Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function.

Human colonic neuromuscular functions decline among the elderly. The aim was to explore the involvement of senescence. A preliminary PCR study looked for age-dependent differences in expression of CDKN1A (encoding the senescence-related p21 protein) and CDKN2A (encoding p16 and p14) in human ascending and descending colon (without mucosa) from 39 (approximately 50: 50 male: female) adult (aged 27-60 years) and elderly donors (70-89 years). Other genes from different aging pathways (e.g., inflammation, oxidative stress, autophagy) and cell-types (e.g., neurons, neuron axonal transport) were also examined. Unlike CDKN1A, CDKN2A (using primers for p16 and p14 but not when using p14-specific primers) was upregulated in both regions of colon. Compared with the number of genes appearing to upregulate in association with temporal age, more genes positively associated with increased CDKN2A expression (respectively, 16 and five of 44 genes studied for ascending and descending colon). Confirmation of increased expression of CDKN2A was sought by immunostaining for p16 in the myenteric plexus of colon from 52 patients, using a semi-automated software protocol. The results showed increased staining not within the glial cells (S100 stained), but in the cytoplasm of myenteric nerve cell bodies (MAP2 stained, with identified nucleus) of ascending, but not descending colon of the elderly, and not in the cell nucleus of either region or age group (5,710 neurons analyzed: n = 12-14 for each group). It was concluded that increased p16 staining within the cytoplasm of myenteric nerve cell bodies of elderly ascending (but not descending) colon, suggests a region-dependent, post-mitotic cellular senescence-like activity, perhaps involved with aging of enteric neurons within the colon.

Changes in neuromuscular structure and functions of human colon during ageing are region-dependent.

OBJECTIVE: To determine if human colonic neuromuscular functions decline with increasing age. DESIGN: Looking for non-specific changes in neuromuscular function, a standard burst of electrical field stimulation (EFS) was used to evoke neuronally mediated (cholinergic/nitrergic) contractions/relaxations in ex vivomuscle strips of human ascending and descending colon, aged 35-91 years (macroscopically normal tissue; 239 patients undergoing cancer resection). Then, to understand mechanisms of change, numbers and phenotype of myenteric neurons (30 306 neurons stained with different markers), densities of intramuscular nerve fibres (51 patients in total) and pathways involved in functional changes were systematically investigated (by immunohistochemistry and use of pharmacological tools) in elderly (≥70 years) and adult (35-60 years) groups. RESULTS: With increasing age, EFS was more likely to evoke muscle relaxation in ascending colon instead of contraction (linear regression: n=109, slope 0.49%±0.21%/year, 95% CI), generally uninfluenced by comorbidity or use of medications. Similar changes were absent in descending colon. In the elderly, overall numbers of myenteric and neuronal nitric oxide synthase-immunoreactive neurons and intramuscular nerve densities were unchanged in ascending and descending colon, compared with adults. In elderly ascending, not descending, colon numbers of cell bodies exhibiting choline acetyltransferase immunoreactivity increased compared with adults (5.0±0.6 vs 2.4±0.3 neurons/mm myenteric plexus, p=0.04). Cholinergically mediated contractions were smaller in elderly ascending colon compared with adults (2.1±0.4 and 4.1±1.1 g-tension/g-tissue during EFS; n=25/14; p=0.04); there were no changes in nitrergic function or in ability of the muscle to contract/relax. Similar changes were absent in descending colon. CONCLUSION: In ascending not descending colon, ageing impairs cholinergic function.

Investigation for Paediatric Cushing's Syndrome Using Twenty-Four-Hour Urinary Free Cortisol Determination.

OBJECTIVE: Paediatric Cushing's syndrome (CS) remains a challenge to diagnose and exclude. We assessed the accuracy of 24-hour urinary free cortisol (UFC) determination in children referred for suspected CS. DESIGN: We conducted a retrospective study of paediatric patients referred to our centre with suspected CS between 1982 and 2014. PATIENTS: Of 66 subjects (mean age 12.9 years; range 4.4-16.9), there were 47 cases of CS (29 males), which included Cushing's disease (CD; 39 patients, 25 males), primary pigmented nodular adrenocortical disease (8 patients, 4 males) and 19 'controls' (6 males) in whom the diagnosis of CS was excluded. MEASUREMENTS: The subjects had between one and five 24-hour UFC collections analysed by radioimmunoassay, chemiluminescent immunoassay or liquid chromatography-mass spectrometry. The data were normalised, corrected for body surface area (m2) and assessed using receiver operating characteristic analysis and an independent two-tailed t test. RESULTS: The diagnostic accuracy of 24-hour UFC for CS was excellent (area under the curve 0.98, 95% CI 0.946-1.00, sensitivity 89%, specificity 100%). CONCLUSIONS: Twenty-four-hour UFC is a reliable and practical investigation with high diagnostic accuracy for paediatric CS. However, further investigations may be required if the UFC is normal but there is a high diagnostic suspicion of CS.