Histochemical and biochemical analysis of collagen content in formalin-fixed, paraffin embedded colonic samples.

Collagen is the most abundant structural protein and extracellular matrix component in mammals. In the colon, collagen fibres reside in all the major sublayers; namely, the mucosa, submucosa, muscularis externa and the serosa. Methods to quantify collagen content in formalin-fixed, paraffin-embedded (FFPE) stained sections are required and image analysis offers a technique by which the spatial distribution and localisation of collagen fibres can be easily measured. This laboratory protocol was developed from established techniques using FFPE colon. Human colonic samples embedded transversally in paraffin wax were serially sectioned and stained with either Masson's trichrome (MT) or Picrosirius red (PSR). Quantitation estimation of collagen content in each sublayer was performed via ImageJ processing. Hydroxyproline content was quantified using a rapid and sensitive assay in sectioned tissue. Either MT or PSR staining followed by morphometric image analysis via ImageJ provided equally appreciable quantitative results. Moreso, analysis of hydroxyproline content in our samples indicate that this protocol could be useful in retrospective studies for FFPE samples. This laboratory protocol provides a systematic and reproducible method that can be utilized to accurately assess collagen content in individual sublayers of the colonic wall as well as detection of overall hydroxyproline content in FFPE specimens.

Effect of old age on the subpopulations of enteric glial cells in human descending colon.

Old age is associated with a higher incidence of lower bowel conditions such as constipation. Recent evidence suggest that colonic motility may be influenced by enteric glial cells (EGCs). Little is known about the effect of aging on the subpopulation of EGCs in the human colon. We assessed and compared the pattern of distribution of EGCs in adult and elderly human colon. Human descending colon were obtained from 23 cancer patients comprising of adults (23-63 years; 6 male, 7 female) and elderly (66-81 year; 6 male, 4 female). Specimens were serially-sectioned and immunolabeled with anti-Sox-10, anti-S100 and anti-GFAP for morphometric analysis. Standardized procedures were utilized to ensure unbiased counting and densitometric evaluation of EGCs. The number of Sox-10 immunoreactive (IR) EGCs were unaltered with age in both the myenteric plexus (MP) (respectively, in adult and elderly patients, 1939 ± 82 and 1760 ± 44/mm length; p > .05) and submucosal plexus; there were no apparent differences between adult males and females. The density of S100-IR EGCs declined among the elderly in the circular muscle and within the MP per ganglionic area. In the adult colon, there were more S100-IR EGCs distributed in the circular muscle per unit area than the Taenia coli. There was little or no GFAP-IR EGCs in both adult and elderly colon. We concluded that aging of the human descending colon does not result in a loss of Sox-10-IR EGCs in the MP and SMP but reduces S100-IR EGCs density within the musculature. This alteration in myenteric EGCs density with age may contribute to colonic dysfunction.

The vulnerability of the human taenia coli to alterations in total collagen within the colon of the elderly.

The structure of the colonic wall relies on collagen, distributed within the submucosa and the muscularis externa. A recent analysis of total collagen in human ascending colon (AC) suggests that the muscularis externa is more susceptible to age-related increases in collagen among the elderly. However, it is not clear if this change also occurs in the descending colon (DC) or if the circular and longitudinal muscle layers are similarly affected in either region of colon. The aim of this study is to determine the total collagen content in the DC and its distribution between the circular muscle (CM) and taenia coli (TC) of the AC and DC of adults and compare the same with tissue from the elderly. Masson's trichrome and Picrosirius red were used to assess total collagen content in the AC and DC; aged 22 - 91 years. Macroscopically normal AC from 22 patients (adults: 22-60 years; 6 male, 6 female; elderly: 70 - 91 years; 6 male, 4 female) and DC from 23 patients (adults: 23-63 years; 6 male, 7 female; elderly: 66 - 88 years; 6 male, 4 female) were obtained following surgery for non-obstructed bowel cancer. The total hydroxyproline content in DC samples was also evaluated. In the DC, tinctorial staining demonstrated an increased occurrence of total collagen fibres in the submucosa of the elderly (159.8 ± 9.6 in elderly vs. 126.9 ± 6.1 in the adults; p 0.05) and in the muscularis externa (respectively 37.4 ± 4.1 vs. 18.8 ± 2.4; p 0.01). In the adult AC and DC, there were no statistically significant differences in the amount of collagen within the CM and TC. In the elderly, the total collagen fibres within the TC was greater in the AC (mean grey intensity: 63.4 ± 3.9% in the elderly vs. 36.6 ± 1.6% in adults; p 0.05) and DC (mean grey intensity: respectively, 59.82 ± 2.4 vs. 40.2 ± 0.9%; p 0.05). In both AC and DC of the elderly samples, several thickened collagen fibrils were microscopically identified within the TC infiltrating to the myenteric plexus. In the TC of the elderly AC, the total collagen fibres were increased by approximately 4% compared to that of the DC. The total collagen concentration in the elderly DC assessed by hydroxyproline assay was increased by approximately 15% compared to the adult. Sex related differences were not found when data combined. We concluded that the total collagen content in the muscularis externa particularly of the TC of human colon increases with age. The subtle change in collagen distribution with age between AC and DC may differentially affect the tensile strength of the colon.

Total collagen content and distribution is increased in human colon during advancing age.

BACKGROUND: The effect of ageing on total collagen content of human colon has been poorly investigated. The aim of this study was to determine if ageing altered total collagen content and distribution in the human colon. METHODS: Macroscopically normal ascending colon was obtained at surgery from cancer patients (n = 31) without diagnosis of diverticular disease or inflammatory bowel disease. Masson's trichrome and Picrosirius red stains were employed to identify the total collagen content and distribution within the sublayers of the colonic wall for adult (22-60 years; 6 males, 6 females) and elderly (70 - 91years; 6 males, 4 female) patients. A hydroxyproline assay evaluated the total collagen concentration for adult (30-64 years; 9 male, 6 female) and elderly (66-91 years; 8 male, 8 female) patients. KEY RESULTS: Histological studies showed that the percentage mean intensity of total collagen staining in the mucosa, submucosa and muscularis externa was, respectively, 14(1.9) %, 74(3.2) % and 12(1.5) % in the adult ascending colon. Compared with the adults, the total collagen fibres content was increased in the submucosa (mean intensity; 163.1 ± 11.1 vs. 124.5 ± 7.8; P < 0.05) and muscularis externa (42.5 ± 8.0 vs. 20.6 ± 2.8; P < 0.01) of the elderly patients. There was no change in collagen content of the mucosa. The total collagen concentration was increased in the elderly by 16%. Sex-related differences were not found, and data were combined for analysis. CONCLUSIONS: Greater total collagen content was found in the submucosa and muscularis externa of the elderly human male and female colon. These changes may contribute to a possible loss of function with ageing.

Functional and anatomical deficits in visceral nociception with age: a mechanism of silent appendicitis in the elderly?

The ability to sense visceral pain during appendicitis is diminished with age leading to delay in seeking health care and poorer clinical outcomes. To understand the mechanistic basis of this phenomenon, we examined visceral nociception in aged mouse and human tissue. Inflamed and noninflamed appendixes were collected from consenting patients undergoing surgery for the treatment of appendicitis or bowel cancer. Supernatants were generated by incubating samples in buffer and used to stimulate multiunit activity in intestinal preparations, or single-unit activity from teased fibres in colonic preparations, of young and old mice. Changes in afferent innervation with age were determined by measuring the density of calcitonin gene-related peptide-positive afferent fibres and by counting dorsal root ganglia back-labelled by injection of tracer dye into the wall of the colon. Finally, the effect of age on nociceptor function was studied in mouse and human colon. Afferent responses to appendicitis supernatants were greatly impaired in old mice. Further investigation revealed this was due to a marked reduction in the afferent innervation of the bowel and a substantial impairment in the ability of the remaining afferent fibres to transduce noxious stimuli. Translational studies in human tissue demonstrated a significant reduction in the multiunit but not the single-unit colonic mesenteric nerve response to capsaicin with age, indicative of a loss of nociceptor innervation. Our data demonstrate that anatomical and functional deficits in nociception occur with age, underpinning the atypical or silent presentation of appendicitis in the elderly.

The psychoneuroimmunological effects of music: a systematic review and a new model.

There has been a growing interest over the past decade into the health benefits of music, in particular examining its psychological and neurological effects. Yet this is the first attempt to systematically review publications on the psychoneuroimmunology of music. Of the selected sixty-three studies published over the past 22 years, a range of effects of music on neurotransmitters, hormones, cytokines, lymphocytes, vital signs and immunoglobulins as well as psychological assessments are cataloged. Research so far points to the pivotal role of stress pathways in linking music to an immune response. However, several challenges to this research are noted: (1) there is very little discussion on the possible mechanisms by which music is achieving its neurological and immunological impact; (2) the studies tend to examine biomarkers in isolation, without taking into consideration the interaction of the biomarkers in question with other physiological or metabolic activities of the body, leading to an unclear understanding of the impact that music may be having; (3) terms are not being defined clearly enough, such as distinctions not being made between different kinds of stress and 'music' being used to encompass a broad spectrum of activities without determining which aspects of musical engagement are responsible for alterations in biomarkers. In light of this, a new model is presented which provides a framework for developing a taxonomy of musical and stress-related variables in research design, and tracing the broad pathways that are involved in its influence on the body.

Smooth muscle cholinergic denervation hypersensitivity in diverticular disease.

BACKGROUND: Evidence from clinical and laboratory investigations into the causes of diverticular disease suggests that disturbances in cholinergic activity are important, the effector mechanisms of which have yet to be established. We aimed to investigate the role of smooth muscle and neural cholinergic activity in the pathogenesis of this disease. METHODS: Two investigators independently did a blinded immunohistochemical image analysis of localising antibodies to choline acetyltransferase, co-localised with protein gene product (PGP)--a marker of general neural tissue-and smooth muscle muscarinic M3 receptors, on three histological sections of sigmoid colons from ten patients with diverticular disease and ten controls, after resections for rectal tumours. We also did isotonic organ bath experiments to assess muscle strip sensitivities to exogenous acetylcholine. FINDINGS: In circular muscle, activity of choline acetyltransferase was lower in patients with diverticular disease than in controls: median percentage surface area of choline acetyltransferase over PGP was 17.5% (range 10.0-37.0) in patients with diverticular disease and 47.0% (29.0-54.0) in controls (p<0.0001). M3 receptors were upregulated in patients with diverticular disease compared with controls: the median surface area was 13.2% (6.0-23.3) in patients with diverticular disease and 2.5% (1.6-3.7) in controls (p<0.0001). The sensitivity to exogenous acetylcholine was increased in patients with diverticular disease (mean -log EC(50) 5.6 [SD 0.3]) compared with controls (4.9 [0.5]; difference 0.7 [95% CI 0.3-1.1], p=0.006). In longitudinal muscle, choline acetyltransferase activity was lower in patients with diverticular disease (median 19.5%, range 12.0-30.0) than in controls (47.0%, 35.0-60.0; p<0.0001), with upregulation of M3 receptors in diverticular disease (diverticular disease 7.8% [1.9-20.4], controls 1.7% [0.8-3.0]; p<0.0001). However, sensitivity to exogenous acetylcholine did not differ between the two groups (diverticular disease mean 5.6% [SD 0.3], controls 5.2% [0.4]; difference 0.4% [95% CI -0.02-0.7], p=0.06). INTERPRETATION: Our results suggest that cholinergic denervation hypersensitivity can affect smooth muscle. Upregulation of smooth muscle M3 receptors might account for specific clinical, physiological, and pharmacological abnormalities associated with diverticular disease.