Alterations in mast cell frequency and relationship to angiogenesis in the rat mammary gland during windows of physiologic tissue remodeling - PubMed (original) (raw)

Alterations in mast cell frequency and relationship to angiogenesis in the rat mammary gland during windows of physiologic tissue remodeling

Robert A Ramirez et al. Dev Dyn. 2012 May.

Abstract

Background: The mammary epithelium undergoes proliferation and regression accompanied by remodeling of the fibrocellular and vascular stroma. Mast cells are abundant in these compartments and have been implicated in remodeling during wound healing and cancer progression. The purpose of this study was to test the hypothesis that mast cell abundance correlates with physiologic mammary tissue remodeling during estrous cycling, lactogenesis (pregnancy and lactation) and involution.

Results: Mast cell and capillary frequency were quantified in the stroma surrounding ducts and lobules from mammary glands of rats. During estrous cycling, periductal mast cell numbers were unchanged, but lobule-associated mast cells significantly increased in the regressive phase of diestrus II. During lactogenesis, lobular stroma mast cells peaked early in pregnancy, at D2, followed by a significant decrease throughout lactation. Involution was associated with a rapid return in mast cell numbers, similar to diestrus II. Lobular vascularization peaked during the state of metestrus, when limited secretory differentiation occurs. Lobular angiogenesis peaked at D7 of pregnancy, regressed, and then returned to high levels during lactation and early involution, when secretory differentiation is high.

Conclusions: These results suggest mast cells are predominantly associated with regressive lobular remodeling during cycling and involution, whereas angiogenesis is predominantly associated with secretory differentiation.

Copyright © 2012 Wiley Periodicals, Inc.

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Figures

Fig. 1

Fig. 1

Mast cell frequency during mammary gland development and lactogenic differentiation. A,B: Terminal ductal lobular units (A) and ducts (B) were analyzed for mast cell number. Panel (A), inset, shows toluidine blue stained mast cells (arrowheads) associated with lobular stroma; the right panel shows the same field under phase contrast. A: Alveolar-associated mast cells were highest in prepubertal (VD28), and slightly but not significantly decreased in peripubertal virgin mammary gland (VD50). Mast cells were initially increased at day 2 of pregnancy, and thereafter significantly and progressively decreased throughout pregnancy. A trend toward increasing mast cell numbers was seen during lactation, with a further progressive increase during early involution. By day 2 of involution, mast cells had returned to resting mammary gland (VD50) levels. Panel (B), inset, shows toluidine blue stained mast cells (arrowheads) surrounding a duct; the right panel shows the same field under phase contrast. B: Mast cell number in ductal stroma was highest in prepubertal (VD28 [virgin day 28]) mammary gland. Thereafter, numbers were decreased and no significant trends were seen during pregnancy (PD18–19), lactation, or involution. Quantitative data shows means ± SEM of total number of fields. P < 0.05 was determined to be significant. Scale bar = 10 μm.

Fig. 2

Fig. 2

Mast cell abundance in toluidine-blue sections throughout postnatal mammary gland development. A–L: Figure 2 demonstrates the histologic appearance of the darkly stained mast cells (arrows) associated with lobular ductules, whether in resting nulliparous (A,B) or parous glands (C) or during pregnancy (D–F), lactation (G–I), or involution (J–L).

Fig. 3

Fig. 3

Capillary frequency in lobules during mammary gland development and lactogenic differentiation. A: Inset shows appearance of hematoxylin and eosin (H&E) -stained paraffin sections of mammary gland under bright field (left panel) and epifluorescent (right panel) illumination. Lobular ductules (arrows) and the number of red blood cell-containing capillaries (arrowhead) were assessed for each lobule, and the number of lobular capillaries was normalized per lobular ductule. Magnification bar represents 20 mm. Asterisks indicate time points where the entire lobule was too large to be imaged in one field, and so data are normalized per field. B: Bars represent average number of ductules either per lobule, or per field at time points when the lobular size exceeded a microscopic field of view (bars with asterisk). C: Capillary number normalized per lobular ductule to remove effects of total lobule size. Capillary number was highest throughout lactation and until D2 involution, significantly decreased at days 4 and 12, and thereafter returned to resting levels at day 90. Statistical analysis was performed using All Pairwise Multiple Comparison Procedures (Dunn’s Method), which sets significance as P < 0.05. Virgin, pregnancy, and parous (d90 involution) time points were not significantly different. Normalized lobular vascularization at day 1 of lactation was significantly greater than both virgin time points, pregnancy time points except day 7, and greater than day 90 involution glands. Day 5–7 lactation was significantly greater than all virgin and pregnancy time points, as well as day 90 involution. Involution at 12 hr, day 2, day 4, and day 12 postweaning were significantly different from all other time points except lactation. Day 12 involution was significantly different from virgin day 50, pregnancy day 2, and day 18–19, and involution day 90; the lobular vascularization of the mammary gland at day 90 postinvolution (i.e., glands from parous rats) was not significantly different from the mammary glands of nulliparous virgin rats. Bars indicate the mean±SEM.

Fig. 4

Fig. 4

A–D: Effect of estrous stage on mast cells associated with (A) ductal stroma, (B) terminal ductule lobular unit, (C) number of terminal ductules per lobule, and (D) mast cells per lobular ductule. A: No significant difference in mast cell number was seen associated with ductal stroma. B: In contrast, mast cells associated with lobules were significantly increased during diestrus II, compared with metestrus and diestrus I (P < 0.05). C: The number of terminal ductules per lobule was assessed, and dropped significantly during diestrus II, compared with all other estrous stages (P < 0.05). D: The number of mast cells normalized per lobular ductule for each field demonstrated that mast cells were significantly greatest in number during diestrus II, compared with all other estrous stages (P < 0.05). Bars indicate mean ± SEM.

Fig. 5

Fig. 5

Quantitative analysis of capillary number associated with lobules in the rat mammary gland throughout the estrous cycle. A: Number of capillaries per entire lobule was greatly increased during metestrus, compared with all other time points (P < 0.05). The capillary number during diestrus II was also significantly less than that during proestrus, estrus, and metestrus (P < 0.05). B: When capillary number was normalized per lobular ductule, capillaries per lobular ductule were significantly increased during metestrus, compared with all other stages; in addition, capillary number during diestrus II was significantly decreased compared with all other time points (P < 0.05). Bars indicate mean ± SEM.

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