The study protocol was approved by the Chang Gung Memorial Hospital’s Institutional Review Committee in Human Research. All participants or their guardians were provided written informed consent prior to participation. Eighty-eight PD patients were prospectively enrolled in the study at the Neurology Department of Kaohsiung Chang Gung Memorial Hospital. Patients were included if they had a definitive diagnosis of idiopathic PD . The disease severity and functional status of each patient were evaluated with the Unified Parkinson Disease Rating Scale (UPDRS) , the modified Hoehn and Yahr stages (HY stage) , and the Schwab and England Activities of Daily Living Scale (SE-ADL) .
PD patients were further separated into three subgroups, classified as PD patients with normal cognition (PDN), PD patients with mild cognitive impairment (PD-MCI), and PD patients with dementia (PDD) in accordance with the Movement Disorder Society Task Force Guidelines . Cognitive impairment was defined as a score 1.5 SD below the normative mean in each of the domains . PDN was defined as less than two domains of cognitive impairment. PD-MCI was defined as one score at − 1.5 SD in each of two or more domains but without dementia. PDD was defined as impairment in more than one cognitive domain with a Mini-Mental State Examination (MMSE) score of less than 26 . In addition, forty-seven healthy subjects without neurological disease, psychiatric illness, alcohol or substance abuse, or head injury, were recruited from the hospital as the normal control (NC) group.
Evaluation of Cognitive Function
Neuropsychological evaluations of five cognitive domains, attention and working memory, executive, language, memory, and visuospatial were conducted using subtests from the Cognitive Ability Screening Instrument (CASI)  and the Wechsler Adult Intelligence Scale-III . The Mini-Mental State Examination (MMSE)  was also performed to assess the general cognitive function of the subjects.
Laboratory Examinations of Plasma DNA in Peripheral Circulation
Plasma levels of nuclear and mitochondrial DNA were assessed in all study participants. Blood was drawn by venipuncture at the forearm on the same day as the MRI study and the neuro-psychological testing. The plasma DNA was measured by a real-time quantitative polymerase chain reaction (RT-PCR) assay (Roche LightCycler, Roche, Grenzach-Wyhlen, Germany) for the β-globin gene (present in all nucleated cells) and ND2 genes (specific for mitochondrial DNA). Quantitative results were expressed as ng/ml. Procedural details are described in a previous study .
DTI-ALPS Index Aquisition
The MRI data were acquired on a 3.0-T MRI scanner (SIGNA, General Electric Healthcare, Milwaukee, WI, USA). The subject’s head was immobilized with foam pillows inside the coil to diminish motion artifacts. DTI scans were obtained using a single-shot echo planar imaging sequence. Images were acquired axially parallel to the anterior-posterior commissure (AC-PC) to cover the entire brain with the following parameters: repetition time (TR)/echo time (TE) = 15800/77 ms; field of view (FOV) = 25.6 cm; matrix size = 128 × 128; voxel size = 2 × 2 × 2.5 mm3; number of excitations (NEX) = 3, 55 slices without gap; b value = 1000s/mm2; 13 non-collinear directions and one nondiffusion-weighted T2 image.
We used dTV. II.13k + software (Dept. Biomedical Information Sciences, Graduate School of Information Sciences, Hiroshima City University)  to calculate the diffusion metric images. The DTI-ALPS method has been detailed in a previous report . The software estimates a diffusion tensor at each voxel of the brain, and outputs a series of images including a color-coded fractional anisotropy (FA), mean diffusivity (MD), and diffusivity maps in the direction of the x-, y-, and z-axis (Dxx, Dyy, Dzz). It is used to evaluate the diffusivity along the direction of the perivascular space by comparing with those of projection fibers and association fibers on a slice at the level of the lateral ventricle body (Fig. 2A). At that level, the direction of the perivascular space is perpendicular to the ventricle wall and is thus mostly in the right-left direction (x-axis) on the axial plane. The direction is also perpendicular to the direction of both the projection fibers (mostly in the z-axis) and the association fibers (mostly in the y-axis) (Fig. 2B). Thus, the diffusivity along the x-axis at regions with projection/association fibers will at least partially represent the diffusivity along the perivascular space. On a color-coded FA map of the plane at the level of the lateral ventricle body, we placed a 5-mm-diameter spherical region of interest (ROI) in the area of the projection fibers (blue on Fig. 2A), the area of the association fibers (green on Fig. 2A), and the area of the subcortical fibers (red on Fig. 2A) in the left hemisphere. In this study, we obtained measurements only in the left hemisphere, as all subjects were right-handed and the SLF is thick enough to place the ROI on the left side.
Further, we calculated an index which we will call as ALPS-index in order to evaluate the activity of the glymphatic system in individual cases. This index is provided by the ratio of two sets of diffusivity value which are perpendicular to dominant fibers in the tissue, that is the ratio of mean of x-axis diffusivity in the area of projection fiber (Dxproj) and x-axis diffusivity in the area of association fibers (Dxassoc) to the mean of y-axis diffusivity in the area of projection fiber (Dyproj) and z-axis diffusivity in the area of association fibers (Dzaccoc) as folows.
ALPS index = mean (Dxxproj, Dxxassoci) / mean (Dyyproj, Dzassoci)
In the area of projection fibers, the dominant fibers runs in the direction of z-axis, and both x-axis and y-axis are perpendicular to the dominant fibers. Similarly, in the area of association fibers, the dominant fibers runs in the direction of y-axis, and both x-axis and z-axis are perpendicular to the dominant fibers. The major difference for the water molecule behavior between x-axis diffusivity in both area (Dxproj and Dxassoc) and the diffusivity which is perpendicular to them (Dyproj and Dzassoc) would be the existence of the perivascular space. The ALPS-index measures the diffusivity from the comportment along the perivascular space direction in those perpendicular projection fibers and association fibers to represent the integrity of the glymphatic system.
The age data were compared by analysis of variance (ANOVA). The sex data were compared using the Pearson chi-square test. The disease severity, MMSE, CASI, neuropsychological test scores, and plasma DNA levels were analyzed using the analysis of covariance (ANCOVA) model with the participant’s age and sex as covariates. The statistical significance was set at p < 0.05.
Analysis of ALPS-index differences between NC and PD subgroups
The ANCOVA was conducted with age and sex as covariates to compare the ALPS-index between the NC, PDN, PD-MCI, and PDD groups. Post hoc tests with Bonferroni correction were performed to investigate which pairs of groups differed after an overall difference had been established.
Correlations among ALPS-index, cognition scores, plasma DNA levels, and disease severity
After confirming data normality, correlations were measured using Spearman’s correlation coefficient to evaluate the relationships among the ALPS-index, plasma DNA levels, and neuropsychological test scores of study subjects. In the PD subgroups, similar analyses were also conducted to evaluate the relationship between the ALPS-index and clinical disease severity. All statistical thresholds were set at p < 0.05 (SPSS V.17, Chicago, IL, USA).