Clinical Investigation And Animal Model Exploration Of Postoperative Delirium

Objective:1, to investigate on clinical epidemiology and to analyze the perioperative factors of postoperative delirium (POD).2, to explore an animal model of POD.Methods:Section 1 Clinical epidemiology investigation of POD and perioperative factors analysis of PODSubjects investigated:Patients who aged over 18, had an ASA classification of Ⅰ～ Ⅲ, scheduled to receive orthopedic, urologic, heart, thoracic, general, or gynecologic surgery, signed to consent entering the program, and accepted interview questionnaires on research purpose in Zhongda Hospital affiliated to Southeast University during June to October 2014 were recruited into the program. Those who had an anesthesia and surgery time over 2 hours, who did not enter ICU in the first or second postoperative days, and who fulfilled the interview questionnaires were enrolled into analysis.Investigation details:The associations of social demographic characteristics, i.e., age, gender, and ASA classifications of patients, and perioperative factors such as anesthesia factors (including anesthesia type, anesthesia and surgery time, and extubation time after general anesthesia), surgery factors (including surgery type, and surgical methods, i.e., video-assisted surgery (VAS) or not), and some serum biochemical index such as postoperative hemoglobin (HB) level and C-reactive protein (CRP) level, with occurrence of POD were analyzed. Further more, the relevance of CRP increases within the first 2 postoperative days from preoperative level (perioperative CRP increase, PCI) with the POD occurrence and severity was also analyzed.Analyze methods:POD (evaluated with CAM method) occurrence and POD severity scores (evaluated with MDAS method) in the first and/or second postoperative day(s) were set as outcome variables. Social demographic characteristics and perioperative factors named in the last paragraph were set as analysis variables.Statistic approaches:Chi-square analyses were used for finding differences between categorical variables. Student’s t-tests were used to analyze the differences between quantitative variables. One-way ANOVA, Post Hoc Multiple Comparison and least-significant difference (LSD) were used to analyze the differences of quantitative variables among multiple groups. Multiple logistic regressions were used to analyze the relevance of surgery types, surgery methods, and PCI with POD occurrence. Multiple linear regressions were used to analyze the relevance between the PCI and POD severity scores. And, confounding factors, i.e., age, gender, ASA classification, anesthesia and surgery time, exbutation time after general anesthesia, and postoperative HB level were controlled in these two regression models. P< 0.05 was considered as statistically significant.Results:814 patients were enrolled into final analysis, with age from 18～93 years old (56.8 +15.3), male 40.2%, and the numbers of ASA classification Ⅰ,Ⅱ and Ⅲ to be 202, 557, and 55. Among them, the numbers of orthopedic, urologic, heart, thoracic, general, gynecologic, and multiple-organ abdominal surgical cases were 411,75,14,47,109,154, and 4, respectively. POD patients had a significantly-higher average age than non-POD patients,66.0±15.3 vs 55.7±14.9 (years old), P<0.0001; Patients aged over 65 had a significantly higher POD occurrence than those aged below 65,20.5% vs 7.2%, P< 0.0001. POD occurrence was associated with higher ASA classification, with Ⅰ,Ⅱ and Ⅲ to be 6.5%,10.4% and 40%, respectively, P=0.0001. POD patients had a significantly longer anesthesia and surgery time than non-POD patients,3.85±1.68 vs 3.33±1.26 (hours), P=0.0003. POD patients had a significantly longer extubation time after general surgery than non-POD patients,44.9±44.8 vs 27.1±24.6 (minuets), P< 0.0001. POD occurrences and severities were significantly different among surgery types, with occurrence and severity of orthopedic, urologic, heart, thoracic, general, and gynecologic surgeries to be 8.8%,13.3%,42.9%,17.0%,19.3%, and 7.1%, respectively, and 0.68±1.98,1.21±3.81,2.86±3.55,1.37±3.05,1.41±3.06, and 0.48±2.05, P< 0.001, respectively. However, the multiple logistic regression analysis with control of patients’age, ASA classification, anesthesia time, and extubation time after general anesthesia denied the significance of differences among surgery types regarding their POD occurrences and severities. POD occurrence was significantly lower in VAS patients compared with non-VAS patients,8.4% vs 17.7%, P=0.0066, however, the multiple logistic regression analysis with control of patients’ age, ASA classification, anesthesia time, and extubation time after general anesthesia denied the significance of differences between these two surgery methods regarding their POD occurrences and severities. POD patients had a significantly higher postoperative CRP level than non-POD patients,60.4±53 vs 41.6±35.7 (mg/L), P=0.0179. Multiple logistic regression analysis found PCI was associated with an increased risk of POD occurrence, OR=1.033 (95% CI:1.007～1.060). Multiple linear regression analysis found PCI was associated with an increased POD severity score, b=0.016(0.007～0.025), P<0.001. ROC of PCI versus POD occurrence showed the AUC was 0.71 (95%CI:0.50～0.93) suggested that PCI had moderate effect in predicting POD. Other factors such as gender, anesthesia type, usage of postoperative analgesia pump, and postoperative HB level were not found to be associated with POD in this investigation.Conclusions:1,The results of clinical epidemiological investigation suggest that patients’ social demographic characteristics such as age and ASA classification, and perioperative factors such as anesthesia and surgery time, extubation time after general anesthesia, and postoperative CRP level were associated with POD.2, Perioperative CRP increase (PCI) had moderate effect in predicting POD.Section 2:Exploration of animal model of PODMethods:Animals:2-8 month-old female C57BL/6J miceBehavioral tests detecting delirious status——Combined Behavioral Tests (CBT):Mice were set to perform light and dark box test (LDBT) for 5 min, open-field test (OFT) for 5 min and non-selective non-sustained attention test (NNAT) for 5 min in a row within 20 minutes individually.5 behavioral indexes including time spent in the light box (from LDBT) by mice, mice movement speed, time spent in the besides wall area by mice, and mice freezing time (from OFT), and mice attention level (from NNAT) were detected.Chapter 1:Behavioral changes induced by scopolamine were detected with CBT in miceInterventions to the mice:mice in the Scopolamine group received 0.06 mg% scopolamine 15 mg/kg intraperitoneally, whereas mice in Control group received isovolemic saline intraperitoneally 30 minutes before the CBT tests.Results:The 5 behavioral indexes of the mice before interventions (Baseline values) were not significantly different between Scopolamine and Control groups. However, all the 5 behavioral indexes of the mice were significantly different between Scopolamine and Control groups 30 minutes after interventions. Compared with mice in Control group, mice in Scopolamine group spent much less time in the light box,0.8±1.13 vs 53.4± 34.0 (%), P< 0.05; had higher movement speed,0.081±0.023 vs 0.037±0.018 (m/s), P <0.05; spent more time in the besides wall area,273.3±9.9 vs 237.6±26.1(s), P<0.05; had less freezing time,10.4±6.7 vs 41.6±16.9 (s), P<0.05; and had lower attention level,38.0±15.4 vs 53.5±7.5 (%), P< 0.05, at 30 minutes after interventions. These data suggest that CBT detected delirium-like behavioral changes in mice such as increase in excitability, decrease in explorative activity, and decrease in attention level, which were induced by scopolamine.Chapter 2:Behavioral changes detected with CBT and brain biochemical changes after anesthesia and surgery in miceInterventions to the mice:Each mice in the anesthesia/surgery (AS) group received a simple laparotomy under 1.4% isoflurane and 100% O2 anesthesia for 2 hours, whereas each mice in the Sham group received 100% O2 only for 2 hours.Indexes detected:12,24, and 48 hours after interventions,5 behavioral indexes of mice including time spent in the light box (from LDBT), movement speed, time spent in the besides wall area, and freezing time (from OFT), and attention level (from NNAT) were detected with CBT.12 hours after interventions, levels of delirium-associated protein in mice brain tissues (cortex) total a-synuclein (T-a-synuclein) and S100P were detected with Western blot analysis.Results:Mice in AS group spent less time in the light box in comparison with mice in Sham group,55.48±12.54 vs 118.9±14.4 (s), P=0.0038, at 12 hours after anesthesia and surgery, but not at 24 and 48 hours after anesthesia and surgery. Mice in AS group had lower attention levels in comparison with mice in Sham group,33%±2.9% vs 49% ±5%, P=0.011, at 24 hours after anesthesia and surgery, but not at 12 and 48 hours after anesthesia and surgery. The movement speeds of mice in both groups were not significantly different from each other at 12,24, and 48 hours after interventions. The T-a-synuclein levels and S100β levels in the cortex were significantly increased at 12 hours after surgery in the mice of AS group compared with those of Sham group,139%± 33.5% vs 100%±13.7%(P=0.037), and 142%±7.7% vs 100%±6%(P=0.002), respectively.Conclusions:1, Combined behavioral tests (CBT) reviewed delirium like behavioral changes such as decreased attention level and decreased exploratory behavior induced by scopolamine in mice.2, Combined behavioral tests (CBT) reviewed delirium like behavioral changes such as decreased attention level and/or decreased exploratory behavior at some postoperative time points in this mice model of anesthesia and surgery.3, Anesthesia and surgery induced delirium-associated neurotoxicity in mice.4, Delirium like behavioral changes and delirium-associated neurotoxicity were detected in this mice model of anesthesia and surgery. These pilot studies facilitate further investigations into animal model of postoperative delirium.