Prediction of Effectiveness of rTMS Application in Alzheimer's Patients

Prediction of Cognitive, Neurotrophic, Anti-Inflammatory, and Antioxidant Effectiveness of rTMS Application in Alzheimer's Patients by Combination of Test-Dose TMS and EEG

Since pharmacological methods are insufficient in the treatment processes of Alzheimer's disease, non-pharmacological methods such as Transcranial Magnetic Stimulation (TMS) have started to be tried as a treatment option as in other neurological and psychiatric diseases. Repeated (rTMS) offers a potential treatment pathway for neurological and psychiatric illnesses. rTMS benefit rate may vary depending on many factors such as the region where it is applied, the progression and the disease degree. This study aim is to predict the benefit rate to be obtained from the treatment by using various evaluation parameters before starting rTMS treatment. Another goal is to develop a personalized TMS treatment protocol based on the effect of Test Dose TMS on EEG and change in brain networks that can be viewed from EEG data generally before and after rTMS treatment. The possible effects of TMS on Alzheimer's pathophysiology and modification of disease process (neuroprotective, anti-inflammatory and antioxidant) will also be revealed through blood samples taken from patients before and after treatment. These approaches also constitute the original value of our study.

Since pharmacological methods are insufficient in the treatment processes of Alzheimer's disease, non-pharmacological methods such as Transcranial Magnetic Stimulation (TMS) have started to be tried as a treatment option as in other neurological and psychiatric diseases. Repeated (rTMS) offers a potential treatment pathway for neurological and psychiatric illnesses. rTMS benefit rate may vary depending on many factors such as the region where it is applied, the progression and the disease degree. This study aim is to predict the benefit rate to be obtained from the treatment by using various evaluation parameters before starting rTMS treatment. Another goal is to develop a personalized TMS treatment protocol based on the effect of Test Dose TMS on EEG and change in brain networks that can be viewed from EEG data generally before and after rTMS treatment. The possible effects of TMS on Alzheimer's pathophysiology and modification of disease process (neuroprotective, anti-inflammatory and antioxidant) will also be revealed through blood samples taken from patients before and after treatment. These approaches also constitute the original value of our study. In our project, 20 people will be included in the study and control groups and electroencephalography (EEG) and TMS will be used together in the study. Before rTMS treatment, resting EEG data will be taken for 5 minutes, eyes open and closed. After then, Test Dose TMS of 150 beats at 20 Hz will be applied to left dorsolateral prefrontal cortex. Immediately after this Test Dose rTMS, the patient's resting state EEG data will be taken again. At the end of all these procedures, rTMS treatment will be started, which will take 5 days. The treatment will consist of two sessions per day with a 20 Hz stimulating protocol, 1500 beats to right-left DLPFC, and totaly 3000 beats. EEG recordings will be taken again from all patients one week after the treatment. Changes in the cognitive functions of Alzheimer's patients will be made through the neuropsychometric test battery taken before and after rTMS. With the analysis of neuropsychometric data, the study group will be divided into two subgroups that benefit from TMS and those who do not, the effect of Test Dose TMS on EEG will be investigated, the algorithm that predicts individuals who will benefit from TMS through pre-TMS EEG data and Test Dose TMS-EEG data will be investigated and The effect of TMS treatment on EEG will be investigated. Therefore, the prediction algorithm will open the door to personalized treatment protocol. In addition, EEG data obtained before and after-TMS will be compared with power spectrum, coherence, functional connectivity and graph methods in both the study and control groups, and information about the electrophysiological effects of TMS will be obtained. Blood samples of the patients before and after rTMS will be taken and the changes in the metabolites given below will be compared: Brain Derived Neurotrophic Factor, Glial Based Neurotrophic Factor, Total Oxidant Level, Total Antioxidant Level, Oxidative Stress Index, Total Thiol, Native Thiol, Disulfide, Exosome, Inflammation Biomarkers (interleukin 1 Beta, interleukin 6, Tumor Necrosis Alpha, Interferon gamma, Nuclear factor kappa ß), Albumin Globulin ratio, Omega 6 and Omega 3. Thus, rTMS has possible neuroprotective, anti-inflammatory and antioxidant effects, consequently modifying the disease process. Additional information will be obtained about. Within the scope of the project, a workshop, sharing of study results in national-international congresses, a patent application originating from algorithm production, production of 3 graduate students' thesis and at least 3 scientific articles will be produced. In addition, the investigators anticipate that TMS prevalence will increase during the treatment process of Alzheimer's patients, and the socio-economic burden caused by this disease will be reduced.

Power Mag TMS device will be used throughout the study, and the excitations will be made with the help of an 8 shaped coil (diameter: 70 mm) with internal cooling. The right-left DLPFC, which is the application area, will be determined with the help of the primary motor hand area and the 10/20 EEG system. Patients will be given two stimulations (pulse duration=3.5 sec, interval between beats=45 sec) consisting of only 75 beats, 100% of the cap threshold value compatible with the magnetic field TMS determined just before the rTMS routine application, and a total of 150 pulses of Test Dose TMS will be applied. A resting state EEG (eyes open-closed) will be taken immediately after (max. 2 minutes later) in the Faraday cage.

.Power Mag TMS device will be used throughout the study, and the excitations will be made with the help of an 8 shaped coil (diameter: 70 mm) with internal cooling. The right-left DLPFC, which is the application area, will be determined with the help of the primary motor hand area and the 10/20 EEG system. Patients will be given two stimulations (pulse duration=3.5 sec, interval between beats=45 sec) consisting of only 75 beats, 100% of the cap threshold value compatible with the magnetic field TMS determined just before the rTMS routine application, and a total of 150 pulses of Test Dose TMS will be applied. A resting state EEG (eyes open-closed) will be taken immediately after (max. 2 minutes later) in the Faraday cage. The same procedures will be done with the sham coil in the control group.

BDNF:measured spectrophotometrically with commercially purchased ELISA kits. GDNF:measured spectrophotometrically with commercially purchased ELISA kits. Exosome: measured spectrophotometrically with commercially available ELISA kits. Anti-inflammatory Cytokines: IL-1β, IL-6, TNF-α, IFNy, NF-kβ values will be measured spectrophotometrically with commercially purchased ELISA kits.

OSI:TOS and TAS will be measured by photometric methods. OSI will be found by mathematical calculation. Total thiol and native thiol concentrations are measured spectrophotometrically in separate solutions prepared for the determination of the thiol-disulfite ratio, which is another indicator of oxidative stress, and the amount of disulfide is determined according to the mathematical ratio between them. Albumin Globulin Ratio:measured by electrophoresis method and albumin globulin ratio will be determined. Omega 6/3 Levels:determined by commercially purchased lipid mediators.

Inflammatory parameters IL-1β, IL-6 and TNF-α will be measured IL-1β, IL-6 and TNF-α levels will be measured photometrically with commercially available ELISA kits.

TAS, TOS, TT and NT levels of oxidative stress parameters in blood samples taken from AD patients will be measured. TAS, TOS, TT, NT levels of blood samples taken will be measured by photometric method with kits to be purchased commercially. The oxidative stress index (OSI) will be found as TOS/TAS, and the amount of dynamic disulfide bonds will be found by determining half of the difference between the TT and NT groups.

Fatty acids comprising more than 95% of the fatty acids detectable in plasma will be measured. Tetradecanoic acid,9(Z)-Tetradecenoic acid,Hexadecanoic acid,9(Z)-Hexadecenoic acid,9(E)-Hexadecenoic acid,Octadecanoic acid,9(Z)-Octadecenoic acid,Methyl 9(E)- Octadecenoate,11(Z)-Octadecenoic acid,Methyl 11(E)- Octadecenoate,Methyl 6(Z)- Octadecenoate,9(Z),12(Z)- Octadecadienoic acid,9(E),12(E)- Octadecadienoic acid,9(Z),12(Z),15(Z)- Octadecatrienoic acid, 6(Z),9(Z),12(Z)- Octadecatrienoic acid,Eicosanoic acid,8(Z),11(Z),14(Z)- Eicosatrienoic acid, 5(Z),8(Z),11(Z),14(Z)-Eicosatetraenoic acid, 11(Z)-Eicosenoic acid, 11(Z),14(Z)- Eicosadienoic acid, 5(Z),8(Z),11(Z),14(Z),17(Z)- Eicosapentaenoic acid, Docosanoic acid, 13(Z)-Docosenoic acid 4(Z),7(Z),10(Z),13(Z),16(Z),19(Z)- Docosahexaenoic acid 7(Z),10(Z),13(Z),16(Z)-Docosatetraenoic acid 7(Z),10(Z),13(Z),16(Z),19(Z)- Docosapentaenoic acid 4(Z),7(Z),10(Z),13(Z),16(Z)- Docosapentaenoic acid Tetracosanoic acid 15(Z)-Tetracosenoic acid

The 41 amino acids to look for by LC-MS/MS are: 1-Methylhistidine 2-Aminoadipic Acid 3-Aminoisobutyric Acid 3-Methylhistidine 4-Hydroxyproline 5-Hydroxylysine Alanine Alloisoleucine Anserine Arginine Argininosuccinic Acid Asparagine β-Alanine Carnosine Citrulline Cystine Cystathionine Ethanolamine Gamma-Aminobutyric Acid Glutamine Glutamic Acid Histidine Homocitrulline Isoleucine Leucine Lysine Methionine Norvaline O-Phosphorylethanolamine O-Phosphoserine Ornithine Phenylanalanine Proline Sarcosine Serine Taurine Threonine Trans-4-Hydroxyproline Tryptophan Tyrosine Valine

EEG data will be separated into one-second epochs after they are cleared of noise. Power spectrums of these data will be obtained in the delta, theta, alpha, beta and gamma frequency bands. Each epoch will be analyzed by Fast Fourier Transform (FFT, Fast Fourier Transform) with 10% Hanning window, then power spectrum analysis will be performed, giving the frequency values for each electrode by averaging all FFTs. Maximum peaks will be determined in the delta (0.5-3.5 Hz), theta (4-7 Hz), alpha (8-13 Hz), beta (15-28 Hz) and gamma (28-48 Hz) frequency bands. these values will be used in statistical analysis for each person and electrode.

Coherence measurements at delta, theta, alpha, beta and gamma frequencies can be analyzed for either intra-hemispheric electrode connections or inter-hemipheric electrode connections.Coherence values take values between 0-1. Values close to 0 indicate that there is no connection at the determined frequency between the two calculated electrode regions, while values close to 1 indicate a high coupling between the two electrode regions. Coherence values will be calculated with the Brain Vision Analyzer program using the formula below.The data obtained during memory and visualization will be separated into one-second epochs after they are cleared of noise.Power spectrums of these data will be obtained in the delta, theta, alpha, beta and gamma frequency bands. Each epoch will be analyzed by Fast Fourier Transform with 10% Hanning window.Then, these data will be calculated for all possible electrode pairs using the brain vision analysis program with the formula given below.

eLORETA software will be used for functional connectivity analysis. sLORETA /eLORETA is an online free software developed by Roberto Pascual-Marqui and his team (http://www.uzh.ch/keyinst/loreta.htm). eLORETA is an algorithm developed to solve the inverse problem and it does not contain localization bias even in the presence of noise (Pascual-Marqui, 2007). In this software, resting state data with eyes closed, separated into 2 second epochs, whose artifacts are cleaned by preprocessing, will be used. The relevant areas to be analyzed in the cortical plane and the relevant frequency band gaps will be determined. The time series containing the eLORETA current source density obtained from these areas will be calculated and a "lagged linear coherence" matrix will be created to be applied in graph theory (Vecchio et al., 2014). "Lagged linear coherence" will give correct physiological connectivity unaffected by volume conduction and low spatial resolution (Pascual-Marqui et al., 2011).

The machine learning method was created based on the current experimental design and the planned number of patients. After the data is cleared and ready for analysis, the differences in the EEG signals of the patients who benefited from rTMS after the Test Dose TMS from the EEG signals after the Test Dose TMS of the patients who did not benefit will be revealed by a classification method. For this purpose, it is planned to implement the Support Vector Machines (SVM) method. The DVM method is the most suitable for our data and the project purpose among the machine learning methods with its high classification success in large-sized data that is difficult to parse.

Inclusion Criteria: Have been diagnosed with clinical Alzheimer's Disease in accordance with the NINCDS-ADRDA diagnostic criteria >55 years old Clinical Dementia Rating Scale (CDR) score in the 1-2 range Not having any other disease that affects their cognitive functions Volunteer to participate in the study Exclusion Criteria: Participant or relative does not give consent The patient's inability to participate in the entire study procedure (eg living in another city) The patient's history of head trauma with alcohol/substance abuse Presence of severe stroke and other neurological sequelae disease in the participant Presence of a metal implant on the head or having a pacemaker and contraindications for other TMS applications During the study or 1 month before, having/been receiving/receiving an investigational drug targeting Alzheimer's disease or neuromodulation treatment such as tDCS and TMS, other than standard treatment for AD symptom control such as acetylcholine esterase and memantine, with the potential to affect the study