2.1 Animals
The 5XFAD mouse model is a
well-characterised double transgenic APP/PSEN1 model, which co-expresses
5 familial AD mutations. This animal model incorporates AD pathological
characteristics including early plaque formation and gliosis starting at
2 months, robust cognitive and behavioural deficits such as memory
impairment, reduced anxiety and social disturbances starting at 4-5
months, and neuronal loss at 6 months. (Oakley et al., 2006; Landel et
al., 2014; Griñán-Ferré et al., 2018). Thus, at the selected age of 7
months, 5XFAD mice provide a severe AD pathological landscape suitable
for the evaluation of the drug effects.
In the present study, 5XFAD (n = 47) and Wild-Type (WT, n = 46) female
mice (7-month-old) were used to perform behavioural and molecular
analyses. Females were used because AD incidence is higher in women and
few studies are available. WT animals were randomly divided into WT
Control (WT Ct ) (n=11), WT treated with donepezil (1
mg-1 kg-1 day-1)
(WT Dp ) (n=12), LSL60101 (WT LSL ) (1
mg-1 kg-1 day-1)
(n=12), and the co-treatment donepezil (1 mg-1kg-1 day-1) and LSL60101 (1
mg-1 kg-1 day-1)
(WT Dp+LSL ). 5XFAD mice were randomly divided into: 5XFAD
Control (5XFAD Ct ) (n=11), 5XFAD treated with Donepezil (1
mg-1 kg-1 day-1)
(5XFAD Dp ) (n=12), LSL60101 (5XFAD LSL ) (1
mg-1 kg-1 day-1)
(n=12), and the co-treatment Donepezil (1 mg-1kg-1 day-1) and LSL60101 (1
mg-1 kg-1 day-1(5XFAD Dp+LSL ). The animals had free access to food and water
and were kept under standard temperature conditions (22 ± 2°C) and
12-h/12-h light/dark cycles (300 lux/0 lux). Compounds were dissolved in
1.8% (2-hydroxypropyl)-β-cyclodextrin and administered through drinking
water for 4 weeks. Control groups received water plus 1.8%
(2-hydroxypropyl)-β-cyclodextrin during the treatment period. The sample
size for the intervention was chosen following previous studies in our
laboratory and using one of the available interactive tools
(http://www.biomath.info/power/index.html).
Moreover, the animal number mismatch among experimental groups was due
to the exclusion of mice by death or ethical reasons according to the
final point indicated in the approved protocol.
After 4 weeks of treatment, behavioural and cognitive tests were
performed to study the effects of treatment on learning, memory, anxiety
behaviour and social interaction (Fig. 1a). Weight and water consumption
were controlled each week, and compounds concentrations were adjusted
accordingly to reach the optimal dose until the euthanasia. All studies
and procedures for the mouse behaviour tests, brain dissection and
extractions followed the ARRIVE (Lilley et al., 2020) and standard
ethical guidelines (European Communities Council Directive 2010/63/EU
and Guidelines for the Care and Use of Mammals in Neuroscience and
Behavioural Research, National Research Council 2003) and were approved
by Bioethical Committees from the University of Barcelona and the
Government of Catalonia.