A Stress Syndrome Prototype Reflects Type 3 Diabetes and Ischemic Stroke Risk: The SABPA Study

Simple Summary The relationship between diabetes, dementia and neuronal injury defines type 3 diabetes (T3D). Similar retinal vessel changes were observed in Alzheimer’s and chronic stressed individuals. The aim was to assess associations of chronic stress with T3D and retina pathology. An established validated chronic stress and stroke risk score (hereafter Stress) stratified participants into Stressed (high stress) and non-Stressed (low stress) groups. Early dementia signs (insulin sensitivity, cognitive dysfunction, neuronal injury) were evident in Stressed compared to non-Stressed individuals. Stress was associated with poor retinal oxygen perfusion, potentiating glaucoma risk. Stress was further related to four dementia risk markers (cognitive dysfunction; telomere shortening, neuronal injury and waist circumference), which comprised a novel Stress syndrome prototype. This prototype showed high risk for T3D as well as retinal vein widening and perfusion deficits, indicating stroke risk. Findings should increase awareness on the detrimental role of chronic stress in the human brain. Abstract Type 3 diabetes (T3D) accurately reflects that dementia, e.g., Alzheimer’s disease, represents insulin resistance and neurodegeneration in the brain. Similar retinal microvascular changes were observed in Alzheimer’s and chronic stressed individuals. Hence, we aimed to show that chronic stress relates to T3D dementia signs and retinopathy, ultimately comprising a Stress syndrome prototype reflecting risk for T3D and stroke. A chronic stress and stroke risk phenotype (Stressed) score, independent of age, race or gender, was applied to stratify participants (N = 264; aged 44 ± 9 years) into high stress risk (Stressed, N = 159) and low stress risk (non-Stressed, N = 105) groups. We determined insulin resistance using the homeostatic model assessment (HOMA-IR), which is interchangeable with T3D, and dementia risk markers (cognitive executive functioning (cognitiveexe-func); telomere length; waist circumference (WC), neuronal glia injury; neuron-specific enolase/NSE, S100B). Retinopathy was determined in the mydriatic eye. The Stressed group had greater incidence of HOMA-IR in the upper quartile (≥5), larger WC, poorer cognitiveexe-func control, shorter telomeres, consistently raised neuronal glia injury, fewer retinal arteries, narrower arteries, wider veins and a larger optic cup/disc ratio (C/D) compared to the non-Stressed group. Furthermore, of the stroke risk markers, arterial narrowing was related to glaucoma risk with a greater C/D, whilst retinal vein widening was related to HOMA-IR, poor cognitiveexe-func control and neuronal glia injury (Adjusted R2 0.30; p ≤ 0.05). These associations were not evident in the non-Stressed group. Logistic regression associations between the Stressed phenotype and four dementia risk markers (cognitiveexe-func, telomere length, NSE and WC) comprised a Stress syndrome prototype (area under the curve 0.80; sensitivity/specificity 85%/58%; p ≤ 0.001). The Stress syndrome prototype reflected risk for HOMA-IR (odds ratio (OR) 7.72) and retinal glia ischemia (OR 1.27) and vein widening (OR 1.03). The Stressed phenotype was associated with neuronal glia injury and retinal ischemia, potentiating glaucoma risk. The detrimental effect of chronic stress exemplified a Stress syndrome prototype reflecting risk for type 3 diabetes, neurodegeneration and ischemic stroke.


Perception of Stroop test stressfulness
Perception of test stressfulness was assessed on a 7-point Likert scale. After completion of each task, participants reported their overall distress according to the question "How stressful was the task you just completed?" on a 7-point Likert scale ranging from not severe to highly severe.

Ambulatory blood pressure
The blood pressure cuff was fitted to the non-dominant arms using an appropriate cuff size. Participants were asked to record occurrences of stress and any disturbances on their 24h ambulatory diary card. In the SABPA study, blood pressure measures were obtained every 30 minutes during the day (08:00-22:00) and hourly during the night (22:00-06:00) to measure blood pressure without too much disturbance/inconvenience during the normal school working day and after school extracurricular activities [26]. Participants were asked to record occurrences of stress and any disturbances on their 24h ambulatory diary card. The 24h successful inflation rate was 77.1% (±12.8) in Stressed individuals and 83.1% (±10.2) in no-Stressed individuals. The data were analyzed with the CardioVisions 1.19 Personal Edition software (Meditech, Budapest, Hungary). Hypertensive status according to the 2018 European Society of Cardiology Guidelines was classified as 24h SBP ≥ 130 mm Hg and/or DBP ≥ 80 mm Hg.

3.
Time-domain heart-rate-variability (HRV) Time-domain HRV analysis was computed to assess spontaneous oscillations resulting from sinus node depolarization. Two-channel ECG recordings over 24h were used to assess time-domain HRV (Cardiotens CE120®, Meditech, Budapest Hungary) were obtained according to a preset program for 20s at 5-min intervals. The software program automatically filtered out ventricular, supraventricular as well as artefacts in RR intervals, and HRV outliers had been manually removed. Time-domain analysis marker included the standard deviation of the normal-to-normal (NN) intervals between adjacent QRS complexes, which equal the square root of variance (SDNN ms). SDNN is regarded as the best overall prognostic tool for values <50 ms are indicative of highly depressed HRV, those between 50-100 ms indicate moderately depressed HRV and those >100 ms are classified as normal. SDNN further reflects vagus nerve-mediated autonomic control of the heart.

Carotid bifurcation stenosis
A trained cardiovascular scientist determined carotid intima media thickness and bifurcation stenosis at 3-year follow-up. Re-evaluation and calculation was done by an independent Echocardiogram Technologist and added as covariate in retinal vessel regression analyses models. Intra-observer variability was 0.04 mm between two measurements made 4 weeks apart on 10 participants. High resolution ultrasound images were acquired from at least two optimum angles of the common carotid artery segments (Sonosite Micromaxx ultrasound system (SonoSite Inc., Bothell, WA, USA) and 6-13 MHz linear array transducer] using the Rudy Meijer protocol. The images were digitized and imported into the Artery Measurement Systems automated software (AMS) II v1.139 (Gothenburg, Sweden) for dedicated analysis of the carotid artery as ischemic stroke risk marker [15].

Telomere length [21]
Before telomere analyses all samples were diluted to the concentration of 10 ng/µl. Reference DNA samples were prepared, and all isolated DNAs were mixed together in equal proportions representing the average of all analyzed patients (N=255). LTL was assessed by multiplex Quantitative Real-Time PCR (Q-PCR) (Fani et al., 2020). Five concentrations of a reference DNA sample spanning a 75-fold range of DNA concentrations were prepared by serial dilution and analyzed in triplicate; these reactions provided the data for the generation of the standard curves used for relative quantification. All experimental DNA samples were assayed in triplicate. All PCRs were performed with CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in a 25 µl volume. PCR reactions comprised of 20 µl master mix (including 5 µl of 5x HOT FIREPol ® EvaGreen ® qPCR Mix Plus (no ROX) (Solis BioDyne, Tartu, Estonia) and 5 µl of each experimental DNA sample.
For multiplex Q-PCR, the telomere primer pair telg: ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT and telc: TGTTAGGTATCCCTATCCCTATCCCTATCCCTATCCCTAACA (final concentrations 900 nM each), were combined with primers for the single copy gene human beta-globin (hbg); hbgu: CGGCGGCGGGCGGCGCGGGCTGGGCGGCTTCATCCACGTTCACCTTG and hbgd: GCCCGGCCCGCCGCGCCCGTCCCGCCGGAGGAGAAGTCTGCCGTT (final concentrations 500 nM each), to give PCR products of 79 bp and 106 bp, respectively. All primers were manufactured by Integrated DNA Technologies (Coralville, IA, USA). The thermal cycling profile was Stage 1: 15 min at 95°C; Stage 2: 2 cycles of 15s at 94°C, 15 s at 49°C; and Stage 3: 32 cycles of 15 s at 94°C, 10 s at 62°C, 15 s at 73°C with signal acquisition, 10 s at 84°C, 15 s at 87°C with signal acquisition. The 73°C reads provided the Ct values for the amplification of the telomere template (in early cycles where the hbg signal is still at baseline); the 87°C reads the Ct values for the amplification of the hbg template (at this temperature there is no signal from the telomere PCR product, because it is fully melted). After thermal cycling and raw data collection were complete, CT values generated by CFX Manager™ Software Version 1.6 (Bio-Rad, Hercules, CA, USA) were exported to REST© software (Relative Expression Software Tool), version REST-384. PCR efficiency for each primer set was calculated by serial dilutions of reference DNA using the REST software tool. The relative LTL per cell was also calculated using the REST software as the ratio (T/S) between relative content of telomere PCR product (T) and human beta-globin PCR product (S). The LTL was calculated for each individual participant relative to the average LTL of the total analyzed group (344 participants).

Mitochondrial DNA (mtDNA) -MutPred Load
DNA for sequencing was extracted from whole blood samples. Sequencing of the entire mtDNA molecule was done using the Ion Torrent PGM, as described elsewhere [42]. Whole mtDNA sequences have been deposited in the sequence read archive (SRA) of NCBI under BioProject accession number PRJNA403942 (https://www.ncbi.nlm.nih.gov/ bioproject/403942). Consensus variants were called against the revised Cambridge reference sequence (rCRS). MtDNA haplogroups were assigned according to the mtDNA variant list of each participant, using Haplogrep 2.0.y We summed the MutPred scores generated for each of the non-synonymous variants after calculating "MutPred mutational loads" on an individual's mtDNA; and adjusted these for the position of the sequence in the phylogeny, ultimately calculating "MutPred adjusted loads". Many different tools for predicting pathogenicity of mtDNA variants exist, but MutPred and SNPs & GO outperformed all other methods, which included PolyPhen2, SIFT and SNAP.T . HRV-SDNN, time-domain 24h heart-rate-variability standard deviation of the normal-to-normal (NN) intervals between adjacent QRS complexes which equal the square root of variance. Figure S1: Presenting the apparatus and method to determine retinal vessel imaging ( Figure S1a,b); and the Stroop test ( Figure S1c,d), which was used as measure of cognitive executive functioning control.