Table 1 shows the demographic and brain AVM characteristics of the study cohort. The mean age (years) for AVM patients was 46 ± 15 and 36 ± 13 for control patients (P=0.11). Mean AVM size was 2.2 ± 1.3 cm.
Presence of inflammatory cells in AVM tissues
To investigate whether inflammatory leukocytes are present in brain AVM tissue, we performed immunohistochemistry on sections from frozen AVM tissues using neutrophil marker MPO, macrophage/microglia marker CD68, T lymphocyte marker CD3, and B lymphocyte marker CD20. We examined unruptured tissue specimens in AVM patients that had not undergone pre-surgical embolization, two potential confounders that might increase the level of inflammatory infiltrates in the lesional tissue. Neutrophils were present in the vascular wall as well as in parenchymal tissues of AVM, demonstrating regional infiltration of neutrophils (). Although a majority of MPO signals were detected in the vascular wall and the parenchymal tissues, some of MPO signals were observed in the lumen of the vessel of AVM (brown color indicated by arrowheads) (). These are presumed to be neutrophils that remained in the vascular lumen during resection. There were no MPO positive cells in epilepsy brain samples ().
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 (more ...)
Macrophages, monocytes, and microglia, identified by CD68 staining, were scattered throughout the AVM tissue specimens such as vascular wall and parenchyma (brown color indicated by arrows, ). are representatives from three brain AVM patients, and g is from an epilepsy patient. are photographed under higher power from areas from respectively (indicated by rectangle).
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 (more ...)
To determine whether neutrophil and macrophage were present in the same vascular loci, serial AVM sections (a and e; c and g) were stained with MPO and CD68 markers respectively. As shown in , a majority of neutrophils and macrophages were present in proximity in the same vessels. However, in some cases, neutrophils and macrophages were present at different locations: neutrophils mainly at the inner wall, and macrophages mainly at the outer vascular wall ().
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 (more ...)
To quantitatively assess leukocyte infilatration in brain AVM tissue, we performed cell counting on slides immunostained with MPO, CD68, or CD31. Table 2 shows results of MPO and CD68 signal representing neutrophils and macrophage indices, respectively, in the vascular wall, lumen, and adjacent parenchyma tissue. As shown in , 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 pixels). In addition, the amount of neutrophils and macrophages in brain AVMs was not closely correlated (R2 = 0.18, y = 0.515× + 230, n = 20, P = 0.06). We found no association between the amount of neutrophil or macrophage and avm location, size, or drainage (data not shown).
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 (more ...)
In contrast to the abundant MPO and CD68 signals present in brain AVM tissues, the signals for T cells (CD3) and B cells (CD20) were rare in brain AVM tissues (data not shown).
Correlation of neutrophils with MPO and MMP-9 levels
To determine whether neutrophils contribute to MMP-9 production in brain AVM tissue, we performed double immunofluorescent staining of MPO and MMP-9 on AVM tissue sections. As represented in , majority of MMP-9 signal (a, green) and MPO signal (b, red) in brain AVM tissue co-localized (c, yellow), indicating that a major source of MMP-9 in AVM tissues was from neutrophils.
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
To validate the use of MPO as a marker for quantitative assessment of neutrophils, we measured MPO and MMP levels in neutrophils isolated from patients with brain AVMs. There was a linear relationship among neutrophil counts and MPO and MMP-9 levels (). Neutrophil count was linearly correlated with MPO and MMP-9 (R2=0.99, R2=0.98). MPO level, in turn, correlated with MMP-9 levels (, R2=0.99). The results indicate that MPO level could be used as an index for assessing the amount of neutrophils, and imply that more neutrophils present in brain avm tissues could be associated with higher MPO and MMP-9 levels.
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 (more ...)
Contribution of the blood pool to tissue measurement of MMP-9
We have previously reported that brain AVM tissues displayed higher levels of inflammatory markers compared to control epilepsy tissues by ELISA (6
). Brain AVM tissues dissected in surgery are heterogeneous, and contain varying amount of blood, both intravascular and extravascular. To assess the amount of blood present in AVM tissues, and the contribution of blood source MMP-9 on measurements of total MMP-9 levels, we measured the amount of hemoglobin in brain AVM tissues and used it as an index for the amount of blood and compared it with MMP-9 tissue levels. MMP-9 level was correlated with Hb (). However, the blood pool, as reflected by Hb content, contributed to only 6% of the variation in the relationship (R2
= 0.06, y = 0.048× + 9.79, n = 77, P = 0.04), suggesting that blood-borne MMP-9 only partially accounted for the amount of total measured MMP-9 activity in surgical brain AVM specimens.
Correlation of MMP-9 with hemoglobin in brain AVM tissue. R2 = 0.057, y = 0.048x + 9.79, n = 77, P = 0.04
To further examine the potential confounding effects of retained blood cells in AVM tissues on MMP-9 tissue levels, we injected blood at varying hematocrits into the mouse brain and measured MMP-9 tissue levels. shows the site of blood infusion and tissue sampling. Blood injection increased MMP-9 level by 22% to 37% (100% blood 137 ± 11 vs
50% blood 131 ± 1.3 vs
25% blood 122 ± 1.8 vs
normal saline 100 ± 10). MMP-2 level increased in a parallel fashion as MMP-9 (). The results indicate that about 78% to 63% MMP-9 level came from parenchyma tissue, and suggest that even large amounts of extravascular blood could not account for the large increases in MMP-9 level that we have previously described (6
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 (more ...)