an ambi-
ent particulate monitor (TEOM, model series 1400a, Rupprecht
and Patashnik, Albany, NY). Six rats were exposed in the first
session at a concentration of 170 µg ultrafine
13
C particles/m
3
(CMD = 37 nm; GSD [geometric standard deviation] = 1.66)
and were sacrificed on days 1 and 3 postexposure. An additional
6 rats were exposed in the second session at a concentration of
150 µg ultrafine
13
C particles/m
3
(CMD = 35 nm; GSD = 1.66)
and were sacrificed on days 5 and 7 postexposure, using 3 rats
per time point. Three unexposed rats served as controls.
Organ Preparation and
13
C Analysis
At 1, 3, 5, and 7 days postexposure, rats were sacrificed and
lung, olfactory bulb, cerebrum, and cerebellum were removed
for
13
C analysis. The excised organs were weighed. Great care
was taken to avoid contamination of the organs with
13
C that
had deposited on the fur of the animals. To achieve this, the ani-
mals were killed by an overdose of intraperitoneal pentobarbital
and the fur was wet-wiped with clean tissues. Subsequently the
animals were completely skinned and the carcasses were rinsed
under water. The carcasses were then moved to a separate clean
room and organs were excised there with different clean sets of
instruments. The cranium was opened and the cerebrum, cere-
bellum, and olfactory bulbs were carefully removed. Each of
the organs was homogenized with different clean homogenizers
and subsequently lyophilized. Unexposed control animals were
treated the same way; they were killed prior to sacrificing the
day 1 exposed rats. Two 1-mg aliquots of each organ sample
were used for δ-
13
C value determinations using continuous-
flow mass spectroscopy for 3–6 replicate measurements. Re-
sults were subjected to a weighted two-way analysis of variance
(ANOVA), with significant increases in δ-
13
C values being de-
fined when p <.05. The two factors in the ANOVA were expo-
sure group and time. The observations were means of replicate
measurements, and the number of replications was used as the
weight.
The difference in δ-
13
C between organs of exposed rats and
the average of respective organs of control rats was used to
calculate the added
13
C organ burden. In order to translate δ-
13
C
values into absolute amounts of
13
C added to each organ (excess
13
C), the baseline carbon content—that is, the sum of
12
C and
13
C—of the different organs needs to be known. Such baseline
levels are summarized for humans as Reference Man values by
ICRP (1992). For example, lung total carbon content is 10% of
lung weight, and an average value for brain total carbon content
is 12.2% of brain weight. Assuming that the carbon content
of organs in rats and humans is the same or very similar, the
baseline
13
C can be determined using the Reference Man data.
These baseline values were used to quantify the added
13
Cin
exposed compared to control animals.
RESULTS
Table 1 shows the results of the δ-
13
C measurements in
lungs and the different sections of the brain of control rats,
and of exposed rats on days 1, 3, 5, and 7 postexposure. The
lungs display the expected significant increase in δ-
13
C from
−20.28 in controls to −19.04 on day 1. A more than 50% de-
crease on subsequent days to −19.75 by day 7 follows, still
significantly above controls. δ-
13
C for the olfactory bulb was
also significantly elevated on all postexposure days, from a
baseline value of −18.91 (controls) to −18.66 (day 1) and
to −18.60 (day 7), reflecting the largest increase on day 1
440 G. OBERD
¨
ORSTER ET AL.
TABLE 1
δ-
13
C Values and excess
13
C levels of lung and brain tissues following6hofultrafine
13
C particle exposure (mean ± SD, n = 3
per time point)
Postexposure
Unexposed control;
day 0 Day 1 Day 3 Day 5 Day 7
Lung
δ-
13
C −20.28 ± 0.27 −19.04 ± 0.23
a
−19.14 ± 0.24
a
−19.54 ± 0.14
a
−19.75 ± 0.14
a
µg 0.01 ± 0.3 1.34 ± 0.22 1.40 ± 0.31 0.80 ± 0.17 0.64 ± 0.16
µg/g 0.0 ± 0.3 1.39 ± 0.26 1.28 ± 0.27 0.83 ± 0.16 0.59 ± 0.15
Olfactory bulb
δ-
13
C −18.91 ± 0.09 −18.66 ± 0.12
a
−18.66 ± 0.01
a
−18.64 ± 0.06
a
−18.60 ± 0.06
a
µg 0.00 ± 0.01 0.03 ± 0.02 0.03 ± 0.00 0.03 ± 0.01 0.04 ± 0.01
µg/g 0.01 ± 0.12 0.35 ± 0.17 0.35 ± 0.02 0.37 ± 0.08 0.43 ± 0.08
Cerebrum
δ-
13
C −19.24 ± 0.04 −19.04 ± 0.02
a
−19.13 ± 0.09 −19.16 ± 0.08 −19.07 ± 0.01
a
µg 0.00 ± 0.07 0.32 ± 0.02 0.19 ± 0.16 0.14 ± 0.13 0.29 ± 0.02
µg/g 0.00 ± 0.05 0.27 ± 0.03 0.15 ± 0.13 0.11 ± 0.11 0.23 ± 0.02
Cerebellum
δ-
13
C −19.46 ± 0.08 −19.14 ± 0.15
a
−19.32 ± 0.07 −19.19 ± 0.10
a
−19.31 ± 0.14
µg 0.00 ± 0.03 0.12 ± 0.04 0.06 ± 0.03 0.10 ± 0.03 0.06 ± 0.06
µg/g 0.00 ± 0.10 0.44 ± 0.20 0.19 ± 0.09 0.37 ± 0.13 0.21 ± 0.19
a
Significantly increased compared to controls, p <.05 (ANOVA).
and continuous slight increases thereafter. Cerebrum and cere-
bellum also showed increases of δ-
13
C postexposure, which,
however, was not consistent, and significantly elevated only
on day 1 (both regions), day 5 (cerebellum), and day 7 (cere-
brum). The higher baseline values for δ-
13
C of the brain re-
gions compared to those in the lungs reflect the greater carbon
content of brain tissues compared to that of the lung (ICRP,
1992).
Table 1 and Figure 2 show the results of excess
13
C in lung,
olfactory bulb, cerebrum, and cerebellum derived from the re-
spective δ-
13
C values. The day following the 6-h
13
C inhala-
tion exposure, an added lung concentration of 1.39 ± 0.26 µg
13
C/g lung was determined. By day 7, this concentration had
decreased to 0.59 ± 0.15 µg added
13
C/g lung. Added
13
C con-
centrations in the olfactory bulb were significantly increased
throughout the postexposure period, increasing to 0.35 ±
0.17 µg/g tissue on day 1 and to 0.43 ± 0.08 µg/g tissue by
day 7. Added
13
C concentrations in cerebellum and cerebrum
were significantly elevated only on 2 of the postexposure days,
reaching concentrations between 0.11 and 0.44 µg/g tissue.
Although by day 7 the olfactory bulb concentration of
13
C ap-
proached day 7
13
C concentrations in the lung, the total amount
accumulating in the olfactory bulb is only small; considering
its average weight of only 85 mg, the amount accumulating
in the olfactory bulb over 7 days was 40 ng (Table 1). This,
however, was achieved after a single exposure only, and since
no decrease in
13
C during the 7 days postexposure occurred,
continuous exposures are likely to reach much higher levels.
Due to the greater weight of cerebrum and cerebellum, their
total
13
C content on day 1 postexposure was calculated to be
10- and 4-fold higher compared to the olfactory bulb (Table 1).
However, this elevation was not consistently significant over the
FIG. 2. Time course of
13
C tissue concentrations in lung, ol-
factory bulb, cerebrum, and cerebellum of rats following a 6-h
inhalation exposure to ultrafine (36 nm CMD) elemental
13
C
particles (n = 3 rats per time point): ––lung;—olfactory—
–—–cerebellum; ··· cerebrum. Asterisk and a, b, c indicate
values significantly greater than controls, p <.05 (ANOVA)
(asterisk for lung, a for olfactory bulb, b for cerebellum, c for
cerebrum).
ULTRAFINE PARTICLE TRANSLOCATION TO THE BRAIN
441
following days; it may well reflect some localized higher accu-
mulation, which needs to be further investigated in subsequent
studies.
DISCUSSION
We found significant and continuous increases of
13
C in the
olfactory bulb throughout the 7-day postexposure period fol-
lowing the 6-h inhalation exposure to ultrafine elemental
13
C
particles with a CMD of 36 nm and GSD of 1.66. Cerebrum
and cerebellum also showed significant increases on 2 of the
postexposure days. Since elemental carbon is insoluble (CRC
Press, 2000), these changes in
13
C concentration reflect the ac-
cumulation and retention of the ultrafine carbon particles in the
respective organs. Translocation of the inhaled ultrafine carbon
particles to the CNS could occur principally via two pathway:
One is through access across the blood–brain barrier (BBB) of
ultrafine particles after their translocation into the blood circula-
tion from deposits anywhere in the respiratory tract; the other—
specific for the olfactory bulb—is via the olfactory nerve from
deposits on the nasal olfactory mucosa the third pathway is via
paracellular or perineural pathways across the olfactory mu-
cosa and ethmoid bone into cerebrospinal fluid (CSF) (Illum,
2000). Although our present study was not designed to differ-
entiate between these three pathways, we offer the following
interpretation of our results based on available literature data.
Supportive of a circulatory route of UFP translocation from
deposits in the respiratory tract are results of our previous study
(Oberd¨orster et al., 2002). Significant amounts of ultrafine
13
C
accumulated in the liver by 24 h postexposure, which must be
a consequence of the ultrafine carbon particles having translo-
cated into the blood circulation. Against the circulatory route
of CNS translocation of UFP speaks the fact that the BBB is
very tight, and studies with iv-injected UFP failed to demon-
strate their access into the CNS across this barrier (Gulyaev
et al., 1999) or across the tight distal barrier of the basal lam-
ina (Muldoon et al., 1999). Only after coating intravenously
injected nanoparticles with polysorbate or apolipoprotein did
they seem to mimic lipoprotein particles that could be taken
up by brain capillary endothelial cells via receptor-mediated
phagocytosis (Kreuter et al., 2002). Yet even then, transcyto-
sis of the nanoparticles across the BBB could not be demon-
strated. Particles translocating along paracellular or perineural
pathways into CSF would have to cross the CSF-brain barrier,
another functional barrier, and would not target specifically the
olfactory bulb. Thus, the consistent postexposure increase of
13
C in the olfactory bulb is unlikely to be of blood-borne CSF
origin. It is conceivable, though, that the increase in
13
C in cere-
brum and cerebellum in our study reflects some translocation
of solid UFP across the BBB in regions where epithelia are less
tight, such as the circumventricular organs, or across the blood-
CSF barrier, such as the choroid plexus, ventricles, brainstem
centers, and hypothalamus (Segal, 2000; Ueno et al., 2000).
Possible translocation from the blood circulation in these areas
may be associated with a very rapid clearance from the lung
early during the exposure of more than 50% of the UFP de-
positing in the lung. Such early rapid clearance was suggested
by the results of our previous study (Oberd¨orster et al., 2002)
based on the difference between measured and predicted lung
burden. The present study, too, shows a large difference be-
tween the predicted deposition of
13
C in the lung (2.93 µg)
∗
and what was actually measured on day 1 (1.34 µg; Table 1),
which suggests, assuming that the model is correct, more than
50% elimination of pulmonary
13
C during the exposure phase.
If most of this is translocated into the blood circulation, some
transport across the less developed sections of the BBB con-
ceivably may occur. This could then account for the increased
13
C levels in cerebrum and cerebellum on day 1 postexposure.
However, since only the olfactory bulbs showed consistent and
increasing
13
C levels on all days in the 7-day postexposure pe-
riod, and cerebellum and cerebrum only on 1 day, we believe
that at least for the olfactory bulb, axonal transport of UFP via
olfactory neurons from nasal deposits into the CNS is the major
translocation mechanism.
The existence of an olfactory nerve pathway as access to
the CNS has been well demonstrated for inhaled or nasally in-
stilled soluble metal compounds (Tj¨alve & Henriksson, 1999;
Arvidson, 1994; Dorman et al., 2002). Since inhalation expo-
sure of UFP results in significant deposition in the nasal area
of the rat, a reasonable interpretation of our result is that in-
haled solid UFP translocate via this route as well. In order to
prove that nasal deposits of UFP translocate to the olfactory
bulb, it would be most convincing to demonstrate the presence
of the ultrafine
13
C particles in the axons of the olfactory neu-
rons. While our present study was limited in its design and
did not include dissection and analysis of olfactory nerves, the
older literature provides convincing evidence for such neuronal
translocation of solid UFP following intranasal instillations in
nonhuman primates and in rats.
De Lorenzo (1970) in a landmark study demonstrated in
squirrel monkeys that intranasally instilled colloidal gold par-
ticles (50 nm) translocated anterogradely in the axons of the
olfactory nerves to the olfactory bulbs. He used transmission
electron microscopy to image the sequence of the passage of
the 50-nm electron-dense particles along the whole pathway:
starting with their localization postinstillation on the olfactory
mucosa, followed by uptake into the olfactory rods of the nasal
mucosa (probably via endocytosis as evidenced by large num-
bers of pinocytotic vacuoles, which led the author to conclude
that “the olfactory rod is busily engaged in “drinking in” from
the extracellular environment”), retrograde translocation within
the olfactory dendrites likely associated with microtubuli,
anterograde movement in the axoplasm along the olfactory fila
of the olfactory nerve, appearance in the olfactory bulb, and then
even showing the 50 nm gold particles having crossed synapses
∗
This prediction is based on the multiple path particle deposition model
(Asgharian et al., 1999) with 0.55 µg
13
C-UFP depositing in the tracheo-
bronchial region and 2.38 µg in the alveolar region of the rats during the
6-h exposure.
442 G. OBERD
¨
ORSTER ET AL.
in the olfactory glomerulus to reach mitral cell dendrites within
1 h after intranasal instillation. An interesting finding in this
study—and important for potential adverse effects—was that
the UFP in the olfactory bulb were no longer freely distributed in
the cytoplasm but were preferentially located in mitochondria.
Remarkable also is the neuronal transport velocity of the 50 nm
gold particles, calculated by De Lorenzo to be 2.5 mm/h.
Other studies on olfactory translocation of solid UFP were
reported in the 1940s and concerned a large series of studies
with 30 nm polio virus intranasally instilled into chimpanzees
and Rhesus monkeys (Bodian & Howe, 1941; Howe & Bodian,
1941). Their studies revealed that the olfactory nerve and ol-
factory bulbs are, indeed, portals of entry to the CNS for the
intranasally-instilled ultrafine polio virus particles, which could
subsequently be recovered from the olfactory bulbs. The trans-
port velocity of the virus in the axoplasm of axons was deter-
mined at 2.4 mm/h, which is very well in agreement with De
Lorenzo’s result, and also with neuronal transport velocities
measured later by Adams and Bray (1983) for solid particles
(up to 500 nm) directly microinjected into giant axons of crabs.
There are additional studies showing that sensory nerves
in the upper and lower respiratory tract can take up solid mi-
cro particles and translocate these. For example, a more re-
cent study by Hunter and Dey (1998) in rats demonstrated the
translocation of intranasally instilled rhodamine-labeled micro-
spheres (20–200 nm) to the trigeminal ganglion inside the cra-
nium via uptake into the ophthalmic and maxillary neurons of
the trigeminus nerve. The trigeminus supplies sensory nerve
endings throughout the nasal mucosa, including the olfactory
mucosa (Shankland, 2001; Schaefer et al., 2002). Hunter and
Undem (1999) in a different study instilled the same micropar-
ticles intratracheally into guinea pigs. They found that sensory
nerves supplying the tracheobronchial region with a dense net-
work can take up and translocate these solid microparticles to
the ganglion nodosum in the neck area, which is networked into
the vagal system. Their method of tracing neuronal pathways
had been developed by Katz et al. (1984), who had shown that
these rhodamine-labeled microparticles (30 nm may be the op-
timal size; Katz, personal communication) translocated along
axons and dendrites when microinjected into cortical sections
of rats and cats.
Collectively, these studies show that solid UFP of different
materials can effectively be taken up by sensory nerve endings
at several sites in the respiratory tract and gain access to the
CNS and ganglionic structures. In particular, the studies by De
Lorenzo (1970) demonstrate unequivocally the existence of a
neuronal olfactory pathway to the CNS for nasally deposited
solid UFP. Since inhaled UFP are deposited to a significant
degree in the nasal region of the respiratory tract of the rat
(Gerde et al., 1991), a most likely mechanism to explain the
13
C accumulation in the olfactory bulb of rats in our inhalation
study is via the olfactory neuronal route. Possibly, differences
in the physicochemical nature of UFP may influence such
translocation.
In an attempt to quantify the translocation of
13
C particles
along this pathway in our study we considered the following:
Nasal deposition studies of ultrafine particles in rats showed that
the deposition efficiency for 42-nm particles is 10.5% (Gerde
et al., 1991). The CMD of the ultrafine
13
C particles in our
study was 36 nm, with a geometric standard deviation of 1.66.
A recently developed particle deposition model (MPPD, multi-
ple path particle deposition; Asgharian et al., 1999) predicts for
the rat that at this particle size distribution 9.4% of the inhaled
particles will deposit in the nasal compartment, including the
nasal olfactory mucosa close to the measured value by Gerde
et al. (1991). Under our specific study conditions, this amounts
to 1.10 µg of ultrafine
13
C particles, which is almost as much
as was measured in the lung. Since only 15% of the total air-
flow is directed to the olfactory mucosa in rats (Kimbell et al.,
1997), the predicted deposited amount of
13
C in this area may
only be 165 ng, 15% of the total nasal deposit. Assuming that
the amount of
13
C measured in the olfactory bulb (30–40 ng,
Table 1) was translocated along the olfactory neuronal route,
this would mean that ∼20% of the deposited amount on the ol-
factory mucosa was translocated to the olfactory bulb over the
7-day postexposure period. There are probably significant un-
certainties around this estimate and therefore it has to be viewed
with caution. Additional studies using more sensitive methods
of detection are required to verify translocation pathways and
more precisely quantitate neuronal translocation efficiency of
inhaled UFP.
Considering that only 5% of the human nasal mucosa is ol-
factory epithelium as opposed to 50% in rats (Keyhani et al.,
1997; Kimbell et al., 1997), one can question the importance of
olfactory nerve translocation for UFP in humans. In addition,
rats are obligatory nose breathers, whereas humans are mixed
oro-nasal/nasal breathers. However, although only 15% of the
total rat nasal airflow is directed to their olfactory mucosa, it is
10% in humans (Keyhani et al., 1997; Kimbell et al., 1997), only
one-third less. Moreover, as pointed out before, the rest of the
nasal mucosa is supplied by sensory nerve endings of the oph-
thalmic and maxillary branches of the trigeminus nerve, which
can also function as a translocation pathway for solid UFP as
discussed earlier (Hunter & Dey, 1998). Some of the trigem-
inal sensory nerve endings in the nasal epithelium also have
branches reaching directly into the olfactory bulb (Schaefer
et al., 2002).
Classical clearance mechanisms for inhaled particles de-
posited in the nasal compartment of the respiratory tract are
via mucociliary transport (toward the oropharynx in the poste-
rior part of the nose, and through sneezing and nose blowing.
Biosoluble components of particles deposited in this region are
subjected to dissolution and subsequent absorption into blood
(U.S. EPA, 1996; Schlesinger et al., 1997). Uptake by sensory
nerve endings (olfactory, trigeminus), which has been the focus
of this discussion, constitutes a clearance pathway to extrapul-
monary organs, that is, the CNS, which has not been appropri-
ately recognized so far, despite several recent reports describing
ULTRAFINE PARTICLE TRANSLOCATION TO THE BRAIN
443
the passage of metals along this route after inhalation or nasal
instillation of biosoluble metal compounds (Gianutsos et al.,
1997; Tj¨alve & Henriksson, 1999; Brenneman et al., 2000;
Dorman et al., 2002). Dorman et al. (2002) pointed out most
recently the potential importance of this pathway for brain deliv-
ery of Mn derived from soluble and slowly soluble inhaled Mn
compounds. That this delivery route exists as well for solid UFP
is clearly demonstrated by the earlier work on olfactory uptake
and transport of 50-nm gold particles and 30-nm virus parti-
cles described above (Bodian & Howe, 1941; Howe & Bodian,
1941; De Lorenzo, 1970); also, our present result with 36-nm
insoluble elemental carbon particles is consistent with this same
translocation mechanism and extends the earlier findings from
nasally instilled to inhaled UFP. Thus, the olfactory route to
the CNS and uptake into other sensory nerve endings in the ex-
trathoracic and tracheobronchial region should be recognized as
mechanisms for extrapulmonary translocation of soluble metal
compounds and UFP (Figure 3), in addition to the “classical”
clearance mechanism in the nasal area already mentioned.
Neuronal uptake and translocation in the respiratory tract
could be especially important for the smallest UFP below 10–
20 nm generated at very high number concentrations in the
FIG. 3. Suggested neuronal translocation pathways in hu-
mans for solid nanosized particles and for soluble components
of larger particles that have been demonstrated in rodents and
nonhuman primates. These include uptake into nerve endings
embedded in mucosa of nasal (a, olfactory; b, trigeminal nerves)
and tracheobronchial (c, afferent vagal nerves) region. The bi-
ological/toxicological importance of these pathways and their
contribution to particle clearance vis-`a-vis the classical clear-
ance pathways of mucociliary and phagocytic cell transport,
dissolution, diffusion, and protein binding remain to be deter-
mined. Drawing courtesy of Dr. J. Harkema.
ambient air from traffic-related sources and photochemical nu-
cleation events (Kittelson, 1998; McMurry et al., 2000; Woo
et al., 2001), or for newly developed nanomaterials (engineered
nanoparticles of carbon, metals, metal oxides, quantum dots
etc.) should they become airborne and be inhaled. The smaller
the inhaled UFP, the more of them could be subjected to a neu-
ronal clearance pathway in the nose, because nasal deposition
increases with decreasing UFP size. Below a size of 5 nm, nasal
deposition efficiency rises rapidly and eventually approaches
100%, whereas particles above 30 nm have only a less than 10%
nasal deposition probability (Figure 1); (ICRP, 1994; Cheng
et al., 1996; Swift et al., 1992). Since the smaller ambient
UFP consist of very little elemental but mostly organic car-
bon (Kittelson, 1998), their fate after deposition depends most
likely on their lipid or water dissolution rate; that is, how long
after deposition will these particles maintain their particulate
state: are they dissolving quickly before translocation, during
translocation, or once they have reached CNS structures? Even
if dissolution occurs during the translocation process, antero-
grade translocation and retrograde translocation of lipids and
organic material along axons of neurons have been shown to
take place as well (Grafstein & Foreman, 1980).
While the early studies—showing nasal to olfactory bulb
translocation of 30-nm virus and 50-nm gold particles in non-
human primates—prove that this transfer mechanism operates
in primates, the significance for humans still needs to be es-
tablished. In general, there are many unanswered questions re-
garding sensory nerve uptake and transport of inhaled UFP de-
posited on the mucosa of the olfactory nasal region and at other
sites of the respiratory tract, including: What is the mechanism
of uptake: receptor-mediated endocytosis, pinocytosis, axonal
transport? How do physicochemical characteristics of UFP and
their surfaces influence uptake and translocation? How far into
the CNS—beyond he olfactory bulb to striatal, frontal cortex,
and other structures—can UFP be translocated? To what extent
is this pathway operable for volatile and semivolatile organics
as major constituents of ambient UFP below ∼20 nm? Are there
toxicological consequences? A recent study described signifi-
cant inflammatory changes of the olfactory mucosa, olfactory
bulb, and cortical and subcortical structures in dogs from a heav-
ily polluted area in Mexico City, changes that were not seen in
dogs from a less polluted control city (Calderon-Garciduenas
et al., 2002). While this study was not designed to investigate a
nasal-CNS transfer route of inhaled ultrafine particulate air pol-
lutants, the observed inflammatory changes are consistent with
such mechanism. Is UFP uptake limited to particles <100 nm,
or does this mechanism also operate for larger particles? Ax-
onal diameter certainly will impose a limit—for example, ol-
factory fila in the squirrel monkey measure 200 nm in diameter
(DeLorenzo, 1970). Anterograde and retrograde transport of
even larger particles directly injected into a giant axon has been
observed (Adams & Bray, 1983), with one suggested mecha-
nism being cytoskeletal movement along an escalator provided
by axonal and dendritic microtubuli (Hirokawa, 1998; Ligon
444 G. OBERD
¨
ORSTER ET AL.
& Steward, 2000) via kinesin and cytoplasmic dynein motor
proteins (Vale et al., 1985; Paschal et al., 1987). Protein coat-
ing of UFP after deposition may be necessary for this neuronal
translocation pathway. In addition, the vascular route of translo-
cation, that is, UFP crossing the BBB after entering the vascular
compartment, needs to be explored as well. Answers to these
questions of are interest not only for ambient UFP but also for
the growing field of biomedical use of engineered nanoparticles
and quantum dots with respect to their kinetics when adminis-
tered in vivo as drug delivery devices or as biosensors.
We conclude from our studies that inhaled ultrafine carbon
particles are to a significant extent translocated to the CNS.
Our findings are consistent with retrograde dendritic and an-
terograde axonal transport via the olfactory nerve, a pathway
whose existence in non-human primates and in rodents has been
proven in earlier nasal instillation studies for solid UFP as well
as for soluble metal compounds. This constitutes a direct portal
of entry for UFP into the CNS, circumventing the tight blood–
brain barrier. This not generally recognized clearance pathway
from the nasal mucosa to the CNS could be of significance
for induction of neurotoxic effects following acute or chronic
inhalation exposures to environmental or occupational UFP. Po-
tential long-term effects of their accumulation in the olfactory
bulb and translocation to other regions of the CNS represent a
new field in PM research.
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