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We recently described a strong association between Table 1: Patient and lesion characteristics
imaging evidence of old silent hemorrhage and Patients Age Sex Size Presentation/
the risk of clinically symptomatic ICH. [21] Further, (years) (cm) clinical details
histological examination demonstrated that the degree 1 38 Female 1
of hemosiderin deposition is positively correlated 2 3 24 Female 3 3
Male
37
with the number of macrophages in the lesion. [21] 4 44 Male 1 Incidental
It is not clear, however, whether the macrophage 5 54 Female 2 Seizure
response is specific or whether other inflammatory 6 7 49 Female 1 1 Seizure
Male
63
cells are also correlated with hemosiderin deposition 8 54 Female 3
and macrophage. Our previous studies demonstrated 9 47 Female 2 Seizure
that both macrophage and neutrophil may play roles in 10 39 Female 4 2
Female
11
53
bAVM pathogenesis. [13‑15] Shi et al. described evidence 12 20 Male 3 Focal deficit
of adaptive immunological responses in cavernous 13 53 Male 4 Incidental
malformation. [22] Although bAVM tissue was used 14 30 Male 3 Headache
as control in Shi’s study and while no oligoclonal 15 49 Female 1 3 Incidental
41
16
Male
Incidental
response was observed, bAVM had a higher polyclonal 17 30 Male 4
response compared to normal brain tissue, suggesting 18 30 Female 3
that lymphocytes may also play a role in bAVM. 19 45 Female 2 Seizure
In this study, we analyzed lymphocytes in addition to activity was quenched by incubating slides in 0.3%
macrophages, and tested the hypothesis that, unlike the H O in phosphate‑buffered saline (PBS) for 15 min.
2
2
innate immune cells (macrophages), adaptive immune After blocking with 10% normal donkey serum,
cell (lymphocytes)‑infiltration is not associated with sections were incubated at 4 °C overnight with primary
micro‑hemorrhage and iron deposition. antibodies diluted in PBS with 1% BSA in the following
concentrations: mouse monoclonal antihuman CD68,
METHODS 1:1000; rabbit monoclonal antihuman CD3, 1:400;
rabbit monoclonal antihuman CD20, 1:200; mouse
All studies involving patients were approved by monoclonal antihuman CD138, 1:800. After washing
the Institutional Review Board of the University of in PBS, the sections were incubated with horseradish
California, San Francisco (UCSF), and patients gave peroxidase labeled antimouse or antirabbit IgG
informed consent. (vector labs) for 1 h at room temperature. The positive
staining was visualized using 3, 3‑diaminobenzidene.
Patients Negative controls were performed by omitting
Patients with AVMs evaluated at UCSF have been the primary antibodies during immunostaining.
entered into an ongoing prospective registry since
2000. [23] We identified 24 unruptured brain AVMs The criteria for identifying hemosiderin were
from patients who did not undergo preoperative birefringent or brownish particles seen in the vascular
embolization or radiosurgery with frozen tissue wall or interstitial tissue between vessels, and were
available in our database; 19 samples were located confirmed by Prussian blue staining on adjacent
and used in this study [Table 1]. Three superficial sections. CD68 , CD3 , CD20 , CD138 cells were
+
+
+
+
temporal arteries (STA), obtained from autopsies of quantified by counting the positively stained cells using
patients who died from nonbrain‑related diseases, were stereological microscopy (Olympus, Japan).
used as control.
Statistical analysis
Histology All data are expressed as mean ± standard deviation
Prussian blue staining was performed using Accustain the differences of means were analyzed using unpaired
Iron Stain kit (Sigma‑Aldrich, St. Louis, MO) according Student’s t‑test. Exact binomial 95% confidence
to the manufacturer’s protocol. intervals (CIs) for proportions are reported. P < 0.05
was considered as statistically significant.
For immunohistochemistry, adjacent sections were
used to stain different surface markers. CD68, CD3, RESULTS
CD20 and CD138‑specific antibodies were purchased
from Abcam (Abcam, Cambridge, MA). Brain AVM Hemosiderin deposition was present in unruptured brain
specimens were embedded in optimum cutting arteriovenous malformations
temperature, sectioned into 8 μm sections, and fixed Consistent with our published data, [21] hemosiderin
with 4% paraformaldehyde. Endogenous peroxidase deposition was found in 8 out of 19 specimens

148 Neuroimmunol Neuroinflammation | Volume 1 | Issue 3 | December 2014