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INTRODUCTION
Polypharmacy is defined as the concurrent use of five or
more drugs [1] and is very common in older adults, who
are the largest consumers of medications. The
prevalence of polypharmacy has increased in many
countries over the last decades [2] and the use of
multiple drugs is becoming a burden on individuals and
healthcare systems worldwide.
Most medications show good safety profiles when taken
alone as monotherapies and correctly. However, the risk
of developing adverse events can raise considerably
when they are combined with other drug therapies, due
for example to drug-drug interactions, prescribing
cascade, and medication errors [3–5]. Furthermore,
older adults are particularly vulnerable to negative
effects of multiple-medication treatments due to both
age-related physiological changes and more frequent
occurrence of multiple pathological conditions (i.e.,
arthritis, cardiovascular diseases, osteoporosis, renal
dysfunction, dyslipidemias) as a consequence of longer
survival with chronic disorders. In the older population
polypharmacy has been associated to a higher risk of
several negative outcomes which comprise falls,
hospitalization, higher frailty, and mortality [6–10].
www.aging-us.com AGING 2021, Vol. 13, No. 11
Research Paper
Long-term exposure to polypharmacy impairs cognitive functions in
young adult female mice
Eroli Francesca1, Johnell Kristina2, Latorre-Leal María1, Hilmer Sarah3, Wastesson Jonas2,4,
Cedazo-Minguez Angel1, Maioli Silvia1
1Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research,
Division of Neurogeriatrics, Solna, Sweden
2Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden
3Kolling Institute, Royal North Shore Hospital and University of Sydney, Sydney, Australia
4Aging Research Center, Karolinska Institutet and Stockholm University, Stockholm, Sweden
Correspondence to: Silvia Maioli; email: silvia.maioli@ki.se
Keywords: polypharmacy, adverse outcomes, memory, female mice, sex-differences
Received: February 10, 2021 Accepted: May 18, 2021 Published: June 2, 2021
Copyright: © 2021 Francesca et al. This is an open access article distributed under the terms of the Creative Commons
Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
ABSTRACT
The potential harmful effects of polypharmacy (concurrent use of 5 or more drugs) are difficult to investigate in
an experimental design in humans. Moreover, there is a lack of knowledge on sex-specific differences on the
outcomes of multiple-drug use. The present study aims to investigate the effects of an eight-week exposure to
a regimen of five different medications (metoprolol, paracetamol, aspirin, simvastatin and citalopram) in young
adult female mice. Polypharmacy-treated animals showed significant impairment in object recognition and fear
associated contextual memory, together with a significant reduction of certain hippocampal proteins involved
in pathways necessary for the consolidation of these types of memories, compared to animals with standard
diet. The impairments in explorative behavior and spatial memory that we reported previously in young adult
male mice administered the same polypharmacy regimen were not observed in females in the current study.
Therefore, the same combination of medications induced different negative outcomes in young adult male and
female mice, causing a significant deficit in non-spatial memory in female animals. Overall, this study strongly
supports the importance of considering sex-specific differences in designing safer and targeted multiple-drug
therapies.
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Nevertheless, there is little experimental data about the
potentially negative effects caused by polypharmacy
and on the mechanisms behind these effects [11]. Drug
safety studies often exclude older patients and are
limited to monotherapies. Another important aspect
which is poorly investigated is the influence of sex in
drug use and response [12]. This is of particular
importance in older adults since they often have altered
pharmacokinetics, pharmacodynamics, efficacy, and
toxicity [13, 14] which have shown to change between
men and women. [15–18]. Therefore, sex represents a
relevant factor to take into account when investigating
adverse events related to polypharmacy.
We recently performed a study to explore the effects of
long-term concomitant administration of five different
medications on locomotion, anxiety, and cognition in
mice [19]. The drugs included in the polypharmacy
treatment were the most frequently used by older adults
in Sweden [20], and among the most frequently used
drug classes also in other European countries [21–24].
Importantly, we observed that polypharmacy impaired
exploration and cognitive functions in young adult wild-
type male mice [19].
In this study, female mice were administered the same
polypharmacy regimen, containing aspirin, paracetamol,
simvastatin, metoprolol and citalopram, with the aim of
investigating the effects of multi medications in female
animals and allow comparison with our previous study
in young adult male mice [19]. Animals were fed with
the polypharmacy diet and then assessed for locomotor
function and coordination, cognitive tests, and anxiety-
like behavior. Hippocampal tissues were analyzed to
measure any changes in protein markers which could be
related to the behavioral outcomes observed in
polypharmacy mice. The following parameters were
monitored as basic health indices: food/water intake,
body weight (BW), serum creatinine and alanine
aminotransferase (ALT) levels.
RESULTS
Treatment tolerance and health parameters
The treatment was well tolerated by the animals and no
increase in mortality was observed in polypharmacy fed
mice compared to controls: all the mice reached the end
of the study in good health. Polypharmacy fed mice
showed a significant BW gain during the study period
while controls did not (week 1 vs week 8: control group
BW= 26 ± 1.2 g vs 28 ± 1.3 g, p= 0.09; polypharmacy
group BW= 26 ± 0.7 g vs 30 ± 1.1 g, p= 0.001, two-way
ANOVA repeated measurements; Figure 1A). No
significant differences in mean food or water intake (FI,
WI) were found between the two groups over the study
period (Figure 1B, 1C), nor in the weekly average
(Figure 1D, 1E). However, both controls and treated
animals revealed a significant reduction of FI during the
last 4 weeks (control group FI, week 3: 4.5 ± 0.2
g/day/mouse, week 8: 2.5 ± 0.1 g/day/mouse, p=0.02;
polypharmacy group FI, week 3: 3.7 ± 0.1 g/day/mouse,
week 8: vs 2.4 ± 0.1 g/day/mouse, p=0.02, two-way
ANOVA repeated measurements; Figure 1D). The
average FI was very close to the estimated one,
therefore the drug concentrations taken by poly-
pharmacy animals corresponded to the expected ones.
Only in the last week the registered FI (2.4 ± 0.1 g,
polypharmacy group, Figure 1D) was about 20% less
than the anticipated one, meaning that the final drug
dosage consumed was: 80 mg/Kg/day metoprolol, 80
mg/Kg/day paracetamol, 16 mg/Kg/day aspirin, 8
mg/Kg/day simvastatin and 8 mg/Kg/day citalopram,
which is within the therapeutic dose range in humans
for these medications [19].
As markers for renal and hepatic health status we
measured serum levels of creatinine and ALT at the
end of the treatment. Dot histograms in Figure 1F
illustrate as there were no significant changes of the
two markers between control and multiple-drug
administered mice.
Polypharmacy diet did not affect locomotor activity
and anxiety-like behavior
We used open field (OF) locomotor cages to study
general locomotor activity over a 30-min free
exploration trial. Horizontal and vertical activity were
analyzed over the total test duration and in time
intervals of 10 min in order to monitor the habituation
phase and the next exploratory patterns. The treatment
did not alter the horizontal or rearing activity analyzed
per time interval (Figure 2A), nor the total locomotion
(horizontal activity: 241.6 ± 24 m vs 182.7 ± 13 m;
rearing: 40.9 ± 6 s vs 32.4 ± 13 s, control vs
polypharmacy group respectively, data not shown). The
map in Figure 2B illustrates that control and
polypharmacy animals showed a similar pattern of
movements in locomotor cages.
Motor coordination and forelimb strength were assessed
through Rotarod and Grip strength tasks. The analysis
of latency to fall over the three Rotarod test trials
showed that control mice significantly improved the
performance on the rotor in trial 3 compared to trial 1
while polypharmacy mice did not (Figure 2C). Despite
this there were no significant differences between the
two groups. The outcomes from Grip strength test did
not highlight relevant differences between control and
treated animals in the front limbs force measured during
the grid pulling (Figure 2D).
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To explore whether the multiple-drug regimen could
affect anxiety-like behavior we performed Dark/Light
Box (DLB) and Elevated Plus Maze (EPM) tests.
The results from DLB experiment showed that
polypharmacy mice displayed a similar time spent in,
and latency to enter, the lit compartment to the controls
(Figure 2E). Likewise, EPM task did not reveal
significant differences in the time spent by the animals
exploring the close arms of the maze (about 80 % of the
total trial duration: dot histogram in Figure 2F and
heatmaps in Figure 2G).
Polypharmacy regimen impaired object recognition
and fear associated contextual memory
Mice underwent cognitive tasks to investigate the effect of
the polypharmacy treatment on different types of memory
and learning. To study spatial working memory, we ran
the Y Maze test. Animals from both the groups performed
a similar number of arm entries and a percentage of
possible alternations above 50 on average (Figure 3A),
suggesting that polypharmacy regimen in young adult
female mice does not affect spatial working memory.
Figure 1. Basic health parameters in control and polypharmacy treated mice: body weight, food and water intake, and
serum proteins. (A) The curves show mouse body weight measured weekly during the two months of control or polypharmacy diet. (B, C)
The histograms represent the total average of food and water intake over the whole study period. (D, E) The curves show the weekly average
of food and water intake monitored during the eight weeks of treatment. (F) Dot histograms express serum creatinine and ALT levels. Ctrl=
control, Poly= polypharmacy, n= 10 animals per group. All data are presented as mean ± SEM.
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Non-spatial memory was investigated via Novel Object
Recognition (NOR) test. On day 3, control mice
exhibited a clear preference in exploring the novel
object compared to the familiar one. Conversely,
polypharmacy animals did not discriminate between the
familiar and the novel object as they spent a similar
time exploring both (Figure 3B, right panel). This was
confirmed by the calculation of the discrimination index
Figure 2. Effect of polypharmacy regimen on locomotion, coordination and strength, and evaluation of anxiety-like behavior.
(A) Locomotor and explorative activity: histograms express horizontal and vertical activity (rearing) assessed in OF cages and analyzed per
time intervals over a total duration of 30 minutes. (B) Representative map of the pattern of movements in a control and polypharmacy
mouse during the 30-min trial in OF cages. (C) average of latency to fall measured over the 3-trial session of Rotarod test. Interaction
between time and treatment groups were analyzed with two-way ANOVA repeated measurements; ***p≤0.001, trial 1 vs trial 3 in control
group. (D) Dot histograms show the forelimb strength average measured by Grip Strength test in control and polypharmacy animals. (E) DLB
test: dot plots express first latency to enter the LB and time spent by the mice moving in that area. (F) EPM test: number of entries and
duration % of time spent in closed arms over the 5 min/trial. (G) Representative heatmaps of the EPM, where red zones display the area that
the mice explored the most (average of control and polypharmacy group maps). Ctrl= control, Poly= polypharmacy, n= 10 animals per group.
All data are presented as mean ± SEM.
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Figure 3. Outcomes of cognitive tests: polypharmacy treatment impaired non-spatial memory. (A) Number of entries and
percentage of spontaneous alternations performed by control and polypharmacy mice in the Y Maze test. (B) NOR test, day 3: the dot plots
express the discrimination index (a score above 0 indicates that the mice explored the novel object more than the familiar one). *p<0.001, t-
Student test. Histograms on the right show the average of time spent in exploring the two objects by control and polypharmacy animals,
***p<0.001, two-way ANOVA repeated measurements. Note that control mice spent about double the time exploring the new object
compared to the familiar one; on the contrary, treated animals did not differentiate between the two objects, as indicated by the
discrimination index. (C) The heatmaps visually represent day 3 of NOR test and specifically the area explored around the objects by the
animals, showing that only in control group there is a clear preference for the novel object compared to the familiar (in red color the most
visited areas). Fam and Nov = familiar and novel object respectively. (D) Contextual and cue FC test: the graph on the left shows the
percentage of freezing time measured on day 1 (habituation phase) vs day 2 (context test); the graph on the right expresses the freezing
percentage measured before vs during the cue stimulus (sound). *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA repeated measurements
test. Ctrl= control, Poly= polypharmacy, n= 10 animals per group. All data are presented as mean ± SEM.
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which was significantly higher for controls compared to
treated mice, that presented an index close to 0 on
average (Figure 3B, left dot plot). Heatmaps in Figure
3C represent by colors as control animals spent more
time on the novel object (in red) while the
polypharmacy mice stayed similarly on both. The
outcomes from NOR test propose that multiple-
medication regimen impaired non-spatial object
recognition memory.
Fear conditioning (FC) test was performed to assess fear
associated memory and learning. Mice were subjected
to an auditive stimulus (cue) paired to a foot shock on
day 1 and then tested for context and cue memory on
day 2 and 3 respectively. The freezing % recorded
during the habituation phase of day 1 (as a measure of
baseline freezing) was compared to freezing % of day 2
to evaluate the context memory. To assess the cue
memory, we measured the freezing % on day 3 before
and during the sound stimulus. During the context test
on day 2, both controls and treated animals showed a
significantly increased freezing behavior compared to
day 1 (Figure 3D, left graph). However, control mice
responded to a greater extent to context recognition
showing a significant higher freezing % than the
polypharmacy ones (Figure 3D, left plot: **p=0.01,
two-way ANOVA repeated measurements), indicating
that the multi-medication treatment may affect FC
contextual memory in young adult female mice. On day
3, we measured freezing % before and during the
delivery of the acoustic stimulus; mice from control and
polypharmacy administered group expressed a
significantly stronger freezing behavior during cue
application compared to before (Figure 3D, right
graph), suggesting that both groups were able to
associate the auditory cue to the adverse stimulus (the
foot shock).
Polypharmacy reduced the levels of memory-related
proteins in hippocampus
Western blotting experiments were performed to
investigate whether the treatment could lead to
changes in levels of hippocampal proteins involved in
regulating synaptic plasticity and memory formation.
We first analyzed the expression of synaptic N-
Methyl-D- aspartate (NMDA) receptors (subunits
NMDAR1 and phospho-NMDAR2A) and postsynaptic
density protein 95 (PSD95), that are known to play a
key role in synaptic transmission and potentiation and
were found to be downregulated in our previous study
on male mice [19]. Interestingly, we did not observe
changes in the hippocampal levels of these markers
between control and polypharmacy animals, as
illustrated by immunoblots and histograms in Figure
4A, 4B. Since the data from NOR test indicated a clear
memory impairment in treated mice, we explored
markers for specific signaling pathways implicated in
recognition memory. In the hippocampi of multiple-
medication fed animals we found a decrease of total
cAMP Response Element-Binding Protein (CREB)
levels compared to controls, although the ratio of
phospho/total CREB remained unchanged between the
two groups (Figure 4C, 4D). The analysis of
Ca2+/calmodulin-dependent protein kinase II
(CaMKII) revealed a significant reduction of
phosphorylated CaMKII in polypharmacy mice, as
shown by the ratio of phospho/total protein (Figure 4E,
4F). The brain-derived neurotrophic factor (BDNF)–
tyrosine kinase B (TrkB) signaling is another
important system that regulates synaptic plasticity and
is involved in recognition memory consolidation [25]
and fear conditioning learning [26]. We quantified the
levels of TrkB and pro-BDNF and we observed a
significant downregulation of TrkB receptor
expression in treated animals compared to controls
(Figure 4G, 4H, left histogram). The levels of
hippocampal pro-BDNF in polypharmacy mice
resulted in a reduction of 25% on average than control
levels (right plot of Figure 4H), albeit not significant
(p=0.12, t-Student test).
DISCUSSION
In this study we performed a preclinical investigation on
the adverse events related to polypharmacy on
locomotion, anxiety, and cognition in female animals.
Previous studies reporting negative outcomes associated
with multiple-drug use on animal models, including our
recent study [19], were performed primarily in males
[27, 28], except for one recently published study on
physical functions in C57BL/6 male and female mice
[29]. In the elderly population, women are more
frequently exposed to polypharmacy and observational
studies have reported a higher risk of receiving
potentially inappropriate prescriptions in women
compared to men [12, 30, 31].
In the current study we found that polypharmacy
treatment significantly impaired object recognition and
affected fear associated contextual memory, together with
a significant decrease of some hippocampal proteins
involved in pathways regulating the formation and
consolidation of these types of memories. Noteworthy, we
did not observe the impairments in explorative behavior
and spatial memory that we previously reported in young
adult male mice administered the same polypharmacy
diet. We believe that the results from this study give
interesting insights about possible sex-specific adverse
effects from multiple-drug use and support the need of
more targeted multi-medication therapies which consider
sex-related differences.
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Animals were administered the polypharmacy regimen
for eight weeks and tested for behavioral experiments
during the last four weeks of treatment. The diet was
well tolerated, serum levels of hepatic and renal
function did not change between control and treated
animals, nor we did observe signs of illness among the
mice. The decrease in FI observed during the last four
weeks in both the control and treated group correlates
with the behavioral assessment period which may
induce stress in mice as we have previously observed
[19]. The average FI was similar between the two
groups over the study period, except for a lower FI
baseline during the first week in the polypharmacy
animals compared to controls. Despite this, we observed
Figure 4. Immunoblotting analysis of hippocampal protein levels in control and polypharmacy fed mice. Hippocampal tissue
lysates of control and polypharmacy mice were analyzed by western blotting experiments. Representative immunoblots and quantification
of: NMDAR1, phospho-NMDAR2A and PSD95 proteins (A, B), phospho- and total-CREB, and ratio (C, D), phospho- and total-CAMKII, and ratio
(E, F), TrkB and pro-BDNF proteins (G, H). *p<0.05, Mann-Whitney (D, H) or t-Student test (F). Total protein levels were normalized with
respect to α tubulin. Ctrl= control, Poly= polypharmacy, n= 10 animals per group. All data are presented as mean ± SEM.
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a significant increase of BW in the polypharmacy fed
mice compared to controls. This BW gain might be due
to a metabolic effect caused by one or more specific
drugs contained in the polypharmacy diet. Mild weight
gain can manifest as a side effect of some beta blockers,
including metoprolol [32], and antidepressants like
citalopram [28, 33]. Also, it might be due to the
presence of simvastatin in the drug combination: use of
statins in adults has been associated with an increase in
body mass index in comparison with statin nonusers
[34]. Interestingly, this increase in BW was not reported
in male mice administered with the same multi-
medication therapy [19]. In this regard it is relevant to
note that side events for statin use, like muscle pain,
have been found to affect women with a higher
prevalence compared to men, together with a lower
efficacy of the lipid lowering action in women than in
men [35, 36]. This supports the fact that drug outcomes
may vary with sex.
When behavior was assessed in mice, no differences
between treatment groups were found in the locomotor
and exploratory patterns recorded in OF cages,
indicating that polypharmacy administration in adult
female mice did not affect exploration and total
locomotor activity. This finding differs from our data in
young adult male mice [19] indicating that female mice
could be more resilient to these effects than males
treated with the same polypharmacy combination at
young adult age. Huizer-Pajkos et al. reported that a
shorter treatment of 4 weeks in young male mice did not
lead to impairments in OF [27], while data from Mach
et al. report reduction of distance traveled in OF after 12
weeks of polypharmacy treatment in middle aged male
mice, as well as after 12 months of low drug burden
index and high drug burden index polypharmacy
treatments in aging male mice [28]. Interestingly,
functional outcomes for motor coordination and balance
in Rotarod did not differ between control and
polypharmacy female mice. However, while control
group improved significantly during the 3 trials of
Rotarod, polypharmacy mice showed no improvement
and unchanged latencies to fall among trials, suggesting
that multi-medications in female mice could start to
affect coordination and balance at young adult age.
Previous studies on aging male mice reported that
performance in Rotarod test was negatively affected by
the polypharmacy treatment [27]. Moreover, the
observed lack of improvement during the Rotarod task
may be also caused by decreased motor learning in the
polypharmacy group rather than coordination deficit
only [37]. OF and Rotarod tests resulted in different
outcomes in adult males and females treated with our
selected drug combination, supporting possible sex-
specific adverse effects in locomotor functions of multi-
medication therapies. A recent study in young and old
male and female C57BL/6 mice found no significant
difference between young males and females in baseline
grip strength, motor coordination, gait speed, distance
travelled in the open field, anxiety or nesting [29],
suggesting that the sex-specific outcomes we observe
here are not related to baseline differences in the
behavioral performance between sexes.
Our previous study was the first to investigate the
effects of polypharmacy on cognitive functions in mice
and we reported that a combination of different
medications had a negative effect on spatial working
memory in Y Maze and reduced hippocampal
postsynaptic proteins already at young age [19].
Interestingly, when the Y Maze test was performed in
female mice no differences were found between groups.
These results were further confirmed by western blot
analyses of proteins mainly involved in formation and
consolidation of spatial memory as NMDA receptors
and PSD95: female mice administered with
polypharmacy did not show a reduction of these
markers in hippocampus when compared to controls. It
is important to mention that NMDAR1 and
NMDAR2A/B expression were reported to be higher in
the hippocampus and in postsynaptic density fractions
of adult female mice than in those of males [38]. This
aspect may influence the sex-specific effect of
polypharmacy on postsynaptic protein levels observed
in our studies.
Noteworthy, the present study shows that multi-
medication therapy in female mice impaired object
recognition memory, measured by the ability to
remember an object previously encountered and
therefore distinguish a novel object from a familiar one
in the NOR test. This type of memory was not affected
by the same treatment in male mice [19]. Several
studies reported that a functional hippocampus is
essential for the formation of recognition memory in
rodents [39, 40]. Within hippocampus, several signaling
cascades have been shown to be critical for
consolidation of this type of memory. Specifically,
CREB inactivation in CA1 and CaMKII inactivation in
mutant mice has been shown to impair long term object
recognition memory [41, 42]. In the hippocampi of
polypharmacy female mice, a decrease in total levels of
CREB as well as a decrease in phosphorylation of p-
CaMKII was shown. These results are consistent with
the behavioral findings in the NOR test.
In addition to Y Maze and NOR we performed FC in
order to assess fear associated memory. In this test,
control and polypharmacy mice learned to associate
both the context and the cue to the adverse event of
the foot shock. However, it must be pointed out that
during the context test polypharmacy mice showed a
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significant lower freezing time than control animals.
This result shows that polypharmacy mice performed
worse when associating the foot shock to the context,
suggesting that the multi-medication treatment
affected the consolidation of fear associated
contextual memory in female mice at young age. The
FC deficits observed in treated mice are not as
consistent as in the NOR test and we may hypothesize
that aging would further lead to greater deficits in fear
associated memories caused by the current
combination of multiple drugs in female mice. This
hypothesis is supported by western blot analyses
revealing a significant reduction of TrkB levels in
hippocampus of treated females as compared to
controls. BDNF–TrkB pathway is a ligand–receptor
system that underlies synaptic plasticity and has been
shown necessary for acquisition and consolidation of
fear conditioning in different brain regions including
hippocampus [43–46]. While we observed a decrease
in TrkB receptors, we did not observe a significant
reduction in BDNF levels in hippocampus of treated
mice, and it is possible that a longer multi-medication
treatment as well as aging would eventually lead to a
greater reduction of BDNF in female mice, followed
by a greater deficit in FC test. Additionally,
phosphorylation of CREB mediated by CaMKII may
as well affect BDNF levels [47]. In this context, it is
important to mention that a large body of evidence
reported that CaMKII-CREB signaling is participating
in estrogen receptor signaling in the brain [48, 49].
Brain estrogen signaling has a neuroprotective role
and is essential for synaptic function. The multi-
medication therapy proposed in this study could affect
estrogen signaling, further supporting the sex-
differences in outcomes as different types of
memories.
To our knowledge, the use of the individual drugs
composing our polypharmacy regimen has not been
reported to induce toxic effects in mice [27, 50–54].
This suggests that the combination of different
medications used in this study causes the negative
outcomes observed. However, only one out of the six
pre-clinical studies cited above has been conducted in
both male and female mice, while the rest used only
male animals. This may lead to a more difficult
interpretation of the data on the effects of
polypharmacy in female mice. For instance, previous
research on monotherapies in rodents did report
different results within male and female animals: a
recent study on treatments for post-traumatic stress
disorder found differential effects caused by
citalopram on fear associated memory in female mice
compared to males [55]; similarly, different outcomes
were found after administration of metoprolol: this
beta-blocker impaired performance in Morris Water
Maze and FC tests in males of the APP Alzheimer’s
Disease mouse model and wild-types but not in
females [56]. Aspirin was reported to increase the
lifespan of male mice but not of females [57], and this
was attributed to different drug metabolism and
disposition between sexes. These observations support
the idea that more targeted research is necessary to
refine appropriate therapies taking into account sex-
specificity.
There are some limitations to consider in this study.
Female and male experiments were not conducted
simultaneously, not allowing a statistical comparison
between male and female groups. While we replicated
laboratory conditions, there may have been
experimental differences that affected the behavioral
outcomes. The study has been conducted at young
adult age. Although there is some evidence of
multiple-drug use in young and adult subjects [58],
and its prevalence over time have been increasing in
younger age groups [59] polypharmacy is more
frequent in old age. Therefore, the use of aged mice
would be of great interest to discuss the effects of
polypharmacy related to older population. To do so,
an optimization of the experimental design will be
necessary for future studies in old animals. A recent
study on the effects of a different polypharmacy
regimen on physical function in young and old male
and female mice has been published, demonstrating a
marked increase in susceptibility to functional
impairment in old age and greater impact on grip
strength in males than in females [29]. The
investigation of the impact of age and sex on
susceptibility to the effects of polypharmacy on
cognitive function can be the subject of future studies.
Taken together, this study is relevant and highlights the
importance of investigating the possible adverse effects
of multiple-medication treatments in female mouse
models in the future. This is one of the first reports of
the effects of polypharmacy in female mice and the first
to study its cognitive effects. The fact that
polypharmacy induces strong impairments in different
types of memory and decreases synaptic proteins
already at young age is significant and support the
importance to further explore adverse effects of the
multiple-drug regimen in old mice. The results from this
study will therefore be useful to design and interpret
future results on aging animals. The same combination
of medications including simvastatin, metoprolol,
aspirin, paracetamol, and citalopram induced clearly
distinguished effects in male and female young adult
mice, that can be translated to humans. In sum, this
study strongly supports the importance of considering
sex-specific differences in designing safer and targeted
multiple-drug therapies for older adults.
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MATERIALS AND METHODS
Animals
In this study we used wild-type C57BL/6J female mice,
which were purchased from Janvier Labs (France) at the
age of 8 weeks and then housed in our animal facility in
groups of five per cage (Karolinska Institutet, Solna,
Sweden) with 12-h light/dark cycle, ad libitum access to
food/water and standard enrichment (cardboard tunnels,
wooden sticks, and tissue paper). A control and a
polypharmacy group of 10 animals each were randomly
constituted in groups of 5 mice per cage, when the mice
were 5.5 months old. We used a standard rodent diet
(control diet) to feed the control group: 18.5 % proteins,
5.5 % oils and fats, 4.5 % fiber (Teklad 2918 diet,
Research Diet Inc., NJ, USA) while the polypharmacy
group was administered with the same diet
supplemented with drugs (polypharmacy diet).
Polypharmacy treatment and study plan
The drugs for the polypharmacy regimen were chosen
based on the most frequently used medications in older
population in Sweden [20]: metoprolol (100
mg/Kg/day; Sigma-Aldrich, USA) [60], paracetamol
(acetaminophen, 100 mg/Kg/day; Sigma-Aldrich, USA)
[61], aspirin(acetylsalicylic acid , 20 mg/Kg/day;
Sigma-Aldrich, USA) [54], simvastatin (10 mg/Kg/day;
Selleck Chemicals, USA) [62] and citalopram (10
mg/Kg/day; Selleck Chemicals, USA) [63].
Paracetamol was selected as analgesic as it is the second
most frequently prescribed drug to older adults with
polypharmacy in Sweden [20]. Many older adults have
chronic pain and paracetamol is considered first line
treatment for acute and chronic pain in older people,
having a more favorable safety profile than non-
steroidal anti-inflammatory drugs (NSAIDs) and
opioids [64]. Aspirin was included in the regiment for
its antiplatelet properties, which is used for prevention
of cardiovascular and cerebrovascular disease and low
dose aspirin is among the three most commonly used
drugs in older adults in Sweden [20].
Compound dosages per Kg/BW were selected after
translation from the human therapeutic range into the
mouse one and according to previous studies where they
did not show toxicity in rodents, as explained in detail
in our polypharmacy pilot study in young wild-type
male mice [19]. Taking into account some variability
between the estimated FI and the real one we decided to
keep the drug concentrations towards the higher
therapeutic dose, with the exception of drugs with
potential dose-dependent toxicity in rodents (i.e.
paracetamol [65, 66]). Medicine concentrations per
Kg/diet were considered based on a FI on average of 0.1
± 0.2 g food/g mouse/day as previously observed for
C57BL/6J mouse strain in our animal facility and
literature [19, 67].
According to our pilot study design [19] the animals
were assessed for behavioral studies after four weeks of
treatment, at 6.5 months of age, while carrying on the
polypharmacy regimen for other four weeks, for a total
duration of eight weeks. Over the study period we
monitored the following parameters weekly: BW, FI (g
food/mouse/day) and WI (ml water/mouse/day). Every
week the chow was replaced with fresh food. At the end
of the two-months treatment period the animals were
sacrificed by cervical dislocation and trunk blood was
collected. After brain dissection, tissues were collected
and immediately snap frozen in dry ice and stored at -80
C until further use.
Ethical statement
All behavioral experiments were run in accordance with
the local national animal care and guidelines and
approved by the local committee of Karolinska
Institutet and the Swedish Board of Agriculture (ethical
permit ID 827). All possible efforts were made to
reduce any suffering or distress to the animals.
Behavioral tests
Mice were evaluated with the following behavioral tests
after four weeks of treatment at 6.5 months of age:
Open Field (OF), Rotarod, Grip Strength, Elevated Plus
Maze (EPM), Dark/Light Box (DLB), Y Maze, Novel
Object Recognition (NOR) and Fear Conditioning (FC).
All the experiments were run between 9:00 and 14:00
by a female researcher (FE) with a break from one to
six days after those tests considered more stressful or
physically demanding to allow the animals to recover.
The order of the tasks was chosen according to the level
of stress caused by the protocol, starting from the least
stressful test: OF, EPM, DLB, Y Maze, NOR, Grip
strength, Rotarod, FC [68]. Mice were allowed to
acclimatize to the experimental room for 45 minutes
prior to starting each test. The experiments were
performed in white light. All the apparatuses were
cleaned with 70% ethanol solution before starting each
test and between animals.
OF activity in locomotor cages, Rotarod, EPM, DLB,
Y Maze and NOR test protocols were run as recently
described in detail [19]. Data of EPM, Y Maze and
NOR experiments were acquired with a camera
installed above the apparatus/boxes, connected to the
video-tracking software Ethovision XT 15 (Noldus
Information Technology, The Netherlands). OF
and DLB tests were performed using 45 x 45 cm
www.aging-us.com 14739 AGING
activity cages where the animal movements were
automatically detected as infrared beam interruptions
by TSE ActiMot software (TSE Systems GmbH,
Germany). Horizontal and vertical activity in OF, as
well as the latency and time spent in the light
compartment in DLB tests were analyzed through the
same software.
Grip strength test
This test was used to evaluate the forelimb grip strength
of the animals. The apparatus consisted of a grid
attached to a force transducer which measured the force
(in grams) applied by the mouse pulling the grid
(Bioseb Instruments) [69, 70]. During the pull the
mouse was held by the tail by the experimenter and only
pulls using both forepaws were considered. The animals
performed three series of 3-pulls each with a short
resting period between each (2 minutes). The final grip
strength was calculated by taking the average of the 9
measurements collected over the 3-pull series
normalized for the BW.
Contextual and cue FC test
This experiment was performed in transparent wall
chambers with a stainless-steel grid floor which were
enclosed in a soundproof apparatus (TSE Multi
Conditioning Systems- TSE Systems GmbH, Germany).
On day 1, mice were allowed to freely explore the
context (a 20 x 20 x 40 cm square base chamber) for 2
minutes (habituation phase) and subsequently were
exposed to a conditioned stimulus (55 dB sound at 5000
Hz, 30 sec duration) followed by a mild foot shock (0.3
mA, 2 sec duration). The sound-shock pairing was
repeated three times in total with a 50-sec interval
between each one. On day 2 (after 24 h) mice were
returned to the same chamber for a period of 3 minutes
to assess contextual fear memory. No sound or shock
were given in this session. On day 3, the context was
altered to evaluate the animals for cue memory [71]: the
squared chamber was replaced with a round one (20 cm
diameter x 40 cm high) and the grid floor was covered
by a black smooth surface. To modify the odor, we
cleaned the chamber with hypochlorous water instead of
70% ethanol. The animals were placed in this “new”
context and after 2-minutes of free exploration they
received the sound stimulus (same as in day 1: 55 dB at
5000 Hz) for a further 2 minutes continuously. The
Freezing behavior (defined as complete absence of
mobility within the same area for a time > 2 seconds)
was measured through TSE Multi Conditioning
software. The freezing % recorded during the
habituation phase of day 1 (as a measure of baseline
freezing) was compared to freezing % of day 2 to
evaluate the context memory. To assess the cue
memory, we measured the freezing % on day 3 before
and during the sound stimulus.
Immunoblotting analysis
We performed western blot experiments on
hippocampal tissue lysates and protein levels were
quantified after separation by acrylamide gel electro-
phoresis (gradient 12-7.5 %) and transfer to a
nitrocellulose membrane, as previously described [19,
72]. Membranes were incubated overnight at 4° C with
the following primary antibodies: rabbit anti-phospho
NMDA receptor 2A (1:250, Abcam), mouse anti-
NMDA receptor 1 (1:2000, BD Bioscience), mouse
anti- PSD95 (1:1000, Abcam), mouse anti-CREB
(1:750, Cell Signaling) and rabbit anti-phospho CREB
(1:1000, Cell Signaling), rabbit anti-CaMKII and rabbit
anti-phospho CaMKII (1:1000, Cell Signaling), rabbit
anti-TrkB (1:1000, Cell signaling), rabbit anti-BDNF
(1:1000, Abcam) and mouse anti-alpha-tubulin
(1:30000; Sigma-Aldrich, USA). Incubations with
secondary antibodies were done for 2 hours at room
temperature with anti-rabbit or anti-mouse immuno-
globulin G (IgG) at 1:10000 dilutions (LI-COR
Biosciences GmbH, Germany). Immunoreactivity was
detected with LI-COR® Odyssey® system (LI-COR
Biosciences, USA) by infrared fluorescence and
quantified with ImageJ 1.48v software (NIH, MA,
USA) by densitometry analysis of the immunoreactive
bands.
Blood analysis
Trunk blood was collected right after the animal sacrifice
and allowed to clot for 30 min at room temperature,
followed by 5000 g centrifugation for 10 minutes at 4° C
to collect the serum fraction [19, 73]. Serum creatinine
and ALT levels were measured using the following assay
kits respectively: DICT-500 (BioAssay Systems) and
MAK052 (Sigma-Aldrich). Assays were performed
according to manufacturer instructions.
Statistical analysis
All data are displayed as mean ± standard error of the
mean (SEM), with n indicating the number of animals.
We used GraphPad Prism 9 software (San Diego, CA,
USA) to perform the statistical analyses. T-Student or
Mann-Whitney tests were used when comparing the
average of two groups for parametric and non-
parametric data respectively. Data distribution was
evaluated with Shapiro-Wilk test. When two
independent variables were present two-way ANOVA
repeated measurements, followed by Tukey’s multiple
comparison test, was used to analyze the data. A P value
≤ 0.05 was considered as index of significance.
www.aging-us.com 14740 AGING
AUTHOR CONTRIBUTIONS
EF: Design and Performing all experiments, Data
collection, Data analysis and interpretation, Writing -
original draft and review and editing. JK:
Conceptualization, Data interpretation, Funding
acquisition, Project administration, Writing – review
and editing. LLM: Performing part of the experiments,
Data collection. HSN: Experiment design, Data
interpretation, writing – review and editing. WJW:
Conceptualization, Data interpretation, writing – review
and editing. CMA: Conceptualization, Funding
acquisition, Writing – review and editing. MS:
Conceptualization, Design and Performing part of
experiments, Supervision, Data interpretation, Writing -
original draft and review and editing.
ACKNOWLEDGMENTS
All the behavioral studies were performed at the Animal
Behavior Core Facility (ABCF) of Karolinska Institutet,
Solna, Sweden.
CONFLICTS OF INTEREST
The authors declare that they have no conflicts of
interest.
FUNDING
This research was supported by the Swedish Research
Council, Margaretha af Ugglas Foundation, the
regional agreement on medical training and clinical
research (ALF) between Stockholm County Council
and Karolinska Institutet, Gun och Bertil Stohnes
Stiftelse, Karolinska Institutet Foundation for geriatric
research, Stiftelsen Gamla Tjänarinnor and Tore
Nilsson Stiftelse.
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