2.1 Study Design and Participants
This is a cross-sectional study with information from the fifth wave of the “Cohort of Obesity, Sarcopenia, and Frailty of Older Mexican Adults” (COSFOMA). COSFOMA procedures and methods have been previously described 9. Briefly, it is a population-based prospective study that began in 2014 with the participation of 1,252 adults aged 60 years or more who were beneficiaries of the Mexican Institute of Social Security (IMSS, by its Spanish acronym) in Mexico City. Participants were randomly selected from administrative records. COSFOMA collected information through interviews and geriatric assessments conducted by previously trained personnel at annual intervals. COSFOMA collected clinical and sociodemographic information annually through interviews carried out by trained health staff. In the fifth wave (the year 2018) of COSFOMA, 523 (41.8%) participants were followed. The present research used the data from this wave.
For the present study, samples were split into four groups as follows: Group 1) No insomnia/no cognitive decline (control group), Group 2) Insomnia-alone, Group 3) Cognitive decline alone, and Group 4) Insomnia and cognitive decline. All cases found from the database were integrated, except in the case of the non-insomnia and non-cognitive decline group, where participants were randomly selected among those negative to both variables, insomnia, and cognitive decline.
Insomnia
The Athens Insomnia Scale (AIS) was used to measure insomnia symptoms through eight items scored on a four-point Likert scale. Total scores range from 0 to 24, with higher values indicating greater insomnia-related issues. The cut-off point ≥ 6 points was used to indicate presence of insomnia 10. AIS has been validated in Mexico 10. The AIS Cronbach alpha value in this study was 0.90.
Cognition
Cognitive status was evaluated with the Mini-Mental State Examination, a 30-item scale that examines different cognitive domains, with higher scores meaning better performance. A cut-off point adjusted by level of schooling (≤ 23) indicated cognitive decline 11,12.
Covariates
Sociodemographic variables included sex, age, years of education, and living arrangement. Health-related variables included current smoking, current alcohol consumption, multimorbidity, number of medications currently used, use of antidepressant medications, and frailty (Linda Fried score index).
Multimorbidity was defined as having two or more diseases out of a predefined list of conditions, namely: 13,14, hypertension, diabetes, heart disease, cancer, kidney failure, cerebrovascular disease, arthritis, chronic liver disease, and chronic pulmonary disease. The use of antidepressant medications was considered separately because of the potential influence of these medications on modifying BDNF concentrations. (7)
Anxiety symptoms were assessed with the Short Anxiety Screening Test (SAST), a scale developed to standardize the detection of anxiety in the elderly, including somatic symptoms, consisting of 10 items, scored from one to four points, with a highest possible score of 40 (highest anxiety level) 15
Depressive symptoms were evaluated using the Center for Epidemiologic Studies Depression Scale (CES-D), a validated 20-item scale consisting of four factors: depressive effect, somatic complaints, positive impact, and interpersonal relations. Scores on the CES-D range from 0 to 60, where scores of 0 to 15 indicate the absence of depression and those of 16 to 60 reflect depressive symptomatology 16.
Frailty was assessed using the phenotype described by Fried et al. 9,17. Briefly described as follows, frail adults are defined as those who show three or more items from the following criteria: Weight loss, self-reported exhaustion, low physical activity, slowness, weakness (low grip strength). Adults who show one or two criteria indicate a pre-frail condition, while the absence of these items means a non-frail state.
2.3 BDNF Immunoblots
As reported in Rivero-Segura et al. Campo (Rivero-Segura et al., 2017), immunoblots were performed. Briefly, protein samples were re-suspended in loading buffer (cat#1610747, BioRad) supplemented with 5% of β-mercaptoethanol and denatured at 95°C for 5 minutes in a block heater. Then, 40ug of protein of each sample were loaded into 4–20% Mini-PROTEAN® TGX Stain-Free™ Protein Gels (cat# 4568093, BioRad) and run under denaturing conditions (90mV during 1 hour at RT°). Proteins were transferred to PVDF membrane (cat# GVWP04700, Merk) at 100mV for 25 min in a cold bath, as previously reported in Rivero-Segura et al. 18. Membranes were blocked with Intercept® (PBS) Blocking Buffer (cat# 927-70001, LI-COR) during 1H at RT°; then membranes were incubated overnight at four °C with blocking buffer containing the corresponding primary antibody: anti-BDNF (cat# SC-546, Santa Cruz Biotechnology) and anti-Trasnferrin (cat# ab82411, Abcam), both kindly brought by Dr. Paola García de la Torre. The hybridized proteins were incubated with the corresponding secondary antibodies: anti-rabbit (cat # 926-32213, or cat#926-68073, LI-COR), conjugated with the fluorophore IRDye 800CW or IRDye® 680RD. The signal was detected by fluorescence using the ODYSSEY blot-scanner (LI-COR). Densitometry analysis was performed with Image J software, and the protein content was normalized against Transferrin.
2.5 Statistical analysis
Group was considered the explanatory variable, whereas the others were considered the control variables. BNDF was regarded as a response. A Kruskal-Wallis test was used to determine whether the response was similarly distributed between groups. Multiple comparisons were assessed through a Dunn’s test.
Generalized linear models were fitted, considering BNDF as the response variable. A gamma distribution was considered since the response is skewed to the left. Identity and logarithmic link functions were used, obtaining similar results; thus, only the analysis using the former is presented. First, we fitted a model in which Group was used as an explanatory variable; after that, we did a model adding all the control variables, including one at a time: frailty, balance, sarcopenia, and gait speed, which present collinearity between them (data not shown). However, since similar results were obtained, only the analysis, including frailty is presented. A similar model was fitted for comparison purposes but using insomnia and cognitive decline instead of Group. A Likelihood Ratio Test (LRT) between the model, including only Group against the model, including all covariates already described, was obtained. A similar test was obtained when insomnia and cognitive decline were used instead of Group.
All participants and their legal guardians were informed of the research procedures and signed a letter of consent before participating. For illiterate participants written informed consent was taken from their legal guardians. The COSFOMA protocol was approved by the IMSS National Committee Research (the National Committee for Scientific Research and the Ethics Committee on Health Research) (Registration No. 2012-785-067). Additionally, the use of samples for this study was approved by the Comité Nacional de Investigación Científica-IMSS with the number R-2017-785-107. All methods employed in the study were in accordance with the Declaration of Helsinki as well as guidelines from the Ley General de Salud of Mexico. The data are available upon express request addressed to the corresponding author and are currently in safekeeping by IMSS.