Role of Hypoxia Ans Sleep Fragmentation in Alzheimer's Disease. and Sleep Fragmentation.
Condition: Alzheimer's Disease; Obstructive Sleep Apnea
MeSH terms: Sleep Apnea Syndromes; Sleep Apnea, Obstructive; Alzheimer Disease; Sleep Deprivation; Hypoxia
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        We prospectively will studied consecutive patients with new diagnosis of mild probable
        Alzheimer's Disease by a neurologist. We will define Alzheimer's Disease according NIA-AA
        criteria.
      
Biospecimen description: 
      APoE4
    
Keywords: biomarkers, sleep fragmentation, intermittent hypoxia