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A distinct subset of submucosal mast cells undergoes hyperplasia following neonatal maternal separation: a role in visceral hypersensitivity?
  1. N P Hyland1,2,
  2. M Julio-Pieper2,
  3. S M O’Mahony2,3,
  4. D C Bulmer3,
  5. K Lee3,
  6. E M Quigley2,4,
  7. T G Dinan2,5,
  8. J F Cryan1,2,6,6
  1. 1
    Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland
  2. 2
    Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
  3. 3
    Immuno-Inflammation CEDD, GlaxoSmithKline Medicines Research Centre, Stevenage, UK
  4. 4
    Department of Medicine, University College Cork, Cork, Ireland
  5. 5
    Department of Psychiatry, University College Cork, Cork, Ireland
  6. 6
    School of Pharmacy, University College Cork, Cork, Ireland
  1. Dr J F Cryan, School of Pharmacy, Cavanagh Pharmacy Building, University College Cork, Cork, Ireland; j.cryan{at}

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We recently read with great interest the article published by Barreau et al (Gut 2008;57:582–90) who report, as one of their primary findings, mast cell hyperplasia and increased colonic rat mast cell protease II (RMCPII) activity following neonatal psychological stress. The authors also persuasively demonstrate a potential role for mast cell-derived nerve growth factor in contributing to increased colonic neural density in this model. In the study we describe here, using a related maternal separation procedure1 we also observed mast cell hyperplasia. However, this was restricted to the colonic submucosa and characterised by RMCPII immunoreactivity and predominantly blue or blue/red positive cells following Alcian blue/safranin staining, without an associated change in mucosal mast cell (MMC) number.

Maternal separation was carried out using a protocol previously described by our group and known to induce a number of behavioural and gastrointestinal (GI) effects,1 and adult male animals, at least 11 weeks of age, were used in subsequent studies. To identify mast cells we used sheep anti-RMCP II (1:500; Moredun, Midlothian, UK) and Alcian blue (1% in 0.7 mol/l HCl)/safranin (0.5% in 0.125 mol/l HCl) staining. Analysis of colonic supernatants for RMCPII release was carried out using an enzyme-linked immunosorbent assay (ELISA; Moredun).

Similarly to Barreau et al we identified extensive RMCPII positive staining along the crypt mucosa axis (non-separated (NS), 49.5 (SEM 3.7) mast cells/mm2, n = 10, maternally separated (MS), 53.1 (SEM 2.5) mast cells/mm2, n = 15, p>0.05) in rat colon; though we did not observe any significant difference between NS and MS animals. We also identified, albeit in much fewer numbers than those recorded in the mucosa, RMCP II positive cells in the submucosa (fig 1B,C). Moreover, the number of submucosal RMCPII immunoreactive cells was significantly increased following neonatal psychological stress relative to NS controls (fig 1A). Although RMCPII is considered a mucosal mast cell (MMC) marker2 it has previously been reported that a small percentage of connective tissue mast cell (CTMC) also stains positively for this chymase in the rat colon.3 Further characterisation of submucosal mast cells revealed that they were insensitive to paraformaldehyde fixation and the majority stained blue or blue/red (fig 1E,F), with few, if any, cells staining red (data not shown) following the Alcian blue/safranin sequence, consistent with a CTMC phenotype.2 3 As observed with RMCPII immunostaining, Alcian blue/safranin positive cells were significantly increased in MS animals (fig 1D).

Figure 1

Significantly more rat mast cell protease II (RMCPII) positive cells were localised in the submucosa of maternally separated (MS) animals compared to non-separated (NS) controls (A, B and C; n = 10, p = 0.05, one-tailed Student t test). As observed with RMCPII immunoreactivity, Alcian blue/safranin positive mast cells were significantly increased in MS colon (D; n = 10, p = 0.02, one-tailed Student t test). Alcian blue (E and F, black arrows) and Alcian blue/safranin (E and F, white arrows) positive mast cells were detected in the submucosa of NS and MS animals. The Alcian blue/safranin staining is not homogenous throughout the tissue section, and in some sections represents the total number of cells counted per section. Goblet cell staining can also be seen in the colonic crypts in both E and F. Values are the means; error bars represent the SEM. Scale bars  = 100 μm. cm  =  circular muscle, lm  =  longitudinal muscle.

As it has been previously demonstrated by others that the mast cell stabiliser doxantrazole reversed visceral hypersensitivity in rat models of early life stress,4 we measured the visceral pain response in our MS animals 30 min following intra-peritoneal administration of doxantrazole (5 mg/kg) or vehicle in order to determine whether increased CTMC may contribute to visceral pain. Colorectal distension was performed by inserting a latex balloon (6 cm) into the colon. The balloon was distended from 0 to 80 mm Hg over 8 min, during which the threshold pressure (mm Hg) and the cumulative number of visceral pain behaviours were visually recorded.1 As expected, MS animals (range, 28.8–71.2 mm Hg) displayed pain behaviours at a lower distension pressure (threshold) than NS animals (range, 55.1–79.6 mm Hg, fig 2A, p⩽0.01); however, this was insensitive to mast cell stabilisation (p⩽0.05, MS + doxantrazole vs NS). In addition, the total number of pain behaviours observed over 8 min from 0 to 80 mm Hg was not significantly altered by doxantrazole pre-treatment (fig 2B). In order to determine whether MS animals displayed increased protease release per se, and to confirm the stabilising effect of doxantrazole, full thickness colonic segments (3 cm) were removed from animals immediately post CRD, were stimulated with anti-rat IgE (1:250; MP Biomedical, Solon, Ohio, USA) for 30 min in Hanks’ balanced salt solution (Sigma, Poole, UK) bubbled with carbogen (95% oxygen/5% carbon dioxide) at 37°C in a shaking water bath and supernatants were analysed by ELISA. MS animals released approximately 50% more RMCPII following anti-IgE stimulation relative to NS animals (NS, 0.07 (SEM 0.02) ng/mg, n = 7; MS, 0.14 (SEM 0.04) ng/mg, n = 9). MS tissues taken from animals treated with doxantrazole displayed similar RMCPII release to NS animals (MS + doxantrazole, 0.07 (SEM 0.01) ng/mg, n = 10). These data suggest that MS tissues, consistent with hyperplasia of RMCPII containing mast cells, display increased protease release, although the exact source, MMC or CTMC, is unclear.

Figure 2

Maternally separated (MS) animals display a decreased threshold, the pressure at which the first pain behaviours were observed, during colorectal distension relative to non-separated (NS) animals. Pre-treatment with the mast cell stabiliser doxantrazole (5 mg/kg, intraperitoneally) did not significantly alter the threshold in MS animals. (A; n = 7–11). Furthermore, doxantrazole had no significant effect on cumulative abdominal contractions observed during ramp distension (0–80 mm Hg, 8 min) (B; n = 7–13). Values are means; error bars represent the SEM. *p⩽0.05; **p⩽0.01; one-way analysis of variance followed by Tukey’s multiple comparison test.

Nonetheless, our data suggest that the role of mast cells in the manifestation of early-life stress-induced hypersensitivity is not sensitive to acute mast cell stabilisation in adulthood. This premise is bolstered by the findings by Barreau et al describing the adolescent period as being critical for GI-associated neuro-immune alterations. Whether this period is also critical in CTMC development or migration or in their elaboration of protease content remains to be determined.

While subtle but significant differences between our maternal separation protocol and that of Barreau et al (eg, litter vs single pup separation and rat strain) may account for the discrepancy in MMC hyperplasia between our studies, our data, in concert with that of Barreau et al, further support a link between detrimental early life events and altered mast cell development or function in adulthood. Moreover, our data emphasise the heterogeneity that exists in the GI tract with respect to mast cell populations, and implicates a novel role for all, or a subset of CTMC (RMCPII containing) in contributing to the development of functional GI disorders as a result of early life stress.


The authors wish to acknowledge C Bongiovanni, B Oschmann and S McSweeney for their assistance with this study.


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  • Funding: The Alimentary Pharmabiotic Centre is a Centre for Science and Technology (CSET) funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI, Industrial Development Agency (Ireland) and GlaxoSmithKline.

  • Competing interests: None declared.

  • Ethics approval: Approval for this study was granted by University College Cork's Animal Experimentation Ethics Committee, Animal Ethical Review Request #2008/11.

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