Evidence of inflammatory cell involvement in brain arteriovenous malformations

Abstract

Objective: Brain arteriovenous malformations (AVM) have high matrix metalloproteinase-9, interleukin-6, and myeloperoxidase (MPO) expression, and polymorphic variations in inflammatory genes are associated with an increased risk of hemorrhage. In this study, we characterized the presence of inflammatory cells in AVM lesional tissue specimens.

Methods: Immunohistochemistry was used to identify and localize neutrophils (MPO as marker), macrophages/microglia (CD68 as marker), T lymphocytes (CD3 as marker), and B lymphocytes (CD20 as marker). Endothelial cell (EC) marker CD31 was used as an index to assess vascular mass (EC mass). Surgical specimens from 20 unruptured, nonembolized AVMs were examined; seven cortical samples from temporal lobectomy were used as controls. Positive signals for inflammatory cell markers were counted and analyzed by normalizing to the area of the tissue section and the amount of endothelial cells (cells/mm/EC mass pixels). Levels of MPO and matrix metalloproteinase 9 were determined by enzyme-linked immunosorbent assay.

Results: Neutrophils and macrophages are all frequently identified in the vascular wall of AVM tissue. In contrast, T and B lymphocytes are rarely observed in AVM tissue. AVM tissue displayed more neutrophil and macrophage/microglia markers than epilepsy control tissue (MPO: 434 +/- 333 versus 5 +/- 4, P = 0.0001; CD68: 454 +/- 404 versus 4 +/- 2, P = 0.0001; cells/mm/EC mass pixels). In ex vivo studies, neutrophil quantity, MPO, and matrix metalloproteinase-9 levels were all colinear (R = 0.98-0.99).

Conclusion: Our study demonstrates that inflammatory cells are present in AVM tissue. Taken together with previous genetic and cytokine studies, these data are consistent with a novel view that inflammation is associated with AVM disease progression and rupture.

Figure 1

Immunohistochemical staining of neutrophil marker MPO on sections from un-ruptured and non-embolized brain AVM tissue (figure a–f) and epilepsy (figure g–h) control tissue. Brown color, indicated by arrows, is positive MPO signal, and blue color is the counter staining with hematoxylin of nuclei. Figure a showed that neutrophils were present in the vascular wall and lumen of AVMs. Figure b was photographed under higher power from selected area of the figure a, indicted by rectangle. Figure c showed neutrophils were present in the vascular wall as well as parenchymal tissue of AVMs, and figure d was photographed under higher power from selected area of c, indicted by rectangle. Figure e showed that neutrophils were predominantly present in the vascular wall, and image f showed selected area from e under higher magnification. Neutrophils were absent in cortical tissue of epilepsy patient (figure g and h). Representative images from 3 AVM patients and an epilepsy patient; size bar for a, c, e, and g: 100μm; size bar for b, d, f, and h: 25μm

Figure 2

Immunohistochemical staining of macrophage/monocyte marker CD68 on sections from un-ruptured and non-embolized brain AVM tissue and epilepsy control tissue. Brown color, shown by arrows, indicates positive CD68 immunoreactivity. Cell nuclei appear blue with hematoxylin counter staining. Figure a showed that macrophages were mainly present in the parenchymal tissue adjacent to the vascular wall, and figure b showed higher magnification of rectangle area from a. Figure c showed that macrophages predominantly reside in the vascular wall and figure d showed higher magnification of rectangle area of c. Similar to figure a, figure e showed that macrophages were mainly present in the parenchymal tissue adjacent to the vascular wall. Figure f was photographed under higher power from selected area, indicted by rectangle, of e. Weak CD68 positive signals were also observed in the cortical tissue from epilepsy patients (figure g and h). Representative images from 3 AVM patients an epilepsy patient; size bar for a, c, e, and g: 100μm; size bar for b, d, f, and h: 25μm

Figure 3

Co-localization of MPO and CD68 signal. Adjacent AVM tissue sections were stained with MPO, or CD68 marker. Brown color, shown by arrows, indicates positive MPO, or CD68 immunoreactivity. Cell nuclei appear blue with hematoxylin counter staining. Figure a and e showed that neutrophils and macrophages were present in proximity at the same vessels. Figure b and f were photographed under higher power from selected area, indicted by rectangle, of a, e respectively. Figure c and g showed that neutrophils and macrophages were present at different locations: neutrophils were mainly present at the inner wall; macrophages were mainly located at the outer vascular wall. Figure d and h were photographed under higher power from selected area, indicted by rectangle, of c, g respectively. Size bar for a, c, e, and g: 50μm; size bar for b, d, f, and h: 50μm

Figure 4

Comparison of the amount of neutrophil and macrophage between brain AVM patients and epilepsy patients. Cell counting was performed on the slides immunostained with MPO, or CD68. Cell counts were from vascular wall and parenchyma, and normalized by tissue section area (mm²) and the amount of endothelial cells (CD31, pixel). Brain AVM tissues had more MPO and CD68 than the cortical tissues from epilepsy patients (MPO: 434 ± 333 vs 5 ± 4, P=0.0001; CD68: 454 ± 404 vs 4 ± 2, P=0.0001; cells/mm²/EC mass pixel).

Figure 5

Double staining of MMP-9 (a, green color) and MPO (b, red color) in brain AVM tissue. Yellow color in c indicates co-localization of MMP-9 with MPO. size bar: 50μm

Figure 6

A. Correlation of neutrophil number with MPO level. R² = 0.99, y = 1.85 – 0.37x, n = 4, P<0.01. B. Correlation of neutrophil number with MMP-9 level. R² = 0.98, y = 0.25 – 0.05x, n = 4, P<0.01. C. Correlation of MMP-9 with MPO. R² = 0.99, y = 0.14x – 0.02, n = 4, P < 0.01. Neutrophils were isolated from the peripheral blood of four AVM patients at the density of 2.5 ×10⁵. Three serial dilution of each sample was: 1.25×10⁵, 0.625 ×10⁵, 0.312 ×10⁵.

Figure 7

Correlation of MMP-9 with hemoglobin in brain AVM tissue. R² = 0.057, y = 0.048x + 9.79, n = 77, P = 0.04

Figure 8

A. coronal section of mouse brain showing the site of blood infusion and tissue sampling. Arrow indicates the position of blood (in red) infusion into striatum of the mouse brain. Yellow circled area represents the area of tissue sampling for assessing the level of MMPs. B. MMP-9 and MMP-2 level assessed by zymography in mice brain 3hr following whole blood injection. N= 3 for each treatment group. S: MMP standards, 1: 100% of original hematocrit, 2: 50% of original hematocrit, 3: 25% of original hematocrit, 4: normal saline. C. Quantitation (Optical density) of MMP-9 and MMP-2 levels.