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, 1294, 183-92

Bone Marrow-Derived Cells Are the Major Source of MMP-9 Contributing to Blood-Brain Barrier Dysfunction and Infarct Formation After Ischemic Stroke in Mice

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Bone Marrow-Derived Cells Are the Major Source of MMP-9 Contributing to Blood-Brain Barrier Dysfunction and Infarct Formation After Ischemic Stroke in Mice

Guangming Wang et al. Brain Res.

Abstract

Matrix metalloproteinase (MMP)-9 has been shown to contribute to blood-brain barrier (BBB) disruption, infarct formation, and hemorrhagic transformation after ischemic stroke. The cellular source of MMP-9 detectable in the ischemic brain remains controversial since extracellular molecules in the brain may be derived from blood. We here demonstrate that bone marrow-derived cells are the major source of MMP-9 in the ischemic brain. We made bone marrow chimeric mice with MMP-9 null and wild-type as donor and recipient. After 90 min of transient focal cerebral ischemia, MMP-9 null mice receiving wild-type bone marrow showed comparable outcomes to wild-type in brain MMP-9 levels and BBB disruption (endogenous albumin extravasation) at 1 h post-reperfusion and infarct size at 24 h post-reperfusion. In contrast, wild-type animals replaced with MMP-9 null bone marrow showed barely detectable levels of MMP-9 in the ischemic brain, with attenuations in BBB disruption and infarct size. MMP-9 null mice receiving wild-type bone marrow showed enhanced Evans blue extravasation as early as 1 h post-reperfusion compared to wild-type mice replaced with MMP-9 null bone marrow. These findings suggest that MMP-9 released from bone marrow-derived cells influences the progression of BBB disruption in the ischemic brain.

Figures

Fig. 1
Fig. 1
Four types of chimeric mice after irradiation (9 Gy) and bone marrow transplantation with various combinations of wild-type (WT) and MMP-9 knockout (KO) as donor and recipient.
Fig. 2
Fig. 2
Zymograms showing gelatinase activity in brain (A), and spleen (B) from mice subjected to 2 h of MCAO followed by 22 h of reperfusion. Compared with irradiation to the head only, whole body irradiation markedly reduced MMP-9 activity in the spleen. Note remarkable reductions in MMP-9 activity levels in the brain and spleen from 9 Gy whole body irradiated mice. In contrast, the lower band presumably reflecting MMP-2 activity was not affected by irradiation. Mice were irradiated 7 days before brain ischemia/reperfusion. (A) Ischemic hemisphere from a non-irradiated mouse subjected only to 2 h MCAO with 22 h reperfusion was loaded on the second lane. C, contralateral hemisphere; I, ischemic hemisphere. B, Two cases are shown in each group.
Fig. 3
Fig. 3
Immunocytochemical detection of MMP-9 in nucleated blood cells from chimeric mice. Nucleated blood cells were counter stained with methyl green. Blood cells from WT/KO (C) are MMP-9 positive as seen in WT (A) whereas blood cells from KO (B) and KO/WT (D) are negative for MMP-9 immunostaining. Note that, in addition to cellular staining, there is strong extracellular MMP-9 immunostaining surrounding methyl green stained cells (A, C). Bar = 20 μm.
Fig. 4
Fig. 4
Gel zymograms (A–C) and Western blot (D) showing MMP-9 in spleen and brain in mice that were subjected to 90 min MCAO followed by either 1 h (A and B) or 24 h (C and D) of reperfusion. Tissue homogenates were treated with gelatin Sepharose 4B and the isolated gelatinases were subjected to zymography or Western blot analysis. C, contralateral hemisphere; I, ischemic hemisphere; hMMP, human recombinant MMP; MW, molecular weight marker.
Fig. 5
Fig. 5
Fluorescent photomicrograph showing in situ gelatinase activity in four types of chimeric animal. A and B, Radiation and bone marrow transplantation from WT to MMP-9 KO mice (B) restored gelatinase activity (green) comparable to WT/WT (A) in the brain after 90 min MCAO with 24 h reperfusion. Double staining with NeuN immunostaining shows that gelatinase activity is primarily associated with neuronal nuclei (orange in the inlets). In situ gelatinase activity was barely detectable in WT mice replaced with KO bone marrow (C) as seen in KO/KO (D). E–G, Confocal laser scanning image showing double labeling of in situ gelatinase activity (E) and MMP-9 immunostaining (F) in WT/KO. Gelatinase activity (green) was also associated with microvasculature (E) while MMP-9 immunostaining (red) was detected in leukocyte-like cells and microvasculature. G, There was association of gelatinase activity and MMP-9 immunostaining in microvasculature. Scale bar = 20 μm.
Fig. 6
Fig. 6
MMP-9 from BMDC contributes to BBB dysfunction and infarct formation. Graphs showing relative protein levels of albumin in the brain (A, B, and C), Evans blue extravasation (D), and infarct size (E, F). The brains were collected from either naïve mice (A) or animals that were subjected to 90 min MCAO followed by 1 h (B, D), 18 h (C) or 24 h (E, F) of reperfusion. A–C, The ratio of Western blot density (albumin/β-actin) was obtained from each brain hemisphere to measure BBB dysfunction. D, In separate animals, Evans blue extravasation corrected for Evans blue blood concentration was measured in each brain hemisphere to evaluate BBB dysfunction. Sham-operated non-ischemic animals injected with Evans blue were used as control. E and F, Infarct area was determined using eight cresyl violet stained coronal brain sections with intervals of 1 mm each and infarct volume was calculated and expressed as direct infarct size (E) or indirect infarct size corrected for brain swelling (F). Cont, contralateral hemisphere; Isch, ischemic hemisphere. Horizontal bars represent mean value. Statistical analysis was made by Mann-Whiteny test (A) or Kruskal-Wallis test followed by Mann-Whiteny test for the comparisons between two groups (B–F). **, p < 0.01 compared with the counterpart in WT (C) or WT/KO (D). The indicated numbers in (E) represent p value between two groups (Mann-Whiteny test). #, p < 0.05 (Mann-Whiteny test) compared with other three groups.

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