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Particulate matter from vehicular exhaust in the setting of chronic cerebral hypoperfusion

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Particulate matter from vehicular exhaust in the setting of chronic cerebral hypoperfusion

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Content Particulate Matter from Vehicular Exhaust in the Setting of Chronic Cerebral Hypoperfusion
William J. Mack, MD
Assistant Professor of Neurosurgery
Keck School of Medicine
University of Southern California

TABLE OF CONTENTS
A. Specific Objectives
B. Anticipated Results and Significance
C. Related Previous Studies
D. Methods and Primary Outcome
E. Power Analysis and Sample Size Calculations
F. References/ Bibliography
A. SPECIFIC OBJECTIVES
Clinical and population-based investigations have established an association between air pollution
and low cognitive performance. However, very little is known about the possible pathobiological
mechanisms. Excess prefrontal white matter hyperintensities on MRI, a marker for cerebral vascular
injury, have been documented in dogs and children residing in high pollution areas in Mexico(Calderon-
Garciduenas et al., 2008a). The hypothesis has been advanced that exposure to particulate matter (PM)
may differentially affect individuals with pre-existing or concurrent neurological disease(Block et al.,
2012), particularly cerebral ischemia(Maheswaran et al., 2010; Wilker et al., 2013). To date, experimental
studies have not been carried out to examine the relationship between PM exposure and cerebral ischemia
and to explore underlying mechanisms. This study is specifically designed to investigate the overlooked
“neurovascular” damage resulting from PM exposure. To address this critical knowledge gap, this
investigation employs a murine experimental model to examine the effects of: 1) particulate matter from
vehicular exhaust and 2) chronic cerebral hypoperfusion (CCH) on white matter injury and
neurocognitive function. The experiments are designed to assess independent effects and to test for
synergism. This proposal will leverage the multidisciplinary expertise of the USC Air Pollution Brain
Network (USC Airpol Brain). We have developed and validated techniques for nanoparticulate matter
(nPM) collection and subsequent rodent exposure. Further, we have refined a highly technical murine
CCH model. Synthesis of these techniques will enable us to examine exposure effects using mutually
relevant outcome measures. To this end, we will pursue the following specific aims and hypotheses:
Specific Aim 1: To examine the timecourse of white matter injury and neurocognitive change
secondary to nPM exposure. We hypothesize that nPM exposures of 45 to 225 cumulative hours will
induce disorganization/ rarefication of white matter in the corpus callosum, neurocognitive deficits, and
reactivity of inflammatory cells in a duration-dependent manner. We hypothesize that significant
differences will be identified between mice exposed to nPM and filtered air at the 150 hour exposure.
Specific Aim 2: To examine the effects of nPM exposure and CCH on white matter injury and
neurocognition. We hypothesize that nPM exposure and experimental induction of CCH will each
produce white matter injury and correponding neurocognitive deficits. When administered in parallel, we
expect these exposures to interact in a synergistic manner.
Specific Aim 3: To examine the impact of nPM exposure and CCH on inflammation, oxidative
stress and Blood-Brain-Barrier (BBB) permeability in the cortical white matter of exposed mice.
We hypothesize that the anticipated synergistic effects of nPM exposure and CCH result from
upregulation of inflammatory cytokines/ chemokines, generation of reactive oxygen species and increased
BBB permeability. We hypothesize that administration of nPM exposure and CCH together will
substantially increase these biomarkers in the white matter of the corpus callosum.

B. ANTICIPATED RESULTS AND SIGNIFICANCE
B1. Air Pollution and Cognitive Impairment/ White Matter Injury
Clinical and epidemiologic studies suggest a relationship between long-term PM exposure and
impaired neurocognitive function. In 2000, Kilburn noted severe neurobehavioral deficits in a small
cohort of men with occupational exposure to confined diesel exhaust(Kilburn, 2000). Larger
investigations have since characterized PM effects across ranges of age, gender, and demographic factors.
Ranft et al. demonstrated impairment on the CERAD-Plus test battery, Stroop color word test, and
olfactory function in older women living near busy roadways(Gehring et al., 2006; Ranft et al., 2009;
Schikowski et al., 2007). In an analysis of the Nurses' Health Study Cognitive Cohort, Weuve et al. found
significant evidence of cognitive decline in older women with long-term PM exposures typically
experienced by individuals in the United States(Weuve et al., 2012). Power et al. established an
association between predicted 1-year average residential black carbon levels and low Mini Mental State
Examination scores in a cohort of men from the Veterans Affair Normative Aging Study(Power et al.,
2011). A secondary analysis of the Neurobehavioral Evaluation System-2 data from the Third National
Health and Nutrition Examination Survey by Chen and Schwartz suggested an association between PM
10

exposure and cognitive function in age- and sex- adjusted models. However, associations did not persist
after controlling for sociodemographic factors(Chen and Schwartz, 2009). Wellenius et al. demonstrated
that residential proximity to a major roadway is associated with poor performance on tests of verbal
learning, memory, psychomotor speed, language and executive functioning(Wellenius et al., 2012). Air
pollution is also associated with impaired neurodevelopment in children(Calderon-Garciduenas et al.,
2008a; Fonken et al., 2011; Suglia et al., 2008; Wang et al., 2009). Excess prefrontal white matter lesions
have been documented on MRI in dogs and children residing in high pollution regions(Calderon-
Garciduenas et al., 2008a). Fonken et al. demonstrated that long-term exposure of mice to fine ambient
PM increased depressive-like responses, impaired spatial learning/ memory and upregulated hippocampal
pro-inflammatory cytokines(Fonken et al., 2011). Knowledge gaps in toxicology remain. Previous in-
vivo studies utilizing high levels of diesel PM and NO2 (a surrogate of traffic-related air pollutants) differ
from real-world exposures. Animal PM-exposure models have not demonstrated the neurobehavioral
changes and memory deficits relevant to the observations of cognitive decline associated with PM. Few
neurotoxicology studies have focused on biological susceptibility. None have examined comorbid
conditions that may impart increased susceptibility.
B2. Air pollution and Inflammation, Oxidative Stress, and BBB Permeability
Accumulating laboratory evidence suggests that traffic pollution results in elevated markers of
inflammation and oxidative stress in multiple brain regions(Bos et al., 2012; Campbell et al., 2009;
Campbell et al., 2005; Gerlofs-Nijland et al., 2010; Levesque et al., 2011a; Levesque et al., 2011b;
Morgan et al., 2011). To date, rodent PM exposure studies have documented increases in inflammatory
cytokines/ chemokines and immune-related transcription factors(Gerlofs-Nijland et al., 2010; Levesque et
al., 2011b). Pronounced inflammatory responses have been documented in the midbrain(Levesque et al.,
2011b) and striatum(Gerlofs-Nijland et al., 2010). Oxidative stress pathways are implicated in
neurotoxicity secondary to PM exposure. In-vitro studies demonstrate that diesel exhaust particles (DEP)
promote release of oxygen free radicals from brain microglia, resulting in superoxide-mediated cellular
injury(Block et al., 2004). Similarly, post-mortem brain tissue from canines exposed to high levels of air
pollution reveal tissue injury consistent with oxidative processes(Calderon-Garciduenas et al., 2002). PM
disrupts BBB integrity. Hartz et al. demonstrated increased expression and activity of p glycoprotein and
elevated brain capillary TNF-alpha levels in mice exposed to PM(Hartz et al., 2008). A series of studies
comparing residents from regions of high and low urban pollution levels found histologic evidence of
cerebral microvascular damage and BBB disruption in those from the high level areas(Calderon-
Garciduenas et al., 2008b). Mounting evidence suggests a strong relationship between inflammation and
cognitive decline/ neurodegenerative disease(de Lima Torres et al., 2013; Fonseca et al., 2013; Heyer et
al., 2013a; Heyer et al., 2013b). Accordingly, associations have been reported between PM exposure and
early markers of dementia and Alzheimer’s disease (AD). Levesque et al. demonstrated that exposure of
rodents to inhalational diesel exhaust resulted in elevated Aβ-42 levels in frontal and temporal cortex,
implicating an association between PM-derived inflammation and preclinical AD markers(Levesque et
al., 2011a). Biomarkers associated with early neurodegenration, including Aβ-42, hyper-phosphorylated
tau, and Alpha-synuclein are also evident in post-mortem brains of individuals living in regions of
elevated pollution(Calderon-Garciduenas et al., 2008b)
B3. Cerebral Hypoperfusion and Cognitive Impairment/ White Matter Injury
Clinical imaging, epidemiology and pharmacotherapy studies verify a strong association between
cortical hypoperfusion and cognitive impairment(de la Torre, 2004). Investigations have established
radiographic evidence of cerebral hypoperfusion in patients with AD, and those with mild cognitive
decline. Decreased resting cerebral blood flow values have been documented in AD patients(Johnson et
al., 2005). Conversely, higher cerebral blood flow measurements have been correlated with reduced
incidence of cognitive decline(Ruitenberg et al., 2005). Studies have demonstrated that carotid
atherosclerosis is independently predictive of dementia(van Oijen et al., 2007). Murine CCH studies have
established an association between white matter injury, astrocyte/ microglial reactivity, and working
memory deficits(Shibata et al., 2007). Sustained CCH results in BBB dysfunction(Nakaji et al., 2006) and
accelerates amyloid beta deposition(Kitaguchi et al., 2009)
B4. Potential Synergies between Air Pollution and Chronic Cerebral Hypoperfusion
The hypothesis has been advanced that exposure to PM may differentially affect individuals with
pre-existing or concurrent neurological disease(Block et al., 2012). Testing for combined exposure effects
may help identify susceptible groups, explain disparities at the population level, and ultimately aid with
risk-factor stratification. Effects of multiple agents are maximized when exposures activate, or have
positive consequences, at different steps of a single mechanistic pathway. Evidence suggests that this may
be the case with PM and CCH. PM exposure and CCH each engender pathophysiological changes that
can amplify neurotoxicity resulting from the other. When intact, the BBB effectively restricts vascular
access to most of the brain, protecting tissue from systemic toxins(Miller, 2010). CCH increases BBB
permeability and upregulates inflammatory mediators(Nakaji et al., 2006). These actions can increase
penetrance and accessibility of PM to otherwise privileged locations. Further, upregulation of regional
inflammatory mediators can enhance susceptibility to the neurotoxic effects of nPM by increasing the
density of reactive astrocytes and microglia. When primed, these cells may generate even more
inflammation in response to nPM. In turn, exposure to PM generates oxidative stress and inflammation.
These alterations can lead to microvascular failure and cerebral no-reflow in the setting of CCH and white
matter ischemic injury. Oxygen species derived from PM exposure may contribute to the overall
oxidative stress burden of the brain(Peters et al., 2006). Processes such as CCH, which consume or hinder
pathways involved in free radical clearance, may increase levels of oxidative stress. Relevant precedents
exist for these types of interactions in experimental and clinical paradigms. A recent experimental study
demonstrated that peripheral administration of Lipopolysaccharide resulted in increased brain
inflammatory cytokines and behavioral changes in Alzheimer's transgenic mice (compromised BBB)
when compared to wild type controls(Takeda et al., 2013). This finding suggests that processes impacting
the BBB integrity could amplify the neurotoxic effects of systematically delivered PM or its bioavailable
components. Multiple studies have found that APO-E deficiency potentiates PM-induced
neuroinflammation in rodents(Campbell et al., 2009; Veronesi et al., 2005). It is postulated that less
efficient repair and glial activation may predispose ApoE-/- animals to more severe PM exposure
effects(Campbell et al., 2009). Synergies between other environmental toxins and carotid disease have
been documented in clinical cohort studies. Barnes et al. demonstrated that high lifetime secondhand
smoke (SHS) exposure may increase the risk of dementia in elderly non-smokers with carotid artery
stenosis(Barnes et al., 2010). The risk of dementia was tripled for participants who had lived with a
smoker for >25 years and also had baseline carotid artery stenosis of >25% (compared to individuals with
0-15 years SHS and <25% carotid stenosis). In contrast, the study showed no elevated risk of incident
dementia as a result of SHS exposure or carotid artery stenosis alone.
B5. Significance of Proposed Research
The proposed research program utilizes nPM exposures that are sampled from a near-roadway
traffic source, representing a real world multi-pollutant exposure, a priority of HEI’s Strategic Plan (2010-
2015). Based on speciation analysis, experimental outcomes can ultimately be explored in relation to the
multi-pollutant components of the nPM exposures. Further, the study targets the health effects of air
pollution in a sensitive/ susceptible population, one of the cross-cutting issues relevant to HEI’s Strategic
Plan. Susceptibility is also important in considering the safety of the National Ambient Air Quality
Standards. The interplay between PM exposure and CCH secondary to cerebrovascular disease is
examined. Stroke and dementia are two of the largest morbidity burdens worldwide(Lopez et al., 2006a;
Lopez et al., 2006b). The overall stroke prevalence was estimated to be 3.0% between 2007 and 2010(Go
et al., 2013). Further, the prevalence of silent cerebral infarction is estimated to range from 6% to 28%,
with higher rates in older populations(Das et al., 2008; Prabhakaran et al., 2008; Vermeer et al., 2007).
Projections show that by 2030, an additional 4 million people will have had stroke, a 21.9% increase from
2013(Heidenreich et al., 2011). The current proposal addresses several additional key research gaps and
priority goals identified during the recent NIH-sponsored workshop on “Outdoor Air Pollution and Brain
Health”: 1. Identify a specific population that is vulnerable to air pollution. 2. Determine how air
pollution induced effects on the CNS and cardiovascular disease interact. 3. Discern the effects of air
pollution on BBB function and 4. Evaluate subclinical outcomes using behavioral endpoints(Block et al.,
2012). That PM affects neurocognition has been established. This proposal employs an experimental
model to evaluate effects on brain white matter and examine joint effects between PM exposure and
compromised cerebral blood flow. Findings can generate a foundation for clinical and translational
studies that ultimately guide public policies and regulation with respect to cognitive health in a vulnerable
population. Understanding the relationship between PM exposure and cerebral ischemia may provide a
critical first step towards individual risk assessment and stratification.

C.RELATED PREVIOUS STUDIES
Dr. Mack (project PI) has devoted substantial research efforts to examining the
role of inflammation and resultant microvascular failure in setting of stroke and
cerebrovascular disease. Since 2010, he has explored the interactions between PM from
vehicular exhaust and cerebral ischemia. Dr. Mack and his mentors collect urban PM
with a particle sampler situated near the CA-110 Freeway in Los Angeles.

Collected

aerosols represent a mix of fresh PM, predominantly from vehicular traffic(Misra et al.,
2002; Morgan et al., 2011). Exposure chambers are utilized to administer PM or filtered
air for study in experimental animal models. The group has demonstrated both in-vitro
and in-vivo cellular reactions in rodent microglia and
neurons following nPM exposure(Morgan et al., 2011).
Dr. Mack’s baseline pilot data suggests that mice exposed
to PM may exhibit white matter rarefication and
structural disorganization in the corpus callosum (n=4
PM/ filtered air; see figure 1). The impact of PM exposure on white matter
injury and neurocognition will be tested in a larger cohort of mice with
differing PM exposure durations in Specific Aim 1 of the proposal. Dr.
Mack’s preliminary data also suggests that PM exposure impacts the
progression and severity of murine reperfused stroke (n=4 PM/ filtered
air; see figure 2). This effect may be associated with inflammation and
microvascular failure, as the
relative amplitude of cerebral
reperfusion (after ischemia) is reduced in the PM-exposed mice.
This suggests an interaction between PM exposure and cerebral
ischemia. Dr. Mack has adapted and refined an experimental
murine model to assess for synergistic effects between PM
exposure and CCH in Specific Aims 2 and 3. His previous studies
demonstrate that animals exposed to CCH via bilateral carotid
artery stenosis (BCAS) exhibit significant white matter injury,
neurocognitive deficits and regional astrocytic/ microglia
reactivity when compared to sham-operated mice (see figure 3).
Dr. Mack has previously demonstrated upregulation of
inflammatory mediators (C5 complement protein) and neuroprotection via genetic C5 modulation in this
CCH model (p<0.05, unpublished data). Inflammatory processes are likely to affect neurotoxicity
secondary to PM exposure. Specific Aim 3 is designed to explore inflammation and oxidative stress,
putative mechanisms of injury in the setting of both PM exposure and CCH. The applicant has established
the temporal characteristics of complement upregulation (potent inflammatory mediator) in experimental
stroke(Mack et al., 2006b). Further, they have demonstrated ischemic protection in complement C3
knockout mice and through pharmacologic inhibition of the C3a and C5a receptors(Kim et al., 2008;
Mocco et al., 2006a). Dr. Mack and colleagues have also established that suppression of excessive
oxidative metabolism can decrease infarct volume and improve neurological function following acute
stroke(Huang et al., 2001; Mack et al., 2006a).

Figure 3: Top left: White matter injury score. Bottom left:
Novel object recognition (percent time at novel object).
Top Right: Reactive astrocytes (GFAP). Bottom Right:
Reactive microglia (IBA-1), (*p<0.05)
Figure 2: Left: 2,3,5-triphenyltetrazolium
chloride stain of mouse brains on post-operative
day 1 following middle cerebral artery stroke
(infarct: white). Right: Percent infarct volume
(top) and reperfusion from baseline (bottom) in
nPM/ ambient exposed mice.
Ambient
nPM
Figure 1:
Representative
Klüver-Barrera stain
in ambient/ nPM mice.
Arrows indicate
increased rarefication/
vacuoles in corpus
callosum of nPM mice
D. METHODS AND PRIMARY OUTCOME
D1. Summary: The study investigates the effects of nPM exposure and CCH on white matter injury and
neurocognition in a mouse model. Specific Aim 1 examines the effects of escalating nPM exposure
durations on white matter injury, neurocognitive outcome and cellular reactivity and determines the
exposure duration for the ensuing aims. Each outcome will be analyzed with a general linear model with
two primary factors (independent variables); one factor is exposure group (nPM, filtered air) and the
second factor is exposure duration. The ages of the mice at the time of exposure in Specific Aim 1 are
selected according to parameters relevant to the ensuing aims. The CCH exposure in Specific Aims 2 and
3 is validated in 12 week old mice. The CCH is designed to overlap with the final 30 days of the nPM
exposure. Therefore, mice will all be 12 weeks and 30 days old at the end of the nPM exposure.
Accordingly, exposures will begin at different ages for the four exposure duration cohorts (13 week old
mice for 3 week exposure; 11 week old mice for 5 week exposure; 6 weeks old mice for 10 week
exposure; 1 week old mice for 15 week exposure) A maximum of 120 mice will be used for this aim (see
section D3). In Specific Aims 2 and 3, main effects of each exposure (nPM, CCH) will be tested. The
primary hypothesis related to synergy of the nPM and CCH exposures on outcomes will be tested with an
interaction term. Specific Aim 2 assesses white matter injury and neurobehavior, while Specific Aim 3
examines biomarkers and mechanism of injury. A total of 88 mice will be used in Specific Aim 2 and 44
additional mice in Specific Aim 3 (see section D4/D5). The age at which the nPM exposure starts will be
selected according to the results of Specific Aim 1. Cognitive assessment will be performed 30 days after
the CCH procedure. Animals will be sacrificed on the same day for tissue analysis. Although experiments
are designed primarily to investigate white matter injury, nPM-induced neurotoxicity and resulting
neurocognitive impairment may be caused by multiple plausible pathways, including neuronal injury.
This study will explore this alternative mechanism by histopathological examination and neurocognitive
testing.

D2. General Methods Certain techniques, models and reagents will be used repeatedly. This section
describes exposure methods (nPM and CCH model), outcome assessments and tissue processing
techniques to be used throughout the aims

D2.A. Wild type mice: Use of laboratory animals will comply with the provisions of the Animal Welfare
Act (7 U.S.C. S 2131 et. seq.) and the guidelines set forth in the Guide for the Care and Use of Laboratory
Animals. Male, C57 Black J6 mice will be used for each aim of this proposal. As the BCAS model
(Specific Aim 2 and 3) has only been validated on C57 black J6 mice(Shibata et al., 2004), the strain was
selected to maintain consistency across the aims and exposures. The effects of CCH could vary
significantly across strains with differing anatomical Circle of Willis configurations(Ward et al., 1990).
Most importantly, large posterior communicating arteries could reduce or eliminate hypoperfusion/
ischemia secondary to experimental carotid artery stenosis. The exclusive use of male mice addresses a
potential effect modifier. Estrogen has been shown to impact functional outcomes following cerebral
ischemia (Gibson et al., 2006).

D2.B. nPM collection: For each aim in this proposal, mice will be exposed to re-aerosolized nPM and/or
filtered air. Following is the exposure protocol: Traffic-generated nano-size particulate matter will be
collected and administered as previously described(Misra et al., 2002; Morgan et al., 2011). Briefly, urban
nPM (aerodynamic diameter <200 nm) will be obtained at 400 L/min flow using a high-volume ultrafine
particle sampler. The sampler incorporates an ultrafine particle multiple rectangular (slit) geometry jet
conventional impactor and an after-filter holder(Misra et al., 2002). nPM will be collected at a single site
situated adjacent to the CA-110 highway in Los Angeles(Morgan et al., 2011). These aerosols represent a
mix of fresh ambient PM mostly from vehicular traffic nearby this freeway(Ning et al., 2007). The
impactor and after-filter holder system employs high flow rates under very low pressure drops, allowing
for animal exposure to ultrafine aerosols at near atmospheric pressure and at significantly higher flow
than the typical human breathing rates(Misra et al., 2002).
The nPM is collected on pretreated Teflon filters (8x10”, PTFE, 2 m pore; Pall Life Sciences)
and then transferred into aqueous suspension by 30 min soaking of filters in Milli-Q deionized water
(resistivity, 18.2 MW; total organic compounds < 10 ppb; particle free; bacteria levels < 1 endotoxin
units/mL; endotoxin-free glass vials), followed by vortexing (5 min) and sonication (30 min)
resuspension. No endotoxin has been detected in these suspensions (Limulus amebocyte lysate assay: LPS
<0.02EU/ml; pilot data not shown). As a control, fresh sterile filters are sham extracted and stored.
Aqueous nPM suspensions are pooled and frozen as a stock at –20°C, which retains chemical stability for
≥ 3 months (Li 2010)
A VORTRAN nebulizer is used to re-aerosolize the particulate suspensions using compressed
particle-free filtered air (see figure 4). Passage through a silica gel will diffusion dry particles and static
charges will be removed by passage over polonium-210 neutralizers. Particle size and concentration
(concentration goal of 300-400 ug/m3- roughly twice as high levels as those in a busy freeway) are
continuously monitored by a scanning mobility particle sizer (SMPS model 3080; TSI Inc., Shoreview,
MN) in parallel with the animal exposure chambers. From the total of 15 l/min of aerosol flow generated,
the majority (10 l/min) is drawn through the exposure chamber. The remaining 5 l/min is diverted to
filters for particle collection and characterization. The mass
concentration of the nPM is determined by pre- and post- weighing
under controlled temperature and relative humidity. Teflon and
quartz filters, sample concurrently the exposure aerosol during the
experiments(Li et al., 2003; Li et al., 2009). The composition of
nPM is monitored. Inorganic ions [ammonium (NH
4
+
), nitrate (NO
3
–
), sulfate (SO
4
2–
)] are analyzed by ion chromatography and PM-
bound metals/ trace elements assayed by magnetic-sector inductively
coupled plasma mass spectroscopy. Water-soluble organic carbon
will be assayed by a GE-Sievers liquid analyzer (GE-Sievers,
Boulder, CO). The inorganic and organic compound contents of these samples have been previously
described(Li et al., 2003; Li et al., 2009). A detailed physical and chemical characterization of the
collected and re-aerosolized nanoparticles will be performed and provided (Morgan et al., 2011) .

D2.C. nPM exposure: Mice will be transferred to whole-body exposure chambers, each sectioned into 9
individual compartments to house a total of 18 mice. Temperature and airflow are controlled for adequate
ventilation and to minimize buildup of animal-generated contaminants. Re-aerosolized nPM or filtered air
will be delivered to the sealed exposure chambers for 5 hours/day, three days/ week(Morgan et al., 2011).
Total duration of the exposure (number of weeks) will vary according to the experimental aim. In prior
studies, mice have not lost weight or shown signs of respiratory distress(Morgan et al., 2011). The
subsequent surgical interventions (12 weeks) and pathological examinations/ neurobehavioral
assessments (12 weeks 30 days) will be performed at the same chronological age of the mouse, regardless
of the exposure scheme.

D2.D. Murine Bilateral Carotid Artery Stenosis (BCAS)
model of Chronic Cerebral Hypoperfusion (CCH): The
BCAS surgery is modified from the protocol originally
described by Shibata et al.(Shibata et al., 2004). Mice are
anesthetized with Ketamine and Xylazine, intraperitoneally.
While positioned prone, a Laser Doppler probe is placed
1mm posterior and 2mm lateral to the bregma, over the left
cerebral hemisphere and affixed with glue. The mouse is
then turned supine. Through a midline cervical incision,
both common carotid arteries are exposed. Two 4–0 silk
sutures are placed around the distal and proximal segments

Figure 5: Left: LDF tracings from a sham (above) and CCH
(below) mouse. Middle: Representative coronal section from
the same mice. Right: Hypoxy probe staining for cerebral
ischemia in the same mice
Figure 4: Schematic of the exposure apparatus
of each common carotid artery. Then, the arteries are lifted by these sutures sequentially and a microcoil
with a diameter of 0.18 mm is applied to each common carotid artery (Sawane Spring Co, Japan). The
skin incision is then closed. Rectal temperature is maintained between 36.5°C and 37.5°C throughout the
procedure. CBF values are recorded in the supine position just prior to surgery, following application of
the first microcoil, and following application of the second microcoil using a Probe 418-1 master probe/
PF 5010 laser Doppler Perfusion Monitoring Unit (Perimed AB, Sweden, see figure 5). Mice that do not
demonstrate greater than fifteen percent blood flow reduction in the left middle cerebral artery territory
following the experimental procedure are excluded from the analysis. Sham-operation (control group)
entails bilateral exposure of the common carotid arteries with no microcoil application. On postoperative
day 30, the mice are deeply anesthetized with Ketamine and Xylazine and perfused transcardially with
phosphate buffered saline (PH 7.4) to clear the vasculature of blood, then with a fixative containing 4%
paraformaldehyde and 0.2% picric acid in 0.1mol/L phosphate buffer (PH7.4). The brains are excised,
and stored for an additional 24 hours in paraformaldehyde at 4C˚, then in 20% sucrose in 0.1mol/L PBS
(PH7.4).

D2.E. White matter ischemic change: Harvested brains are embedded
in paraffin. A section of the brain located from 1 mm anterior to the
bregma to 2 mm posterior to the bregma (adjusted according to mouse
atlas) is then sliced into 10 serial 3µm-thick coronal sections. The
remainder of the section is saved. Klüver-barrera (KB) staining is
performed on the slice located at the bregma and the slice just posterior
to the bregma. White matter integrity is evaluated in the medial region of
the corpus callosum (see figure 6) according to a previously described
four point scale(Shibata et al., 2004; Wakita et al., 1994): normal (grade
0), disarrangement of the nerve fibers (grade 1), formation of marked
vacuoles (grade 2), and disappearance of myelinated fibers (grade 3).
Analysis is performed by two, blinded-to-exposure status, and previously
trained, independent observers. Scores from each of the observers is averaged to generate a final score for
each section. The scores from the two sections are then averaged, generating a final score

D2.F Neurobehavioral analysis: In the BCAS model utilized for this study, Y-maze(Dong et al., 2011;
Maki et al., 2011; Washida et al., 2010) and Novel Object Recognition (unpublished data, see section C)
tests have demonstrated strong correlations with white matter ischemic change.

Y Maze Task: The Y maze task assesses spatial working memory. The maze consists of three divergent
arms (34 cm long, 6 cm wide and 14.5 cm deep, labeled A, B, or C) 120° angles from a central point. The
experiments are performed in a dimly illuminated room, and the floor of the maze is cleaned with ethanol
following testing of each mouse. Each mouse is placed at the end of one arm and allowed to move freely
in the maze during an 8 minute session. The sequence of arm entries is recorded manually on video. An
alternation is defined as entries into the three arms on consecutive occasions. The maximum alteration is
the total number of arm entries minus two. The percentage of alternation is calculated as (actual
alternations / maximum alternation) × 100. The total number of arms entered during the sessions is also
recorded(Dong et al., 2011; Maki et al., 2011; Makinodan et al., 2009; Washida et al., 2010).

Novel Object Recognition Test: Novel object recognition test evaluates declarative memory. The protocol
is based on three phases: in the first phase (habituation to arena), mice are placed in an open-field arena
(20 x 20 x 20 cm), made in black Plexiglas. Each mouse is left in the apparatus and allowed to explore for
15 min. Twenty-four hours later, mice undergo the sample trial, consisting of the exploration (for 15 min)
of two identical plastic-made objects (O1 and O2), situated inside the arena at equivalent distance from
the center and the corners, in opposite positions. Objects are affixed to the floor with double-sided tape,
so that they cannot be moved by the animals. The third phase consists of the object recognition trial: mice
Figure 6: Klüver-barrera stain of coronal
sections at the bregma in sham (top) and
BCAS (bottom) mice. Red box indicates the
medial region of the corpus callosum. Top
row represents a score of 0 (from sham
animal). Bottom row represents a score of 2
(from BCAS animal)
are returned to the arena 24 h after the sample trial, and observed under equivalent conditions, in the
presence of one familiar object (O3, identical to those used in the sample trial), and one novel object
(N1). Location of the objects is counterbalanced across the trial. Between each trial, both the arena and
the objects are washed with 95% ethanol solution and dried. All sessions are video recorded with a
camera positioned above the arena, and analyzed by an experimenter blind to treatment conditions.
Exploration is defined as sniffing or touching the object with the snout; sitting on the object is not
considered exploration. Analysis of object exploration in the sample and recognition trials includes
frequency (number of exploratory approaches), duration (total time spent in exploration) and latency
(time elapsed between the beginning of the session and the first exploration). Object recognition is
calculated for each animal as a novelty exploration index, with the following formulas: T
N1
/ (T
N1
+T
O3
),
with T indicating the duration of object exploration(Bortolato et al., 2009; Makinodan et al., 2009). The
three testing phases can be administered on the final three days of the exposure (nPM, CCH) so that a
separate cohort of mice is not needed for behavioral testing.

D2.G. Quantitative immunohistochemistry: Harvested brains are embedded in paraffin. A section of
the brain located from 1 mm anterior to the bregma to 2 mm posterior to the bregma is then sliced into
serial 3µm-thick coronal sections. Immunohistochemistry is performed on the slices posterior to that used
for KB staining. After immunohistochemistry is performed, slides are deparaffined, and then hydrated by
a series of different concentration alcohol (from 100% to 70%). Antigen is retrieved by microwave,
dipped in 3% H2O2 for 10 min, and then blocked with serum. Slides are incubated overnight with a rabbit
anti- glial fibrillary acidic protein (GFAP) antibody (diluted 1:10,000; Dako, Denmark), rabbit anti-
ionized calcium-binding adapter molecule 1(IBA1) antibody (1:200; Wako, Japan), HNE antibody
(1:400; Alpha Diagnostic International, San Antonio, TX, USA) or MPO antibody (MPO, diluted 1:50,
#A0398, DAKO, Glostrup, Denmark). Subsequently, sections are treated with the appropriate biotinylated
secondary antibody Vectastain Elite ABC kit (Vector Laboratories, Burlingame, California, USA) and
visualized with diaminobenzidine (DAB). Photos of the immunostained slices are captured by a LAS AF
microscope (Leica, Germany). The optical density of DAB signal is analyzed and quantified using NIH
Image J software (rsbweb.nih.gov/ij/). The images are converted to 8 bit and adjusted to threshold to
count the positive cells per 0.1mm square. Protocols follow the NIH Image J user guide.

D2H. MDA assay: MDA levels are measured using commercially available Bioxytech LPO-586 assay
kits (OxisResearch, Foster City, CA, USA) according to the manufacturer’s specifications Concentration
is estimated by measuring the absorbance at 586 nm on a spectrophotometer (Molecular Devices,
Sunnyvale, CA, USA).

D2I. Neuroinflammatory gene expression: Gene expression is assessed by real-time PCR measurement
of cytokine/ chemokine upregulation. Fresh whole brain tissue is collected at the time of sacrifice, flash
frozen and stored at −80 °C. Total RNA is extracted using a homogenizer (Ultra-Turrax T8, IKAWorks,
Wilmington, NC, USA) and an RNeasy Mini Kit (Qiagen, Gaithersburg, MD, USA). RNA is reverse
transcribed into complementary DNA using the iScript cDNA Synthesis kit (Bio-Rad Laboratories). The
cDNA products of the RT reaction are stored at –80°C or used immediately for QPCR. QPCR, using iQ
SYBR Green Supermix (Bio-Rad Laboratories) as the fluorescent DNA intercalating agent, is analyzed
using a IQ4 multicolor detection QPCR system (Bio-Rad Laboratories). The relative abundance of target
mRNA is normalized to β-actin. The following cytokines/ chemokines will be assessed: Interleukin-1
Beta (IL1β), Tumor Necrosis Factor-alpha (TNFα), Interleukin-6 (IL-6), Intracellular adhesion molecule-
1 (ICAM-1), and Chemokine C-C motif ligand-2 (ccl2).

D2J. Blood-brain Barrier permeability To assess BBB permeability mouse endogenous IgG and fibrin
will be detected as we previously described(Bell et al., 2012). Brain sections (see above
immunohistochemistry) are removed and embedded into O.C.T. compound (Tissue-Tek) on dry ice,
cryosectioned at a thickness of 14-18 μm, and fixed in acetone. Sections are subsequently be blocked with
5% normal swine serum (Vector Laboratories) for 1 h and incubated in the following primary antibodies
diluted in blocking solution overnight at 4° C:goat anti-PDGFR (R&D, AF1042, 1:200), mouse anti-
NeuN (Millipore, MAB377, 1:250) using the M.O.M. kit (Vector Laboratories), goat anti-Thrombin
(Santa Cruz Biotechnology, sc-23355, 1:100), rabbit anti-fibrin (Dako, A0080, 1:1000). To visualize
immunofluorescent signal the following fluorophore-conjugated secondary antibodies will be used: Cy3-
conjugated bovine anti-goat (Jackson ImmunoResearch, 805-165-180, 1:100) to detect PDGFR, Dylight-
649-conjugated streptavidin (Vector Laboratories, SA-5649, 1:100) to detect NeuN, Alexa fluor 488-
conjugated donkey anti-goat IgG (Invitrogen, A-11055, 1:100) to detect Thrombin and/or Alexa fluor
546-conjugated donkey anti- rabbit (Invitrogen, A10040, 1:200) to detect fibrin. For endogenous IgG
detection the sections are incubated for 48 h with Cy3-conjugated donkey anti-mouse IgG in PBS at 4° C.
To visualize brain microvessels, sections are incubated with fluorescein-conjugated tomato lectin (Vector
Laboratories FL-1171, 1:200) or biotinylated tomato-lectin (Vector Laboratories, B-1175, 1:1000) flowed
by incubation with Dylight 649-conjugated streptavidin (Vector Laboratories, SA-5649, 1:1000). Sections
are cover slipped using fluorescent mounting medium (Dako) and scanned using a Zeiss 510 meta
confocal microscope. Z-stack projections and pseudo-coloring is performed using ZEN software (Carl
Zeiss Microimaging). To quantify extravascular accumulations, the IgG- or fibrin-positive
immunofluorescent signals are subjected to threshold processing and measured using the NIH Image J
software Integrated Density analysis measurement tool.

D3. Specific Aim 1: To examine the timecourse of white matter injury and neurocognitive change
secondary to nPM exposure, and to establish an experimentally relevant exposure duration for specific
aim 2 and 3 of this proposal. We hypothesize that nPM exposures of 45 to 225 cumulative hours will
induce disorganization and rarefication of white matter in the corpus callosum and neurocognitive deficits
in a duration-dependent manner. We hypothesize that significant differences will exist between mice
exposed to nPM and filtered air at the 150 hour and greater exposure durations (See figure 7)

D3.A. Rationale: This aim examines the
effects of nPM exposure on white matter
injury, neurocognition, and cellular
reactivity. A dose (duration) escalation
design is utilized to determine an
exposure duration for Specific Aims 2 and
3. The number of mice affected by the
exposure (white matter grade > 0) will be
determined for nPM and filtered air-
exposed mice in each exposure duration
group. The minimal duration at which 5
more nPM-exposed mice (compared to
filtered-air exposed mice) demonstrate
white matter change will be selected as
the prescribed exposure duration for the subsequent aims. As nPM has not been extensively studied in
this experimental model, exposure durations are based on data generated from pilot studies and other
paradigms in our laboratory. Our team has demonstrated both in-vitro and in-vivo cellular reactions in
rodent microglia and neurons following a ten week (150 total hours) exposure(Morgan et al., 2011).
Inflammatory and oxidative mechanisms were implicated. Preliminary studies suggest that nPM affects
white matter integrity in the corpus callosum and neurocognitive outcomes at this exposure duration (see
section C). Further, pilot data from the principal investigator’s laboratory suggests detrimental effects of a
3 week exposure (45 total hours) in the setting of acute large vessel murine stroke. We have, therefore
selected these two exposure durations for our current study and have incorporated an intermediate time
point (5 weeks) and a prolonged subchronic exposure (15 weeks). The first exposure examined will be the
Figure 7: Pathogenesis schematic of Specific Aim 1
150 hour duration (hypothesized to demonstrate injury based on preliminary data). If the threshold white
matter injury is achieved, the lower doses will be tested. If the threshold injury is not achieved, the longer
dosing duration will be tested. Once a group achieves the threshold number affected, the subsequent
exposure durations will have only an nPM exposure group (not filtered air). These cohorts will undergo
behavioral testing and assessment for astrocyte/ microglial reactivity as described below. The nPM
exposures will be continued after achieving the threshold number to provide important toxicology data. If
white matter ischemic changes are not evident in any of the groups, all exposure durations will include
both nPM and filtered air groups.

D3.B. Experimental exposures: 120 (potentially fewer, see below) mice will be transferred to whole-
body exposure chambers. All exposures (nPM/, filtered air) will occur for five hours a day, three days a
week. Total exposure times will be adjusted by altering the number of exposure weeks. Four cohorts of
mice (n=30 in each group, 15nPM/ 15 filtered air) will be examined in this specific aim 1) mice exposed
to nPM/ filtered air for three weeks (total duration 45 hours), 2) mice exposed to nPM/ filtered air for five
weeks (total duration 75 hours), 3) mice exposed to nPM/ filtered air for ten weeks (total duration 150
hours), 4) mice exposed to nPM/ filtered air for fifteen weeks (total duration 225 hours). Exposures will
be performed according to the outline in the Rationale section (D.3.A)

D3.C. White matter injury (primary outcome): For mice in each exposure duration cohort, white
matter injury will be quantified in the medial region of the corpus callosum by KB staining. Means and
standard deviations will be calculated and compared between mice exposed to nPM and filtered air. As
described above, the minimal exposure duration at which 5 more nPM-exposed mice (compared to
filtered-air exposed mice) demonstrate white matter change (score >0) will be selected as the prescribed
dose duration for the subsequent aims. Secondary analyses will be performed in the same manner on other
white matter regions (optic tract, caudoputamen). Additionally, adjacent sections will be stained with
Hematoxylin and Eosin to assess for cortical/ hippocampal damage secondary to neuronal injury(Sun et
al., 2006).

D3.D. Neurobehavioral analysis: For mice in each exposure duration cohort, Y-maze and novel object
recognition testing will be performed. Means and standard deviations will be calculated and compared
between mice exposed to nPM and filtered air.

D3.E. Reactivity of microglia and astrocytes: For mice in each exposure cohort, quantitative
immunohistochemistry will be employed to assess for reactive microglia and astrocyte cell counts in the
medial region of the corpus callosum (per 0.1mm
2
), in addition to the frontal cortex and hippocampus.
Means and standard deviations will be calculated and compared between mice exposed to nPM and
filtered air.

D3.F. Statistical analysis: Each outcome (white matter injury, neurobehavior, reactive microglia and cell
counts) will be analyzed with a general linear model with two primary factors (independent variables);
one factor is exposure group (nPM, filtered air) and the second factor is exposure duration. The exposure
duration factor will be modeled first as a series of indicator (class) variables; the interaction of exposure
group by duration will test for a differential effect of nPM as exposure duration increases. Post hoc
comparisons will test for exposure group differences on outcomes at each exposure duration; multiple
hypothesis testing will be controlled. To evaluate dose (duration)-response trends, the exposure duration
variable will also be modeled as a single independent variable (total hours of exposure); the interaction
term of exposure group by duration will test for increasing (or decreasing) exposure group differences on
outcomes as duration increases. Because the filtered air group will only be included in these experiments
up to the identification of the duration/dose threshold, it is possible that a significant range of exposure
duration will not be achieved in this exposure group. The dose-response trend analyses will be completed
in the nPM group only (for whom data will be collected across all planned exposure durations). Among
the nPM animals, each outcome will be evaluated for a trend in response by duration of nPM exposure;
different forms of dose-response will be tested, including linear, quadratic, and exponential relationships.

D4. Specific Aim 2: To examine the independent and joint effects of nPM exposure and CCH on white
matter injury and neurocognition. We hypothesize that upregulation of inflammatory mediators and
oxidative stress secondary to nPM exposure can promote microvascular failure in the setting of chronic
cerebral hypoperfusion. Further, BBB breakdown secondary to CCH can allow increased nPM penetrance
and direct toxicity. We hypothesize that nPM exposure (for duration established in specific aim 1) and
experimental induction of CCH will each produce white matter injury, neurocognitive deficits and
regional reactivity of inflammatory cells (astrocytes, microglia). When administered together, we expect
these exposures to interact in a synergistic manner (see figure 8).

D4.A. Rationale: A randomized,
four group, blinded study design will
be used to assess the effects of each
of two exposures (nPM, CCH) and
determine whether they act in a
synergistic manner. The nPM dose
duration will be based upon results of
Specific Aim 1. A factorial design
will be used. The two exposures will
be administered in parallel, with the
CCH occurring for the final 30 days
of the nPM exposure. The CCH
surgeries will be performed during
the final 30 days of the nPM exposure to
allow for outcome assessment at the previously validated time point (day 30).

D4.B. Experimental exposure: Eighty-eight male C57 black J6 mice (age at the beginning of exposure
will be determined by the exposure duration, See D1) will be randomized to one of four experimental
groups (see below for diagrammatic representation, n=22 in each group): The groups are: 1) filtered air
and sham surgery (-nPM/ -CCH) or 2) filtered air and chronic cerebral hypoperfusion (-nPM/ +CCH) or
3) particulate matter and sham surgery (+nPM/ -CCH) or 4) particulate matter and chronic cerebral
hypoperfusion (+nPM/ +CCH) The mice will be exposed to aerosolized particulate matter or filtered air
for 5 hours a day, 3 days a week. The duration of the exposure will depend on the results in Specific Aim
1. 30 days from the end of the prescribed exposure, the mice will undergo CCH or sham surgery (see
figure 9).

Figure 9: Schematic representation of experimental design for Specific Aim 2
Figure 8: Pathogenesis schematic of Specific Aim 2

D4.C. Outcome analysis: After completion of the exposure(s), quantification of white matter injury and
neurobehavioral analysis will be performed. In the analysis stage, main effects (of nPM, CCH) will be
assessed, as well as synergy (see figure 10).

1. White matter injury (Primary Outcome): For mice in each exposure cohort, white matter injury will
be quantified in the medial region of the corpus callosum by KB staining. Means and standard
deviations will be calculated and compared. Secondary analyses will be performed in the same
manner on other white matter regions (optic tract, caudoputamen).

2. Neurobehavioral Analysis: For mice in each exposure cohort, Y-maze and Novel Object
Recognition testing will be performed. Means and standard deviations will be calculated and
compared between groups

Null hypothesis: C1/C4 = C2/C4 + C3/C4
Alternate hypothesis: C1/C4 ≠ C2/C4 + C3/C4

D4.D. Statistical analysis: All outcomes will be analyzed with a 2x2 ANOVA (nPM exposure by CCH).
Main effects of each exposure (nPM, CCH) will be tested. The primary hypothesis related to synergy of
the nPM and CCH exposures on outcomes will be tested with the interaction term (testing the hypothesis
that the mean outcome in group C1 above will be greater than that predicted from the summed main
effects of CCH+ and nPM+). Post hoc analyses will perform pairwise group comparisons with
adjustment for multiple testing.

D5. Specific Aim 3: To examine the impact of nPM exposure and CCH on inflammation, oxidative
stress and BBB permeability in the cortical white matter of exposed mice. We hypothesize that the
combined (synergistic) effects of nPM exposure and CCH result from upregulation of inflammatory
cytokines/ chemokines, generation of reactive oxygen species and increased BBB permeability. We
hypothesize that administration of nPM exposure and CCH together will substantially increase these
biomarkers in the white matter of the corpus callosum relative to levels predicted for nPM or CCH alone.
This aim will leverage the brain specimens harvested from the Specific Aim 2 (see figure 11).

D5.A. Rationale: Brain specimens
will be leveraged from Specific Aim
2 to assess for mechanism of injury.
Individual exposure effects and
synergy for each biomarker assay
will be determined by methods
outlined in Specific Aim 2.
Assessment of inflammatory gene
expression and Malondialdehyde
(MDA) assays will require additional
Figure 10: Schematic representation of the analysis method for
synergy between particulate matter/ CCH
Figure 11: Pathogenesis schematic for Specific Aim 3
exposure groups, as the preparation of harvested brains differs from that of KB staining and
immunohistochemical analysis.

D5.B. Inflammatory mediators:
1. Neuroinflammatory gene expression will be assessed by real-time PCR measurement of cytokine/
chemokine upregulation. Gene expression will be examined for a tailored battery of cytokines and
inflammatory mediators: Interleukin-1 Beta (IL1β), Tumor Necrosis Factor-alpha (TNFα),
Interleukin-6 (IL-6), Intracellular adhesion molecule-1 (ICAM-1), and Chemokine C-C motif ligand-
2 (ccl2) gene expression. Prior studies have demonstrated that the chosen markers are affected by
particulate matter and/or cerebral ischemia(Campbell et al., 2005; Gerlofs-Nijland et al., 2010;
Levesque et al., 2011a; Levesque et al., 2011b; Zhong et al., 2009). However, upregulation in white
matter/ corpus callosum has not been examined following these exposures. As brain homogenates are
required for this assay, an additional 20 mice will undergo nPM/ filtered air exposure and BCAS/
sham surgery according to the above prescribed protocol (n=5 in each group). Once harvested, brains
will be sectioned sagitally. The corpus callosum will be dissected out (en bloc) from each of the left
hemispheres. Gene expression studies will be performed on corpus callosum homogenates (5 corpus
callosum samples combined for each homogenate analysis due to limited quantity of tissue from each
mouse; total n=4 homogenates). We have successfully dissected corpus callosum samples and
performed different analyses in previous studies (unpublished data). The entire right hemisphere of
each mouse brain will undergo gene expression analysis. These dissections will allow gene profiling
of both whole brain and focused analysis of the corpus callosum in each mouse
.
2. Immunohistochemistry will be performed on brain sections from eighty mice (n=20 from each of
the four groups) obtained in Specific Aim 2. Cells will be counted and densities quantified in the same
region (medial) of the corpus callosum (per 0.1mm
2
) that was graded for white matter ischemic
change in Specific Aim 2, in addition to the frontal cortex and hippocampus. Numbers of reactive
microglia (IBA-1) and astrocytes (GFAP) will be quantified as previously described in the BCAS
model(Dong et al., 2011; Maki et al., 2011; Shibata et al., 2004; Washida et al., 2010). Reactive
leukocytes (MPO) will be counted. Deposition and density of pro-inflammatory mediators will also
be determined (three markers will be assessed). The inflammatory mediators chosen for this section
will be those that exhibit highest levels of gene expression in the corpus callosum as determined in
the previous section (selected from: Interleukin-1 Beta (IL1β), Tumor Necrosis Factor-alpha (TNFα),
Interleukin-6 (IL-6), Intracellular adhesion molecule-1 (ICAM-1), and Chemokine C-C motif ligand-
2 (ccl2)).

D5.C. Oxidative injury: Quantitative immunohistochemical analysis of 4-hydroxy-2-nonenal (HNE) will
be performed in the medial corpus callosum, hippocampus and frontal cortex. Oxidative stress will be
examined by quantification of MDA, a lipid peroxidation product. As brain homogenates are required for
this assay, an additional twenty mice will undergo nPM/ filtered air exposure and BCAS/ sham surgery
(n=5 in each group) as in the gene expression study (above). Once harvested, brains will be sectioned
sagitally, as they were in the gene expression studies. Again, the corpus callosum will be dissected out (en
bloc) from each of the left hemispheres. MDA assays will be performed on corpus callosum homogenates
(5 corpus callosum samples combined for each homogenate analysis; total n=4 homogenates). The entire
right hemisphere of each mouse brain will undergo gene MDA analysis (n=5 in each group).

D5.D.BBB permeability: Endogenous IgG and Fibrin deposition will be examined by confocal
microscopy. We will analyze 6 randomly selected fields from the corpus callosum, frontal cortex and
hippocampus and in 6 non-adjacent sections (~100 m apart) in the mice each from each of the four
exposure groups in Specific Aim 2.

D5.E. Statistical analysis: All outcomes will be analyzed with a 2x2 ANOVA (nPM exposure by CCH).
Main effects of each exposure (nPM, CCH) will be tested. The primary hypothesis related to synergy of
the nPM and CCH exposures on outcomes will be tested with the interaction term (testing the hypothesis
that the mean outcome in group C1 above will be greater than that predicted from the summed main
effects of CCH+ and nPM+). Post hoc analyses will perform pairwise group comparisons with
adjustment for multiple testing.

D6. Interpretation of Results, Limitations and Alternative Approaches:

D6.A Strengths and interpretation of results
The application has the following major strengths:

1. This study focuses on a PM target, cortical white matter, which has not been previously studied in an
experimental system. The protocol incorporates refined neurocognitive testing and assessment of cellular
reactivity and relevant biomarkers. The outcome measures employed in Specific Aim 1 are those that have
been previously validated in our murine CCH model. This allows establishment of baseline levels of
injury secondary to nPM exposure and consistency across experimental groups (Specific Aim 1-3).

2. The proposal extends previous findings into a new research area. Studies have recognized an important
role for PM exposure in the setting of cognitive decline. These effects do not occur in isolation and are
likely modified by other neurological disease processes. It is now critical to assess pathologies that may
contribute to neurotoxicity. Specific Aim 2 establishes a reproducible model for assessing the complex
interplay between PM exposure and chronic cerebrovascular disease. The findings in Specific Aim 3 will
complement the observational studies by addressing mechanisms and biological plausibility

3. The methods aim to be efficient. In Specific Aim 1, we will limit experimental sample size in light of
resources and ethical issues. Once a desired effect is noted, we will only expose mice in the subsequent
exposure durations to nPM (not filtered air), thus decreasing the remaining sample size by fifty percent.
Specific Aim 3 leverages samples obtained from the four exposure groups in Specific Aim 2 to explore
putative mechanisms of injury that are highly relevant to both nPM exposure and CCH. This will greatly
reduce the total number of mice needed for the experiments. Studies conducted for Specific Aim 3
examine inflammatory mediators, byproducts of oxidative stress and regional BBB integrity in cortical
white matter. These experiments are expected to generate valuable nPM neurotoxicity data as previous
studies have primarily focused on oxidative stress pathways/neuroinflammation, largely ignoring
interplay with cerebral microvascular damage. These results may help better understand the neurotoxic
effects of nPM exposure in a susceptible population (CCH).

4. We have assembled a very strong research team of investigators with scientific expertise needed for
this multidisciplinary study. The study combines an innovative means of PM collection/ exposure with a
highly technical surgical model and correlative outcome measures. All procedures and assessment tools
incorporated in this proposal have been utilized successfully by our research team.

5. Dr. Mack’s work and expertise will ultimately allow for future studies of PM and CCH synergy in
relevant translational models. While at Columbia University, he and his mentors established a clinical
carotid endarterectomy model of cerebral hypoperfusion. Their previous research has demonstrated a
roughly 25% incidence of subtle cognitive decline in the absence of overt neurologic change or
radiographic evidence of stroke(Heyer et al., 2002). The studies have demonstrated that neurocognitive
change is associated with elevations in biochemical markers of cerebral injury(Connolly et al., 2001) and
upregulation of leukocytes(Mocco et al., 2006b) and inflammatory mediators(Mack et al., 2008). Risk
factors for cognitive decline included advanced age and diabetes, in addition to the apoE- e4 allele(Heyer
et al., 2005). These putative risk factors parallel those of PM exposure.

D6.B. Limitations and alternate approaches:
The following are the potential limitations to the study and the measures taken to address them:

1. nPM exposure composition: A limitation (across the aims of this study) is the use of re-aerosolized
nPM as opposed to a direct exposure. Our exposure is an aerosol that has substantial similarities in size
and chemical composition to what we typically see in an urban area, at highly increased, but still
environmentally realistic exposure levels. While water soluble species are captured at 100%, insoluble
PM species are not captured as well due to loss of volatiles and semivolatile organic compounds. Clearly,
some toxicity is missed as a result of the lower insoluble PM content. However, our overall effects seen in
vivo and in vitro, at relatively realistic exposure scenarios, are clear. Another advantage of this approach,
which is not available when using concentrators, is a consistent aerosol across the entire exposure period.
This is not a possibility when working with real world PM which varies temporally and spatially.

2. nPM mechanism of brain entry: These experiments do not attempt to distinguish the mechanism by
which nPM exposure affects the brain (e.g.: directly via transport via the olfactory nerve, indirectly
through the systemic inflammatory markers, or indirectly through infiltration of peripheral monocytes)
Our experimental design aims to assess the impact of nPM exposure on white matter injury and
neurocognition and ultimately assess the interaction between nPM exposure and chronic cerebral
hypoperfusion (Specific Aim 2). Future studies will allow us to address route of entry into the brain
parenchyma.

3. nPM exposure duration: We will select the minimal duration necessary to achieve desired white matter
changes in the medial corpus callosum (from Specific Aim 1) for our prescribed exposure in aim 2.
However, Specific Aims 2 and 3 are not contingent upon the results from Specific Aim 1. It is possible that
white matter ischemic changes and/ or neurocognitive decline could be absent, even in the cohort with the
longest exposure latency. The ensuing aims/ experiments are still highly relevant. Mice subjected to
surgically-induced CCH model are presumably more vulnerable to neurovascular damage, making it
possible that the PM toxicity, even if absent in Specific Aim 1, may become measurable in the sensitive
animals. nPM exposure could engender subclinical biochemical changes that, in the setting of BBB
permeability and pro-inflammatory alterations secondary to CCH, might translate to direct neurotoxicity.
This would create a plausible mechanism for synergy in Specific Aim 2. As stated in our second aim, we
hypothesize that increased BBB permeability and inflammatory upregulation secondary to CCH will
augment the toxic effects of nPM exposure. BBB breakdown can enable translocation and entrance of
toxins into the otherwise privileged neurovascular territory. White matter injury secondary to CCH may,
in turn, be enhanced by failure of collateral perfusion in a region already primed by oxidative stress and
pro-inflammatory effects of nPM exposure.

4. nPM exposure timing: The two exposures (nPM/CCH) in Specific Aim 2 will be implemented in
parallel, with the CCH/ sham procedure occurring 30 days prior to the culmination of the nPM/ filtered air
exposure (if the nPM exposure is scheduled for less than 30 days, CCH will be initiated in advance).
Clinically, the two exposures do not always occur in this temporal pattern. However, this exposure
sequence was chosen to optimize both technical feasibility and generalizability. Relevant outcome
measures (white matter injury, neurocognitive outcomes) have been validated in the CCH model at thirty
days. Longer periods of experimental CCH have been shown to affect brain regions outside of the white
matter (cortical tissue, hippocampus). As our CCH model is not reversible, utility of performing the CCH
at an earlier time points is limited (CCH durations would exceed thirty days). This exposure schedule
closely represents the most common clinical paradigm. Generally, individuals are exposed to air pollution
earlier in life and develop cerebrovascular disease at a later time point. Exposure to air pollution typically
persists during the time period that the individual is affected by cerebrovascular disease.

5. Biomarkers/ white matter injury: It is possible that biomarkers selected for examination in Specific Aim
3 will not demonstrate upregulation in the corpus callosum following nPM and/or CCH exposure. This
has not previously been studied. For this reason, we will also assess each of these outcome measures in
the cortical tissue. Further, we will tailor our immunohistochemical analysis to the results of our gene
profiling studies. This will optimize chances of finding the inflammatory biomarker most relevant to our
exposures. It is also possible that the two exposures will not affect the biomarkers in a synergistic manner,
even if they do act synergistically with respect to white matter injury and/or neurocognitive change.

6. Neurocognitive assessment: Y-maze and Novel Object Recognition testing are chosen as outcome
measures for this study as they have both been previously validated in the BCAS model. In Y-maze
testing of spatial working memory tasks, CCH mice made significantly more errors than control mice
following one month of hypoperfusion(Dong et al., 2011; Maki et al., 2011; Washida et al., 2010). While
spatial reference memory tasks are related to cognitive domains that likely rely on hippocampal integrity,
working memory impairment may be attributable to either frontal white matter and/or the hippocampus.
Previous studies employing this CCH model have demonstrated that there is a selective impairment in
spatial working memory, with all other measures of spatial memory intact(Coltman et al., 2011). 8-arm
radial maze testing, which also assesses spatial memory, has been validated in this CCH model(Coltman
et al., 2011; Shibata et al., 2007). Y-maze testing was chosen for the current study as 8-arm radial maze
testing requires extensive training of the mice and, thus, cannot be performed on the same group of
animals assessed for white matter injury. Use of the 8-arm radial maze test would significantly increase
the number of mice needed in Specific Aims 1 and 2. If white matter injury is demonstrated, but no
neurocognitive deficits evident, on Y-maze testing in Specific Aim 1,
consideration will be given to including a cohort for 8-arm radial
maze testing in Specific Aim 2. The principal investigator has
experience with 8-arm radial testing in the CCH model (Figure 12).
Novel Object Recognition testing is classically used in studies of
hippocampal damage/deficits. However, significant differences are
noted in the CCH model, in which structural injury is restricted to the
white matter (unpublished data, see section C). Declarative memory,
tested by Novel Object Recognition, is clearly affected by damage of
commissural structures, albeit not in a direct way. Alterations of this
task can be sensitive to perturbations of white matter insofar as they
connect also the hippocampi and perirhinal cortices and convey
reciprocal information to each of these formations. This study
leverages the ability of Novel Object Recognition testing to reflect
both hippocampal and white matter pathology. Although the
experiments are designed primarily to investigate white matter injury,
nPM-induced neurotoxicity and resulting neurocognitive impairment
may be caused by multiple plausible mechanisms, including neuronal
injury. Novel Object Recognition testing (with hippocampal and
cortical neuropathological analyses) assesses each of these possibilities.

E. POWER ANALYSIS AND SAMPLE SIZE CALCULATIONS
E.1. Specific Aim 1
In Specific Aim 1, we have calculated sample size according to the primary endpoint, white matter injury.
The assumed mean± standard deviation for white matter injury in the CCH model based on pilot data is
0.89 (CCH) vs. 0.30 (sham); pooled SD = 0.445. We believe that the effect size from nPM exposure will
be similar to that of CCH, a mild ischemic insult. Conservatively, we have estimated at 80% of the CCH
effect (table lists sensitivity analysis according to varying predicted percent effect) for the ten week
exposure (duration assessed in our related studies). The effect size will likely vary with exposure
Figure 12: In the 8-arm radial maze working memory
test, BCAS mice showed more total entries and more
reentry errors with each consecutive training session
than sham mice.
duration. To demonstrate the expected mean group difference (two-sided alpha= 0.05, 80% power), each
arm of the study (nPM/ filtered air) would require roughly 15 animals. Total maximum sample size for
this dose escalation exposure aim is 120 mice. However, we will limit experimental sample size in light
of ethical and resource issues. Once a desired effect is noted, we will only expose mice in the subsequent
escalated time groups to nPM (not filtered air), thus decreasing the remaining sample size by fifty percent.
Once an effect is demonstrated, we will continue the nPM exposures, as dose escalation can provide
important toxicity data. Based on prior studies, we do not expect animal deaths due to the nPM exposure.

Aim 1 sample size estimates
nPM Effect Mean Group
Difference
Effect Size (Mean
Diff/SD)
n/group
CCH model 0.59 1.326 10
Percent CCH model:
90% 0.531 1.193 13
80% 0.472 1.061 15
75% 0.442 0.994 17
50% 0.295 0.663 37

E.2. Specific Aim 2
In Specific Aim 2, we will use the same estimates (0.89 vs. 0.30; mean group difference 0.59, pooled SD =
0.445, CCH effect size 1.33) for white matter injury in the CCH/ sham cohort as in Specific Aim 1.
Accordingly, we will assume a more conservative nPM effect size of 1.061 (80% of CCH model – see
above estimates), yielding an estimated white matter injury of 0.772 in the nPM+/CCH-. We expect a
white matter ischemia scale score of 2.0 in the cohort of mice exposed to both nPM and CCH. This yields
an effect size for nPM exposure in the CCH+ cohort that is 2.25-times larger relative to the nPM effect in
the sham-operated cohort. To demonstrate the expected mean group differences of a synergistic effect
between nPM exposure and CCH (two-sided alpha= 0.05, 80% power), roughly 20 animals will be
needed in each cell. A total sample size of 80 mice is therefore required. Accounting for a roughly 10%
mortality in the CCH + cohorts (as a result of the surgery), 88 mice are therefore required for this aim.

Assuming nPM effect size = 1.061 (80% of CCH model – see Aim 1 estimates)

CCH + CCH -
nPM + ??? 0.772
nPM - 0.89 0.30

nPM effect size in CCH +
(relative to nPM effect size in
CCH -)

Mean in CCH+/nPM+ n/group
1.75 x 1.716 51
2 x 1.834 29
2.25 x 1.952 19
2.5 x 2.07 13

nPM+ vs. nPM- mean group difference among CCH- = 0.472 (effect size = 1.061)
CCH + vs. CCH- mean group difference among nPM- = 0.59 (from existing data)
alpha=0.05, 80% power

E.3. Specific Aim 3
In Specific Aim 3, we expect to use tissue obtained from the mice in specific aim 2 for quantitative
immunohistochemistry. These analyses will not require additional exposed mice. Prior CCH experiments
have suggested adequate power to detect a significant difference in reactive astrocytes (GFAP) and
microglia (IBA-1) between CCH and sham-operated mice at the sample sizes calculated in specific aim 2
Specifically, CCH effect sizes (mean group difference in CCH vs. sham, divided by SD) in these
experiments are very large (effect size=2.77 for GFAP and 2.59 for IBA-1). Assuming that the nPM
main effect size will be a somewhat smaller 80% of these (i.e., 2.22 for GFAP and 2.07 for IBA-1), we
calculated the power to detect such group differences, given 20 animals per group. Power to detect the
main effects of nPM and CCH will be greater than 99%. Of more interest to the proposed work, we will
have sufficient power to detect synergistic relationships between CCH and nPM on these variables. We
will have 80% power to detect differences in nPM effects that are 1.6 times higher in CCH+ groups
compared to CCH- groups; we will have 93% power to detect nPM effects that are 1.75 times greater in
CCH+ compared to CCH- groups. We believe differences and effect size will be similar for the other
immunohistochemical biomarkers of inflammation and BBB permeability (MPO, ICAM, IgG, Fibrin).
Real time PCR cytokine and chemokine measurement and MDA assays will require additional brain
specimens as the chemical and structural preparation for analysis is different. Based on prior experience,
we believe that 5 mice per group should be sufficient to provide statistical power for these assays. We
expect a roughly 10% mortality in the CCH+ groups. Therefore, 44 additional mice will be required for
this aim (22 each for the cytokine/ chemokine measurement and MDA analysis).

Timeline
The timeline of proposed major research activities is summarized below by fiscal year

1 2 3 4 1 2 3 4 1 2 3 4
Specific Aim 1
Collection of nPM for Specific Aim 1 X X X X
Exposure for Specific Aim 1 X X X
KB staining/ immunohistochemistry for Specific Aim 1 X X X
Neurocognitive testing for Specific Aim 1 X X X
Statistical Analysis for Specific Aim 1 X
Specific Aim 2
Collection of nPM for Specific Aim 2 X
Exposure for Specific aim 2 X
CCH for Specific Aim 2 X
KB staining for Specific Aim 2 X
Neurocognitive testing for Specific Aim 2 X
Statistical Analysis for Specific Aim 2 X
Specific Aim 3
Collection of nPM for Specific Aim 3 X
Exposure for Specific Aim 3 X
CCH for Specific Aim 3 X
Gene profiling studies Specific Aim 3 X
Immunohistochemistry: Inflammation Specific Aim3 X X X
Immunohistochemistry: Oxidative stress Specific Aim3 X X X
Immunohistochemistry: BBB Specific Aim3 X X X
MDA analysis Specific Aim 3 X
Statistical Analysis for Specifi Aim 3 X
Presentations/ Submissions
Submit/ Present abstract/ manuscripts X X X
Progress Report X X X X X X
FY1 FY2 FY3

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119, 3437-3449.

Abstract (if available)

Abstract The proposed research program seeks to determine the impact of particulate matter (PM) exposure on white matter injury and neurocognitive decline. These associations are further examined in the setting of underlying cerebrovascular disease (chronic cerebral hypoperfusion). Studies have established a strong relationship between PM exposure and atherosclerotic cardiovascular disease. Clinical imaging, epidemiology and pharmacotherapy studies have verified a critical role for cerebral vascular dysfunction in the onset and progression of dementia and cognitive deficits. Investigations suggest a relationship between long-term PM exposure and low cognitive performance, however, very little is known about underlying pathophysiology or putative mechanisms. Our preliminary experimental data suggests an association between PM exposure and white matter injury in the corpus callosum of mice. Cerebrovascular disease may affect this process. Experimental studies examining the effects of air pollution in the setting of cerebrovascular disease are lacking. The proposed investigation utilizes an experimental murine model to address theses knowledge gaps through the following specific aims: 1) To examine the time course of white matter injury secondary to PM exposure. 2) To examine the effects of PM exposure and chronic cerebral hypoperfusion (CCH) on white matter injury and neurocognition and, 3) To examine the impact of PM exposure and CCH on inflammation, oxidative stress and BBB permeability. Urban PM will be collected with a particle sampler situated near the CA 110 Freeway in Los Angeles. Collected aerosols represent a mix of fresh PM, predominantly from vehicular traffic. These samples will then be distilled to nanoparticles and re-aerosolized for administration to mice through exposure chambers. The principal investigator has refined a Bilateral Carotid Stenosis model of CCH which generates reproducible white matter injury and behavioral deficits. A factorial design will be used to assess the independent and combined effects of PM exposure and CCH on white matter injury and neurocognitive decline. When administered together, we expect these exposures to exhibit synergy. Putative mechanisms of injury including inflammation, oxidative stress and blood-brain barrier breakdown, will be examined. The proposed research program utilizes nanoparticulate matter exposures sampled from a near-roadway traffic source, representing real world, multi-pollutant exposures. Scientific knowledge obtained from this study will advance our understanding of the relationship between PM exposure and white matter injury and neurocognitive decline. Insight into the role of underlying cerebrovascular disease will be gained. Results could ultimately impact public policies and regulation with respect to cognitive health in a vulnerable population and provide a critical first step towards individual risk assessment and stratification.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Mack, William Jacob (author)

Core Title Particulate matter from vehicular exhaust in the setting of chronic cerebral hypoperfusion

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical, Biomedical and Translational Investigations

Publication Date 11/05/2015

Defense Date 11/05/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cerebral,hypoperfusion,OAI-PMH Harvest,particulate,stenosis

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zlokovic, Berislav V. (committee chair), Mack, Wendy Jean (committee member), Samet, Jonathan M. (committee member)

Creator Email mackw@usc.edu,wjmack@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-343212

Unique identifier UC11296432

Identifier etd-MackWillia-2132.pdf (filename),usctheses-c3-343212 (legacy record id)

Legacy Identifier etd-MackWillia-2132.pdf

Dmrecord 343212

Document Type Thesis

Format application/pdf (imt)

Rights Mack, William Jacob

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA