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Wu et al. Mast cells and vein graft remodelling

the donor mouse was a male mouse of the same graft, serial sections of 5 µm thickness were cut from
genotype. In the recipient mouse, the right common 5 evenly divided regions from the proximal to the
carotid artery was prepared by isolating it, tying two distal end. In all cases, 5 slides (1 slide from each
sutures around the middle and cutting. A nylon cuff region) were stained as outlined below and the values
was then sleeved onto the distal arterial end. In order averaged for each vein graft. The advantage of this
to graft the vein onto artery rather than nylon cuff, the method is that the average value gives an indication
artery was everted back over the cuff and ligated using of the value from the whole of the graft rather than one
8/0 silk suture (Ethicon, Livingston, UK). The proximal discrete area which may not be representative of the
end of the carotid artery was prepared in an identical graft as a whole. For general and gross morphology
fashion. The vena cava to be grafted was then sleeved and for planimetry studies, haematoxylin and eosin
onto each arterial end in turn and tied into position with staining was used and the slides were photographed
8/0 suture. Twenty-eight days after grafting, mice were and analysed using Image pro plus software (Media
euthanised by a rising concentration of CO . The neck Cybernetics, Marlow, UK). Collagen was stained by
2
was opened and the vein graft removed and placed in picrosirius red and visualised under polarised light.
physiological bathing solution. As with other stains used, at least 5 slides were used
from the length of the graft to give a mean intensity
Perivascular mast cell reconstitution of the total fluorescent signal and this value was used
In a previous study from our laboratory, we established for comparison of collagen content between groups.
a reliable method for reconstituting mast cells locally Elastin was identified by Verhoeff Van Gieson staining
to the perivascular region. The advantage of this and mean intensity of light absorption of the bluish
method is that it re-establishes a local mast cell dark stain within the neointima was quantified. To avoid
reconstitution in the Kit W-sh/W-sh mice without mast cells batch-to-batch variation, for each staining protocol, all
being present elsewhere. Thus, this method allows the vein graft samples were stained at one time. For
[14]
the role of perivascular mast cells to be investigated immunostaining, slides were de-waxed and pressure-
without the consequences of systemic mast cell cooked in citrate buffer to retrieve antigens of interest.
reconstitution, which can include ectopic mast cell To study proliferation in vein grafts, one slide per vein
accumulation and abnormal distribution of mast cells graft was stained using an antibody to the marker Ki67
in target organs/tissues. [10,14] Briefly, C57BL/6J mice (rabbit anti-Ki67 antibody, Abcam). The percentage cell
were used as the source of bone marrow cells and proliferation was presented as the ratio of Ki67 positive
these were cultured with mast cell-differentiating nuclei over total nuclei in the wall of the vein graft.
media (containing murine IL-3 and stem cell factor;
PeproTech, New Jersey, USA) for 4 weeks as we Statistics
have described previously. To confirm that the In all experiments, data are presented as mean
[14]
cultured cells were in fact differentiated into mast ± standard error of the mean where n refers to the
cells, flow cytometry with anti-c-Kit and anti-FcεRI number of mice. To make comparisons between two
antibodies (eBioscience, Hatfield, UK) was used. To groups an unpaired Student’s t-test was used as long
[14]
investigate the impact of local reconstitution of mast as the data had a normal distribution and, for data
cells on neointima formation within the vein graft, the not normally distributed, the non-parametric Mann
perivascular area of the right common carotid artery Whitney test was used. To compare data from multiple
was injected with the bone marrow-derived mast cells groups a one-way analysis of variance (ANOVA)
(BMMCs) as follows. Briefly, the recipient Kit W-sh/W-sh with Tukey’s post hoc test was used as appropriate.
mouse was anaesthetised with sodium pentobarbital Statistical analyses were performed using Graph
as previously described and the common right carotid Pad Prism v6.04. In all comparisons, a significant
artery was exposed. BMMCs were injected around the difference was assumed when P < 0.05.
artery (1 million cells per mouse). A suture of size 6/0
was used to close the wound and the animal was kept RESULTS
for 4 weeks to allow repopulation of the perivascular
area with a mast cell population similar to that seen Mast cells promote elastin but not collagen
in wild type (C57BL/6) mice. Vein graft surgery was deposition in vein grafts by 4 weeks
[14]
performed 4 weeks after mast cell reconstitution. Significant collagen deposition was identified in all
vein grafts at 4 weeks. The majority of collagen was
Histology and immunostaining present in the neo-adventitia and perivascular area
Vein grafts were perfusion-fixed to maintain graft [Figure 1A]. In contrast, elastin was only detected in
patency and then stored overnight in 10% formalin the neointima [Figure 1B and C]. Mast cell deficiency
before being embedded in paraffin. For each vein (Kit W-sh/W-sh ) had no impact on collagen deposition,
Vessel Plus ¦ Volume 1 ¦ September 26, 2017 139