Twenty-Six Individuals from the University of Washington ADRC Patient Registry, Who Had

(E) Methods

Twenty-six individuals from the University of Washington ADRC Patient Registry, who had agreed to be contacted in regards to research projects, were recruited to participate in this study. Due to problems with data collection, we were ultimately able to examine fifteen Caucasian individuals (7 female, 8 male) diagnosed with probable AD using NINCDS-ADRDA criteria,1 aged 68 ± 10 years. They had 16 ± 2.4 years of education and scored 18 ± 7.5 on their Mini-Mental State Examination (n = 14, all <26).2 Subjects had experienced initial AD symptoms for 5.6 ± 2.7 years and been diagnosed 3.0 ± 1.6 years prior to participating in this study. CSF was collected, using a Sprotte 24 g atraumatic spinal needle, between 0900 and 1100 hours after an overnight fast. Samples with >500 RBCs/mL were excluded. Samples were frozen immediately on dry ice and stored at -80 °C until assay. CSF hypocretin-1 was measured from the 14th-21st mL of CSF collected.

Sleep/wake data were collected using wrist actigraphy (Actiwatch-L, MiniMitter-Respironics, BendOR) and an accompanying sleep log, filled out by a caregiver. The Actiwatch-L is a small, wrist-worn device that, through the use of an accelerometer, records three-dimensional movement. Activity data obtained from such a device has been validated as a useful proxy for sleep-wake patterns3 and has been used to detect such patterns in those with AD.4 Actiwatch-L data were analyzed for sleep and napping patterns using Actiware software (v.3.1, Cambridge Neurotechnology, UK) and for rhythmicity in locomotor patterns using version 5.0 of the same software. Using the nap analysis sub-program, we defined naps as being at least 10 minutes in length, yet not greater than 180 minutes, and having fewer than 10 activity counts occurring during the time frame. We also examined nocturnal sleep fragmentation using the sleep analysis sub-program set at medium sensitivity.

Crude CSF was analyzed for concentrations of hypocretin-1 using a modification of a commercially available radioimmunoassay (Phoenix Pharmaceuticals, BelmontCA) and a custom primary antibody. In duplicate, 50 µL of CSF diluted to 100 µL were assayed with a detection limit of 4 pg/tube, an intra-assay CV of 2.9% and an inter-assay CV adjusted through the use of an internal reference sample (134 pg/mL).

All methodology was approved by the University of Washington and Stanford University Institutional Review Boards. Due to the cognitive impairment of our subjects, extra precautions were used in obtaining consent for participation in this study from both the individual with AD and an authorized legal representative. Actigraphy data were scored blind to hypocretin-1 concentrations. Summary statistics and Student’s T test were done using Microsoft Excel (v.10, RedmondWA). Regression and curve fitting analyses were done with Microcal Origin (v.6.1, NorthamptonMA). Data are presented as average ± SD.

References

1. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of the Department of Health and Human Services Task Force on Alzheimer's Disease. Neurol 1984;34:939-944.

2. Folstein MF, Folstein SE, McHugh PR. "Mini-mental state". A practical method for grading the cognitive state of patients for the clinician. J Psych Res 1975;12:189-198.

3. Ancoli-Israel S, Cole R, Alessi C, Chambers M, Moorcroft W, Pollack CP. The role of actigraphy in the study of sleep and circadian rhythms. Sleep 2003;26:342-392.

4. Yesavage JA, Friedman L, Kraemer H et al. Sleep/wake disruption in Alzheimer's disease: APOE status and longitudinal course. J Geriatric Psychiatry Neurol 2004;17:20-24.