This retrospective study was approved by the local ethics committee. Written informed consent was obtained from all participants before the study. Fifty patients with diagnosis of idiopathic PD were enrolled at the Neurology Department of Kaohsiung Chang Gung Memorial Hospital. The patients were divided into two groups according to their pulmonary function test results: 25 patients were included in the abnormal pulmonary function (APF) group (13 men, 12 women; mean age: 62.9 ± 10.8 years) and 25 sex- and age-matched patients were included in the normal pulmonary function (NPF) group (14 men, 11 women; mean age: 62.3 ± 6.9 years). Two patients had a known history of cigarette smoking (one in the APF group, the other in the NPF group). The exclusion criteria included a known history of psychiatric and neurologic disorders, psychotropic medication usage, and structural abnormalities of the chest wall or a recent upper respiratory tract infection.
All the patients were diagnosed as having idiopathic PD by an experienced neurologist based on the Parkinson Disease Society’s criteria . The disease severity and functional status of each patient were evaluated using the Unified Parkinson Disease Rating Scale (UPDRS), modified Hoehn and Yahr staging (H & Y) scale, and Schwab and England (S & E) activities of daily living scale during the “OFF” state.
Pulmonary function testing
The pulmonary studies included spirometry, lung volume and pulse oximetry testing. All the pulmonary function tests were performed according to American Thoracic Society/European Respiratory Society criteria [20, 21]. Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV 1), maximal expiratory flow after expiration of 50% of FVC (MEF50) and oxygen saturation (SpO2) values were measured using a spirometer (MasterScreen PFT; Jaeger, Hoechberg, Germany). All measurements of pulmonary function test were expressed as percentages of predicted normal values.
Patients were diagnosed as having central obstructive pattern with FEV1 < 80% and FEV1/FVC < 80%, peripheric obstructive pattern with MEF50 < 70%, and restrictive pattern with FVC < 80% and FEV1/FVC > 80% . Patients with these abnormal pulmonary functional parameters were assigned to the APF group.
Blood sampling and assessment of inflammatory markers
All the patients underwent blood sampling by venipuncture of forearm veins.
Assessment of inflammatory markers
The serum concentrations of TBARS and thiol were measured in all the patients in order to detect lipid peroxidation and determine anti-oxidative defense capability, respectively . In addition, the level of EPCs was measured by flow cytometry based on a previous report . First, mononuclear cells (106) were incubated for 30 min at 4 °C in a dark room with monoclonal antibodies against kinase insert domain-conjugating receptor (KDR) (Miltenyi Biotec, Bergisch Gladbach, Germany) and fluorescein isothiocyanate-conjugated CD34 and CD133, by which the EPC surface markers of CD133/CD34 and KDR/CD34 were determined. The control ligand (IgG-fluorescein isothiocyanate conjugate) was then added. Quantitative two-color flow cytometric analysis was performed using an Epics XL flow cytometer (Beckman Coulter). In these arrays, each analysis included 10,000 cells per sample and was performed in duplicate, with mean level reported.
Assessment of serum adhesion molecules
To assess serum sICAM-1, sE-selectin, and sP-selectin levels, commercially available enzyme-linked immunosorbent assays (R & D Systems, Minneapolis, MN, USA) were used . The dual wavelength absorbance, from which the degree of enzymatic turnover of the substrate was estimated, was measured at 450 and 620 nm. Absorbance was directly proportional to the concentration of antigens present. To determine the antigen concentrations of the unknowns, a standard curve of absorbance of standard antigen versus the given antigen concentration was plotted.
A GE Signa 3 T whole-body MRI scanner (General Electric Healthcare, Milwaukee, WI) using an 8-channel phase array head coil was used to perform the volumetric structural MRI scans. With 110 contiguous axial slices aligned to the anterior and posterior commissure, whole-brain 3-dimensional T1-weighted images of all participants were collected using an axial inversion-recovery prepared fast-spoiled gradient-recalled echo pulse sequence. The scanning parameters were as follows: repetition time = 9.5 ms, echo time = 3.9 ms; inversion time = 450 ms, flip angle = 15o; number of excitations = 1; field of view = 240 × 240 mm2; matrix size = 512 × 512; and voxel size = 0.47 × 0.47 × 1.3 mm3 (without inter-slice gap and interpolation).
Voxel-based morphometry analysis
Voxel-based morphometry analysis was performed using the Statistical Parametric Mapping software (SPM12 version 7219, Wellcome Institute of Neurology, University College London, UK, http://www.fil.ion.ucl.ac.uk/spm/) and Matlab R2010a (Mathworks, Natick, MA). The default settings were used unless otherwise specified. First, whole brain T1-weighted images were bias-corrected and segmented into gray matter (GM), white matter (WM), and cerebrospinal fluid using the New Segment Toolbox of SPM12. Then, the GM images were rigid aligned to the tissue probability maps in the Montreal Neurological Institute (MNI) standard space and averaged to create the study-specific tissue template using the high dimensional Diffeomorphic Anatomical Registration Exponentiated Lie (DARTEL) algorithm . Subsequently, all native space GM images were registered to this study-specific template and further spatially normalized into standard MNI space (1.5 mm isotropic voxel). The resulting GM images were modulated by Jacobian determinant of the corresponded deformation filed to correct for volume changes. Finally, the modulated GM images were smoothed using an isotropic Gaussian kernel of 8 mm full-width at half maximum.
Analysis of demographic data, pulmonary function parameters, global brain volumes, and inflammatory markers
All statistical analyses of demographic data and global tissue volumes were performed using SPSS software, version 22, for Windows (SPSS, Chicago, IL). Age and sex were compared between groups by the 2-sample Student t test and Pearson chi-squared test, respectively. Analysis of covariance (ANCOVA) was used to analyze differences in pulmonary functional parameters, clinical severity, inflammatory markers, and global brain volume with the participant’s age and sex as covariates. All data were reported as the mean ± standard deviation (SD). The P value for statistical significance was set at < 0.05.
Analysis of between-group regional GMV differences
To examine between-group differences in regional GM volume, a voxel-wise general linear model was used to compare GM volume between APF group and NPF group using 1-factor 2-level ANCOVA design with age, sex and total intracranial volume as covariates. The statistic threshold was set at cluster-level family-wise error (FWE) corrected P-value< 0.05, with a cluster size of at least 287 voxels, based on the results of a Monte Carlo simulation using the command-line tool of Analysis of Functional NeuroImages software (AFNI; Version AFNI_17.1.04; http://afni.nimh.nih.gov/afni/; 3dClusterSim with the following parameters: voxel P-value< 0.005, with explicit GM mask and 10,000 simulations). The regional GM volume of clusters with significant between-group differences were extracted and averaged for further correlation analysis.
Correlations between regional GMVs and pulmonary functional parameters
Partial correlation analysis was performed to correlate the pulmonary functional parameters with regional GMs of reduced volumes, inflammatory parameters, and disease severity after controlling for age, sex and TIV. The threshold for statistical significance was set at P-value < 0.05 with Bonferroni correction for multiple comparisons.