Protocol for generating airway organoids from 2D air liquid interface-differentiated nasal epithelia for use in a functional CFTR assay.

We present a protocol to generate organoids from air-liquid-interface (ALI)-differentiated nasal epithelia. We detail their application as cystic fibrosis (CF) disease model in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent forskolin-induced swelling (FIS) assay. We describe steps for isolation, expansion and cryostorage of nasal brushing-derived basal progenitor cells, and their differentiation in ALI cultures. Furthermore, we detail the conversion of differentiated epithelial fragments into organoids of healthy controls and CF subjects for validating CFTR function and modulator responses. For complete details on the use and execution of this protocol, please refer to Amatngalim et al.1.

Functional brain network and trail making test changes following major surgery and postoperative delirium: a prospective, multicentre, observational cohort study.

BACKGROUND: Delirium is a frequent complication after surgery in older adults and is associated with an increased risk of long-term cognitive impairment and dementia. Disturbances in functional brain networks were previously reported during delirium. We hypothesised that alterations in functional brain networks persist after remission of postoperative delirium and that functional brain network alterations are associated with long-term cognitive impairment. METHODS: In this prospective, multicentre, observational cohort study, we included older patients who underwent clinical assessments (including the Trail Making Test B [TMT-B]) and resting-state functional MRI (rs-fMRI) before and 3 months after elective surgery. Delirium was assessed on the first seven postoperative days. RESULTS: Of the 554 enrolled patients, 246 remained after strict motion correction, of whom 38 (16%) developed postoperative delirium. The rs-fMRI functional connectivity strength increased 3 months after surgery in the total study population (ß=0.006; 95% confidence interval [CI]: 0.001-0.011; P=0.013), but it decreased after postoperative delirium (ß=-0.015; 95% CI: -0.028 to 0.002; P=0.023). No difference in TMT-B scores was found at follow-up between patients with and without postoperative delirium. Patients with decreased functional connectivity strength declined in TMT-B scores compared with those who did not (ß=11.04; 95% CI: 0.85-21.2; P=0.034). CONCLUSIONS: Postoperative delirium was associated with decreased brain functional connectivity strength after 3 months, suggesting that delirium has a long-lasting impact on brain networks. The decreased connectivity strength was associated with significant cognitive deterioration after major surgery. CLINICAL TRIAL REGISTRATION: NCT02265263.

Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia.

Cystic fibrosis is caused by genetic defects that impair the CFTR channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described CFTR function assays in 3D airway organoids were not fully optimal, because of inefficient organoid differentiation and limited scalability. In this report, we therefore describe an alternative method of culturing nasal-brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1<i>ß</i> and interleukin-1<i>ß</i>, which enabled consistent detection of CFTR modulator responses in nasal-airway organoid cultures from subjects with cystic fibrosis.

Stability of neuropsychological test performance in older adults serving as normative controls for a study on postoperative cognitive dysfunction.

OBJECTIVE: Studies of postoperative cognitive dysfunction (POCD) rely on repeat neuropsychological testing. The stability of the applied instruments, which are affected by natural variability in performance and measurement imprecision, is often unclear. We determined the stability of a neuropsychological test battery using a sample of older adults from the general population. Forty-five participants aged 65 to 89 years performed six computerized and non-computerized neuropsychological tests at baseline and again at 7 day and 3 months follow-up sessions. Mean scores on each test were compared across time points using repeated measures analyses of variance (ANOVA) with pairwise comparison. Two-way mixed effects, absolute agreement analyses of variance intra-class correlation coefficients (ICC) determined test-retest reliability. RESULTS: All tests had moderate to excellent test-retest reliability during 7-day (ICC range 0.63 to 0.94; all p < 0.01) and 3-month intervals (ICC range 0.60 to 0.92; all p < 0.01) though confidence intervals of ICC estimates were large throughout. Practice effects apparent at 7 days eased off by 3 months. No substantial differences between computerized and non-computerized tests were observed. We conclude that the present six-test neuropsychological test battery is appropriate for use in POCD research though small sample size of our study needs to be recognized as a limitation. Trial registration ClinicalTrials.gov Identifier NCT02265263 (15th October 2014).

The association between brain volume, cortical brain infarcts, and physical frailty.

Physical frailty is an age-associated syndrome of decreased reserve leading to vulnerability to physiological stressors and associated with negative outcomes. The underlying structural brain abnormalities of physical frailty are unclear. We investigated the association between brain volume, cortical brain infarcts, and physical frailty. In this multicenter study, 214 nondemented participants were classified as frail (n = 32), prefrail (n = 107), or nonfrail (n = 75) based on the Fried frailty phenotype. The associations between frailty and brain volumes and cortical brain infarcts were investigated by linear or logistic regression analyses. Participants in the frail group showed a lower total brain volume (-19.67 mL [95% confidence interval -37.84 to -1.50]) and lower gray matter volume (-12.19 mL [95% confidence interval -23.84 to -0.54]) compared to nonfrail participants. Frailty was associated with cortical brain infarcts [frail 16% [n = 5], prefrail 11% [n = 12], and nonfrail 3% [n = 2]). Reduced total brain volume and gray matter volume and increased cortical brain infarcts seem therefore to be part of the structural substrate of the physical frailty phenotype.