Protein-retention expansion microscopy for visualizing subcellular organelles in fixed brain tissue.

BACKGROUND: Protein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduce fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. NEW METHOD: Here we compare the staining and digestion conditions of existing proExM workflows to identify the optimal protocol for visualizing subcellular organelles (mitochondria and the Golgi apparatus) within reporter-labeled neurons in fixed mouse brain tissue. RESULTS: We found that immunostaining before proExM and using a proteinase K based digestion for 8 h consistently resulted in robust fluorescence retention for immunolabeled subcellular organelles and genetically-encoded reporters. COMPARISON WITH EXISTING METHODS: With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in individual CA2 neurons in the mouse hippocampus. CONCLUSIONS: This organelle optimized proExM protocol will be broadly useful for investigators interested in visualizing the spatial distribution of immunolabeled subcellular organelles in various reporter mouse lines, reducing effort, time and resources on the optimization process.

Quality of life after oesophageal stenting in patients with palliative oesophageal cancer.

AIM: Patients with incurable oesophageal cancer have poor outcomes, with disabling symptoms and a poor quality of life (QOL), which may be improved by oesophageal stenting. We aimed to measure change in symptoms related specifically to oesophageal cancer and overall QOL before and 30 days after stent insertion, to measure adverse effects and to define any patient factors that may be significant in predicting patients who may benefit most. METHODS: We prospectively enrolled patients in an observational study at Middlemore Hospital, New Zealand, and administered validated QOL- and symptomatology-based questionnaires before and 30 days after stent insertion. Additional patient-related demographics, procedural characteristics, adverse events and outcomes were collected. RESULTS: Between 31 March 2014 and 3 July 2020, 57 patients were initially recruited. Four patients withdrew from the study, and 13 patients died before 30 days. Forty patients (29 males; mean±SD age, 72±12 years) completed the study. A significant improvement was noted at one-month post stent insertion in the overall global QOL score (mean 35 to 46, p=0.01). The most significant score improvements were seen in dysphagia, trouble eating, trouble swallowing saliva and dry mouth (p<0.001). Physical, emotional, cognitive and social functioning did not change. Post-procedural adverse events occurred in 17 patients (43%). A poorer initial level of functioning was associated with reduced improvement in global QOL (p≤0.04). Patients followed-up died a mean of 2.8 months after insertion. CONCLUSION: In patients surviving longer than 30 days, there is significant improvement of overall QOL and dysphagia one-month post oesophageal stent insertion for malignant, palliative dysphagia. Multiple psychosocial facets were unchanged with this intervention. Stent-related adverse events were common.

Chromoendoscopy versus standard colonoscopy for detection of nonpolypoid dysplasia in patients with inflammatory bowel disease.

AIM: Inflammatory bowel disease (IBD) is associated with an increased risk of colorectal cancer. Studies show that chromoendoscopy (CE) can increase the detection of dysplasia at surveillance colonoscopy, compared to standard white light endoscopy (WLE). We performed a retrospective cohort study to compare standard WLE to CE with targeted biopsies in detecting nonpolypoid dysplasia in IBD patients undergoing surveillance colonoscopy at a single tertiary centre. METHOD: Data was collected on 110 consecutive patients with IBD who underwent surveillance colonoscopy from 1 August 2015 to 31 July 2017 at Counties Manukau District Health Board, Auckland. Patients had either WLE or CE. Patient characteristics, endoscopic and histologic descriptions were reviewed. Rates of dysplasia detection by the different endoscopic techniques were compared using an exact Poisson test. RESULTS: 76/110 (69%) had WLE (mean age 56y; median disease duration 18y) and 34/110 (31%) had CE (median age 59y; median disease duration 19y). Nonpolypoid dysplasia was detected in 0/76 (0%) patients who had WLE. Seven nonpolypoid dysplastic lesions were detected in 4/34 (11.8%) patients who had CE. Dysplasia pick up rate was significantly higher in the CE group with a risk difference of 11.8%, 95% confidence interval (0.93, 22.59), p=0.008. Dysplasia detection rate per patient was also significantly higher in the CE group with a rate difference of 20.6 lesions per 100 patients, 95% confidence interval (5.3, 35.8), p=0.0003. As expected, there was no difference between the number of polypoid dysplastic lesions found between the two groups (p=0.12). CONCLUSION: In our cohort of IBD patients undergoing surveillance colonoscopy, CE with targeted biopsy is associated with a significantly increased nonpolypoid dysplasia detection rate when compared to WLE. These results are comparable to studies performed in the rest of the world.

Efficacy and safety of TC-325 (HemosprayTM) for non-variceal upper gastrointestinal bleeding at Middlemore Hospital: the early New Zealand experience.

AIMS: A case series to review early experiences with HemosprayTM for a variety of non-variceal upper gastrointestinal bleeding (UGIB) at Middlemore Hospital. METHODS: HemosprayTM was administered therapeutically as first line or rescue at the discretion of the endoscopist. All cases of UGIB requiring HemosprayTM at Middlemore Hospital were identified to the investigator who undertook analysis of electronic and hard copy notes. RESULTS: Between October 2013 and July 2016, 36 patients were treated endoscopically with HemosprayTM. Source of bleeding was predominantly gastric in 17, 15 were duodenal and four oesophageal. The majority of lesions were peptic ulcer or post-intervention (78%), with others being Mallory Weiss tear (MWT), gastric mass, Dieulafoy lesion, portal hypertensive gastropathy and post-biopsy. Thirty-one were actively bleeding with mostly oozing haemorrhage (75%). Twenty-three patients were on antithrombotic therapy (ATT), two each on warfarin and low molecular weight heparin (LMWH) and 19 on antiplatelet agents. HemosprayTM was administered therapeutically in all cases, as first line or rescue. Acute haemostasis was achieved in all patients; four (11%) episodes of re-bleeding occurred within seven days, with average follow-up of 16 months. There were no instances of equipment malfunction or adverse events specific to use of HemosprayTM. CONCLUSIONS: Our early experience with HemosprayTM is very promising and there is clear role for HemosprayTM as a rescue therapy when standard methods have failed to achieve haemostasis and possibly as first line in cases of diffuse bleeding not amenable to standard interventions. However, HemosprayTM is not recommended as a standalone therapy for spurting haemorrhage due to the increased frequency of re-bleeding.

Symptom presentations and other characteristics of colorectal cancer patients and the diagnostic performance of the Auckland Regional Grading Criteria for Suspected Colorectal Cancer in the South Auckland population.

AIM: This study reviews the presenting symptoms of colorectal cancer in the ethnically diverse Middlemore Hospital referral population of South Auckland, New Zealand. The performance of the newly introduced Auckland Regional Grading Criteria as prediction tool for selecting colorectal cancer cases referred from primary care was evaluated in this group. METHOD: Retrospective review of all colorectal cancer (CRC) cases diagnosed between January 2006 and January 2011. Information extracted from case note review was used to grade patients using the Auckland Regional Grading Criteria. RESULTS: A total of 799 patients were included. The commonest symptoms were: rectal bleeding (25.5-42.3%) and change in bowel habit (20.6-26.8%). Low-risk symptoms including abdominal pain (16.3-46.8%) and weight loss (18.4-26.1%) were not uncommon. 64.4% of Maori and 64.9% of Pacific patients had stage III or IV cancers. Pacific patients had more stage IV disease, 37.7% (p<0.001) and were less likely to undergo tumour resection, 26.0% (p<0.001). The Auckland Regional Grading Criteria would miss 24.7% of the patients with CRC in the referral population. CONCLUSION: While rectal bleeding and change in bowel habit are frequent presenting symptoms, low-risk atypical symptoms including constipation, weight loss and abdominal pain were not uncommon. Significant proportion of Pacific patients present with late-stage disease. The current Auckland Regional grading criteria would miss significant proportion of our study population with colorectal cancer.

Complications of endoscopic retrograde cholangiopancreatography.

AIM: The aim of this prospective study was to audit all endoscopic retrograde cholangiopancreatographic (ERCP) procedures performed between July 1999 and November 1999, in particular to determine the spectrum and rate of complications. METHODS: An audit data sheet was designed with 100 variables on which the endoscopist recorded all the relevant details of the procedure, from patient demographics to overall success of the procedure. Data were collected at the time of ERCP and before discharge. Any complications within 30 days of ERCP were also recorded. RESULTS: Of 210 consecutive patients audited, successful diagnostic and therapeutic ERCP was performed in 93% with an overall complication rate of 9.5%. The incidence of pancreatitis was 4.76% and haemorrhage 2.38%. No severe complication or procedure-related mortality occurred. CONCLUSION: The most common complications of ERCP were pancreatitis and haemorrhage.