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.