Subject enrollment and study design
A total of 458 patients who had experienced acute ischemic stroke onset less than 24 h before treatment were eligible for recruitment into the study. Routine cerebral computer tomography (CT) diagnostic images were acquired in all patients on a 64-row, multi-slice CT scanner (Siemens Medical Systems, München, Germany) and 163 patients also underwent 3.0 T MRI within 24 h (GE Healthcare, Marlborough, USA). All enrolled patients were over 18 years of age and gave informed consent before entering the study. In addition, enrolled patients had clinical symptom scores of ≥5 in the National Institutes of Health Stroke Scale (NIHSS). Exclusion criteria were trauma or a previous invasive procedure, cerebral hemorrhage, history of malignant tumor, chronic inflammatory disease, any infection before acute ischemic stroke, autoimmune disease, or a coagulation disorder. None of the patients had received t-PA thrombolytic treatment. The enrolled stroke patients were evaluated using the Oxfordshire Community Stroke Project (OCSP) classification criteria: lacunar infarct (LACI), total anterior circulation infarct (TACI), partial anterior circulation infarct (PACI), and posterior circulation infarct (POCI) [11]. Pneumonia was the only post-stroke infection noted, and was defined by the following two positive criteria during in-hospital stay: (1) presence of clinical and laboratory or radiological signs of pneumonia (productive cough, fever, apnea, leukopenia of < 4 × 109/L, or leukocyte count of > 12 × 109/L) and infiltration confirmed by chest X-ray; and (2) plasma high sensitivity C-reactive protein (hsCRP) of more than 5 mg/L. To compare peripheral cholinergic activity in acute ischemic stroke (within 24 h of onset) with that in recovery phase of stroke, lymphocyte-derived ACh was also determined on day 10 after stroke onset in the same patient. As control subjects, we recruited 216 healthy individuals who were neurologically intact and lacked any history of stroke, myocardial infarction, or peripheral artery disease. Controls approximately matched stroke patients for age and sex. This study was approved by Medical Ethical Committee and the Scientific Research Committee of Tianjin Medical University General Hospital. Written informed consent was obtained from all subjects.
Brain MRI assessment
3 T MRI was performed with an eight-element phased-array torso coil. Lesion volumes were measured using diffusion-weighted imaging (DWI). The parameters for brain DWI at b = 1000 s/mm2 were as follows: TR =7059/7059 ms; TE = 45.7/64.9 ms; DFOV = 42 cm; 128 × 128 matrix directions of the motion-probing gradient, three orthogonal axes. Infarction areas were selected as regions of interest (ROIs). The images were transmitted to a workstation and FuncTool GE Software was used for image processing.
Lymphocyte-derived ACh determination using UPLC-MS/MS
We have successfully used performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with Waters CORTECS chromatographic column previously to determine intracellular ACh in human PBMCs with detection limits of up to 0.005 ng/106 PBMCs [10]. In brief, in this study fresh PBMCs were prepared from blood and added to 100 μL deionized H2O/0.1% formic acid (vol:vol), and then vortexed to produce homogenates. The homogenates were further broken down ultrasonically and were de-proteinized by adding 300 μL acetonitrile containing the internal standard (D9-ACh, C/D/N, Quebec, Canada). The sample was vortexed again for 1 min before being centrifuged at 15,000 rpm for 10 min [12]. The supernatant was then filtered and transferred to an autosampler glass vial for ACh determination. UPLC–MS/MS analyses were carried out using an Acquity UPLC system coupled to a Xevo TQ-S (Waters Corporation, Milford, MA, USA) consisting of a binary pump, an automatic sampler, and a column compartment. An Acquity UPLC CORTECS HILIC column (1.6 μm, 100 mm × 2.1 mm) was used to ensure sufficient efficacy of isolation and ionization of ACh. The column temperature was set to 45 °C and the flow rate was 0.4 mL/min. The solvents were 50 mmol/L ammonium formate (solvent A, pH adjusted to 3 with formic acid) and acetonitrile (solvent B) with a gradient over the 3.5 min run time as follows: 20% A (initial), 20–50% A (0–0.75 min), 50–70% A (0.75–1.25 min), 70–70% A (1.25–1.7 min), 70–20% A (1.7–2.1 min), 20–20% A (2.1–3.5 min). The sample injection volume was 5 μL. ACh and D9-ACh were identified by the triple quadrupole mass spectrometer using positive electrospray ionization (ESI) mode. Assays were validated according to the FDA Bioanalytical Method Validation Guidance for Industry [13].
Assays of lymphocytic vesicular ACh transporter (VAChT), acetylcholinesterase (AChE), and choline acetyltransferase (ChAT)
PBMC total mRNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription reactions were prepared using the SYBR Premix Ex Taq System (Takara, Japan). Real-time PCR was performed with the IQ5 System (Bio-Rad, US). Cycle conditions and relative quantification were carried out following the manufacturer’s instructions. Expression of VAChT, AChE, and ChAT were calculated using the comparative computerized tomography method with efficiency calculations, and with all mRNA levels normalized to endogenous GAPDH mRNA. Amplification of the targets was performed using the following primers: 5′- GGCATAGCCCTAGTCGACAC − 3′ (forward) and 5′- CGTAGGCCACCGAATAGGAG − 3′ (reverse) for VAChT, 5′- GGGTGGTAGACGCTACAACC − 3′ (forward) and 5′- GTGCCCTCAAAACCTGGGTAT − 3′ (reverse) for AChE, 5′- AACCACGGAGATGTTCTGCTGCTAT − 3′ (forward) and 5′- TTGTTGCCAATGGCTTGCTCTCAG − 3′ (reverse) for ChAT. All reactions were run in triplicate. Protein was obtained using protein extraction reagent containing protease inhibitors. SDS protein electrophoresis and western blotting were performed following standard protocols. The antibodies used were rabbit anti-AChE (1:1000, Abcam, Cambridge, USA), goat anti-ChAT (1:500, Merck Millipore (Chemicon), Darmstadt, Germany), and goat anti-VAChT (1:1000, Abcam). Blots were visualized using the SYNGENE imaging system (UK) and analyzed with Image J software. AChE catalytic activity was measured with reference to Ofek’s method [14]. To eliminate any assay-by-assay variation, we reanalyzed 10 arbitrary control samples from each cohort in different plates and at different measurement times. The capacity of hydrolyzing substrate was represented by OD value per 100 nmol substrate/min × mL.
Statistical analysis
Statistical analysis was performed using SPSS Statistics 20 (IBM Corp. Released 2011; IBM SPSS Statistics for Windows, Version 20.0, Armonk, NY, USA). Subject characteristics are presented as means ± SD; all other results are expressed as means ± SEM. To test for significant differences between the groups, a one-way ANOVA was applied. Post hoc testing was performed using the Bonferroni correction. From individual subject data, correlation coefficients were calculated to test for associations between selected parameters (i.e., ACh concentration, age, sex percentage). We used a logistic regression model to estimate the impact of clinical factors on OCSP stroke subtypes. The Kaplan–Meier estimator was used to determine the outcome risk at 1 year after the onset of acute ischemic stroke. In all tests, the threshold for significance was set at P < 0.05.