Quantitative micro CT analysis of bone vasculature.

<p>Quantitative micro CT analysis of bone vasculature.</p

Macroscopic evaluation of bone repair over time.

<p>2D micro CT images (A-H) were compared with 5 μm histological sections of un-decalcified bone stained with von Kossa and toluidine blue (VK/TB) to distinguish mineralised (black) from un-mineralized tissue (blue). Representative mid-sagittal images show shards of old bone (A, E arrows) remaining in the defect at 5d PO and significant new bone at 14d PO in the medullary canal and on the periosteal surface opposite the defect in both <i>WT</i> (B) and <i>Cpa3</i>^<i>Cre/+</i> (F) femora. At 28 days PO, the defect is bridged with primary bone in many <i>WT</i> (C), but not <i>Cpa3</i>^<i>Cre/+</i> (G asterix) mice. By 56d PO the majority of <i>WT</i> femora have assumed their pre-operative anatomy (D), whereas most of those from <i>Cpa3</i>^<i>Cre/+</i> mice exhibit mal-union on the defect side (H asterix) and large channels separating old from new bone on the contralateral cortex (H arrows). Images are representative of N = 7 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 5d PO; N = 16 <i>WT</i> and N = 11 <i>Cpa3</i>^<i>Cre/+</i> at 14d PO; N = 8 <i>WT</i> and N = 10 <i>Cpa3</i>^<i>Cre/+</i> at 28d PO and N = 6 <i>WT</i> and N = 8 <i>Cpa3</i>^<i>Cre/+</i> at 56d PO.</p

CD34 immunohistochemistry in regenerating bone.

<p>Bones were decalcified, embedded in paraffin and 5μm sections stained immunochemically for CD34 expression. Representative images of the defect, medulla and contralateral cortex show robust staining of cells lining vessels at 5d PO in <i>WT</i> bone compared with weak, disorganised staining in <i>Cpa3</i>^<i>Cre/+</i> bone. By 28 days PO CD34 immunoreactivity is restricted primarily to the periosteum in <i>WT</i> bone, but persists in the remaining fibrous tissue in <i>Cpa3</i>^<i>Cre/+</i> bone (asterix). Images are representative of N = 7 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 5d PO; N = 11 <i>WT</i> and N = 9 <i>Cpa3</i>^<i>Cre/+</i> at 14d PO and N = 8 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 28d PO.</p

Histochemical analysis of bone in contralateral cortex.

<p>Representative images of 5 μm sections of von Kossa stained un-decalcified bone (A-H) were compared with 5μm sections of decalcified bone stained with ALP. A thick, fibrous periosteum is apparent in <i>Cpa3</i>^<i>Cre/+</i> bones (E, G asterix) in the absence of any significant difference in ALP activity. Bone formation with large osteoblasts adjacent to osteoid is apparent at 14d PO in <i>WT</i> (B) and <i>Cpa3</i>^<i>Cre/+</i> (F) bones, accompanied by high ALP activity. Active bone formation is sustained at 28d (G) and 56d (H) PO in <i>Cpa3</i>^<i>Cre/+</i> mice, but is less apparent in <i>WT</i> mice (C, D). Images are representative of N = 7 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 5d PO; N = 16 <i>WT</i> and N = 11 <i>Cpa3</i>^<i>Cre/+</i> at 14d PO; N = 8 <i>WT</i> and N = 10 <i>Cpa3</i>^<i>Cre/+</i> at 28d PO and N = 6 <i>WT</i> and N = 8 <i>Cpa3</i>^<i>Cre/+</i> at 56d PO.</p

Mast Cells Play No Role in the Pathogenesis of Postoperative Ileus Induced by Intestinal Manipulation

<div><p>Introduction</p><p>Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, <i>Cpa3^Cre/+</i>, devoid of mast cells but with intact Kit signaling.</p><p>Design</p><p>The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. <i>Kit^W-sh/W-sh</i> and <i>Cpa3^Cre/+</i> mice, and by use of the mast cell stabilizer cromolyn.</p><p>Results</p><p><i>Kit^W-sh/W-sh</i> mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, <i>Cpa3^Cre/+</i> mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in <i>Cpa3^Cre/+</i> mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (<i>Cpa3^+/+</i>). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI.</p><p>Conclusions</p><p>Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.</p></div

Defective bone repair in mast cell-deficient <i>Cpa3^Cre/+</i> mice

<div><p>In the adult skeleton, cells of the immune system interact with those of the skeleton during all phases of bone repair to influence the outcome. Mast cells are immune cells best known for their pathologic role in allergy, and may be involved in chronic inflammatory and fibrotic disorders. Potential roles for mast cells in tissue homeostasis, vascularization and repair remain enigmatic. Previous studies in combined mast cell- and Kit-deficient <i>Kit</i>^{<i>W-sh/W-sh</i>} mice <i>(Kit</i>^<i>W-sh</i>) implicated mast cells in bone repair but <i>Kit</i>^<i>W-sh</i> mice suffer from additional Kit-dependent hematopoietic and non- hematopoietic deficiencies that could have confounded the outcome. The goal of the current study was to compare bone repair in normal wild type (<i>WT</i>) and <i>Cpa3</i>^<i>Cre/+</i> mice, which lack mast cells in the absence of any other hematopoietic or non- hematopoietic deficiencies. Repair of a femoral window defect was characterized using micro CT imaging and histological analyses from the early inflammatory phase, through soft and hard callus formation, and finally the remodeling phase. The data indicate 1) mast cells appear in healing bone of <i>WT</i> mice but not <i>Cpa3</i>^<i>Cre/+</i> mice, beginning 14 days after surgery; 2) re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organised in <i>Cpa3</i>^<i>Cre/+</i> mice compared with <i>WT</i> mice; 3) the defects in <i>Cpa3</i>^<i>Cre/+</i> mice were associated with little change in anabolic activity and biphasic alterations in osteoclast and macrophage activity. The outcome at 56 days postoperative was complete bridging of the defect in most <i>WT</i> mice and fibrous mal-union in most <i>Cpa3</i>^<i>Cre/+</i> mice. The results indicate that mast cells promote bone healing, possibly by recruiting vascular endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair.</p></div

Quantitative staining analysis of cellular activity.

<p>Quantitative staining analysis of cellular activity.</p

Identification of osteoclasts and macrophages in regenerating bone.

<p>5 μm sections of decalcified bone were stained with tartrate resistant acid phosphatase (TRAP) or immunochemically with the macrophage marker F4/80. Representative images show more TRAP activity in <i>WT</i> than in <i>Cpa3</i>^<i>Cre/+</i> bones at 5d (A vs B) and 14d (C vs D) PO, and less at 28d (E vs F) PO. F4/80 positive macrophages were seen in condensed mesenchyme filling the defect/medulla at 5d PO in both <i>WT</i> (A1) and <i>Cpa3</i>^<i>Cre/+</i> (B1) bones. In <i>WT</i> bone, F4/80 positive cells can be seen lining vessels at 14d (C1) PO and scattered throughout bone marrow at 28d (E1) PO, whereas they were embedded in fibrous tissue in <i>Cpa3</i>^<i>Cre/+</i> bone (F1). Images are representative of N = 7 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 5d PO; N = 16 <i>WT</i> and N = 11 <i>Cpa3</i>^<i>Cre/+</i> at 14d PO; N = 8 <i>WT</i> and N = 10 <i>Cpa3</i>^<i>Cre/+</i> at 28d PO and N = 6 <i>WT</i> and N = 8 <i>Cpa3</i>^<i>Cre/+</i> at 56d PO.</p

Increased resistance to S. <i>ratti</i> infection in Treg-depleted BALB/c DEREG but not in C57BL/6 DEREG mice.

<p>BALB/c (white bars), BALB/c DEREG (black bars), C57BL/6 (light grey bars), and C57BL/6 DEREG (dark grey bars) mice were treated with DT and infected s.c. with 2000 <i>S. ratti</i> iL3. <b>A:</b> Experimental setup is shown. <b>B:</b> Number of parasitic adults in the small intestine was counted on day 6 p.i. Shown are the combined results of two independent experiments (n = 10). Numbers show difference of the mean analyzed by students <i>t</i> test. <b>CD:</b> release of <i>S. ratti</i>-derived DNA in the feces over 24 h was quantified at the indicated time points in DT treated BALB/c and BALB/c DEREG (<b>C</b>) or C57BL/6 and C57BL/6 DEREG (<b>D</b>). Shown are the means of 5 mice per time point and group. This result is representative for two independent experiments. Asterisks show significant difference of the mean analyzed by students <i>t</i> test (* p≤0.05). <b>E:</b> MMCP-1 in the serum of infected mice was quantified at the indicated time points. Shown are the combined results of three independent experiments (n = 10 for day 7; n = 9 for days 6 and 3; n = 8 for day 5). Asterisks indicate significant difference of the mean analyzed by one-way ANOVA with Bonferroni post test (* p≤0.05, *** p≤0.001).</p

Role of mast cells during <i>S. ratti i</i>nfection in Treg-depleted BALB/c mice.

<p><b>AB:</b> BALB/c Cpa3^WT (white bars), BALB/c DEREG Cpa3^WT (black bars), BALB/c Cpa3^CRE (light green bars) and BALB/c DEREG Cpa3^CRE (dark green bars) mice were treated with DT on three consecutive days starting one day before s.c infection with 2000 <i>S. ratti</i> iL3. <b>A:</b> Number of parasitic adults in the small intestine was counted on day 6 p.i. Depicted are the combined results of three independent experiments (n≥12) and error bars show SEM. <b>B:</b> Splenocytes were prepared at day 6 p.i. and cultured in the presence of α-CD3 for 72 h. IL-9 concentration in the supernatant was quantified by ELISA. Unstimulated splenocytes did not secrete detectable IL-9. Shown are the combined results of three independent experiments (n≥12). <b>AB:</b> Asterisks indicate significant difference of the mean analyzed by students <i>t</i> test (*** p≤0.001).</p