PNDABLE study
The Perioperative Neurocognitive Disorder and Biomarker Lifestyle Study (PNDABLE) is intended to explore the pathogenesis, risk factors and biomarkers of perioperative neurocognitive disorders in the northern Chinese Han population. PNDABLE is aimed to identify lifestyle factors that may affect the risk of PND in the non-demented northern Chinese Han population in order to provide a basis for disease prevention and early diagnosis. This study has important scientific and practical values for establishing the standardized model of early diagnosis and prevention for PND in China.Informed consent was obtained from all the included patients before we extracted preoperative cerebrospinal fluid and blood of the patients. This study has been registered in the Chinese Clinical Trial Registry (clinical registration number ChiCTR2000033439) and approved by the Ethics Committee of Qingdao Municipal Hospital.
Participants
This study has been registered in the Chinese Clinical Trial Registry (clinical registration number ChiCTR2000033439) and approved by the Ethics Committee of Qingdao Municipal Hospital.
The Han Chinese patients undergoing unilateral total knee arthroplasty (no gender limitations, aged 65 ~ 90, weight 50–80 kg, ASAⅠ~ Ⅱ) combined with epidural anesthesia were enrolled in the PNDABLE study at Qingdao Municipal Hospital (East Hospital) from June 2020 to November 2020. The exclusion criteria include: (1) Preoperative MMSE score < 24 points; (2) A history of neurological and mental diseases such as Alzheimer's disease, Parkinson's disease, and cerebrovascular accident, etc.; (3) Drug or psychotropic substance abuse, as well as long-term use of steroid drugs and hormone drugs; (4) preoperative Ⅲ-Ⅳ hepatic encephalopathy; (5) Recent major surgery; (6) Severe visual and hearing impairments; (7) Abnormal coagulation function before surgery.
A total of 600 cognitively normal participants from PNDABLE had available information on covariates. We excluded 25 participants who had no information about MMSE, 5 participants without available CSF PGRN data, 17 participants who had no CSF biomarker data or had data outside four standard deviations (SD) of the mean, and 8 participants whose surgeries were suspended. Finally, 545 participants were included in this analysis and they were divided into two groups according to whether POD occurred or not: POD group and non-POD group. POD cases and non-POD controls were frequency-matched (1:1) on five variables using incidence density sampling. Specifically, one non-delirium control was randomly selected for each POD case from the source population according to the five matched variables, including age, diagnosis, American Society of Anesthesiologist’ (ASA) physical status, duration of surgery, and intraoperative blood loss. These variables were listed in the European Society of Anesthesiology evidence-based and consensus-based guideline on POD and were considered to be risk factors for POD after hip fracture surgery. A patient recruitment flowchart is shown in Fig. 1.
The participants did not receive preoperative medications, and they were instructed not to drink for 6h and not to eat for 8h before surgery. After entering the operating room, we routinely monitored ECG, SpO2 and NBP, opened vein access and extracted 3ml of whole venous blood. All patients underwent combined spinal-epidural block, with the space between lumbar 3–4 spinous processes (L3-4) as the puncture site. After successful puncture, 2ml of cerebrospinal fluid was extracted from the subarachnoid space, followed by injection of 2-2.5ml ropivacaine (0.66%) for about 30s. After anesthesia, the sensory level was controlled below the T8 level. During the surgery, oxygen was inhaled via mask at 5 L/min to maintain blood pressure within +/- 20% of the baseline value. If intraoperative NBP < 90 mmHg (1mmHg = 0.133kPa) or it decreased by more than 20% of the baseline value, ephedrine 5mg was injected intravenously. If HR < 50 beats/min, atropine 0.5 mg was injected intravenously. Intravenous patient-controlled analgesia (butorphanol 0.1mg/ml + tropisetron 50 g/ml, diluted with normal saline to a total volume of 100 ml) was used in acute postoperative pain management. After the operation, the patient was sent to the anesthesia resuscitation room (PACU). If no abnormalities were found during a 30-minute observation period, then the patient could return to the ward with low-flow oxygen and continuous monitoring of vital signs.
We interviewed all the patients the day before surgery and collected their baseline data, including age, gender, body mass index (BMI), ASA physical status, years of education. Other information including comorbidities, past medical history, fracture classification, and types of anesthesia and surgery were also collected according to the patients’ medical records. All the history collection, physical evaluation and cognitive assessment related to dementia were conducted by neurologists.
CSF core biomarker and CSF PGRN measurements
CSF samples were processed immediately within 2 h after standard lumbar puncture. Each sample was centrifuged at 2000×g for 10 min, and CSF samples were separated and stored in an enzyme-free EP (Eppendorf) tube (AXYGEN; PCR-02-C) at − 80°C for further use in the subsequent steps of this study. The samples were subjected to a maximum of two freeze-thaw cycles.
CSF PGRN and core biomarkers (Aβ1−42, Aβ1−40 ,T-tau and P-tau)were measured by ELISA using the microplate reader (X) (Thermo Scientific Multiskan MK3). CSF PGRN measurements were done with ELISA kits (Human PGRN SimpleStep ELISA kit; BioVendor, no. RMEE103R) and CSF core biomarker measurements were done with other ELISA kits (INNOTEST; FUJIREBIO). All ELISA measurements were performed by experienced technicians in strict accordance with the manufacturer’s instructions. They were blinded to the clinical information. The samples and standards were measured in duplicate, and the means of duplicates were used for the statistical analyses. All the antibodies and plates were from a single lot to exclude variability between batches. Moreover, the within-batch CV was < 5% and the inter-batch CV was < 15%.
APOE ε4 in serum measurements
APOEε4 gene was PCR amplification primer sequence is: upstream, downstream of the 5 '- ACAGAATTCGCCCCGGCCTGGTACACTGCCA − 3', 5 '- TAAGCTTGGCACGGCTGTCCAAGGA − 3', PCR amplification reaction volume of 25 mu, L reaction conditions for and steps for:Pre-denaturation at 95℃ for 5.0 min, denaturation at 95℃ for 1.0 min, annealing at 58℃ for 1.0 min, extension at 72℃ for 1.0 min, 32 successive cycles were carried out, and finally extension at 72℃ for 5.0 min.Restriction enzymes of enzyme reaction for 20 muL volume, reaction conditions and steps for: 10 mu LPCR amplification products, 5 ucfol, 2 muL10 * BSA, Buffer, 0.2 mu L100 digest 6.0 Gh at 37 ℃, on 8.0% polyacrylamide gel electrophoresis separation of restriction enzymes enzyme products, in the uv gel electrophoresis under the imager observations, reference standard and according to the length of the segments in APOEε4 sample under test.
Neuropsychological Tests
The Mini-Mental State Examination (MMSE) was completed by neurologists 1d before surgery to assess the preoperative cognitive status and record relevant medical history. Patients whose MMSE scores < 23 points were excluded. Participants received interview preoperatively .
The assessment of delirium was performed in PACU, on the first, second, third and seventh days (or before discharge) after surgery at 9:00 am and at 17:00 pm by neurologists. We used the visual analog scale (VAS) score of 0–10 (lower scores indicating lower levels of pain) [9] to assess pain at the same time. POD was defined by the Confusion Assessment Method (CAM)[10], and POD severity was measured using the Memorial Delirium Assessment Scale (MDAS)[11]. The Chinese versions of CAM and MDAS have been proven to have good reliability and validity in the Chinese elderly population[12–13]. Therefore, CAM-positive and MDA-positive patients postoperatively in PACU and on the first, second, third and seventh days (or before discharge) were recorded.
In the six month,cognitive function was assessed with the modified Telephone Interview for Cognitive Status (TICS-m)-a 12-item questionnaire that provides an assessment of global cognitive function by verbal communication via telephone; scores range from 0 to 50, with higher score indicating better function 23. The quality of life was assessed with the World Health Organization Quality of Life brief version (WHOQOLBREF)-a 24-item questionnaire that provides assessments of the quality of life in physical, psychological, and social relationship, and environmental domains. For each domain, the score ranges from 0 to100, with higher score indicating better function.
Statistical Analysis
The scheme comprises 4 biomarkers: aggregated Aβ (Aβ1−42), Aβ (Aβ1−40), aggregated P-tau and neurodegeneration (T-tau). And each biomarker is binarized based on whether they are normal or abnormal.
CSF PGRN didn’t follow a normal distribution as assessed by Kolmogorov-Smirnov test (P < 0.001) and visual inspection of the Q-Q plot (Fig. 1S). Therefore, they were log-transformed to obtain a normal distribution. All the statistical analyses described in this study are performed on the log10-transformed values. We performed the analysis after excluding outliers (defined as 4 SD below or above the group mean) in order to exclude the influence of extreme values. Two independent-samples’ t tests were used for the comparisons between POD and NPOD groups. We used the Correlation analysis to explore whether CSF PGRN is related to CAM score and MDAS score. Given the different trends of PGRN at different ages in the biomarker framework, we applied a one-way ANCOVA followed by Bonferroni post hoc analyses.
We also studied the associations between CSF PGRN and the CSF core biomarkers for POD, using a multiple linear regression adjusted for age, gender, years of education, and APOE ε4 carrier status. The analyses were performed in the total sample and then in subgroups stratified by age, gender, years of education and APOE ε4 carrier status.
ROC curve analysis was used to evaluate the clinical diagnostic value of PGRN in POD. Statistical significance was set at P < 0.05. SPSS statistical software, version 21.0 (SPSS, Inc. Chicago, IL, USA), and GraphPad Prism software, version 6.01 (GraphPad Software, Inc., La Jolla, CA, USA), were used for data analysis.