Histomorphometry of mast cells in the convexity of human intracranial dura mater

Abstract

Mast cells, known as pro-inflammatory effector cells, are immunocytes present in the meninges and may be involved in the pathophysiology of migraine. This study aims to evaluate the histomorphometric parameters of mast cells located in the convexity of the human intracranial dura mater. For this, samples of intracranial dura mater from eight human fresh cadavers were collected between 8- and 24-h post-mortem. The whole samples were fixed and, subsequently, two fragments of 1.5 cm² each were cut from four different areas of the dura mater convexity, containing a segment of the middle meningeal artery, totaling 64 fragments. After histological processing, the fragments were submitted to microtomy (5 and 10 µm), stained with toluidine blue (0.1%), or immunohistochemically labeled for tryptase, and analyzed using optical microscopy. The following histomorphometric parameters were evaluated: distance from mast cells to vessels, the density of mast cells, and percentage of mast cells with degranulation. Histomorphometric analyzes showed a higher density of mast cells in the vicinity of blood vessels (arterial and venous), with distances around 0-150 µm. A greater number of mast cells was detected near venous vessels in the periosteal layer (17.0 ± 10.1 cells/mm²) than in the meningeal layer (14.1 ± 7.0 cells/mm²) (p < 0.05). Mast cells from the region close to the superior sagittal sinus were found in greater quantity close to the venous vessels (16.7 ± 10.1 cells/mm²) than to the arterial vessels (11.2 ± 7.5 cells/mm²) (p < 0.05). In short, in the convexity of the human intracranial dura mater, mast cells are located close to blood vessels, with a greater number of cells next to the venous vessels of the periosteal layer and in the proximal region of the superior sagittal sinus.

Keywords: cadaver; dura mater; mast cells; meningeal arteries; meninges; microscopy; migraine disorders; morphology; superior sagittal sinus.

FIGURE 1

Location of the areas analyzed by histomorphometry of mast cells in human intracranial dura mater. (a) Superior view of human intracranial dura mater with right and left middle meningeal arteries, superior sagittal sinus (*asterisk), anterior part (a), posterior part (p) after fixation. (1) Region distal to the superior sagittal sinus and periosteal layer; (3) Region proximal to the superior sagittal sinus and periosteal layer; (b) Inferior view of the intracranial dura mater with the superior sagittal sinus (*asterisk) after fixation. (2) Region distal to the superior sagittal sinus and meningeal layer; (4) Region proximal to the superior sagittal sinus and meningeal layer

FIGURE 2

Schematic summary of histomorphometric analyses. (a) Photomicrographs of random areas with mast cells and dural vessels (arterial and/or venous) of magnification 100×, stained with toluidine blue (0.1%), were stored in the Motic Images Plus program (2.0) Software. (b) Mast cells were identified and enumerated at 400× magnification. Measurements of distances from mast cells to venous and/or arterial vessels were performed with the ImageJ^® 1.52a Software (2019). Each image was calibrated from the standard scale (100 µm) and later measurements were taken of the distance (yellow line) from the mast cell membrane to the arterial vessel (tunica media of the middle meningeal artery and/or descending arteriole) and/or venous vessel (tunica media of veins and/or endothelial cells of venules). Data from distance measurements and quantification of mast cells were entered into Windows Excel spreadsheets.

FIGURE 3

Photomicrographs of degranulated (two black arrows) or granulated (one black arrow) mast cells immunohistochemically labeled for tryptase near blood vessels in the convexity of human intracranial dura mater. (a) Region distal to the superior sagittal sinus and the periosteal layer, with the presence of six mast cells (2 granulated; 4 degranulated); (b) Region proximal to the sagittal sinus and the periosteal layer, with the presence of one degranulated mast cell; (c) Region distal to the superior sagittal sinus and of the meningeal layer, with presence of three degranulated mast cells; (d) Region proximal to the superior sagittal sinus and of the meningeal layer, the presence of two mast cells (1 degranulated; 1 granulated). Scale bar 10 µm

FIGURE 4

Photomicrographs of degranulated (two black arrows) or granulated (one black arrow) mast cells stained with toluidine blue (0.1%) near the arterial vessels (VA) in the convexity of the human intracranial dura mater. (a) Region distal to the superior sagittal sinus and the periosteal layer, with the presence of three granulated mast cells; (b) Region proximal to the superior sagittal sinus and the periosteal layer, with the presence of two mast cells (one degranulated and one granulated); (c) Region distal to the sagittal sinus and of the meningeal layer, with the presence of five mast cells (one granulated and four degranulated); (d) Region proximal to the superior sagittal sinus and of the meningeal layer, with the presence of one degranulated mast cell. Scale bar 10 µm

FIGURE 5

Photomicrographs of degranulated (two black arrows) or granulated (one black arrow) mast cells stained with toluidine blue (0.1%) near venous vessels (VV) in the convexity of human intracranial dura mater. (a) Region distal to the superior sagittal sinus and the periosteal layer, with the presence of two mast cells (1 granulated; 1 degranulated); (b) Region proximal to the superior sagittal sinus and the periosteal layer, with the presence of five mast cells (1 granulated; 4 degranulated); (c) Region distal to the sagittal sinus and meningeal layer, with the presence of seven mast cells (1 degranulated; 6 granulated); (d) Region proximal to the superior sagittal sinus and meningeal layer, presence of five mast cells (2 degranulated; 3 granulated). Scale bar 10 µm

FIGURE 6

Photomicrograph of degranulated mast cell. (a) Degranulated mast cell (black arrow) near blood vessel (BV). (b) Degranulated mast cell (MC) with greater magnification, showing the released granules. Toluidine blue stain (0.1%), 400x magnification