Role of Hypoxia Ans Sleep Fragmentation in Alzheimer's Disease. and Sleep Fragmentation.
Role of Hypoxia Ans Sleep Fragmentation in Alzheimer's Disease. and Sleep Fragmentation.
Alzheimer's Disease (AD) is the most prevalent neurodegenerative disease, manifested as an
 initial deficit of episodic memory that evolves into a global cognitive and psychosocial
 dysfunction and which prevalence is increasing around the world. Sleep disturbance is
 frequent since early stages of the disease and sleep fragmentation had been demonstrated
 increase the production of amyloid peptide (AB) (main pathological hallmark) in non-demented
 population. Obstructive Sleep Apnea (OSA), which consist in intermittent hypoxia and sleep
 fragmentation, is a major health problem with multiple systemic effects and it's very
 prevalent in AD. However, the influence of this comorbidity on the cognitive evolution of AD
 patients remains unknown. The investigation of neurobiological markers and sleep recording
 may reveal potential mechanisms of neurodegeneration and explain the influence of sleep
 fragmentation and/or hypoxia on cognitive decline.

 To fill those gaps, investigators will perform a multidisciplinary and translational project
 to assess the progression of symptoms in AD patients, diagnosis of sleep disturbance and new
 biomarkers of progression of the disease.

 The present proposal is going to be developed by coordination of different expertises that
 will be range from the clinical research conducted by a medical neurologist, to the animal
 model and most molecular work, to be done by an experimented group in mouse work.
As AD and OSA have a bidirectional relationship, OSA causes cerebral hypoxia and sleep
 fragmentation favouring the deposition of AB and AD causes alterations in sleep quality,
 investigators will develop a comprehensive project with human patients and an animal model of
 AD with sleep fragmentation (SF) and intermittent hypoxia (IH).

 Investigators prospectively will study consecutive patients with new diagnosis of mild
 probable Alzheimer's Disease by a neurologist. Investigators will define Alzheimer's Disease
 according to National Institute of Aging-Alzheimer's Association criteria (NIA-AA).

 All patients undergo routine neuropsychological battery, sleep polysomnography and actigraphy
 registry, brain MRI, lumbar puncture and blood biochemistry.

 Neuropsychological battery The following assessments will be used at baseline and at 12
 months: Alzheimer's Disease Assessment Scale-cognitive (ADAS-cog), Mini-Mental State Exam
 (MMSE), Hachinski Scale, Digit Wechsler Adult Intelligence Scale(WAIS- III); Stroop
 Color-Word Interference Test (Stroop); Verbal fluency test; Trail Making Test (TMT) A and B;
 California verbal learning test (CVLT), Rey-Osterrieth Complex Figure Test (RCFT), Cornell
 depression scale, neuropsychiatric inventory, caregiver burden scale and EuroQol Test.

 CSF Cerebrospinal fluid will be obtained by lumbar punction at 8 am, after overnight fasting.
 Amyloid beta 42 (AB42), tau and phosphotau will be measured using enzyme-linked
 immunoabsorbent assay (ELISA)(INNOTEST, Innogenetics). The cut-off will be based on prior
 studies of our laboratory. Also, ELISA will be employed to detect the levels of
 hypoxia-inducible factor 1-alpha (HIF-1alpha vascular endothelial growth factor (VEGF), nerve
 growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the cerebrospinal fluid
 (CSF).

 Sleep measurement To evaluate the sleep characteristics of patients a PSG will be done to all
 patients at baseline. The duration of each PSG will be about 8 hours, from about 23 pm until
 7 pm. 6 channels of EEG will be recorded with reference to the two ears.

 Channels included 2 central electroencephalograms (EEG), bilateral electrooculogram (EOG),
 chin electromyogram (EMG), thoracic and abdominal respiratory effort, airflow (using
 nasal-oral thermocouple and nasal pressure recording), finger pulse oximetry,
 electrocardiogram (ECG), body position, and bilateral piezoelectric sensors to detect leg
 movements. Data will be evaluated by trained technicians, and sleep stage will be assessed in
 30-second epochs according to standard criteria. Apnea (complete cessation of airflow) and
 hypopnea (discernible > 30% reduction in airflow) will be defined if occurring for 10 seconds
 or longer and accompanied by a 4% or greater oxygen desaturation. Arousals from sleep will be
 defined as an abrupt shift in EEG frequency of 3 seconds or longer; arousals during rapid eye
 movement (REM) sleep required an increase in chin EMG activity. Sleep disordered breathing
 will be measured by the apnea-hypopnea index (AHI; the number of apnea plus hypopnea events
 per hour of sleep), and prevalent sleep-disordered breathing coded as the apnea -hypopnea
 index of 15 or more events per hour. Calculated variables used as indices of hypoxia included
 the oxygen 4%15 or < 15 events per hour; the percentage of sleep time with oxygen saturation
 (SaO2) < 90% 1% of sleep time or < 1% sleep time with SaO2 < 90%; and the percentage of sleep
 time in apnea or hypopnea (>4% of oxygen desaturation coded into tertiles). Calculated
 variables of sleep fragmentation will include arousal index (AI) defined as the number of
 arousals per hour of sleep and minutes of wake after sleep onset (WASO) (both coded into
 tertiles). Sleep duration will be measured as the total sleep time (TST) coded into tertile.
 Investigators also measured REM and stage I sleep duration.Patterns of sleep and circadian
 rhythms will be measured with an actigraph (Actiwatch 2; Philips). Participants will be
 instructed to wear an actigraph on the nondominant wrist for 14 days and to push a marker on
 the actigraph whenever getting in and out of bed. The quantity of sleep will be measured with
 total sleep time. The quality of sleep will be measured with sleep efficiency, which is total
 sleep time divided by time in bed, expressed as a percentage. A secondary measure of sleep
 quality will be wake time after sleep onset. Concurrently, participants filled out a sleep
 diary each morning. The sleep diary queried for naps the previous day, bedtime, sleep
 latency, nighttime awakenings, wake time, and opened comment. The number of days per week
 that at least 1 nap will be taken was calculated as "nap days per week." Brain MRI

 All patients will be evaluated by MRI performed using a Siemens Avant 1.5 T, MRI scanner. The
 protocol performed will include:

 1. Volumetric T1-weighted 3D multiplanar reconstruction, FoV 256 mm, slice thickness 1 mm,
 Time relapse (TR) 1600 ms, TE 3,01 ms, distance factor 50%. T2 tse axial, FoV 230 mm,
 slice thickness 5 mm, TR 3560 ms, TE 89 ms, distance factor 30%. Flair axial, FoV 230
 mm, slice thickness 5 mm, TR 8000 ms, Time exposition (TE) 94 ms, distance factor 30%.
 T2 gradient echo axial, FoV 230 mm, slice thickness 5 mm, TR 800 ms, TE 26 ms, distance
 factor 30%. Echoplanar diffusion images, FoV 230 mm, slice thickness 5 mm, Time relapse
 3902 ms, TE 102 ms, distance factor 40%. T2 Turbo spin echo coronal, FoV 210 mm, slice
 thickness 5 mm, TR 3250 ms, TE 100 ms, distance factor 20%.

 2. Perfusion - weighted images (PWI) will be obtained using an echo-planar imaging sequence
 (FoV 230 mm, slice thickness 5 mm, TR 1439 ms, TE 30 ms) after the injection of a
 gadolinium chelate (Gadovist) using an automatic injector with a flow of 4 ml/s.

 3. Spectroscopy Study evaluating the cortical gray matter of the posterior cingulate and
 right middle temporal gyrus with the use of two echo times (31 and 136 ms) and the same
 TR 2000 ms. Dimensions of the volume in posterior cingulate 20 x 20 x 20 mm (8 cc) and
 right middle temporal gyrus 18 x 18 x 18 mm (5,8 cc). The posterior cingulate will be
 taken as the reference due to it is the most affected region in patients with Alzheimer
 in SPECT studies.

 Metabolomic and lipidaemic analyses. Lipidaemic and metabolomic analyses will be performed on
 the blood sample and CSF. For metabolites extraction, samples will be deproteinized using
 cold methanol in the presence of antioxidants and internal standards. Lipids will be
 extracted using chloroform/methanol in the presence of antioxidants and class-specific
 internal standards. Metabolite and lipid extracts will be subjected to mass spectrometry
 using an High pressure liquid chromatography 1290 series coupled to an Electrospray
 ionization-quadrupole reflection time-of-flight (ESI-QTOF) Mass spectrometry/Mass
 Spectrometry 6520 (Agilent Technologies, Santa Clara, California, USA). The liquid
 chromatography-mass spectrometry metabolomic and lipidemic analyses will be based on
 previously described methods (Jove et al., 2013; Jove et al., 2014).

 Blood samples Blood samples will be obtained by a venous puncture at 8 am after PSG. The
 samples will be processed and stored in accordance with Royal Decree 1716/2011 of 18th
 November 2011 by authorization requirements and operation of biobanks for biomedical research
 and treatment of biological samples down of human origin (BOE No. 290 of 12.12.2011).

 Determination of molecular markers, metabolomics, lipidemic will be drawn. Commercial
 biomarkers (TNF-alpha, Interleukine-6 and Interleukine-8), the Thiobarbituric Acid Reactive
 Substances (TBARS) will be measured. Will be performed genotyping APOE4.

 Sample size calculation This is an observational cohort study, which will include a group of
 72 patients with OSA and another 72 patients without OSA from the same population of patients
 with mild initial AD after making PSG. Patients will be recruited consecutively. If more
 patients with AD that the ability to carry out the study of sleep, investigators will apply a
 random selection (web based tool).

 The sample size has been calculated accepting an alpha risk of 0.05 and a beta risk of 0.2 in
 a bilateral contrast to detect a difference equal to or greater than 3.5 units on the
 ADAS-cog scale. It is assumed that the common standard deviation is 7. It has been estimated
 a loss rate of 12% follow-up.

 Statistical analysis The standard statistical analysis will be performed with the SPSS
 statistical package, version 15.0 (SPSS, Chicago, Illinois,USA). The results of the different
 variables will be expressed as means ± standard deviation value. The statistical significance
 of differences between groups and variables assessed by analysis of variance (ANOVA)
 considering the practice of repeated measurements of the same variable. To investigate the
 existence of correlations between clinical variables will use in bivariate Spearman
 correlation test and multivariate analysis.
Inclusion Criteria:

 1. Patients diagnosed of mild probable AD according to NIA-AA criteria (MMSE>20).

 2. Informed Consent Form signed by the patient (and/or if applicable the legal
 representative if different from the responsible caregiver) and the responsible
 caregiver.

 3. The patient has a knowledgeable and reliable caregiver who will accompany the patient
 to all clinic visits during the study.

 4. Absence of visual and hearing problems that, in the investigator's judgement,
 difficult for compliance with the study procedures.

 Exclusion Criteria:

 1. Previous diagnosis of OSA.

 2. Severe Alzheimer's disease, other types of dementia or patients with mild Alzheimer's
 disease with current acetylcholinesterase inhibitors or memantine treatment.

 3. Presence of any previously diagnosed sleep disorder: narcolepsy, insomnia, chronic
 lack of sleep.

 4. Having serious comorbidities: cancer, excessive intake of alcohol (>280 gr/week),
 severe depression, severe renal or hepatic insufficiency, severe cardiac or
 respiratory failure.

 5. Currently receiving an investigational drug or device.

 6. Patient or family declining to take part.

 7. Disabling drowsiness not justifiable by any other cause.

 8. The patient has MRI evidence of hydrocephalus, stroke, a space-occupying lesion,
 cerebral infection or any clinically significant central nervous system disease other
 than AD.

 9. The patient suffers from mental retardation, organic mental disorders, or mental
 disorders due to a general medical condition (DSM-IV-TR™ criteria).

 10. The patient has an untreated vitamin B12 or folate deficiency that is considered
 clinically significant, or has clinical and laboratory evidence of untreated thyroid
 disease. Patients with vitamin B12 or folate deficiency may be enrolled in the study
 provided they have been on a supplement therapy for >3 months prior to the Screening
 Visit and are stable. Patients with thyroid disease may be enrolled in the study
 provided they are stable and euthyroid.

 11. Patient on betablockers, antidepressants, neuroleptics, hypnotics, or they have been
 removed 15 days before conducting polysomnography (PSG).