Increased plasma and tissue MMP levels are associated with BCSFB and BBB disruption evident on post-contrast FLAIR after experimental stroke

Abstract

In this study, we examined the relationship between tissue and blood levels of matrix metalloproteinase (MMP)-2 and MMP-9 through gelatin zymography at multiple time points after experimental stroke. We additionally investigated the association between these levels and the evidence of blood-cerebrospinal fluid (CSF) barrier (BCSFB) and blood-brain barrier (BBB) disruption on post-contrast fluid-attenuated inversion-recovery (FLAIR) imaging. Increased plasma MMP-9 was associated with BCSFB disruption at 1h post-reperfusion. Ventricular enhancement ipsilateral to the stroke was 500+/-100%, significantly higher than sham, 24, and 48 h groups. Increased tissue MMP-2 and MMP-9 were associated with BBB disruption at 48 h post-reperfusion. Parenchymal enhancement was 60+/-20% for a volume equivalent to 260+/-80 mm(3). Although the percent enhancement was comparable across groups, the volume of enhancing lesion was significantly higher at 48 h (260+/-80 mm(3), 100%) in comparison to 1 h (8+/-3 mm(3), 3%) and 24 h (51 mm(3), 18%). These findings support the use of imaging markers of BCSFB and BBB status as indirect measures of MMP regulation in the blood and brain tissue. The methods presented herein should be useful in understanding the link between MMPs, barrier integrity, and subsequent hemorrhagic transformation.

Figure 1

Experimental design. Imaging experiments were performed according to the above timeline. Animals were separated into five groups (naive, sham, 1, 24, 48 h groups). Naive animals received no imaging and were strictly used as blood and brain tissue controls. All remaining animals received a baseline scan 30 mins after sham surgery or MCAO. Follow-up imaging was performed immediately after reperfusion (REP) for sham and 1 h groups, at 24 h for the 24 h group, and at 48 h for the 48 h group. Blood and brain tissue samples were taken immediately after final imaging.

Figure 2

FLAIR imaging of barrier disruption. Blood–CSF barrier (BCSFB) and blood–brain barrier (BBB) disruption were quantified through FLAIR MRI with and without Gd-DTPA. The outlined region (dotted line) for the 1 h group represents the ADC lesion at baseline before reperfusion. The outlined regions (solid lines) represent the ADC lesion for the 1 h group and T2 lesion for the 24 and 48 h groups post-reperfusion. Post-Gd FLAIR scans were standardized to be exactly 10 mins after Gd-DTPA administration. The 1 h group showed evidence of BCSFB disruption (large arrows) on post-Gd FLAIR and dFLAIR (difference map, pre-Gd versus post-Gd). Ventricular enhancement ipsilateral to the stroke was 500±100%, significantly higher than sham, 24 (bracket), and 48 h groups (P<0.01). The 48 h group showed evidence of BBB disruption (small arrows) on post-Gd FLAIR and dFLAIR (difference map, pre-Gd versus post-Gd). Parenchymal enhancement present throughout the infarct was 60±20% for a volume equivalent to 260±80 mm³ (100±20% of T2 lesion volume). This was significantly higher than both 1 and 24 h groups (P<0.01).

Figure 3

Plasma zymography. (A) Representative zymogram comparing plasma MMP-2 and MMP-9 levels across naive, sham, 1, 24, and 48 h groups. A bacterial collagenase control was used to standardize band intensities across gels. (B) Quantified densitometry of total MMP-9 (pro+active). MMP-9 levels were increased within the 1 h group in comparison to the naive group. (C) Quantified densitometry of total MMP-2 (pro+active). There were no changes in MMP-2 levels. Values are presented as mean±standard error of the mean (s.e.m.). N=6 for individual groups. ^**P<0.01.

Figure 4

Brain tissue zymography. (A) Representative zymogram comparing brain tissue MMP-2 and MMP-9 levels across naive, sham, 1, 24, and 48 h groups. A bacterial collagenase control was used to standardize band intensities across gels. (B) Quantified densitometry of total MMP-9 (pro+active). MMP-9 levels were increased for the 48 h group in comparison to naive, sham, and 1 h groups. (C) Quantified densitometry of total MMP-2 (pro+active). MMP-2 levels were increased for the 48 h group in comparison to all others. Values are presented as mean±standard error of the mean (s.e.m.). N=5 for individual groups. ^*P<0.05, ^**P<0.01.

Figure 5

Enzyme immunohistochemistry. Images (40 × ) of representative brain tissue from sham, 1, 24, and 48 h groups. Top: ventricles and choroid plexus. For the 1 h group (black asterisks), cresyl violet staining showed intense cytoplasmic vacuolization (swelling), indistinct epithelial membranes, and varying degrees of pyknosis. MMP-2 staining was almost nonexistent, whereas MMP-9 staining was increased in comparison to sham. The morphological changes observed at 1 h were less obvious for the 24 and 48 h groups, suggesting partial recovery. Bottom: brain parenchyma. For the 48 h group (yellow asterisks), the lesion core showed frank necrosis, intense IgG staining with perivascular extension into the parenchyma, and MMP-2 and MMP-9 staining of vessels, microglia, and macrophages. Further positive staining included a limited number of neurons. Similar observations were made along the lesion periphery. The changes observed at 48 h were more severe than both the 1 and 24 h groups.