Cerebrovascular reactivity: a new frontier for measuring cognitive health in models of accelerated

aging?

David A. Hutton, Alyssa N. Cavalier, Zachary S. Clayton

Cognitive decline naturally occurs with healthy ageing. However, accelerated decline can
have grave implications, as increased cognitive decline is associated with increased risk of
developing Alzheimer’s disease related dementias (ADRDs). Currently, ADRDs are a key research
priority for the National Institutes on Aging. As the United States’ population continues to grow
older, the prevalence of cognitive decline is likely to rise. Studies support a direct correlation
between cerebrovascular function and cognition (Thorin-Trescases et al., 2018). This is plausibly due
to age-related changes in large elastic artery stiffness and vascular endothelial function. In young
adults, large peripheral elastic arteries (e.g., the aorta & carotids) serve to dampen large swings in
arterial blood pressure produced with each heartbeat. This, in turn, slows the velocity of blood as it
flows towards the resistance arteries of the brain. The endothelium and vascular smooth muscle
cells within these resistance vessels then sense this change in pulse pressure, dilating in response
(Thorin-Trescases et al., 2018). However, these mechanisms become impaired with advancing age.

A common hallmark of vascular ageing is increased large elastic artery stiffness. Vascular
hypertrophy derived from lifelong distension and recoil of the vessels induces arterial remodeling,
whereby elastin fibers within the vascular walls become fragmented and are replaced with stiffer
collagen fibers, decreasing the ability of the arteries to absorb changes in pulse pressure (Thorin-
Trescases et al., 2018). Subsequently, pulse pressure increases and impairs vascular endothelial
function, altering the ability of the endothelium to respond to vasodilatory stimuli, such as CO2.
Along with being an independent risk factor for cardiovascular disease, increased arterial stiffness is
associated with increased risk of cognitive decline. One measure of cerebrovascular function which
is sensitive to changes in arterial compliance is cerebrovascular reactivity (CR), or the ratio of
cerebral artery blood flow response to a CO 2 stimulus. Importantly, CR decreases with advancing age
and is directly associated with cognitive decline (Thorin-Trescases et al., 2018). Therefore, CR may
not just be a measure of cerebrovascular function; it may provide insight into current and future
cognitive health.

This is an Accepted Article that has been peer-reviewed and approved for publication in The Journal
of Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an
'Accepted Article'; doi: 10.1113/JP279949.

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 Spaceflight is believed to be an accelerated model of vascular aging. Indeed, data from
different studies support age-related morphological and functional changes to astronaut vasculature
over the course of long duration (6 months) spaceflight missions. For one 6mo mission on the
International Space Station (ISS), echography was used to measure the intima-media thickness (IMT)
in the carotid and femoral arteries of astronauts preflight, inflight, and postflight (Arbeille et al.,
2016). Increased IMT is considered a risk factor for cardiovascular diseases (e.g., atherosclerosis) and
increases progressively with age (Arbeille et al., 2016). For these astronauts, the average increase in
IMT for was 12%. This increase in IMT is indicative of accelerated aging, as a 12% increase in IMT is
equivalent to 20-30 years of vascular aging (Arbeille et al., 2016). Although this increase in IMT is
alarming, it is unclear whether these changes to IMT will persist long-term, as Arbeille et al. (2016)
only noted elevated IMT 4 days postflight.

In another study, Hughson et al. (2016) investigated how spaceflight alters carotid artery
stiffness via carotid artery distensibility coefficient and -stiffness index (two indicators of arterial
stiffness). After 6 months of spaceflight aboard the ISS, all astronauts had a decreased carotid artery
distensibility coefficient (20% change, preflight to postflight) and increased -stiffness index (25%
change, preflight to postflight), indicating increased arterial stiffness (Hughson et al., 2016).
Importantly, this study demonstrated that even with unaffected blood pressure regulation,
spaceflight was able to induce age-related increases in arterial stiffness, equivalent to 10-20 years of
normal vascular aging (Hughson et al., 2016). However, similar to IMT, these changes may be short-
lived, as arterial distensibility rebounded 4 days postflight to similar preflight values (Arbeille et al.,
2017). This recovery suggests that vascular remodeling (e.g. elastin fragmentation and collagen
deposition) likely did not occur over the course of 6mo. Additionally, Hughson et al. (2016) only
performed pre- and postflight measures and, as such, no inferences can be made about whether
functional changes to carotid arterial stiffness occurred during spaceflight.

As the authors of these studies suggest, this increase in IMT and vascular stiffness may be
due to a secondary mechanism, which remains to be elucidated. Although the mechanism driving
these changes is unclear and no firm conclusions can be made about whether these changes persist
longitudinally, results from Arbeille and Hughson suggest that spaceflight-related headward fluid
shift appears to induce age-related changes to astronaut vasculature for the duration of spaceflight.

Along with accelerated vascular aging, long-duration spaceflight (or spaceflight analog) has
been associated with other negative sequelae, such as the development of optic disc edema and
thickening of the retina surrounding the optic nerve head, characteristics of Spaceflight Associated
Neuro-ocular Syndrome (SANS) (Laurie et al., 2020). Similar to accelerated vascular aging, the

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weightlessness and subsequent cephalic fluid shift astronauts experience is believed to contribute to
this syndrome by increasing intracranial pressure (ICP). Additionally, due to confined quarters, most
astronauts are exposed to mildly elevated (3-5 mmHg) ambient CO2 levels (hypercapnia) during long
periods of spaceflight. For Laurie et al. (2020), this chronic exposure is a concern, as some research
has suggested that elevated ambient PCO 2 increases arterial PCO2, which vasodilates the cerebral
arteries and increases ICP (Laurie et al., 2020). Together, this elevation in ambient CO 2 and
headward fluid shift are believed to contribute to spaceflight-related ocular impairments via
increased ICP.

CR can provide information about cerebral artery blood flow velocity and cerebrovascular
conductance (how well blood flows through cerebrovascular circulation at a given arterial blood
pressure), including under extreme conditions such as spaceflight or spaceflight analogs. However,
whether a headward fluid shift and elevated ambient PCO 2 together affect cerebrovascular
hemodynamics is unclear. Thus, Laurie et al. (2020) sought to investigate the relationship between
chronic hypercapnia and 6° head-down tilt bed rest (HDTBR; a common spaceflight analog) on CR. To
test whether mildly elevated PCO2 and HDTBR can change cerebrovascular dynamics, eleven healthy
subjects (5 females, 6 males) were recruited to participate in a 30-day HDTBR study. For the duration
of the study, research subjects remained in a strict, 6° head-down tilt position in a mild hypercapnic
(4 mmHg PCO2) environment. At four different time points during the 30-day HDTBR, middle
cerebral artery blood flow velocity and cerebrovascular conductance were measured to calculate
cerebrovascular reactivity. When the authors compared pre-hypercapnic/HDTBR measurements to
those of post-hypercapnic/HDTBR recovery, they observed no significant changes in CR at any time
point (Laurie et al., 2020).

Even under extreme conditions, CR appears to be unaffected by mildly hypercapnic

environments and chronic HDTBR. Given that a headward fluid shift can increase carotid artery
stiffness and CR is sensitive to changes in arterial compliance, it would logically follow that CR would
decrease over the course of the study. Thus, this null result is rather perplexing. One potential
explanation is that actual spaceflight elicits greater changes in CR than that of spaceflight analogs,
such as HDTBR. Furthermore, a longer latency period may be required to observe significant
augmentation. For a short (1- to 2-week) Neurolab (STS-90) Space Shuttle mission, Iwasaki et al.
(2007) observed improvements in cerebrovascular autoregulation in astronauts (Iwasaki et al.,
2007); however, during long duration spaceflight, this was not the case. When astronauts returned
from a 147 (± 49)-day mission aboard the ISS, astronauts presented with impaired autoregulation
and CO2 reactivity (Zuj et al., 2012). Importantly, for the short and long-duration spaceflight studies,

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changes in cerebrovascular dynamics were only observed postflight, whereas Laurie et al. (2020)
demonstrated no longitudinal change in CR whilst subjects underwent chronic HDBTR and mild
hypercapnia. Moreover, it may be that cerebrovascular dynamics can adapt to an increased ICP
from spaceflight, albeit for short durations. Whether there is a particular time point at which CR
begins to decline during spaceflight is still unclear; however, if feasible, a follow-up study in which
inflight changes to CR were tracked longitudinally aboard a future ISS mission could answer this
lingering question.

Although cerebrovascular autoregulation appeared unaffected by sustained HDBTR and

chronically elevated mild hypercapnia, CR may still be a useful measure for other models of
accelerated aging, such as anthracycline-based chemotherapy. Compared to spaceflight, cancer is a
regular occurrence, with ~1.8 million new cancer cases projected to occur in 2020. Similar to
cognitive decline, cancer risk also increases with advanced age. Anthracyclines are the front-line
chemotherapeutic agent for various forms of common cancers and, sadly, the same mechanisms
that make anthracyclines such effective chemotherapeutics are also the reason they are considered
models of accelerated ageing. Similar to ageing adults, patients who undergo anthracycline-based
chemotherapy also experience an ADRD-like form of cognitive impairment.

More colloquially known as “chemo brain,” patients who undergo anthracycline treatment
exhibit cognitive impairments normally associated with advancing age, such as memory and learning
deficits. Although anthracyclines are considered accelerators of ageing in multiple cell types, the
effects of anthracyclines on measures of cerebrovascular function (i.e., CR) are understudied. Given
that CR decreases with advancing age and is associated with cognitive decline, understanding how
CR is influenced by anthracyclines may allow us to understand the underlying mechanism(s) behind
anthracycline-associated cognitive decline.

Previous studies suggest that anthracyclines increase reactive oxygen species (ROS) and
inflammation in multiple cell types. Excessive oxidative stress and inflammation can lead to
increased large elastic artery (aorta) stiffening, which can elevate systolic blood pressure. Indeed,
many studies support that, increased arterial stiffness and high blood pressure can increase the risk
of cognitive decline (Thorin-Trescases et al., 2018). Given that CR is highly dependent on arterial
elasticity, it may be that anthracycline-induced ROS and inflammation is affecting cerebrovascular
function, ultimately contributing to cognitive impairment.

To explore the relationship between anthracycline-derived chemo brain and cerebrovascular

function further, certain clinical and preclinical outcomes for oxidative stress, inflammation, vascular

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health, and cognitive function could be measured. To identify anthracycline-related changes,
physiological phenotyping could be performed on survivors of anthracycline-based chemotherapy
and compared to age-matched, untreated controls. Ideally, this would be done a few ways. Cultured
cerebrovascular smooth muscle cells (cVSMC) would be exposed to plasma from patients and
controls; from there, antioxidant enzyme (e.g., superoxide dismutases [SODs], catalase) abundance
could be measured. Since excessive ROS is generally accompanied by a compensatory increase in
expression of antioxidant enzymes, this measure could serve as a surrogate marker of oxidative
stress. Additionally, following plasma exposure, these cVSMC could be washed and cultured again to
collect the cultured media, which could be used to determine how plasma exposure influences
cytokine (IL-1 , TNF, etc.) secretion from these cells. Finally, atomic force microscopy could be
performed on these cVSMC to see if increases in ROS and inflammation from anthracycline patient
plasma increase cellular stiffness.

To correlate these changes to cerebrovascular function, these assays would be accompanied

by complete macrovascular phenotyping, including carotid-femoral pulse wave velocity (cfPWV; a
measure of large elastic artery stiffness) and blood pressure. Additionally, cerebral blood flow
(surrogate measure to total brain blood flow) and CR (flow response to a stimulus [i.e., CO 2], as
described in Laurie et al., 2020) would provide a comprehensive measure of the cerebral
vasculature. Lastly, cognitive function would be measured using the NIH toolbox, a validated
modality to measure cognitive function.

To gain mechanistic insight, preclinical studies could then reverse translate these clinical
observations to a mouse model. Three groups could be used: old control (as a reference group),
young control, and young (age-matched) treated mice receiving a dose of anthracycline (e.g.,
Doxorubicin/Adriamycin). Ideally, measures of arterial stiffness (in vivo aortic pulse wave velocity, ex
vivo intrinsic mechanical stiffness [elastic modulus] of the arterial wall), and cognitive function
(Barnes maze and novel object recognition) would be performed. Unfortunately, CR cannot be
measured in mice, per se, which would be a limitation in the translatability of preclinical findings.

Since the hippocampus is the memory center of the brain and is thought to be impacted by
anthracyclines, the results from the vascular phenotyping could then be correlated to hippocampal
neuroinflammation. This could be done by measuring ROS and inflammatory cytokine abundance in
middle cerebral artery via electron paramagnetic resonance (EPR) and ELISA [IL-1 , TNF, etc.]),
respectively. Another measure of hippocampal neuroinflammation that may be employed is
hippocampal transcriptomics, whereby gene expression related to cognition and learning/memory
could be measured in old, young, and young anthracycline -treated mice. The hippocampal

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transcriptome in the anthracycline mice could then be compared to the old (reference) group of
mice, which could validate that anthracycline chemotherapy can be a model of brain aging. Finally,
an additional measure would be anthracycline accumulation within peripheral and neural tissues.

Currently, results are mixed about whether therapeutic doses of anthracyclines cross the
blood-brain barrier. Anthracyclines such as Doxorubicin/Adriamycin are rapidly cleared from the
blood within 24-72h, making them difficult to measure in circulation. Thus, performing these
measures in peripheral tissues could further elucidate whether anthracyclines act in a direct or
systemic fashion, potentially providing therapeutic targets. Hypothetically, if the deleterious
phenotypes observed in chemo brain are effects derived from peripheral insults (e.g., oxidative
stress and inflammation in vascular smooth muscle cells), treatment with therapeutics such as
exogenous antioxidants (e.g., ascorbic acid) or anti-inflammatories (e.g., NSAIDs) may provide
feasible modalities to prevent or ameliorate the downstream phenotypes of chemo brain.

In conclusion, CR as an in vivo measure of cognitive function may help to elucidate how

anthracyclines contribute to cognitive decline in cancer patients. As industrialized societies continue
to age, the prevalence of cognitive decline, cancer, and anthracycline-induced cognitive impairment
are likely to rise. A primary risk factor for cancer development is ageing and, with projections of
close to two million new cancer cases in 2020 alone, CR may become an essential measure within
this clinical population. Although no changes were observed in subjects undergoing chronic
hypercapnic conditions in the Laurie et al. (2020) study, it may be that some modalities are more
potent evokers of change in CR than others. Age-accelerating anthracyclines may be one of those
potent stimuli and, as such, should be the subject of future investigations.

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This article is protected by copyright. All rights reserved.