The experiments were performed in accordance to European guidelines for the care and use of laboratory animals (Directive 2010/63/UE) as described by Lilli et al. [13]. Briefly according to the decree 2013-118 of February 1, 2013 on the protection of animal used for scientific purposes, animal suffering has been minimized and the number of animals used in the experiments reduced. Animal sacrifice with the sole purpose of using their organs or their tissues is not considered as an experimental procedure (https://www.recherche-animale.org/sites/default/files/decret_2013-118, Pdf). For this reason, no authorization was required for this project. Prior to the administration of drugs, animals used to prepare mesencephalic cell cultures were naive. After receipt at the animal house at the latest the day before the sacrifice, pregnant mice were euthanized with CO₂, according to the authorized procedures. The nervous tissues of interest were then recovered from the embryos and cultured. Central nervous system (CNS) cultures of mice embryos were not subject to any authorization.

As described by Harrison [14], mice hippocampal neurons were cultured. Briefly pregnant female mice (Swiss mice; Janvier) of 15 days gestation were euthanized with CO₂. The foetuses were removed from the uterus and placed in ice-cold medium of Leibovitz 15 (L15; PanBiotech, Germany) containing 2% of Penicillin-Streptomycin (PS; PanBiotech, Germany) and 1% of bovine serum albumin (BSA; PanBiotech, Germany). The hippocampi were dissociated by trypsinisation for 20 min at 37˚C (Trypsin EDTA 1X; PanBiotech, Germany). The reaction was stopped by the addition of Dulbecco’s modified Eagle’s medium (DMEM; PanBiotech, Germany) containing DNAase I grade II (0.1 mg/ml; Roche Diagnostic, France) and 10% of foetal calf serum (FCS; Invitrogen, USA). Cells were then mechanically dissociated by 3 passages through a 10 ml pipette and then centrifuged at 180x g for 10 min at 4˚C on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded and the cells of pellet were re-suspended in a culture medium consisting of Neurobasal (Invitrogen, USA) supplemented with B27 (2%; Invitrogen, USA), L-glutamine (2 mM; PanBiotech, Germany), 2% of PS solution and 10 ng/ml of brain-derived neurotrophic factor (BDNF; PanBiotech, Germany). Viable cells were counted in a Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a density of 20.000 cells/well in 96 well-plates (pre-coated with poly-D-lysine; Greiner, Austria) and were cultured at 37˚C in a humidified air (95%)/CO₂ (5%) atmosphere. Half of the medium was changed every 2 days with fresh medium. After 12 days of culture, astrocytes are present in the culture and release growth factor allowing neurons differentiation. Hippocampal neuron cultures (6 wells par condition) were treated with MIM (2LMISEN^®; 11 mM) or vehicle (11 mM) from 22 days in vitro (DIV) until 27 DIV and senescence was evaluated at 22, 25 and 27 DIV (Figure 1).

Cells were fixed at 15 (control condition), 22, 25 and 27 DIV by a solution of paraformaldehyde 4% (Sigma-Aldrich, USA) for 25 min at room temperature and washed two times with phosphate buffered saline (PBS, PanBiotech, Germany). Cells were then permeabilized and non-specific sites were blocked with a solution of PBS containing 0.1% of saponin (Sigma Aldrich, USA) and 1% of FCS for 15 min at room temperature. Cells were then incubated with primary antibodies p16^INK4a produced in mouse (Thermo Scientific, USA), MAP2 produced in chicken (Abcam, France) and Caspase 3 produced in rabbit (Abcam, France). These antibodies were revealed with Alexa Fluor 488 goat anti-mouse

IgG (Molecular probe, USA), Alexa Fluor 568 goat anti-rabbit IgG (Molecular probe, USA) and Alexa Fluor 633 goat anti-chicken (Molecular probe, USA). Nuclei of neurons were labeled by a fluorescent marker (Hoechst solution, Sigma-Aldrich, USA). Quantification of p16^INK4a and Caspase 3-positive neurons was done using Developer software (GE Healthcare, France). For each well of culture, 20 pictures (20x magnification) per well were taken in the same conditions using InCell Analyzer^TM 2200 (GE Healthcare, France).

The MIM is notified to the Belgian Federal Agency for Medicines and Health Products under notification number 1507CH47F1. As reported by Lilli et al. [13], MI medicines for oromucosal administration are composed of lactose-saccharose pillules impregnated with LD and/or ULD of ethanolic preparations of immune mediators and nucleic acids. Active substances are obtained through a “serial kinetic process” reproduced a defined number of times, consisting of a 1/100 dilution process followed by vertical shaking. Centesimal Hahnemannian (CH) dilutions are used to express medicines composition indicating the number of times by which the two proceedings are carried out for each active substance. The composition of the MIM tested capsule is as follow: recombinant human (rh) interleukin-2 (IL-2; 10CH); rh epidermal growth factor (EGF; 10CH); dehydroepiandrosterone (DHEA; 10CH); dimethylsulfoxide (DMSO; 10CH); ribonucleic acid (RNA; 10CH); specific nucleic acid^® (SNA) targeting human leukocyte antigen (HLA) class I (SNA-HLA I; 10CH); SNA^® HLA class II (SNA-HLA II; 10CH); SNA^® targeting human telomerase reverse transcriptase (TERT) (SNA-MISEN; 16CH). Vehicle used as control consists of lactose-saccharose pillules impregnated with the vehicle solution without any active substance.

The pillules contained in one capsule of MIM or vehicle control (380 mg) were dissolved in cell culture media (100 ml). Indeed, the tested lactose-saccharose concentration was 11 mM.

Statistical analyses and graphs have been performed using SAS software 9.4 (SAS Institute, Cary NC). The data were represented by box plot with scatter diagram (of 6 data per condition, 1 culture). The comparison between groups was performed using a one-way analysis of variance (ANOVA) following by Dunnett’s test to compare control 15 DIV vs controls at 22, 25 and 27 DIV and Tukey test to compare vehicle vs MIM at 22, 25 and 27 DIV. The level of significance is set at p ≤ 0.05.

We showed that an increase of p16^INK4a immunostaining appeared at 22 DIV in hippocampal neurons culture and remained at 25 DIV and 27 DIV (Figure 2). We found that vehicle had no effect on p16^INK4a expression, whereas MIM was able to decrease p16^INK4a levels at 22 DIV, 25 DIV and 27 DIV in a statistical significant manner (control 15 DIV vs controls 22, 25, 27 DIV [p ≤ 0.0001]; control vs MIM 22 DIV [p = 0.0004], 25 DIV [p = 0.0004] and 27 DIV [p = 0.0272]; vehicle vs MIM 22 DIV [p = 0.0017], 25 DIV [p = 0.0037], 27 DIV [p = 0.1333]). Representative pictures of hippocampal neurons staining with primary antibodies p16^INK4a are reported in Figure 4.

We showed that a statistically significant increase of caspase 3 immunostaining appeared at 22 DIV in culture (Figure 3). We found that neither vehicle nor MIM had effect on caspase 3 level at any time of culture (control 15 DIV vs control 22 DIV [p = 0.0098]; control vs MIM 22 DIV [p = 0.8077], 25 DIV [p = 0.8655] and 27 DIV [p = 0.7868]; vehicle vs MIM 22 DIV [p = 0.7156], 25 DIV [p = 0.6677], 27 DIV [p = 0.9886]). Representative pictures of hippocampal neurons staining with primary antibodies caspase 3 are reported in Figure 4.