Cancer Treatment Reviews 69 (2018) 72–83

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Cancer Treatment Reviews

journal homepage: www.elsevier.com/locate/ctrv

Complications of Treatment

Chemotherapy and cognition: International cognition and cancer task force T

recommendations for harmonising preclinical research
⁎
Gordon Winocura,b,c, , Ian Johnstond, Hélène Castele,f,g
a
Rotman Research Institute, Baycrest Centre, Toronto, Canada
b
Department of Psychology, Trent University, Peterborough, Canada
c
Departments of Psychology and Psychiatry, University of Toronto, Toronto, Canada
d
School of Psychology, The University of Sydney, Sydney, Australia
e
Normandie Univ, UNIROUEN, INSERM, DC2N, 76000 Rouen, France
f
Institute for Research and Innovation in Biomedicine (IRIB), 76000 Rouen, France
g
Cancer and Cognition Platform, Ligue Nationale contre le Cancer, Caen, France

A R T I C LE I N FO A B S T R A C T

Keywords: Cancer survivors who undergo chemotherapy for non-CNS tumours often report substantial cognitive dis-
Chemotherapy turbances that adversely affect quality of life, during and after treatment. The neurotoxic effects of anti-cancer
Cancer drugs have been confirmed in clinical and pre-clinical research. Work with animals has also identified a range of
Preclinical research factors and underlying mechanisms that contribute to chemotherapy-induced cognitive impairment. However,
Cognitive function
there is a continuing need to develop standard cognitive testing procedures for validation and comparison
purposes, broaden the search for biological and neurochemical mechanisms, and develop improved animal
models for investigating the combined effects of treatment, the disease, and other potential factors (e.g., age,
stress). In this paper, a working group, formed under the auspices of the International Cognition and Cancer Task
Force, reviews the state of pre-clinical research, formulates strategic priorities, and provides recommendations to
guide animal research that meaningfully informs clinical investigations.

Introduction investigating the effects of chemotherapy on cognition in animal

Significant advances in cancer treatment in recent years have en-
abled cancer survivors to live longer and with improved prospects for
better quality of life. At the same time, increased survival rates have
focussed attention on side effects of powerful drugs that can undermine
day-to-day function. In particular, a substantial number of cancer pa-
tients receiving chemotherapy report cognitive disturbances that in-
clude attentional problems, memory loss, and confused thought pro-
cesses (‘chemobrain’ or ‘cognitive fog’), that are often accompanied by
mood disorders and fatigue see [1–5]. Although patient complaints of
chemotherapy-induced cognitive impairment (CICI) have been sup-
ported by neuropsychological investigations, their importance was not
fully appreciated when considered in the context of living with a life-
threatening disease. Moreover, when the condition was acknowledged,
there was uncertainty over the extent to which cognitive deficits should
be attributed to changes in brain function, or to the psychological
challenges of coping with cancer and a difficult treatment.
The developing controversy underscored the need for preclinical
research and, by the mid-2000s, labs around the world were
models see [6]. Very quickly, studies confirmed that commonly used
anti-cancer drugs reliably produced cognitive impairment in healthy
adult rodents [e.g., 7–11]. Significantly, the most frequently reported
deficits were related to attentional processes, learning, memory, and
executive function – essentially the same pattern as that observed in
clinical investigations. These findings undermined the argument that
cognitive impairment is secondary to stress-related factors and pointed
to a direct toxic effect on neurocognitive function. As well, through
preclinical research, there have been significant advances in our ap-
preciation of lost and spared cognitive abilities, recovery over time,
biological mechanisms, as well as associated non-cognitive abnormal-
ities (e.g., fatigue, emotional reactivity).
The International Cancer and Cognition task Force (ICCTF), re-
cognizing the importance of preclinical research for advancing the field,
determined that the time is right to examine progress made by animal
researchers, identify priorities for future directions, and develop stan-
dardized experimental procedures. As a first step, a meeting of clinical
scientists and basic researchers was convened at its 2016 conference in
Amsterdam, from which emerged a broad consensus that a coordinated

⁎
Corresponding author at: Rotman Research Institute, Baycrest Health Sciences, 3560 Bathurst St, Toronto, Ontario M6A 2E1, Canada.
E-mail address: gwinocur@research.baycrest.org (G. Winocur).

https://doi.org/10.1016/j.ctrv.2018.05.017
Received 22 February 2018; Received in revised form 30 May 2018; Accepted 31 May 2018
0305-7372/ © 2018 Elsevier Ltd. All rights reserved.
G. Winocur et al.
Table 1
Common tests used in preclinical research to assess chemotherapy effects on cognitive functions mediated by hippocampus or frontal lobes.
Cognitive function Test
Hippocampus – sensitive Spatial memory Morris water maze
Novel location recognition
Barnes maze
Context memory Context fear conditioning
Conditioned emotional
response
Recognition memory Object recognitionπ
Internal inhibition Passive avoidance
Retroactive interference
Frontal lobe-sensitive Rule learning Operant nose-poke
Non-matching-to-sample
Temporal order Temporal familiarity
Conditional learning Conditional associative
learning
η
Normal spatial learning and recent memory.
π
Impaired remote spatial memory.
λ
Hyper-reactivity to novelty interpreted as defect in flexibility.
ϕ
Normal recognition memory.
and collaborative research effort was needed. The result, at the in-
vitation of the ICCTF executive, is the following review which includes
a formulation of strategic priorities and specific recommendations to
guide animal research as it strives to complement and meaningfully
inform clinical science and practice. See [5] for a similar review of
clinical research].
Cognitive assessment
Recommendation: As a validation of new research programs into CICI
in animals, initial investigations should include reliable tests of memory
and executive function in accordance with standard operating proce-
dures. It is further recommended that subsequent investigations within
the program include at least one such reliable test.
Clinical neuropsychological studies and preclinical research into the
behavioural effects of anti-cancer drugs have converged in identifying a
reliable pattern of core cognitive deficits that reflect dysfunction in
hippocampal and frontal-lobe brain regions [6,12–15]. Table 1 provides
a summary of tests commonly used in animal research to assess the
effects of chemotherapy on cognitive functions mediated by these re-
gions. As a validation of research strategies, particularly as they relate
to new programs, and to ensure meaningful comparison of results
across labs, it is strongly recommended that investigators include, in
their test batteries, a hippocampus-sensitive test of memory and a test
of executive function that depends on the frontal lobes. It is further
recommended that subsequent investigations within the program in-
clude at least one such validation measure.
The Morris Water Maze (MWM) test of spatial memory, contextual
fear conditioning (CFC), and location and object recognition memory
tests are widely accepted tests of hippocampal memory. These straight-
forward tests are well-characterized, and can be administered re-
peatedly. The latter is particularly important when longitudinal as-
sessment is desirable, or when potential treatments are being evaluated
and it is necessary to compare performance before treatment, im-
mediately after, and over the long term.
The MWM [16] is a test of context-dependent spatial memory that is
conducted in a circular pool filled with opaque water. The animal is
required to use environmental cues to learn and remember the fixed
location of a submerged platform on which it can climb and escape the
water. CFC is a test of non-spatial memory in which an association is
formed between a shock-induced fear response and the configuration of
Cancer Treatment Reviews 69 (2018) 72–83
References
Dubois et al. [22]η,π, [23]η; Fardell et al. [7,24]; Rendeiro et al. [55]; Seigers et al. [20]; Winocur
et al. [11,15,57,58,89]
Acharya et al. [126]; Christie et al. [127]; Li et al. [79]; Lyons et al. [25,128]; Mustafa et al. [9];
Seigers et al. [117]; Callaghan & O’Mara [40]; Salas-Ramirez et al. [42]; Kitamura et al. [129]
Seigers et al. [117]
Acharya et al. [126]; Christie et al. [127]; Fremouw et al. [130]; Macleod et al. [21]; Seigers et al.
[20,117]
ElBeltagy et al. [90]
Acharya et al. [126]; Dubois et al.[22,23]λ; Fardell et al. [7,24,131]; Fremouw et al. [130]ϕ;
Seigers et al. [117]; Yang et al. [132]; Callaghan & O’Mara [40]ϕ
Konat et al. [91]; Yang et al. [132]
Winocur et al. [56]
Walker et al. [118]
Winocur et al. [11,15,57,58,61,89]
Acharya et al. [126]
Winocur et al. [15]
contextual cues in the test environment [17]. The location recognition
memory test [18] compares animals’ responses to identical objects in
familiar or new locations, while the object recognition memory test
[19] compares responses to familiar and unfamiliar objects. Animals
exhibit recognition memory when they spend more time exploring
unfamiliar locations or objects, respectively. Rats and mice with hip-
pocampal dysfunction are reliably impaired on all three tasks and,
significantly, similar deficits on the MWM [11,20], CFC [21], and lo-
cation and object recognition tests [22–25] have been reported in ro-
dents administered anti-cancer drugs1. Standard operating procedures
for these tasks and others to follow are provided in Appendix A.
Frontal lobe impairment is associated with disruption of a wide
range of attentional and executive functions. In the animal literature,
such processes are often measured by tests of non-matching-to-sample
(NMTS) and conditional associative (CAL) learning. Both tasks in-
corporate working memory and rule-learning components in success-
fully making goal-oriented decisions. The NMTS test consists of a series
of paired trials in which, initially, a distinctive stimulus signals reward,
followed immediately by a test trial in which the familiar stimulus is
paired with a novel stimulus. On the test trial, the animal must respond
to the new stimulus to obtain reward. In CAL, the animal is trained to
associate different stimuli with different responses to obtain rewards.
Animals with frontal lobe damage are impaired on both tasks [26,27].
Developed as land-based tests, they have been adapted for use in a
water-filled pool in which animals are highly motivated to perform.
These versions require less training than land-based tests and reliably
yield deficits in chemotherapy-treated rodents [11,15]; see Appendix A
for standard operating procedures].
Assessment of non-cognitive responses
Recommendation: Preclinical research into CICI should be broadened
to include greater assessment of non-cognitive responses (e.g., stress,
emotional reactivity) to drugs.
Cancer patients undergoing chemotherapy must cope with many
challenges. Not surprisingly, therefore, they often exhibit signs of
emotional instability that adversely affect their quality of life. Clinical
1
As noted in Table 1, the effects of chemotherapy on the object recognition test are
somewhat more variable than the new location test.
73
G. Winocur et al.
investigators have argued justifiably that such responses contribute to
impaired cognitive function in this population. Indeed, current thinking
acknowledges that ‘chemobrain’ is multifactorial and impacted by
clusters of symptoms that include heightened stress, fatigue, mood al-
terations, and sleep disturbances, amongst others [28,29]. Che-
motherapeutic agents themselves can produce many of these non-cog-
nitive symptoms, which can interact with the drugs’ effects on cognitive
systems, and possibly through common neural pathways [30,31].
Preclinical research can play an important role in achieving a better
understanding of co-occurring symptoms associated with che-
motherapy, and their relationship to cognitive performance. In rodents,
emotionality and stress-related responses have been tested reliably in
the elevated plus maze [32,33]. This test evaluates stress avoidance by
requiring the animal to choose between secure parts of the maze (en-
closed arms) and aversive parts (open arms). Anxiety-related behaviour
is assessed by the number of entries and time spent in the open arms,
relative to the enclosed arms. Other reliable tests of emotional re-
activity in rodents include the sucrose preference test [34], a functional
measure of reward systems that models anhedonia, and the forced swim
test [35], which models depressive-like behaviour by measuring im-
mobility in inescapable stressful situations. The tail suspension [36] is a
common alternative to the forced swimming test, but it is more suitable
for use with mice than rats. These and other tests commonly used to
measure emotional reactivity are cited in Table 2. Animal researchers
have begun to use some of these tests and the general finding has been
that drug-induced cognitive deficits are often accompanied by increases
in anxiety or depressive-like behaviour e.g., [37–40]; see Appendix A
for standard operating procedures].
Fatigue, possibly related to loss of muscle strength, sleep dis-
turbances, or over-exertion, is a common complaint of cancer survivors
following chemotherapy. Fatigue in rodents can be assessed in
straightforward ways by measuring activity in their home cages, open
field environments, or running wheels. For example, reductions in
spontaneous locomotion and running wheel activity have been reported
in mice treated with paclitaxel [41], and in rats receiving a combination
of cyclophosphamide and doxorubicin [42].
It is worth mentioning an ingenious test of ‘behavioural economics’
developed by Salamone and colleagues [43] to measure fatigue and
effort-related symptoms. In this task, the rodent is placed in an operant
conditioning chamber where it can obtain a desirable food reward by
pressing a lever a fixed number of times. Alternatively, it can directly
access less palatable lab chow with minimal effort. The degree of effort
the animal is prepared to put forth to obtain reward can be determined
by varying the lever-press/reward ratio. When confronted with a
choice, well-trained normal rats will get most of their food by lever-
pressing for the desirable food pellets, and will consume relatively little
lab chow. Animals that are less motivated to work for food, as a result
of stress or emotional unresponsiveness, prefer the freely available, but
less palatable, lab chow. This test has the added advantage of tapping
into related affective processes, such as motivation and attaching value
to rewards, that factor into symptom clusters that contribute to CICI.
While adapted for use in animal models of anhedonia and related
conditions [44], it has not been used in a model of chemobrain, al-
though it appears well suited for this purpose (see Appendix A).
Table 2
Common tests used in preclinical research to assess emotional reactivity.
Behaviour Test
Emotionality, anxiety, conflict resolution Elevated plus maze
Light-dark emergence test
Tail suspension test
Stress, depressive behaviour Forced swim test
Sucrose preference test
Impulsive behaviour Resident intruder aggression test
Compulsive behaviour Marble burying test
74
Cancer Treatment Reviews 69 (2018) 72–83
Mechanisms of CICI
Biological
Recommendation: Animal investigators are urged to broaden the
search for biological mechanisms (e.g., neuroinflammation, HPA-axis
system dysfunction, mitochondrial damage), underlying CICI.
The identification of biological mechanisms that underlie CICI is
essential to [1] understanding the full effects of the drugs, [2] estab-
lishing causal links between such changes and treatment-related cog-
nitive deficits and [3] the development of cognitive treatment strate-
gies. While the search for causal links is still in early stages,
considerable progress has been made in recent years [28,45,46]. To a
large extent, attention has focussed on neuroinflammation, with nu-
merous clinical studies pointing to an association between the release of
pro-inflammatory cytokines (e.g., IL-1, IL-6, TNF-α) and behavioural
symptoms (e.g., mood changes, sleep disturbances, cognitive impair-
ment, fatigue) that have come to be identified with CICI. There is also
evidence that elevated cortisol levels and general malfunction of the
HPA axis system contribute to a similar range of symptoms. Biological
changes associated with disruption of the two systems interact with
each other and there is growing consensus that chronically high stress
levels associated with cancer and treatment commonly trigger both sets
of responses [47].
Other potential biomarkers of CICI include disruption of the
monoamine neurotransmitter system [48], mitochondrial impairment
and oxidative stress [49], and changes in brain structure [50] and
function [51] that may reflect disruption of functional brain networks
[52]. Recently, attention has been drawn to chemotherapy-induced
changes in cerebrospinal fluid (CSF) compounds (e.g., phospholipids,
lysophosphatidyl-acetylcholine) that serve as markers of white matter
integrity and, potentially, indicators of cognitive impairment. Altera-
tions in CSF resulting from chemotherapy have also been associated
with significant increases in tau production which are reliably linked to
neurodegenerative disease [51]. Many of these biological changes (e.g.,
neuroinflammation, CSF and mitochondrial disruptions) have been as-
sociated with both chemotherapy and tumour growth, reinforcing the
notion that the search for mechanisms underlying CICI must be con-
sidered in terms of their relationship to multiple factors.
Relationships between biological changes and cancer-related cog-
nitive impairment are being vigorously pursued in clinical investiga-
tions. As Seigers and Fardell [6] pointed out, preclinical research is also
essential to this process and, in at least one important area, has led the
way. Animal studies have shown that anti-cancer drugs suppress the
production of new cells and their functional integration into the adult
hippocampus [neurogenesis – 20,23,53–56]. This significant finding
helps to explain the loss of hippocampus-dependent memory and re-
lated functions that are reliably observed following chemotherapy. This
relationship was confirmed in complementary animal studies showing
that recovery from chemotherapy-induced memory loss following
physical exercise [57] and environmental enrichment [58] is positively
correlated with restoration of new cell production.
Preclinical research has linked CICI to increased cell death and al-
terations in synaptic plasticity in specific brain regions (e.g.,
References
Lister et al. [32]; Pellow et al. [33]
Smith et al. [133]; Kitamura et al. [129]
Can et al. [134]; Steru et al. [36]
Can et al. [135]; Porsolt et al. [136]; Callaghan & O’Mara [40]
Willner et al. [34]; Krishnan et al. [137]; El Yacoubi et al. [138]
Mineur et al. [139]
Nicolas et al. [140]
G. Winocur et al.
hippocampus, prefrontal cortex), as well as mitochondrial damage,
neuroinflammation, and HPA-system dysfunction [6,59]. The meth-
odologies for assessing co-varying changes in cognitive and biological
systems are clearly available. For many labs, the challenge will be to
formulate multi-disciplinary, comprehensive research programs that
take into account multiple operative components (e.g., psychosocial,
cognitive, neurobiological) and enable meaningful distinctions between
correlational and causative relations.
Cognitive
Recommendation: Animal scientists should intensify the investiga-
tion of basic cognitive mechanisms (e.g., attentional processes) asso-
ciated with broad categories of behavioural symptoms resulting from
chemotherapy.
Research into basic cognitive operations that are affected by anti-
cancer drugs has lagged behind the search for biological mechanisms. It
is clear from subjective reports and neuropsychological investigations
that, following chemotherapy, cancer survivors have difficulty focusing
and sustaining attention. This condition, sometimes termed, ‘atten-
tional fatigue’ [60], is manifested in various ways including heightened
distractibility, repetitive thought processes, and poor multi-tasking.
These symptoms suggest a breakdown in an internal regulatory system
that, in situations involving competing demands, serves to inhibit in-
fluences that interfere with performing the task at hand. Damage to
such a system could account for many, if not all, of the cognitive deficits
exhibited by such individuals.
Winocur et al. [56] tested the hypothesis that chemotherapy induces
a state of internal disinhibition and exaggerated susceptibility to in-
terference in rats. A retroactive interference paradigm was used in
which animals were initially trained on a discrimination task and, fol-
lowing an interpolated high- or low-interference experience, were re-
tested on the same task. Chemotherapy and control groups did not
differ in original learning, but drug-treated rats in the high-interference
condition were severely impaired in relearning the discrimination. The
deficit was correlated with suppression of neurogenesis levels, a finding
in line with reports of hippocampal involvement in this and other high-
interference tasks [61]. Given the pervasiveness of attentional deficits
in patients receiving chemotherapy, it is likely that other brain regions
are involved as part of a complex neural circuitry. Indeed, Wimmer
et al. [62], using a cross-modal divided attention task designed for
mice, identified a network involving the thalamic reticular system,
under prefrontal cortical control, that appears to support information
filtering in high-interference situations.
Inhibitory control is a complex process that involves several fun-
damental components. In addition to sensory gating, as demonstrated
by Wimmer et al. [62], the ability to evaluate conflicting information
and exercise response control is required, and chemotherapy could
adversely affect any or all of these operations. In an initial attempt to
address these questions, Winocur (in preparation) examined the effects
of chemotherapy in three passive avoidance tasks. Each task had an
essential inhibitory component but they differed in critical ways. One
was a basic test of inhibitory avoidance in which the animal had to
suppress the tendency to step down from a platform onto a grid floor
that delivered a foot shock; a second task, conducted in a runway, re-
quired the animal to suppress a learned approach response to avoid
shock; the third task was similar to the second but, before testing, the
animal was cued with the distinctive features of the shock-associated
goal box. Chemotherapy-treated rats exhibited normal response in-
hibition in tasks 1 and 3 but were impaired on task 2, indicating that
the problem was likely related to a deficit in processing competing
information associated with approach and avoidance responses.
These findings provide direction for further investigation of the
nature of chemotherapy-induced attentional deficits. It has also been
suggested that the cognitive aging process offers a model for CICI [2,63]
and that generalised cognitive slowing can account for some of the
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Cancer Treatment Reviews 69 (2018) 72–83
observed deficits (e.g., skill acquisition, problem solving). With respect
to memory loss, chemotherapy affects autobiographical episodic
memory, especially when it is necessary to suppress related, but in-
appropriate, memories that arise during the retrieval process. However,
memory for semantic or schematic information (general knowledge,
well-learned habits and routines) has not been investigated to the same
degree. This type of memory entails other forms of processing and re-
presentation, and it is not clear how it is impacted by anti-cancer drugs.
Tasks for addressing such issues are available, or can be developed,
thereby enabling preclinical research to play a central role in describing
fundamental cognitive processes that are directly affected by che-
motherapeutic agents.
Risk/protective factors and treatment
Recommendation: Preclinical researchers should attach priority to
identifying risk factors for CICI and contributing to the development of
interventions aimed at minimizing cognitive loss following treatment.
In their review of candidate mechanisms of chemobrain, Ahles and
Saykin [1] outlined various genetic variabilities that could increase the
risk of cognitive impairment following chemotherapy. Particular at-
tention has focussed on Apoliprotein-E (APO-E), a class of apoliproteins
that is responsible for transporting cholesterol and other fats through
the bloodstream. Individuals carrying an APOE-4 allele are at increased
risk for age-related cognitive decline and dementia of the Alzheimer’s
type [64]. Ahles et al. [65] found that cancer survivors with the APOE-4
allele experienced more cognitive impairment following chemotherapy
than those who did not. Other genetic polymorphisms involving, for
example, the catechol-o-methyltransferase gene have also been linked
to cognitive deficits in chemotherapy-treated cancer patients [66].
A number of psychosocial and lifestyle variables influence the re-
sponse of cancer survivors to chemotherapy. Cognitive reserve refers to
the brain’s capacity to draw on enriching experiences during youth to
protect against later assaults on neurocognitive function [67]. Built up
through stimulating experiences, such as education and environmental
enrichment, cognitive reserve enables compensatory mechanisms to
reduce the damaging effects of neurodegenerative disease, traumatic
brain injury, and vascular damage. Ahles et al. [2] found that low
cognitive reserve, as measured by the Wide Range Achievement Test,
was a major factor in the poor performance on standard neuropsycho-
logical tests by breast cancer patients who had received chemotherapy.
In the same study, Ahles et al. [2] found that the effects of che-
motherapy on cognitive performance were exacerbated in older parti-
cipants. Age is known to affect recovery from cancer and, in particular,
acute lymphoblastic leukemia (ALL), which occurs predominantly in
children under the age of five and adults over 50. Five-year survival
rates following intrathecal treatment that prevents spread of disease or
neurotoxic effects of other forms of treatment (e.g., cranial radio-
therapy) to the brain, are as high as 80% in children but only between
30% and 40% in adults [68]. These are significant findings in that age is
the single most important risk factor for the development of many
forms of cancer. The aging brain is susceptible to insult so the combined
effects of disease, treatment, and age are likely to produce exaggerated
cognitive deficits. It is surprising, therefore, that there has been so little
research into this question. Among the few studies, a large prospective
trial of older breast cancer patients found that cognitive decline fol-
lowing chemotherapy was greatest in the oldest individuals, particu-
larly when the regimen included docetaxel [69]. In another study also
involving breast cancer patients, cognitive function recovered more
slowly in older patients [70]. An appreciation of the impact of aging is
needed to better understand the dynamics of CICI and is crucial for its
prevention and treatment.
Physical activity is widely advocated in promoting brain health and
in protecting against cognitive losses resulting from various conditions,
including stroke and normal and pathological aging. Cancer survivors,
who engage in regular exercise, consistently experience better physical
G. Winocur et al.
and mental health, longer survival times, superior cognitive perfor-
mance, and overall higher quality of life [71–76].
As indicated above, stress associated with a cancer diagnosis is a
contributing factor to CICI. Cancer patients are prone to post-traumatic
stress following diagnosis [77] and at least one study has demonstrated
that this form of stress exacerbates the cognitive impairment subse-
quently experienced by breast cancer patients following chemotherapy
[78]. It follows that interventions aimed at reducing post-traumatic
stress could ameliorate behavioural symptoms resulting from treat-
ment.
Most of the risk factors identified in clinical studies are being in-
vestigated in preclinical research, and with similar results. For example,
following chemotherapy, rats, like humans, perform better on cognitive
tests when allowed to engage in physical activity [24,57]. Similarly,
chemotherapy-treated rats reared in diverse environments that provide
opportunities for social stimulation and new learning, exhibit less
cognitive impairment than similarly treated rats housed in standard lab
cages [58].
In a recently completed study, Winocur et al. (in preparation) ex-
amined the effects of building up cognitive reserve and chemotherapy
in a transgenic model of breast cancer (see next section for a description
of the model). At the age of three months, transgenic and control mice
were trained on a battery of cognitive tests and then, six months later,
after tumours had developed and the mice received chemotherapy, a
new set of tests was administered. Tumour growth and chemotherapy
were each associated with cognitive impairment in mice not receiving
early cognitive training. At test, all the groups in the early training
condition (tumourigenic mice receiving saline, chemotherapy-treated
normal mice, and tumourigenic mice that had received chemotherapy)
out-performed their counterparts that had not received training. The
results confirm the benefits of early enrichment and point to cognitive
reserve as a powerful protector of neurocognitive function from the
later effects of peripheral cancers and chemotherapy treatment.
There has been little preclinical research into age as a factor in CICI.
In a notable exception, Dubois et al. [23] compared the impact of
chemotherapy on cognitive function in young (8 weeks) and aged
(20 months) mice in relation to the proliferation of hippocampal neural
stem cells (NSC). An interesting result was that age-related emotional
and cognitive deficits were associated with a significant decrease of
hippocampal-dividing NSC. Chemotherapy equally affected the pool of
NSC in both young and aged mice, but there was evidence that di-
minished cognition in the aged animals affected their ability to with-
stand the drug-induced effects. Recently, Winocur (in preparation) ex-
amined the effects of chemotherapy and aging in young (6 months) and
older (16 months) rats on cognition. Age and chemotherapy each con-
tributed to cognitive impairment but the combined effects were ex-
ponentially greater than the singular effects. The limited evidence from
animal studies is consistent with the view that normal aging exacer-
bates the neurotoxic effects of chemotherapy, and that the two factors
interact with each other.
Similarly, there has been little investigation into the effects of
chemotherapy on developing brains. One study examined the potential
for early life low-dose chemotherapy to induce long-term cognitive
impairments. Li et al. [79] administered low-dose methotrexate (MTX;
1 mg/kg) to juvenile rats beginning just after weaning (post-natal day
16) for 8 weeks. When tested as adults, MTX-treated rats demonstrated
impairments in location recognition tests but not in object recognition
tests. Moreover, analysis of CSF showed suppressed folate persisting for
at least 3 weeks after treatment. These results point to the value of
preclinical models in examining the effects of anti-cancer drugs on the
cognitive development of children, and the potential effects on life-long
cognitive function
The discovery of risk/protective factors is a reminder that cancer-
related cognitive impairment is not immutable and that, even when
spontaneous recovery is limited, interventions are possible. The degree
of impairment in cancer survivors is usually described as mild to
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Cancer Treatment Reviews 69 (2018) 72–83
moderate, suggesting that such individuals may be good candidates for
cognitive rehabilitation programs. Few such programs have been de-
signed specifically for this population but there have been promising
developments. For example, Ferguson et al. [80] have developed a
behavioural training program (Memory and Attention Adaptation
Training – MATT) in which patients learn compensatory strategies for
improving cognitive function and coping with the challenges of daily
living. In a trial involving breast cancer patients, these investigators
reported that training resulted in significant improvement on various
measures of cognitive performance and quality-of-life. Wolf et al. [81]
developed a metacognitive strategy training program (MCST) that tar-
gets primarily executive function. In a preliminary pre-post, within-
subjects study of 14 breast cancer patients, the group receiving training
improved significantly on several cognitive tests including the Cogni-
tive Failures Test, the Dysexecutive Questionnaire, and the Delis-Kaplan
Executive Function Test, as well as several psychosocial measures. It is
noteworthy that behavioural improvements were accompanied by en-
hanced functional connectivity in 6/10 patients who underwent func-
tional neuroimaging. Kesler at al. [82] also reported significant cogni-
tive recovery in breast cancer patients who underwent training that,
like the MCST, targeted executive functions.
These studies represent progress but, in absolute terms, the ob-
served benefits were modest. As well, it is not clear if they were long-
lasting or how well they generalised to real-world situations.
Nevertheless, the results provide direction and a foundation for a
broader approach in developing interventions for cancer survivors. For
example, the results of clinical and animal studies involving physical
activity and social stimulation point to the potential gains of in-
corporating exercise regimens and techniques for enhancing lifestyle
into rehabilitation programs that emphasize the application of atten-
tional and information-processing strategies.
No pharmacotherapies have been approved specifically for CICI
although several drugs have been tested [83,84], with mixed results.
For example, the stimulant, modafinil, was shown to reduce fatigue and
improve cognitive function in breast cancer patients treated with che-
motherapy [85], but another stimulant, methylphenidate, was not ef-
fective [86]. As noted above, patients with ALL often receive intrathecal
treatment prophylactically to protect the brain but cognitive loss is
known to occur whether or not cranial radiation is also administered.
There is clinical evidence that the glutamate receptor antagonist, dex-
tromethorphan, can counteract the secondary effects of intrathecal
MTX treatment [87] and, in an animal model, Vijayanathan et al. [88]
showed that the reversal extends to cognitive deficits.
The anticholinesterase drug, donepezil, extensively used to slow the
progression of dementia in early Alzheimer’s Disease, has been shown
to reduce cognitive deficits resulting from a combination of MTX + 5-
FU in rats [89]. However, there is little clinical evidence of this effect.
Other drugs that have been shown to reduce cognitive impairment
following chemotherapy in animals include the SSRI antidepressant,
fluoxetine [90], the anti-oxidant, N-acetylcysteine [91], and the ribo-
nucleotide reductase inhibitor, PAN-811[92] but again, they have not
been tested adequately in clinical trials
Preclinical research has demonstrated that it has a role to play in
finding ways to prevent and treat cancer-related cognitive impairment.
Ultimately, real progress will depend on the coordinated efforts of in-
dividuals with a broad range of expertise in cancer pathology, neu-
roscience, neuropsychology, and patient care. These efforts must in-
clude developing strategies for designing clinical studies to validate the
findings of animal studies that identify cognitive changes and under-
lying mechanisms. The best outcomes are likely to be realized through
close cooperation between clinical and basic researchers, and with
treatment programs that combine the strengths of pharmacological and
behavioural approaches. With that in mind, it may be informative to
examine cognitive recovery programs that have had success with other
compromised populations – e.g., normal older adults, patients with
Mild Cognitive Impairment, individuals with traumatic brain injury
G. Winocur et al.
[e.g., 93–95].
Creating better animal models
Recommendation: In developing animal models of CICI, greater use
should be made of tumour-bearing rodents.
In order to determine unconfounded effects of chemotherapy on
neurocognitive function, animal researchers typically experiment with
healthy, young adult rodents. A major limitation of this approach is that
anti-cancer drugs are never prescribed for healthy people – only for
people who have been diagnosed with cancer. Thus, the most com-
monly used models do not take into account the potential effects of the
disease itself on cognitive function. The importance of considering tu-
mour development as a contributing factor in cognitive impairment is
underscored by mounting clinical evidence that cancerous patients can
exhibit cognitive deficits at the time of diagnosis, before receiving any
form of treatment [96–100].
There has been progress in developing cancer-bearing mouse
models, although such models are just beginning to be incorporated
into behavioural research. Transgenic mice that are genetically en-
gineered to develop human forms of cancer are especially promising.
One transgenic model that has received considerable attention in the
biological literature is the well-characterized and commercially avail-
able FVB/N-Tg (MMTV-neu) 202 Mul/J mouse that expresses similar
pathology to that seen in breast cancer in women [101]. By
8–10 months, tumours typically appear in 70–80% of the animals. Life
expectancy, when untreated, is an additional 4–6 months, which makes
them good candidates for cognitive assessment. Winocur and colleagues
[102] confirmed CICI in this model and, consistent with clinical reports,
also observed cognitive deficits in tumour-bearing mice that had not
received chemotherapy. An investigation of underlying biological me-
chanisms showed that impaired cognition in treated and untreated
animals was related, in varying degrees, to changes in neurogenesis
levels, brain volume (particularly in regions implicated in cognitive
performance), and dysregulation of cytokine activity. These results re-
inforce the potential of this transgenic mouse for studying CICI in breast
cancer, and underscore the need to develop animal models that in-
corporate disease-related factors as well as other potential variables
that influence CICI in humans.
Another commercially available transgenic breast cancer model is
the MMTV-PyMT mouse [103], which develops highly aggressive
mammary tumours. By 14–16 weeks, females develop tumours in
mammary glands which rapidly metastasize to lung. This model can be
used for certain types of cognitive assessment, but a limiting factor is
that the animals generally exhibit high tumour load and metastatic
disease within 8–12 weeks of initial tumours.
Another way to mimic human cancer in animals is to utilise tumour
transplantation techniques in which cancer cells are implanted under
the skin or into a specific tissue/organ in immunocompromised mice
tumour xenograft model – [104]. Yang et al. [105], using this tech-
nique, found that tumour-bearing mice (xenografted with a colon car-
cinoma cell line CT26) exhibited depressive behaviour and impaired
object recognition memory. These effects were associated with a
number of biological changes including increased levels of mRNA en-
coding pro-inflammatory cytokines in the hippocampus, plasma glu-
cocorticoid level, along with reduced levels of hippocampal brain-de-
rived neurotrophic factor mRNA, and hippocampal neurogenesis.
Seigers et al. [106] compared biological and behavioural changes in
Buffalo rats that were subcutaneously injected with Morris Hepatoma
7777 cells or a phosphate buffered saline (PBS) control solution. Tu-
mour development was associated with decreased hippocampal cell
proliferation, reduced food intake and body weight. However, there
were differences between rats receiving PBS and normal or cancerous
rats receiving methotrexate, suggesting that, in the heterotopic tumour
model, inhibition of hippocampal proliferation is mediated by che-
motherapy.
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Cancer Treatment Reviews 69 (2018) 72–83
An important advantage of tumour transplantation is that it can be
performed with either established human tumour cell lines or human
tumour cells. Examples include patient-derived xenograft models of
breast cancer in which a patient’s tumour cells are propagated through
successive generations of mice and then implanted orthotopically into
the mammary fat pad of murine hosts. This allows the investigation of
interactive effects of human cancer and chemotherapy on cognition
under controlled conditions that are not possible in clinical studies.
However, severely compromised immunodeficient mouse strains must
be used in human xenograft tumour models so that their immune
system does not reject the tumour cells. Thus, in addition to being ex-
pensive and time-consuming, human xenograft tumour models have the
disadvantage of requiring animals to be continuously maintained in a
completely sterile environment. The necessity of sterility and the dif-
ficulties with animal handling pose serious challenges for some cogni-
tive testing protocols. A major advantage of transgenic or genetically
modified models is that the animals are immunocompetent and can be
exposed to the environments which are routine in established cognitive
testing protocols.
Other therapies
This review has focused on cognitive changes associated with che-
motherapy but many other cancer treatments are available and, for the
most part, their effects on cognitive function have been insufficiently
studied. It is well known that cranial radiotherapy seriously impairs
neurocognitive function in children and adults [59], but peripheral
radiotherapy can also affect cognition. Shibayam et al. [107] showed
that breast cancer patients, who received adjuvant regional radio-
therapy, exhibited significant memory loss and related cognitive im-
pairment, relative to patients not exposed to radiotherapy. Interest-
ingly, the authors identified elevated levels of pro-inflammatory
cytokines, specifically IL-6, as a factor in the deficits. To our knowledge,
there have been no investigations of cognitive changes following per-
ipheral radiotherapy in animals.
Targeted therapies target specific molecules within cancer cells that
send signals in support of cell division and tumour growth. For ex-
ample, anti-angiogenic therapy (AAT) is designed to suppress the
growth of new blood vessels that are needed for tumour development.
Joly et al. [3] reported that patients with metastatic renal cell carci-
noma, who received AAT [vascular endothelial growth factor (VEGF)
inhibitor] exhibited cognitive deficits, as well as deterioration in quality
of life. In parallel work, Castel and her colleagues (in preparation)
observed spatial learning deficits in mice following VEGF inhibitor
treatment. Finally, Dubois et al. [23] tested the effects of AAT in a study
of mice receiving the mTOR inhibitor, everolimus. They found no al-
terations in cognitive performance or emotional reactivity. Nor was
there an effect on neural cell proliferation in the hippocampus. How-
ever, everolimus-treated mice did experience significant fatigue, weight
loss, and altered activity in brain areas involved in the sleep/wake
cycle. Although the results of the Dubois et al. [22] study were essen-
tially negative with respect to changes in learning and memory, on
balance, the evidence does point to the likelihood that cognition is
compromised following AAT.
New generation hormonal therapies, which are used increasingly in
cancer treatment, can also lead to cognitive decline. Recent reviews
indicate that men receiving androgen deprivation therapy (ADT) for
prostate cancer perform worse than controls on tests of cognitive
function [108,109]. However, other investigations have failed to yield
such effects [110].
Anti-estrogen drugs, such as tamoxifen, are commonly administered
to prevent or treat breast cancer. Reduced estrogen levels have been
linked to cognitive losses in post-menopausal women [111] and in early
stage Alzheimer Disease [112]. It follows that tamoxifen could impair
cognitive performance in cancer patients but the evidence is mixed
[113,114]. Aromatase inhibitors, enzymes that block the production of
G. Winocur et al.
estrogen, are commonly used in conjunction with or as a replacement
for tamoxifen. While aromatase inhibitors are potentially effective as a
cancer treatment, their effects on cognitive function are uncertain
[113,115,116].
Even less is known conclusively about the impact of other cancer
treatments (e.g., surgery, immunotherapy, neural stem cell transplan-
tation) on cognition and psychosocial activities. As these therapies
improve and survival rates increase, greater appreciation of secondary
effects will be needed to ensure quality of life. It is likely that different
treatments affect cognition in different ways and, since patients often
receive more than one treatment, an understanding of singular and
combined effects of the various treatments will be required. As with
chemotherapy, strategies that incorporate preclinical and clinical re-
search in collaborative ways will be needed. With respect to the con-
tributions of preclinical research, the issues identified in this review are
as relevant to the other therapies as they are to chemotherapy, and
should provide useful direction in developing appropriate and com-
plementary animal models.
Summary and other considerations
Preclinical research has played a significant role in establishing CICI
as a legitimate clinical problem that should be taken into account in
treatment programs. Work with animal models has confirmed the pat-
tern of drug-induced cognitive deficits first described by clinical sci-
entists, and has led the way in identifying putative biological me-
chanisms that result in impaired neurocognitive function. It is
encouraging that many labs are becoming increasingly engaged in this
field of research but, at the same time, heterogeneity in methodology
and redundancies in strategy could undermine continued progress.
The present review includes a number of key recommendations,
notably the adoption of standard cognitive testing procedures, parti-
cularly as new programs are being launched. In light of evidence that a
range of non-cognitive behavioural changes often accompany cognitive
deficits, it was recommended that tests of emotional reactivity (e.g.,
stress, fatigue) also be included in standard protocols. As well, the need
to accelerate the search for risk factors and underlying mechanisms in
order to better understanding the disorder and develop effective
treatments, was emphasized.
A number of issues, considered beyond the scope of this review,
deserve mention. For example, whereas some chemotherapeutic agents
have been extensively examined with respect to their effects on cog-
nition (e.g., MTX, 5-FU, doxorubicin, docetaxel), most have not. Studies
that investigated multiple drugs typically found that cognitive functions
under hippocampal or frontal-lobe control are most often affected. In
what appears to be the most ambitious study of this type, Seigers et al.
[117] treated mice with cyclophosphamide, docetaxel, doxorubicin, 5-
FU, MTX, or topotecan, and tested them on several learning and
memory tasks. All drugs affected hippocampus-sensitive, object and
spatial recognition memory, and three (cyclophosphamide, docetaxel,
doxorubicin) resulted in impairment on the Barnes test of spatial
memory, another test of hippocampal memory. Performance on a
choice reaction-time test that required directed and sustained attention
as well as inhibitory control, frontal-lobe mediated processes, were
disrupted by cyclophosphamide, docetaxel, and topotecan. Walker et al.
[118] administered tamoxifen, MTX, and 5-FU, individually or in
combination, to mice and tested them on a variable-interval operant
conditioning task that also depended on frontal-lobe controlled execu-
tive processes. All groups were impaired on this task with the extent of
impairment related to drug and dosage. Clearly, it is necessary to cast a
wider net in studying the impact of commonly-used anti-cancer drugs
on specific cognitive domains.
The importance of paying close attention to drug dosage to ensure
valid comparisons between preclinical findings and clinical cases
cannot be overestimated. Even a cursory review of the literature reveals
that drug dosages vary widely in animal studies. For example, 5-FU, on
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Cancer Treatment Reviews 69 (2018) 72–83
its own or in combination with other drugs, has been administered to
rodents at dosages that vary between 50 mg/kg [15] and 150 mg/kg
[119]. In general, decisions regarding dosages are often made on the
basis of dose–response tests that identify a threshold at which sickness
behaviour is observed. Alternatively, estimates of clinically relevant
dosages are made but they are not necessarily reliable. The conversion
of drug dosages to animals must take into account several factors, in-
cluding differences in pharamacokinetics and metabolism, that could
affect toxicity. Calculating dosage on the basis of the animal’s estimated
surface area can help to account for differences in metabolic activity
[120]. As well, Nair & Jacob [121] have devised a promising conver-
sion algorithm, based on allometric scaling methods, that appears
promising for extrapolating clinically relevant drug dosages from hu-
mans to animals.
Another important consideration is the scheduling of treatments.
Many animal studies examine the effect of a single acute dose of che-
motherapy drugs on subsequent behaviour and cognitive abilities.
While these studies provide important information about the potential
for anti-cancer drugs to cause neurocognitive insult, they fail to address
the potential of repeated cycles of chemotherapy treatment for causing
progressive and long-term damage to the nervous system. For instance,
a single acute dose of oxaliplatin produces impairment on the location
recognition test that typically resolves after a month (Johnston, in
preparation), whereas 3 cycles of the same dose produces impairment
that persists for at least 11 months after treatment [122].
Finally, much of the preclinical research into CICI has been con-
ducted on female animals and that is because, for some time, patient
complaints were mainly from female survivors of breast cancer, and
most of the clinical research was conducted on this population. It is now
clear that CICI occurs in patients with other types of cancer, including
prostate [123] and testicular [124], which of course, affect only men, as
well as colorectal [100] and lymphoma [125], which affect both men
and women in large numbers. Moreover, many drugs are used to treat
several forms of cancer. Unless there is a specific reason for not doing
so, it is advisable that male and female animals be used in preclinical
research to assess sex differences in response to the drugs.
The development of improved animal models that accurately reflect
CICI in humans will enhance our appreciation of the disorder and ad-
vance the field in practical terms. Also needed, particularly in the
current era of limited funding opportunities, is the formation of colla-
borative networks of basic and clinical scientists, as well as open
communication and the sharing of resources and expertise between
research centres. The guidelines outlined here are not intended to be
exhaustive – rather, along with the proposed recommendations, they
are intended to highlight the central role of preclinical research in
understanding, preventing, and treating CICI, and to help in the plan-
ning and implementation of sound, translationally relevant, research
strategies.
Acknowledgements
The authors are grateful to the ICCTF executive, Dr. Tim Ahles, Dr.
Sanne Schagen, Dr. Janette Vardy, and Dr. Jeffrey Wefel for their
support and for having commented on earlier versions of this paper.
Thanks also to Dr. Melanie Sekeres, Dr. Peter Cole, and Dr. Jeanne
Mandelblatt for their helpful contributions to the final draft. The
technical assistance of Nick Hoang is gratefully acknowledged.
The preparation of the paper was supported by grants to GW from
the Canadian Institutes for Health Research and the Canadian Cancer
Society, to HC from the French National Institute for Health, the
Northwest Cancéropôle, the Ligue Nationale contre le Cancer, and the
French National Cancer Institute, and to IJ from the New South Wales
Cancer Council.
G. Winocur et al.
Conflict of interest
The authors declare that they have no conflict of interest. All au-
thors have read and approved the final version of the review.
Author’s Contributions
GW is the lead author of the paper. HC and IJ helped conceptualize
the review, made original contributions to various sections, and helped
with revisions.
Appendix A. Standard operating procedures for behavioural tests
Hippocampus-sensitive memory tests
Contextual fear conditioningApparatus. Contextual Fear Conditioning
(CFC) can be conducted in a chamber (e.g., 50 × 40 × 18-cm) that
consists of four clear Plexiglas walls, a hinged clear Plexiglas roof with
holes to allow ventilation, and a floor made of metal rods, spaced
1.3 cm apart. During fear-conditioning, a tone, presented through a
centrally mounted speaker attached to the roof of the box, is paired
with a 1 s, 1 mA foot shock that is delivered by a shock generator. A
video camera and recording equipment mounted above the test
chamber records freezing behaviour.
The fear conditioning chamber is positioned on a table, at least 1 m
above the floor, and situated in the centre of a standard laboratory
room. Illumination is provided by overhead lighting and all environ-
mental cues (e.g., pictures, bookshelves) must be located in the same
locations throughout the experiment.Fear conditioning training. On
Day 1, the animal is transferred to the test room, placed in the con-
ditioning chamber, and allowed 30 min to explore. This exposure is
important as it enables normal animals to become familiar with the
chamber and form a strong representation of contextual cues within the
environment. The animal is then returned to its home cage.
The next day, the animal is transferred to the test room, placed in
the conditioning chamber, and allowed 3 min to settle down. At the
2 min mark, its freezing behaviour is recorded every 5 s for 60 s
Freezing is defined by an immobilized crouching response in which the
only detectable movement is the animal’s breathing. Ten tone (CS) –
shock (US) pairings (tone: 2000 Hz; 80–90 db, 30 s; shock: 1 mA, 1 s)
are then administered with a variable interval (30–120 s) between
pairings. After the last shock, freezing behaviour is recorded every 5 s
for 60 s. The animal is then returned to its home cage.Fear conditioning
testing. Testing is conducted 24 h after training. Shock is not ad-
ministered during testing.
Context-only. The animal is placed in the conditioning chamber for
5 min and its freezing behaviour recorded every 5 s. The animal is then
returned to its home cage. The amount of time spent freezing is the
measure of fear conditioning to the contextual environment. Animals
with hippocampal impairment typically freeze less than normal ani-
mals.
Context + CS. One hour later, the animal is returned to the con-
ditioning chamber.
After 60 s, the tone is sounded for 5 min during which freezing
behaviour is recorded every 5 s. After the tone has ceased, the animal's
freezing behaviour is recorded every 5 s for 120 s. The purpose of this
test is to assess fear conditioning to a discrete conditional stimulus –
hippocampally-impaired and normal animals typically do not differ on
this test. The animal is then returned to its home cage.
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Cancer Treatment Reviews 69 (2018) 72–83
Morris water maze (MWM) test of spatial memoryApparatus. The MWM
spatial memory test is administered in a circular pool located in the
centre of a standard testing room. The pool is filled with opaque water
and maintained at room temperature (21 °C). An inverted flower pot,
situated a few cm below the surface, can serve as a platform on which
the animal can climb to escape the water. Throughout testing, the water
should be cleaned after each trial and changed every 2–3 days.
The pool can be divided into six zones of approximately equal size.
Swimming patterns and escape latencies are monitored by a centrally-
located overhead camera connected to a data processing system. The
system enables computation of the time required to mount the platform
(latency) as well as swimming patterns. An error is recorded each time
the animal enters a zone not containing the platform.Procedure. The
task begins with two days of orientation to the maze (5 trials/day) in
which the animal is individually placed in the pool and allowed to swim
to a visible platform. The start point and the location of the platform are
randomized on each trial. By the end of the second day all animals
should be swimming directly to the platform.
Training begins the following day. The platform is now positioned a
few cm below the surface and fixed in the centre of one of the zones of
the pool (e.g., north-east). To start a trial, the animal is placed in the
water, facing the wall at the edge of one of 5 zones. Animals are never
placed in the zone containing the platform. Each trial continues until
the animal mounts the platform with all four paws, or until 60 s have
elapsed. If the animal fails to find the platform within 60 sec, it is
manually guided to the platform. After 20 s on the platform, the animal
is placed in a holding cage that contains a heat lamp to await the next
trial. Five such trials, with an approximate inter-trial interval of
4–5 min, are administered each day for 5 consecutive days. Escape la-
tency and errors are recorded for each trial. If the mouse fails to find the
platform within 60 s, it can be given an arbitrary error score of 15 and a
latency score of 60 s for that trial.
On the sixth day of training, trials 1 & 2 are administered in the
usual manner. The third trial serves as a probe trial. For the probe trial,
the platform is removed, and the amount of time the animal spends
swimming in the platform target zone (north-east zone) is recorded and
used as an index of the rat’s memory of the platform’s location. After 60
s, the rat is removed and returned to its home cage.
Novel location recognition (NLR)2Apparatus. The NLR test is typically
conducted in a plastic box (e.g., 49-cm wide × 66-cm long × 40-cm
high). Stimuli can consist of small ceramic or plastic objects (e.g., for
rats – 7–12 cm high × 7–9 cm wide; smaller objects would apply for
mice) that vary in colour and size. The objects are secured to the floor of
the box with Velcro strips. A video camera mounted on the wall directly
above the box can be used to record the testing session for off-line
analysis.
Familiarisation training. Initially, animals receive two sessions in
which they spend 45 min in the testing room and 5 min exploring the
empty apparatus. Three days of familiarisation training follow. On each
day, the animal is placed in the testing room for 45 min and then in the
empty box for 1 min. The animal is then removed and two identical
objects are placed in different corners of the apparatus. The animal is
returned to the box and allowed to explore for 5 min. This procedure is
repeated two more times on each day (mean interval between ex-
posures is about 60 min).
NLR testing. At the beginning of the test session, the animal is
placed in the empty apparatus for 2 min. It is then placed in a holding
cage for 10 min before returned to the apparatus for a 10-minute choice
trial. In this trial, one of the objects from the familiarisation sessions
remains in the same corner (‘familiar’ location) and the other is moved
2
Dimensions pertaining to apparatus and objects are intended for testing rats. The New
Location and Object Recognition tests can be administered to mice but dimensions must
be scaled down accordingly.
G. Winocur et al.
to a different corner (‘new’ location), and the animal is allowed to ex-
plore the two objects. Object exploration is scored from video record-
ings. The animal is said to be exploring an object when its nose is within
2 cm of the object and the vibrissae were moving. Object exploration is
not scored when the animal gnaws the object or uses it to rear upward
with its nose facing the ceiling.
A second test can be conducted 24 h later. Following a 2-minute re-
familiarisation with the empty apparatus and a 10-minute period in a
holding cage, the animal is returned to the apparatus. For this test, the
‘familiar location’ object remains in the same corner but the ‘new lo-
cation’ object is again moved, this time to the remaining open corner.
Object recognition (OR)Apparatus. The type of apparatus and materials
described for the NLR test can be used for the OR test.
Familiarisation training. Familiarisation training is the same as in
the NLR test.
OR testing. The animal is rehabituated to the empty test apparatus
for 2 min. It is then removed and two objects (one novel object and an
object from the familiarization phase) are placed in the box. The animal
is allowed to explore the objects for 10 min. Object exploration is scored
as in the NLR test from video recordings. Records of object exploration
should be organized in 1-min blocks, as the most sensitive measures of
object recognition occur early in the test session when the novel object
is most unfamiliar. The OR test can be repeated 24 h later, using the
same familiar object paired with a new, novel object.
Frontal Lobe-sensitive tests of strategic learning (Executive
Function)
Non-Matching-to-Sample (NMTS)
Apparatus
Same as MWM Spatial Memory.
Procedure
This test begins with two days of orientation to the maze (5 trials/
day) in which the animals is placed in the pool and allowed to swim to a
visible platform, which is located beneath a grey cylinder (e.g., 30 cm
long × 3 cm in diameter). The start point and the location of the plat-
form are randomized for each trial. By the end of the second day all rats
should be swimming directly to the platform.
The NMTS task consists of a series of paired sample and test trials
during which the platform is submerged a few cm below the surface of
the water. In the sample trials, a black or white cylinder (30 cm
long × 3 cm in diameter), suspended 5 cm above the submerged plat-
form, signals the platform’s location. In the subsequent test trial, both
cylinders are present in different locations. The cylinder that was not
present during the preceding sample trial now signals the platform’s
location.
For each sample trial of the NMTS test, the animal is placed in the
same (e.g., south-east) zone of the pool and allowed to swim to the
submerged platform under the sample cylinder. The animal remains on
the platform for 20 s and is then transferred to a heated holding cage
while the platform is moved and the cylinders repositioned for the test
trial. The animal is then placed in the pool and allowed to swim to the
submerged platform or until 60 s has elapsed. If the animal fails to find
the platform within 60 s, it is manually guided to the platform. After 20
s on the platform the animal is transferred again to the holding cage, to
await the next pair of trials. Ten daily sessions, each consisting of 8
pairs of sample and test trials, are administered.
As in spatial memory, the data recording system records the time
required to mount the platform (latency) as well as swimming patterns.
An error is recorded each time the animal enters a zone not containing
the platform.
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Cancer Treatment Reviews 69 (2018) 72–83
Conditional associative learning (CAL)
Apparatus
The CAL task is administered in a circular pool (as above), that is
fitted with a cross-maze.
Procedure
Training begins with three days of orientation to the maze (10
trials/day) in which the animal is placed in one of the arms facing the
edge of the pool. At the centre of the maze, a grey cylinder, suspended
5 cm above the water and near the entrance of the arm containing a
visible platform, signals the location of the platform at the end of the
arm. The start point and the arm containing the platform are rando-
mized on each trial. If the animal fails to find the platform within 60 s,
it is manually guided to the platform, where it remains for 20 s. By the
end of the third day all animals should be swimming well and finding
the platform within 60 s. Animals that take longer than 60 s may be
given an additional day of familiarisation training.
For the CAL task, the animal is started in a different arm on each
trial according to a random schedule. On each trial, a black or white
cylinder is suspended over the centre of the maze, equidistant from the
entrance to each arm. Half of the animals are trained to associate the
black cylinder with turning right, and the white cylinder with turning
left, to find the platform at the end of the arm. For the other half, the
opposite is the case. Presentation of the cylinders is organized ac-
cording to a semi-random schedule that ensures an equal number of
trials with both cylinders over 10 daily trials. For each trial, animals are
given a maximum of 60 s to find and mount the platform with all four
paws. Animals are allowed 20 s at the end of each trial to rest atop the
platform. Ten such trials are administered each day for 10 days.
Error sequence and latency to find and mount the platform are re-
corded via the data processing system located centrally above the
centre of the maze. An error is recorded when the animal enters an
incorrect arm with all four paws, or leaves the goal arm without suc-
cessfully mounting the platform. Typically, error scores and latencies
are highly correlated.
Non-cognitive tests
Elevated plus maze
Apparatus
A typical plus-maze for this test consists of two closed arms and two
open arms of equal length (25 cm). The wall surrounding the closed
arms is 20 cm high. Each open arm is surrounded by a rim about 0.5 cm
high. The maze is located in a quiet room and elevated 50 cm above the
floor. The animal’s behaviour can be recorded with a video tracking
system.
Procedure
At the beginning of the test, which lasts for 5 min, the animal is
placed in the centre of the maze with its head in the direction of an
open arm. The important measures are the number of entries (defined
as having all four legs in the arm) into, and time spent in each open and
closed arm. Time spent in enclosed arms is considered a sign of high
anxiety. It is also possible to measure the number of head-dips, which
are an indicator of exploration. A large number of head-dips reflect low
anxiety. Stretch-attend-postures can also be measured and interpreted
as anxiety-induced information-taking about the environment.
Sucrose preference test
Apparatus
Animals, housed in individual cages, are given access to 2 bottles;
one contains water and the other contains a 2% sucrose solution.
G. Winocur et al.
Procedure
Initially, animals are habituated for 3 days to the presence of the
two drinking bottles in their home cage. After this period, they have the
choice of drinking the 2% sucrose solution or water for a period of
3 days. During this time, the volume of the solution consumed in each
bottle is measured and the sucrose preference is calculated as the per-
cent of the volume of the sucrose solution drunk relative to the total
volume of liquid drunk. Normal animals prefer the sweetened drink;
failure to do so is a measure of anhedonia/depression.
Forced swim test
Apparatus
A 2 Liter beaker, filled with 1.500 L of water (25 °C), is located in a
quiet testing area that is equipped with a video tracking system. Water
is changed between each animal.
Procedure
The animal is placed in the water for 5 min and allowed to swim
freely. The animal is considered to be immobile when there is no
movement, except for those necessary to keep its head above the water
surface. The animal’s behaviour is analyzed through video records.
Animals that make little effort to escape and quickly become immobile
are thought to exhibit signs of depression.
Tail suspension test (for mice only)
Apparatus
The mouse is suspended by the tail from an elevated bar. Its activity,
while dangling, can be recorded with a video tracking system.
Procedure
The test consists of a single 6 min session. Records are kept of la-
tency to first bout of immobility (as defined by passive hanging with no
attempt to right itself), and number and duration of immobility bouts.
Animals that exhibit few escape oriented behaviours and become im-
mobile are thought to exhibit signs of depression.
Food choice test
Apparatus
A standard operant chamber (30 × 25 × 30 cm) located in a quiet
room or preferably in a sound-proof chamber. The chamber contains a
lever that is centrally located in one wall and an adjacent food dis-
penser that delivers commercially available 45 mg food pellets.
Procedure
Before beginning behavioural training, rats are placed on a 23.5
food-deprivation schedule during which they receive 30 min daily
feeding sessions with both standard lab chow and food pellets available.
After several days, rats receive 30 min daily familiarization training
sessions in the operant chamber in which they learn to associate the
lever and dispenser with food pellets. Rats are then trained in 30 min
sessions to press the lever to obtain food pellets on a continuous re-
inforcement (CRF schedule in which each lever press produces a single
pellet. Following familiarization and CRF training, rats are allowed
20 min access to lab chow, to ensure that they will have consumed
adequate amounts of food each day.
When CRF lever pressing has stabilized, the food-choice condition is
introduced. For this condition the reinforcement schedule is changed to
fixed ratio (FR) 5 in which the rat must press the lever 5 times to receive
a single pellet. After several days, when the rat is pressing consistently
on this schedule, 15 g of lab chow is introduced into the chamber and
made freely available. Thus, the rat now has a choice of pressing the
lever on FR5 reinforcement schedule to receive desirable food pellets or
simply eating the less preferred lab chow. Testing should be continued
81
Cancer Treatment Reviews 69 (2018) 72–83
until the number of lever-press responses does not vary by more than
25% from the mean over a 3-day period. (The operant test can be made
more demanding by increasing the response/reinforcement ratio –
Salamone et al. [44] reported that normal rats will continue to prefer
the pellets and press the lever at high rates at FR schedules that exceed
60.) At the end of each food-choice session, the number of lever-press
responses and the amount of lab chow consumed (taking into account
spillage) are recorded. Animals that are less motivated to work for food,
as a result of fatigue, stress, or emotional unresponsiveness, prefer the
freely available, but less palatable, lab chow.
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