FUNDAMENTAL AND APPLIED TOXICOLOGY 4 0 , 2 2 0 - 2 2 7 (1997)
ARTICLE NO. FA972390
Intratracheal Inhalation vs Intratracheal Instillation:
Differences in Particle Effects
M. Osier and G. Oberdorster
Department of Environmental Medicine, University of Rochester School of Medicine, 575 Elmwood Avenue, Box EHSC, Rochester, New York 14642
Received April 1, 1997; accepted October 9, 1997
strated an increased particle deposition in the basal regions
of the lung following instillation. Pritchard et al. (1985) and
Domes et al. (1992) have both demonstrated a significantly
less homogeneous particle distribution of a single instilled
Our laboratory has developed a method of intratracheal inhala- dose, when compared to an inhalation exposure. Ferin and
tion whereby rats can be exposed to high aerosol concentrations, Feldstein (1978) showed an increased clearance to the hilar
resulting in high lung particle burdens in a short time period with lymph nodes following instillation. Also, Henderson et al.
deposition occurring directly in the lower respiratory tract, thus
(1995) showed different patterns of injury in rats following
avoiding many drawbacks of larger nose-only or whole body inhainstillation
and inhalation exposures to a-quartz, with interlation systems. In this report, we compare the response of rats
stitial
granulomas
being seen only in those animals exposed
exposed by intratracheal inhalation to "fine" (~250 nm) and "ulby
inhalation.
The
same study also suggested that the use
trafine" (~21 nm) titanium dioxide particles with rats exposed
to similar doses by intratracheal instillation. Animals receiving of a vehicle, as is required for intratracheal instillation, may
particles through inhalation showed a decreased pulmonary re- alter the study results or sensitivity. Vogel et al. (1996) have
sponse, measured by bronchoalveolar lavage parameters, in both also shown instillation to be a less preferable method for
severity and persistence, when compared with those receiving par- the administration of therapeutic agents to the respiratory
ticles through instillation. These results demonstrate a difference tract, with an instilled dose of antibodies less effectively
in pulmonary response to an inhaled vs an instilled dose, which
ameliorating ricin toxicity than an inhaled dose.
may be due to differences in dose rate, particle distribution, or
While providing a more homogeneous dose as well as a
altered clearance between the two methods, c 1997 society of Toxicology.
more physiological method of exposure, inhalation exposure
methodologies are not without their disadvantages. First,
Intratracheal instillation is used frequently for the expo- they tend to involve large pieces of equipment, particularly
sure of animals to particles, both soluble and insoluble. It is the exposure chambers themselves, which makes cleaning
a relatively inexpensive method of administration that allows and decontamination involved and costly. These larger sysfor the instantaneous delivery of a known amount of test tems also generally require significantly more test material
material, suspended in a small volume of vehicle, directly than instillation, adding to costs of performing a study. Expoto the lung, or even to a single lobe within the lung. Because sures using standard inhalation exposure systems also do not
it administers the material directly to the lower respiratory bypass upper respiratory defenses, as is done in instillation,
tract, it avoids deposition in the nasal passages and/or on resulting in large amounts of material deposited in the nasal
the fur as can occur in nose-only or whole-body inhalation passages and, in whole-body inhalation systems, on the fur.
exposure systems, thus making it very useful for studying Thus ingestion, due to upper respiratory clearance or matethe effects of test materials on the lower respiratory tract. rial ingested from skin and fur, particularly in the case of a
This method is particularly well-suited for the administration whole-body exposure, can become a serious consideration
of highly toxic or radioactive materials, as the dose is con- when interpreting effects, and a confounding factor when
fined to a small volume, eliminating the need for decontami- attempting to study direct effects on the lung. Additionally,
nation of large pieces of equipment.
some larger particles may be respirable in humans but not
However, intratracheal instillation is not without its disad- in rodents (Raabe et al., 1988; Technical Committee, ISS,
vantages. Instillation is not a physiological route for human SOT, 1992), making their study in a rodent inhalation model
exposure. Additionally, several investigators have shown difficult.
discrepancies, including differences in particle distribution,
We have developed a method of intratracheal inhalation
clearance, and pattern of injury, between inhalation and in- (Oberdorster et al., 1997), whereby rats are ventilated at a
stillation routes of exposure. Brain et al. (1976) demon- constant rate with an aerosol of the test material. It has the
Intratracheal Inhalation vs Intratracheal Instillation: Differences in Particle Effects. Osier, M., and Oberdorster, G. (1997)
Fundam. Appl. Toxicol. 40, 220-227.
0272-0590/97 $25.00
Copyright B 1997 by the Society of Toxicology.
All rights of reproduction in any form reserved.
220
INTRATRACHEAL INHALATION VS INSTILLATION
advantages of an intratracheal administration, such as a direct deposition of particles into the lower respiratory tract,
bypassing upper respiratory defenses, but is an inhalation
method, and would be expected to deposit particles in a
pattern similar to other inhalation exposure methods. Additionally, it consists of a closed system, allowing for the
filtration and removal of potentially hazardous or radioactive
particles prior to exhaust, thus making decontamination procedures significantly simpler and faster than those for standard inhalation systems.
A transoral/intratracheal inhalation method has been reported previously (Drew et al, 1987). In this system, anesthetized rats were intubated intratracheally and exposed to
an aerosol of fibers. Unlike our system, the animals were
allowed to breathe normally. As anesthetized rats tend to
breathe more shallowly, which would be expected to decrease deep lung deposition, we chose to ventilate our animals. This gave us greater control of the breathing patterns
of the animals during exposure, allowing us to maximize
deposition in the lower respiratory tract. Additionally, the
system reported by Drew et al was not a closed system,
and was thus lacking the advantages mentioned above.
Several reports (Ferin and Feldstein, 1978; Henderson et
al, 1995; Ferin et al, 1994; Hirano et al, 1994; Ritz et al,
1993; Oberdorster et al, 1980) have compared the effects
of test compounds administered by intratracheal instillation
and other inhalation systems. Ferin and co-workers (Ferin
and Feldstein, 1978; Ferin et al, 1994) reported increases
in unlavagable lung content and clearance to the hilar lymph
nodes following an instilled dose, when compared to an
inhalation exposure. Additionally, Henderson et al. (1995)
showed differences in the pattern of a-quartz histopathology
following instillation or 4-week inhalation, with interstitial
granulomas being seen only in the inhalation-exposed animals. However, more soluble particles, like nickel sulfate
or enzymatic detergents, appear to be less affected by the
exposure method (Hirano et al, 1994; Ritz et al, 1993;
Oberdorster et al, 1980).
Titanium dioxide has been used in the past by many investigators as a negative control particle due to its low reactivity.
For example, Henderson et al (1995) reported no significantly different responses from controls in rats receiving
TiO2 by single intratracheal instillation or 4-week inhalation.
Other studies (Driscoll et al, 1990a,b; Lee et al, 1986)
found significant changes, including increases in lung
weight, inflammation, fibronectin production, and tumor formation, as a result of TiO 2 exposure, but only at extremely
high lung particle burdens, where prolonged particle clearance due to overload would be expected to occur. Still others
have shown that TiO2 particles with a very small mean primary particle size (~21 nm diameter), referred to as "ultrafine" particles, are much more reactive, at similar mass
doses, than the larger-sized TiO2 particles, showing altered
221
BAL parameters, including increases in total cells, PMN
numbers, and total protein (Oberdorster et al, 1990), as well
as increased interstitialization (Ferin et al, 1992), alveolar
macrophage cytokine release (Driscoll and Maurer, 1991),
fibrosis, and tumor formation (Heinrich et al, 1995) when
compared to the larger-sized ("fine") particles.
The primary objective of these studies was to compare
the response of animals to particles administered by the two
exposure methods, with a secondary goal of comparing the
responses of animals exposed to fine and ultrafine TiO 2 . We
hypothesized that animals exposed to TiO2 particles by the
different exposure methods of intratracheal instillation and
intratracheal inhalation would show differences in their pulmonary responses.
MATERIALS AND METHODS
Animals.
Specific pathogen-free male Fisher 344 rats weighing 175—
225 g were group-housed in wire-bottom cages. Animals were fed Purina
Laboratory Chow and water ad libitum. Groups of six animals were used
for each exposure time point.
Selection of particles. For this study, two types of titanium dioxide
were used. The first had a mean primary particle size of 0.25 /im, and will
be referred to as fine TiO2 (Fisher Scientific, Springfield, NJ). The second
had a mean primary particle size of ~0.021 fim and will be referred to as
ultrafine TiO2 (Degussa A. G., Frankfurt am Main, Germany). Both particle
types were of the anatase crystalline form.
Intratracheal inhalation exposures. Intratracheal inhalation exposures
were performed as previously reported by our laboratory (OberdOrster el
al., 1997). Briefly, animals were anesthetized with halothane and intubated
intratracheally with a modified 14-gauge cannula. The open end of the
cannula was attached to a port on the inhalation system (see Fig. 1) such
that an air-tight seal was formed. The animals were then ventilated at a
frequency of 30 breaths per minute (1.5 s inhalation, 0.5 s exhalation) at a
maximum inhalation pressure of 15 cm H2O with an aerosol of either water
or test material.
TiO2 aerosols were generated from a sonicated aqueous suspension (20
mg/ml) using a Lovelace nebulizer and dried through a drying tube. The
TiO2 aerosols for both particle types had a mass median aerodynamic diameters (MMAD) ranging from 1.0 to 1.2 fim and geometric standard deviation
(a,) values of 1.6-2.2, as determined by Mercer cascade impactor measurements. Concentration was monitored with a light-scatter particle sensor
connected to a chart recorder. Target concentration was 125 mg/m3. Additional material was added as needed to the nebulizer cup through a custominstalled inlet, allowing either water or particle suspension to be added
as required. Halothane (0.75% final concentration) was used to maintain
anesthesia during exposure. Animals were placed on a heating pad to prevent a reduction in body temperature during exposure. Exposure duration
was 2 h. No more than six animals were exposed at any one time. Following
exposure the tracheal catheter was removed and the animals were allowed
to recover on a heating pad, usually 5-15 min, before being returned to
their cages. Animals (six/group) were euthanized at day 0, 1,3, and 7
postexposure to evaluate pulmonary responses. A separate group of animals
(n = 3) was exposed similarly and used to evaluate evenness of particle
distribution.
Intratracheal instillation exposures. Animals were anesthetized with
halothane and a small catheter attached to a 1-ml syringe was inserted into
the trachea via a modified otoscope. An amount of the appropriate particles
equivalent to that determined, by atomic emission spectroscopy (see below),
to be present in the day 0 ITIH group (500 fig fine TiO 2 , 750 fig ultrafine
222
OSIER AND OBERDORSTER
Halothane
Generator
Relief
Valve
15cm
Magnehelic
Compressed
Air
Filter
Aerosol
Particle
Sensor
\ \ \ \
Ports for Animal
Exposure
Vacuum
Drying
Tube
Solenoid
Valve
Compressed
Air
Syringe
Interval
Timer
Jet Nebulizer
FIG. 1.
1.5 M C Inhalation
0.5 »oc Exhalation
Schematic of intratracheal inhalation exposure system.
TiO 2 ) was instilled suspended in a volume of 0.2 ml saline. Particle suspensions were placed in an ultrasonic bath for 10 min prior to instillation to
ensure an even suspension. These doses resulted in lung particle burdens
(compared at day 1) that were slightly, though not significantly, below those
seen from the ITIH exposures. Animals were allowed to recover and returned to their cages. Groups of animals (six/group) were euthanized at day
0, 1,3, and 7 postexposure to evaluate pulmonary responses.
Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was performed as previously described (OberdOrster et al., 1990). Briefly, animals
were anesthetized with sodium pentobarbital and euthanized by exsanguination via the carotid artery. The trachea was cannulated and the trachea,
heart, and lungs were removed en bloc. The heart and fascia were removed,
and the lungs lavaged 10 times with 5 ml each of sterile saline at room
temperature. The first two washes were pooled, as were the final eight. The
washes were centrifuged at 400g for 10 min at 4°C in a swinging-bucket
centrifuge. The supernatant from the first two washes was saved for protein
analysis, and the cells from all washes were pooled and resuspended in
saline to 15 ml final volume. Cell number and viability using trypan blue
exclusion were determined with a hemocytometer. A small aliquot of cells
was spun onto slides and stained with the Diff-Quick staining system (Baxter Scientific, Edison, NY) for determination of cell differential. The remainder of the cell pellet was saved for later analysis. Following completion of
these procedures, the lungs and aliquots of the cell pellet were saved for
analysis of titanium dioxide content
Tissue titanium dioxide determination.
Analysis was performed using
techniques previously described (OberdOrster et al., 1992). Samples of lung
tissue, lavage supernatant, or lavage cell pellet were dried overnight, ashed,
and then fused in platinum crucibles with a 2:1 (w/w) mixture of sodium
carbonate and sodium borate. The melt was dissolved in 7% (vol/vol)
sulfuric acid and titanium content determined by plasma atomic emission
spectroscopy, reading emission at the 334.91-nm emission line. Titanium
dioxide concentration was calculated from the titanium content. Recovery
from spiked samples was greater than 909b. For the distribution studies,
the lung lobes were analyzed individually, except for die two smallest lobes
which were combined.
Statistical analysis. Data were analyzed with the SigmaStat statistics
package (Jandel, Inc.). An analysis of variance was performed, and Tukey's
test used to determine significance of individual groups, with comparisons
being made between unexposed, sham-exposed, and particle-exposed
groups as well as between similarly exposed groups at different timepoints.
A group was to be considered significantly different if the p value was less
than 0.05.
RESULTS
TiO 2 lung burdens, measured at day 1 postexposure, are
shown in Table 1. For both particle sizes, there was slightly
less TiO2 at day 1 in the lungs of instilled animals. These
differences were not statistically significant. The lungs of
ultrafine TiO2-exposed rats contained approximately 50%
more TiO2 following ITIH exposure than those of rats exposed to fine TiO 2 .
Figure 2 shows the ratio of TiO2 content within individual
TABLE 1
Pulmonary Particle Burdens at Day 1 Postexposure
Day 1 lung burden
(mean ± STDS)
Particle type
Fine TiO2
Fine TiO2
Ultrafine TiO2
Ultrafine TiO2
Exposure method
Intratracheal
Intratracheal
Intratracheal
Intratracheal
instillation
inhalation
instillation
inhalation
(MS)
393.8
498.5
559.4
690.6
± 43.97
± 58.76
± 34.59
± 133.5
223
INTRATRACHEAL INHALATION VS INSTILLATION
40
10
20
30
40
Percent of Total Lung Weight
FIG. 2. TiO2 distribution following ITIH within the lobes of the lung.
The regression line coincides with the line where Lobe Weight (percentage
of total lung wet weight) equals Lobe TiO 2 Content (percentage of total
lung TiOj).
lung lobes to lobe wet weight. The line represents where the
percentage TiO2 content for a given lobe equals the percentage of the total lung wet weight the lobe, as would be expected if the particles were evenly distributed among the
lobes during distribution. The regression line from the exposure points is identical to the line where lobe wet weight is
proportional to lobe TiO2 content.
Total cell numbers in the lavage cell pellet are shown in
Figs. 3a and 3b. Sham-exposed animals for either instillation
or ITIH groups showed no significant differences from unexposed animals. Likewise, animals exposed to fine TiO 2 ,
whether by instillation or ITIH, showed no differences from
either unexposed animals or their respective sham-exposed
group. However, animals from both groups exposed to ultrafine TiO 2 showed increases in total cell number over
sham-exposed and unexposed animals at the day 1 timepoint.
In those animals exposed by instillation, this level remained
elevated throughout day 7, while ITTH-exposed animals returned to control levels by day 3. The difference between
instilled and ITIH-exposed animals was significant at days
3 and 7. No significant changes in viability were observed
(data not shown).
Figure 4 shows the total numbers of macrophages in the
cell pellet, with those animals exposed to fine TiO2 in Fig.
4a, and the ultrafine animals in Fig. 4b. No significant
changes were noted in those animals exposed to fine TiO 2 ,
regardless of the method of administration. In contrast, animals exposed to ultrafine TiO2 by instillation showed a significant increase in macrophage number over both controls
and ITTH-exposed animals at days 3 and 7.
The percentages of polymorphonuclear leukocytes
(PMN's) in the lavage cell pellet are shown in Figs. 5a and
5b. Figure 5a shows that animals instilled with fine TiO 2
showed an influx of PMN's at day 1, which decreased by
day 3 and remained at control levels through day 7. By
contrast, animals exposed by FTTH showed no significant
alterations in PMN percentages following exposure to fine
TiO 2 . Results from the ultrafine TiO2-exposed groups are
shown in Fig. 5b. While both methods of administration
caused significantly elevated levels of PMN's at day 1, those
of the instilled group were significantly higher than those of
the ITIH group. Additionally, the instilled ultrafine TiO 2
group continued to show an increased PMN response
throughout days 3 and 7, while the ITIH group returned to
control levels by day 3.
Total protein levels in the lavage supernatant are shown
in Figs. 6a and 6b. Both particle types showed significantly
elevated levels of total protein, as compared to unexposed
animals, immediately following ITIH exposure. Sham-exposed ITIH animals showed elevated, but not statistically
significant, protein levels as well. Ultrafine TiO2-exposed
animals showed a continued elevation in total protein at day
1, and returned to normal levels by day 3. No further alterations in total protein levels were observed.
DISCUSSION
Previously in our laboratory, we reported the administration of particles to rats by intratracheal inhalation (Oberdors•Control
EaSham ITIH
13IT Instilled
• IT Inhaled
a 4
£2
5
i
DayO
Day1
Day3
I
Day 7
t
1
t
•Control
EOSham ITIH
S I T Instilled
HIT Inhaled
52
8
DayO
Day1
Day3
Day7
FIG. 3. Total cell numbers in BAL pellet for fine TiOrexposed (a) or
ultrafine TiOr-exposed (b) animals. Data are means ± SD. ^Significantly
(p < 0.05) different from unexposed controls. tSignificantly different from
unexposed controls and ITIH group of the same time point.
224
OSIER AND OBERDORSTER
•Control
CDSham ITIH
• FT Instilled
• n* Inhaled
r*
1
x i
DayO
Day1
Day 3
Day 7
•Control
EDSham rpx
M r r Instiled
• IT Inhaled
1
o
I
DayO
Day1
Day3
instillation. This was the case even when multiple instillations were used as the exposure regimen. Pritchard et al.
(1985) reported that the distribution following instillation
was four times less homogeneous than that following inhalation. Domes et al. (1992) showed that, under conditions
similar to those used in the present study, 70% of BAL
macrophages contained no detectable amounts of test material following a single intratracheal dose, whereas those animals exposed via inhalation showed a more homogeneous
distribution. Additionally, Henderson et al. (1995) showed
differences in BAL parameters and histologic injury pattern
following instillation or inhalation of crystalline SiO2 or fine
TiO 2 . A difference in efficacy of instilled vs inhaled antibodies was shown by Vogel et al. (1996), with an inhaled dose
more effectively ameliorating ricin toxicity than an instilled
dose.
Our particle distribution data suggest that an intratracheal
inhalation exposure deposits particles in an even deposition
pattern, similar to other inhalation exposure methods. If our
data are converted to the Evenness Index (El) used by Brain
et al. (1976), defined as the percentage of total lung particle
content contained in a given lung lobe divided by the percentage of the total lung weight the lobe represents, with
100% being an even distribution, the El would range from
89.1 to 124.5%. They reported a lobar El range of 88 to
Day7
FIG. 4. Total macrophage numbers in BAL pellet for fine TiO2-exposed
(a) or ultrafine TiO2-exposed (b) animals. Data are means ± SD. tSignificantly different from unexposed controls and ITIH group of the same time
point
ter et al., 1997). Here we report the results of studies comparing the response of rats to two sizes of titanium dioxide
particles when administered by intratracheal instillation or
intratracheal inhalation.
Our results appear to show an increased response, as indicated by alterations in BAL parameters, of both sizes of
TiO 2 particles when administered by single intratracheal instillation as compared to those administered by intratracheal
inhalation. These differences were most dramatic with the
more reactive ultrafine particles, where significant differences in total cell number, macrophage numbers, PMN percentage, and BAL total protein levels were seen. However,
even in the less reactive fine particle size groups, where a
lower deposited dose was observed, significant differences
were observed.
There are a number of factors that could explain the increased responses of the instilled group over the IT inhaled.
The distribution patterns of particle deposition have been
shown to be different between inhalation and instillation
modes of exposure. Brain et al. (1976) reported that inhaled
particles deposited in a more even pattern and with significantly less variation between lobes than animals exposed by
a 4O,
• Control
EDSham ITIH
• FT Instill
• IT Inhaled
: 20-
10-
. aDayO
. i
Alii
Day1
Day 3
Day 7
b 4Oi
•Control
EDSham FTIH
• IT Instill
CBfT Inhaled
a
t
I
10-
DayO
Day1
Day 3
Day7
FIG. 5. Numbers of polymorphonuclear leukocytse (PMN's) in BAL
pellet for fine TiO2-exposed (a) or ultrafine TiO2-exposed (b) animals. Data
are mean ± SD. 'Significantly (p < 0.05) different from unexposed controls. tSignificantly different from unexposed controls and ITIH group of
the same time point
225
INTRATRACHEAL INHALATION VS INSTILLATION
•Control
•Sham ITIH
SIT Instilled
• IT Inhaled
DayO
b
Day1
ill
Day3
Day 7
0.9
0.8
•Control
(EDSham ITIH
• IT Instilled
• IT Inhaled
f 0.7
"Bb 0.6
§0.6
0.3
0.2
0.1
0.0
n
DayO
El
Day1
Day3
Day 7
FIG. 6. Total protein levels in BAL fluid. Data are means ± SD. •Significantly (p < 0.05) different from unexposed controls.
151 % for the rat following a single 1 -h inhalation, and 44
to 112% for a single instillation exposure, with 100% being
a uniform distribution. As the instillation procedure used in
these studies is essentially the same as that used by Brain
et al., it is reasonable to expect a similar pattern in our
studies. These differences in distribution of dose may play
an important role in the differences in effects we observed.
An altered distribution of particles between the two dosing
methods would have the potential to influence the response
of the lung to particles. A less homogeneous distribution
pattern, as has been shown to be the case following an instilled dose, would tend to create areas of high and low
particle burden within the lung, as is illustrated in Fig. 7.
Thus, while the total pulmonary particle burden would be
the same for the two exposure methods, an instilled dose
may show an increased response due to these focal areas of
high particle burden. This would be difficult to distinguish
using BAL parameters, which tend to evaluate general pulmonary trends rather than localized phenomena. Histopathological techniques would be useful for studying this issue
further.
In addition to differences in particle distribution, the dose
rate may be an important factor in explaining the differences
observed in these studies. Ferin et al. (1994) reported sig-
nificant differences in interstitialization of particles administered by intratracheal instillation or inhalation, and claimed
that the difference in dose delivery rates was a decisive
factor. However, Henderson et al. (1992) found no differences in BAL parameters among animals exposed by inhalation to carbon black at different exposure rates but with
similar particle burdens. Perhaps the difference between an
instantaneous dose, as is seen in instillation, and a dose that
accumulates over time, as is seen by inhalation, is a more
biologically significant difference than variations in inhalation exposure concentration/time. Additional studies will be
needed to further clarify this area.
Several studies have addressed the possibility that particle
clearance may be different following an instilled or inhaled
dose. Ferin and Feldstein (1978) reported that while total
lung particle burden does not appear to be different following
exposure of rats via the two exposure methods, animals exposed to particles by instillation showed increased clearance
to the hUar lymph nodes. In another study, Ferin et al. (1994)
reported an increased percentage of unlavagable ultrafine
particles following intratracheal instillation, when compared
to a multiexposure inhalation dose. In a study whose conditions more closely compare to the present one, Hirano et al.
(1994) showed no significant difference in clearance of
NiSO4 in rats when administered through instillation or a
single, 2-h inhalation. However, because of the high solubility of NiSO 4 , it is likely that much of it was absorbed into
the lung, adding an additional factor to the clearance of the
particle that would not be present with an insoluble particle,
like TiO 2 .
Several groups have examined the differences between
fine and ultrafine TiO2 particles, with the general finding that
the ultrafine particles show a significantly greater response at
similar pulmonary mass doses. Our studies would tend to
support these previous data, with the ultrafine TiO2 particles
showing increased total cell numbers (Oberdorster et al.,
1990, 1992, 1994a,b), macrophages (Oberdorster et al.,
Instillation
Inhalation
FIG. 7. Differences in particle distribution following an instillation or
inhalation exposure, illustrating the decreased homogeneity of an instilled
dose.
226
OSIER AND OBERDORSTER
1994b; Ferin et al., 1991), and increased PMN influx (Oberdorster et al., 1990, 1992, 1994a,b; Driscoll and Maurer,
1991; Ferin et al., 1991) over the fine particle exposures. It
must be cautioned, however, that a direct comparison between fine and ultrafine TiO 2 cannot be made from our data
due to the difference in retained dose following intratracheal
inhalation of the two particle types. An interesting observation in the present studies was a lack of increase in BAL
total protein levels following instillation of ultrafine TiO 2 .
Previous studies (Oberdorster et al., 1990, 1992, 1994a,b)
have reported increases in BAL protein levels following instillation of ultrafine TiO 2 . The reason for this discrepancy
is unknown.
Intratracheal inhalation has a number of characteristics
which could make it a useful tool in studying the effects of
inhaled materials. Like other inhalation exposure systems,
it delivers the test material to the lungs with an even distribution. This has been shown to be more effective to delivering
therapeutic antibodies in mice (Vogel et al., 1996) than intratracheal instillation. However, unlike standard inhalation
systems, ITIH bypasses the upper respiratory tract defenses,
depositing the aerosol directly in the lower respiratory tract.
Thus, deposition on the fur and in the nasal passages, which
can be a major source of difficulty in standard inhalation
systems, is eliminated. The ITIH system is also considerably
smaller than even many small whole-body and nose-only
inhalation systems, so that a smaller amount of test material
would be needed to achieve a given aerosol concentration.
Additionally, the ITIH system is closed, with all air and
test material remaining enclosed within the relatively small
apparatus and being passed through a filter before exhaust,
thus facilitating its use in the study of highly toxic or radioactive substances.
Intratracheal inhalation is not without its disadvantages,
which must also be considered when utilizing this technique.
While the intubation procedure is similar to that of intratracheal instillation, we have found that inserting a catheter in
order for it to remain properly in the trachea is more technically complex than temporarily inserting a catheter for instillation. During the ITIH procedure, the animals are lightly
anesthetized, which may alter enzyme levels within the lung
or other tissues (Raucy et al., 1993). Care must be taken not
to over- or underanesthetize the animals during the procedure. FTTH also requires more time and test material than
intratracheal instillation techniques, which may make it less
practical for very large studies.
Our studies have demonstrated our ability to expose rats
to a particle aerosol by intratracheal inhalation. Animals so
exposed exhibited a decreased response to the particles when
compared to intratracheal instillation, as measured by BAL
parameters. These studies support the utility of intratracheal
inhalation as a short-term inhalation exposure method. Further characterization and comparison of the responses be-
tween intratracheal inhalation and intratracheal instillation
should provide useful information on the role of dose rate
and particle deposition patterns in the pulmonary response
to insoluble particles.
ACKNOWLEDGMENTS
The authors thank Pamela Wade-Mercer, Nancy Corson, Kiem Nguyen,
and Robert Gelein for technical advice and assistance and Christopher
Cox for statistical advice. These studies were supported by NIEHS Grants
ES04872, ES0I247, and HL07216.
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