Brain mast cell relationship to neurovasculature during development

Abstract

Mast cells, derived from the hematopoietic stem cell, are present in the brain from birth. During development, mast cells occur in two locations, namely the pia and the brain parenchyma. The current hypothesis regarding their origin states that brain mast cells (or their precursors) enter the pia and access the thalamus by traveling along the abluminal wall of penetrating blood vessels. The population in the pia reaches a maximum at postnatal (PN) day 11, and declines rapidly thereafter. Chromatin fragmentation suggests that this cell loss is due to apoptosis. In contrast, the thalamic population expands from PN8 to reach adult levels at PN30. Stereological analysis demonstrates that mast cells home to blood vessels. More than 96% of mast cells are inside the blood-brain barrier, with ~90% contacting the blood vessel wall or its extracellular matrix. Mast cells express alpha4 integrins -- a potential mechanism for adhesion to the vascular wall. Despite the steady increase in the volume of microvasculature, at all ages studied, mast cells are preferentially located on large diameter vessels (>16 microm; possibly arteries), and contact only those maturing blood vessels that are ensheathed by astroglial processes. Mast cells not only home to large vessels but also maintain a preferential position at branch points, sites of vessel growth. This observation presents the possibility that mast cells participate in and/or regulate vasculature growth or differentiation. The biochemical and molecular signals that induce mast cell homing in the CNS is an area of active investigation.

Figure 1. Developmental changes in mast cell number

A. Pial mast cell population was 3558 ± 404 at P0, increased to 4958 ± 844 at P8, peaked at 5550 ± 764 at P11, then decreased by 74% to 1466 ± 220 at P15, and reached adult levels of 46 ± 14 at P30. B. Thalamic mast cells first appear at P8 (138 ± 38), increase rapidly between P15 and 21, and reach adult levels by P30 (1424 ± 267 at P30; 1566 ± 267 at P112). C, D. Distribution of mast cells in the pia and thalamus respectively, along the anterior - posterior axis with reference to bregma (n = 5-8 animals/age).

Figure 2

DAPI stained tissue to test for nuclear fragmentation in pial mast cells. (A-C) Pial mast cell with its characteristic pattern of eu- and heterochromatin. D-F. Apoptotic pial mast cell with fragmented chromatin (white arrows). Optical slices (z axis, 1 μm). Scale bar: 5 μm.

Figure 3

Localization of mast cells in relation to the blood vessel wall. Mast cells are labeled with avidin (red) and endothelia are labeled with RECA-1 (white). (A-C) This is an example of a mast cell localized on the brain side of the endothelial cell. (D-F) This mast cell lies in the lumen of the blood vessel. (D-E) Composite image of 7 1-μm serial optical slices. Scale bars; 10 μm

Figure 4

To determine the cellular localization of mast cells relative to the smooth muscle of the blood vessel and its basal lamina, triple-label immunocytochemistry was used. Mast cells are located between the smooth muscle actin positive cell (red) and the laminin-delineated basal lamina (white). (A-D) At PN11 thalamic mast cells (A) are associated with large blood vessels with smooth muscle containing α-SMA (B). Laminin (C) is present in the basal lamina of the smooth muscle and surrounds the mast cell. (D) Overlay of A-C. (E-G) A similar arrangement is shown for PN30 as in P11. Additionally, at this age, laminin is present in neuronal cell bodies (G). Images are composites of 4 (A-D) and 6 (E-H) optical slices (z axis, 1 μm). Scale bars, 20 μm.

Figure 5

Mast cells in the thalamus seen express the integrin α_4. (A-C) Mast cells in the P11 pia are labeled with avidin (A) contain α₄ integrin immunoreactivity (B). (C) Overlay. Image is a composite of 10 1-micron optical sections acquired using confocal microscopy. A similar observation was made in the PN30 thalamus (D-F). Scale bar 20 μm.

Figure 6

Mast cells are associated with blood vessels which have acquired astrocytic endfeet. Triple label immunocytochemistry is used on tissue from P8, P15 and P30 to examine the physical interaction of mast cells and astrocytes. A1-A4 illustrate a large blood vessel (bv) just penetrating of the thalamus and a portion of the glial limitans and pia (indicated by the double headed yellow arrow in A2). Mast cells in the PN8 pia (yellow arrows, A1) are surrounded by GFAP positive processes (A2), These processes comprise much of the pia mater. The blood vessel, in addition to its ensheathment with glial endfeet, is surrounded by several layers of smooth muscle (A3). At P15 (B1-B4) and PN 30 (C1-C4) mast cells are again seen in the thalamus is association with glial endfeet. At higher magnification (Figure 6D and 6E) the GFAP processes cup the mast call. Scale bars A-C= 20μm, D, E=10μm.

Figure 7

The figure shows the frequency with which mast cells contact GFAP-positive processes from PN8-30. The percent mast cells contacted by GFAP-positive processes (circles) remains at about 80% at all ages. The OD of GFAP-positive processes (diamonds) increases with age.

Figure 8

Stacked bar graphs reveal properties of vasculature in relation to localization of mast cells in development. A. The small blood vessels significantly increase in density from P8 to P15 and P30. B. The percent of blood vessels in each size category is shown over development. C. Mast cells are preferentially located on large blood vessels at all ages.

Figure 9

Perivascular mast cells are preferentially located at blood vessel branch points. (A-C) (A) Mast cells at P15 are located at (B) endothelial (RECA-1, white) branch points (arrows). C. Overlay of (A) and (B). Images are composites of 13 optical slices (z axis, 1 μm) acquired using confocal microscopy. Scale bar, 20 μm. (D) Mast cells are preferentially located at blood vessel branch points at PN15 and PN30. Blood vessel density increases significantly from PN8 to PN15 and P30 (Tukey Test, P = 0.008 for PN8 vs. P15, and P = 0.001 for P8 vs P30; n =3 per age group). Branch points compose between 2.4-6.9% of the blood vessel volume from PN8 to PN30, whereas the proportion of mast cells located at branch points increases from 6.4 at PN8 to 36.2 % at PN30. E) The proportion of mast cells at branch points (MC^BP) vs. the proportion of branch points of all blood vessels indicates that mast cells are localized to branch points at P15 and P30. N = 3 animals/group. BP, branch point; MC, mast cells. Scale bar = 30μm