Astrocytic induction of matrix metalloproteinase-9 and edema in brain hemorrhage

Transcription

1 & 2007 ISCBFM All rights reserved X/07 $ Astrocytic induction of matrix metalloproteinase-9 and edema in brain hemorrhage Emiri Tejima 1, Bing-Qiao Zhao 1, Kiyoshi Tsuji 1, Anna Rosell 2, Klaus van Leyen 1, R Gilberto Gonzalez 3, Joan Montaner 2, Xiaoying Wang 1 and Eng H Lo 1 1 Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, USA; 2 Laboratorio de Investigacio Neurovascular, Vall d Hebron Hospital, Barcelona, Spain; 3 Division of Neuroradiology, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts, USA We tested the hypothesis that astrocytic matrix metalloproteinase-9 (MMP-9) mediates hemorrhagic brain edema. In a clinical case of hemorrhagic stroke, MMP-9 co-localized with astrocytes and neurons in peri-hematoma areas. In a mouse model where blood was injected into striatum, MMP-9 was colocalized with astrocytes surrounding the hemorrhagic lesion. Because MMP-9 is present in blood as well as brain, we compared four groups of wild type (WT) and MMP-9 knockout (KO) mice: WT blood injected into WT brain, KO blood into KO brain, WT blood into KO brain, and KO blood into WT brain. Gel zymography showed that MMP-9 was elevated in WT hemorrhagic brain tissue but absent from KO hemorrhagic brain tissue. Edematous water content was elevated when WT blood was injected into WT brain. However, edema was ameliorated when MMP-9 was absent in either blood or brain or both. To further assess the mechanisms involved in astrocytic induction of MMP-9, we next examined primary mouse astrocyte cultures. Exposure to hemoglobin rapidly upregulated MMP-9 in conditioned media within 1 to 24 h. Hemoglobin-induced MMP-9 was reduced by the free radical scavenger U83836E. Taken together, these data suggest that although there are large amounts of MMP-9 in blood, hemoglobin-induced oxidative stress can trigger MMP-9 in astrocytes and these parenchymal sources of matrix degradation may also be an important factor in the pathogenesis of hemorrhagic brain edema.. doi: /sj.jcbfm ; published online 21 June 2006 Introduction Intracerebral hemorrhage (ICH) accounts for up to 15% of all strokes (Ariesen et al, 2003), and it has been estimated that 30% to 40% of ischemic strokes will undergo some degree of hemorrhagic conversion (Lyden and Zivin, 1993). The prognosis after ICH is poor and treatment options are limited. Yet, in comparison to ischemic stroke, pathogenic mechanisms of ICH remain relatively unclear. In experimental models, recent data suggest that neuronal injury and cell death evolves in Correspondence: Dr EH Lo, Neuroprotection Research Laboratory, Massachusetts General Hospital, CNY 2401, East , Charlestown, Massachusetts 2129, USA. Lo@helix.mgh.harvard.edu This work was supported in part by NIH Grants R01-NS37074, R01-NS38731, R01-NS40529, P50-NS10828, and a Bugher Stroke award (EHL) and Scientist Development award (XW) from the American Heart Association Received 3 January 2006; revised 5 May 2006; accepted 10 May 2006; published online 21 June 2006 peri-hematoma areas via multifactorial mechanisms. These pathways include damaging cytokines and inflammatory mediators (Castillo et al, 2002; Hua et al, 2000; Peeling et al, 2001a, b), apoptosis (Felberg et al, 2002; Gong et al, 2001; Matsushita et al, 2000; Matz et al, 2000), and microglial activation (Wang et al, 2003; Wang and Tsirka, 2005a, b). Blood flow reductions have also been documented in some models of ICH (Kobari et al, 1988; Yang et al, 1994), although the question of whether perfusion deficits truly reach ischemic thresholds remains to be confirmed (Rosand et al, 2002; Zazulia et al, 2001). As these molecular pathways become better defined, new therapeutic opportunities for preventing neuronal death after ICH may eventually be found. In the meantime, one of the biggest challenges from a clinical perspective are related to the brain edema that accompanies ICH (Hoff and Xi, 2003). Edema and increases in intracranial pressure typically occur between 24 to 48 h post-ictus, and are, suggested to be a major cause of clinical deterioration and death. Emerging data suggest an important

2 role for extracellular proteases that are abundant in blood that has leaked into brain (Gingrich and Traynelis, 2000). For example, thrombin amplifies edema adjacent to ICH (Lee et al, 1996; Xi et al, 1998), and treatment with thrombin inhibitors can reduce edema (Kitaoka et al, 2002). More recently, the large family of matrix metalloproteinases (MMPs) have also been implicated. Matrix metalloproteinases have already been shown to mediate blood brain barrier (BBB) injury after cerebral ischemia (Asahi et al, 2001a, b; Gasche et al, 2001; Rosenberg et al, 1998). Since MMPs are also upregulated after ICH (Power et al, 2003; Wang and Tsirka, 2005a, b), these proteases may also trigger peri-hematoma edema via similar effects on the neurovascular matrix and BBB. In this study, we specifically ask whether MMP-9 mediates brain edema after ICH using a combination of clinical case material, knockout (KO) mouse models, and cell culture systems. Our data show that MMP-9 may indeed be upregulated in reactive astrocytes surrounding an ICH, and although large amounts of MMP-9 are already present in blood, these endogenous astrocytic sources of MMP-9 may still play a significant role in mediating brain edema that accompanies ICH. period. The needle was left in place for 10 mins after completion of the infusion to prevent leakage of blood out of the brain, and then removed slowly. The craniotomy was sealed with bone wax, the scalp sutured closed, and mice were allowed to recover. Immunohistochemistry At 48 h after ICH, mice (n = 4) were transcardially perfused with ice-cold phosphate-buffered saline (PBS), ph 7.4, followed with ice-cold 4% paraformaldehyde in PBS (ph 7.4). Mouse brains were removed, immersed with 4% paraformaldehyde in PBS overnight at 41C, and then cryoprotected in 30% sucrose in PBS at 41C. Frozen coronal sections (20 mm) were prepared using a cryostat. The following antibodies were used: rabbit anti-mmp-9 polyclonal antibody (1:200; gift from Dr Robert Senior, Washington University, St Louis, MO, USA), fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (IgG) (1:100; Jackson Immuno Research), and anti-grail fibrillary acidic protein (GFAP) antibody (1:100; Chemicon, USA). Negative control sections received identical treatment except for the primary antibody. Immunostainings were analyzed with a fluorescence microscope interfaced with a digital charge-coupled device camera and an image analysis system. 461 Materials and methods Clinical Intracerebral Hemorrhage Case Brain tissue was obtained from a patient who suffered a massive hemorrhagic stroke involving the right hemisphere, and died approximately 3 h after stroke onset. Sampling was performed within 6 h after onset. Computed tomography (CT) scans obtained at admission were used to guide tissue sampling during autopsy. Blood mass was removed and adjacent parenchyma was obtained along with matching contralateral tissue. All samples were snapfrozen and stored at 801C until immunohistochemistry was performed. This study was approved by the ethics and human research committee of the Vall d Hebron Hospital in Barcelona, Spain. Informed consent was obtained from relatives before autopsy. Mouse Intracerebral Hemorrhage Model All experiments were performed following an institutionally approved protocol in accordance with the National Institute of Health s Guide for the Care and Use of Laboratory Animals. Matrix metalloproteinase-9 KO and background-matched wild-type (WT) mice (male, 24 to 30 g, 10 to 12 weeks old) were used. Mice were anesthetized with halothane (1% to 1.5%) under spontaneous respiration in a nitrous oxide/oxygen mixture and placed in a stereotactic frame. A midline scalp incision was made, and a craniotomy was performed. A 30-gauge needle was lowered into the center of striatum (from bregma: 1 mm anterior, 2 mm lateral, 3.5 mm depth), and 20 ml of whole blood was infused into over a 10 mins Measurement of Brain Edema A standard wet dry method was used for the measurement of total brain water content as an indicator of edema. At 48 h after ICH, mice were euthanized and brains were removed immediately, without fixation. A 2 mm thick coronal slice located at the center of the hemorrhagic lesion was cut then divided into ipsilateral and contralateral sides. Tissue samples were immediately weighed to obtain the wet weight (WW), dried in a dessicator oven at 1001C for 24 h, then weighed again to obtain the dry weight (DW). Total water contents (%) was calculated as follows: (WW DW)/WW 100. To assess the role of MMP-9 in the development of ICH brain edema in our mouse model, we studied four different sets of mice (n =8 per group): WT blood injected into WT brain, KO blood injected into KO brain, WT blood injected into KO brain, and KO blood injected into WT brain. Primary Culture of Mouse Cortical Astrocytes Primary astrocyte cultures were prepared from cerebral cortices of 1-day-old neonatal mouse after standard procedures. Briefly, dissociated cortical cells were suspended in Dulbecco s modified Eagle s medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 25 mmol/l glucose, 4 mmol/l glutamine, 1 mmol/l sodium pyruvate, and supplemented with 10% fetal bovine serum. Isolated cells were plated into 25 cm 2 flasks at a density of one cortex per flask. Monolayers of type I astrocytes were obtained at 12 to 14 days after initial seeding. Astrocytes were dissociated by trypsinization and then reseeded into 6-, 12-, or 24-well plates or slide chambers at a density of

3 462 20,000 cells/cm 2. After cells reached confluence (7 to 8 days post-seeding), cultures were switched to serum-free DMEM medium, and experiments were initiated 24 h later. In this system, more than 95% of the cells were type I astrocytes identified by GFAP and Diamidino-2- phenylindole (DAPI) double staining (data not shown). Purified (clinical grade endotoxin-free) human hemoglobin was obtained from Hemosol Inc., Canada. The combination of rodent or murine cell cultures to human hemoglobin has been previously validated (Wang et al, 1999, 2002). Gelatin Zymography Matrix metalloproteinase-2 and -9 levels in conditioned media were assessed with standard gelatin zymography. After treatments, the culture medium was collected and centrifuged at 14,000 r.p.m. for 5 mins at 41C to remove cells and debris. The cleared medium was concentrated 10-fold using Microcon (Millipore, Billerca, MA, USA) with a 10 kda pore diameter cutoff, then each sample was mixed with equal amounts of sodium dodecyl sulfate (SDS) sample buffer (Invitrogen) and electrophoresed on 10% SDS-polyacrylamide gels (Invitrogen) containing 1 mg/ml gelatin as the protease substrate. After electrophoresis, gels were placed in 2.7% Triton X-100 for 1 h to remove SDS, and then incubated for 20 h at 371C in developing buffer (50 mmol/l Tris base, 40 mmol/l HCl, 200 mmol/l NaCl, 5 mmol/l CaCl 2, and 0.2% Briji 35; Invitrogen) on a rotary shaker. After incubation, gels were stained in 30% methanol, 10% acetic acid, and 0.5% w/v Coomassie brilliant blue for 2 h followed by destaining. Gelatinolytic activity was manifested as horizontal white bands on a blue background. Matrix metalloproteinase bands were identified after standard techniques (Arai et al, 2003; Asahi et al, 2001a, b; Wang et al, 2000), using molecular weight criteria and the loading of mouse MMP-2 and MMP-9 purchased from Chemicon. To further confirm that active forms of MMP-9 were involved, we also incubated mouse MMP-9 standards with trypsin to achieve proteolytic cleavage/activation. The multiple lower MW bands of MMP-9 that emerged were consistent with previously published results (Fisher et al, 2002). Relative gelatinolytic activity (MMP-2 and MMP-9) was quantified via measurement of optical density using the NIH image analysis software. Statistical Analysis Data were analyzed using analysis of variance followed by Tukey s honestly significant difference tests between individual groups. Data were expressed as mean7s.d. Differences at P < 0.05 were considered significant. Results Matrix Metalloproteinase-9 is Upregulated in Peri-Hematomal Astrocytes Brain samples were rapidly obtained in a clinical case of hemorrhagic stroke within 6 h of death. This Figure 1 Clinical case of spontaneous hemorrhagic stroke in a 74 year old male. (A) Computed tomography scan showed a massive hematoma in the right hemisphere with surrounding edema, as reflected by the clear hypodense rim along with a prominent midline shift. (B) Brain was removed at 6 h for histologic analysis, which confirmed the presence of a large hematoma. Hematoxylin eosin stains suggested that no neutrophils were detected in the peri-hematoma areas, perhaps consistent with the relatively early timepoint involved. Arrow indicates neuron. Arrowheads indicate astrocytes. (C) Double label immunohistochemistry showed that MMP-9 was increased in peri-hematomal areas (middle and lowest rows) compared with contralateral cortex (uppermost row). Matrix metalloproteinase-9 was strongly colocalized with GFAP-positive astrocytes (middle row). Arrow indicates single astrocyte depicted in inset photo. Bar indicates 100 mm. In addition, MMP-9 was also colocalized with many NeuN-positive neurons (lowest row). Bar indicates 50 mm. patient suffered from a large spontaneous ICH in the right hemisphere. Computed tomography scans revealed a massive hematoma with leakage into the ventricles and gross midline shift (Figure 1A). Hematoxylin eosin histopathology showed that at this relatively early timepoint, no neutrophil infiltration in peri-hematomal areas were detectable (Figure 1B). Brain sections from areas adjacent to the hematoma showed clear signs of upregulated MMP-9. Although multiple cell types were involved, the strongest signals appeared to come from GFAP-positive astrocytes and NeuN-positive neurons (Figure 1C).

4 463 Figure 3 Brain edema in ipsilateral hemipsheres after ICH in the mouse model. WT blood injected into WT brain resulted in a 2% increase in water content, compared with normal values in contralateral brain (76% to 77%). Deletion of MMP-9 gene in either blood or brain or both compartments significantly reduced edema. *P < 0.05 versus mice with WT blood injected into WT brains, n = 8 per group. Figure 2 MMP-9 in a mouse model of ICH. (A) Immunostaining showed low baseline levels of MMP-9 in contralateral striatum (upper left panel), whereas MMP-9 was clearly increased in ipsilateral striatum adjacent to the ICH lesion (upper right panel). Increased MMP-9 was colocalized with GFAP-positive astrocytes (lower left and right panels), bar indicates 50 mm. (B) Gel zymography showed that MMP-9 was elevated in hemorrhagic brain tissue from WT brains and not MMP-9 KO brains. Rightmost lanes were loaded with mouse MMP-2 and MMP-9 standards. The position of the bands suggested that active MMP-9 was detected in this model. To further assess this idea of MMP-9 upregulation after ICH, we turned to an experimental mouse model where 20 ml of homologous blood was stereotactically injected into the striatum. Brain sections obtained at 48 h post-ich showed that MMP-9 signals were primarily colocalized with GFAP-positive astrocytes (Figure 2A). Gel zymograms showed that MMP- 9 levels were increased in hemorrhagic brain tissue when WT blood was injected into WT brain (Figure 2B). The MW position of the bands suggested that active forms of MMP-9 may be involved. No MMP-9 bands were detected when KO blood was injected into KO brain (Figure 2B). Edema After Intracerebral Hemorrhage is Reduced by Matrix Metalloproteinase-9 Gene Knockout in Blood or Brain To assess the role of MMP-9 in our model of ICH, we compared four groups of WT and MMP-9 KO mice: WT blood injected into WT brain, KO blood injected into KO brain, WT blood injected into KO brain, and KO blood injected into WT brain. At 48 h post-ich, brains were removed and edema was quantified as the total water content using the wet/dry weight assay. In all groups, water content in contralateral hemisphere was within normal range (76% to 77%) compared with sham operated controls. In the ipsilateral hemisphere, edema was clearly induced when WT blood was injected into WT brain (79.5% 70.2%). However, when MMP-9 was missing from either blood or brain or both compartments, the elevation in ipsilateral brain water content was significantly ameliorated (Figure 3). Nevertheless, there were no differences between the three groups (KO blood into KO brain, WT blood into KO brain, or KO blood into WT brain), regardless of whether MMP-9 was deleted from blood or brain or both. Hemoglobin Induces Matrix Metalloproteinase-9 in Astrocytes via Oxidative Stress To further assess the role of astrocytes as a source for MMP-9 after ICH, we turned to a cell culture system. Primary mouse cortical astrocytes were exposed to hemoglobin. Matrix metalloproteinase-9 levels in conditioned media were examined at 1 to 24 h. Hemoglobin (0.5 to 10 mmol/l) clearly upregulated MMP-9 (5- to 20-fold) in a concentration-dependent manner (Figure 4A). This response in MMP-9 was rapid, occurring within 1 h and was sustained for up to 24 h (Figure 4B). No clear changes in MMP-2 were detected in our system. As MMP-9 can be induced by oxidative stress and hemoglobin is a potent trigger of oxidative stress, we sought to pharmacologically validate our results with a free radical scavenger. Co-incubating

5 464 Figure 4 Induction of MMP-9 by hemoglobin in primary mouse astrocytes. Upper panels show representative gelatin zymograms. Lower panels show quantified densitometry. (A) Hemoglobin exposure (0.5 to 10 mmol/l) for 24 h induced a concentrationdependent response in MMP-9 within conditioned media. The position of the band appeared consistent with a lower MW active form of MMP-9. Matrix metalloproteinase-2 was detected as a strong band at 72 kda. In all cases, an unidentified band above MMP-2 was also seen, but no clear changes in this band occured. The three rightmost lanes indicate mouse MMP standards loaded as controls: SD1 is MMP-9; SD2 indicates multiple bands of protease-cleaved/activated MMP-9; SD3 is MMP-2. *P < 0.05 versus control cultures not exposed to hemoglobin, n = 6 per group. (B) Hemoglobin (1 mmol/l) induced a rapid upregulation in astrocytic MMP-9 within 1 h that persisted for 24 h. *P < 0.05 versus control cultures not exposed to hemoglobin, n = 6 per group. (C) Co-incubation with the free radical scavenger U83836E significantly blocked the MMP-9 response induced by 1 mmol/l hemoglobin. *P < 0.05 versus untreated cultures exposed to hemoglobin alone, n = 6 per group. the astrocytes with the lazaroid compound U83836E (5 to 40 mmol/l) significantly reduced the hemoglobin-induced MMP-9 response (Figure 4C). No effects on MMP-2 were detected. Discussion Matrix metalloproteinases comprise a large family of zinc endopeptidases that can collectively modify almost all components of extracellular matrix in brain (Yong et al, 2001). The gelatinase MMP-9, in particular, has been implicated in central nervous system (CNS) injury (Lo et al, 2002; Rosenberg, 2002; Yong et al, 2001). Matrix metalloproteinase-9 is upregulated in human stroke (Clark et al, 1997) and many animal models of cerebral ischemia (Asahi et al, 2000, 2001a, b; Fujimura et al, 1999; Gasche et al, 1999; Heo et al, 1999; Mun-Bryce and Rosenberg, 1998; Romanic et al, 1998; Rosenberg et al, 1996, 2001) and CNS trauma (Noble et al, 2002; Wang et al, 2000). By degrading homeostatic cellmatrix signaling, MMP-9 can induce anoikis-like neuronal death (Gu et al, 2002; Lee and Lo, 2004;

6 Lee et al, 2004). Hence, MMP inhibitors have been shown to be neuroprotective in animal models of CNS ischemia, hemorrhage and trauma (Asahi et al, 2000; Gu et al, 2005; Jiang et al, 2001; Lee et al, 2004; Romanic et al, 1998; Wells et al, 2003). By degrading the integrity of extracellular matrix, especially within neurovascular basal lamina, MMP-9 can also disrupt the BBB thus causing edema and hemorrhagic conversion in models of cerebral ischemia (Aoki et al, 2002; Asahi et al, 2001a, b; del Zoppo and Mabuchi, 2003; Gasche et al, 2001; Sumii and Lo, 2002). In the present study, we found that MMP- 9 was increased in reactive astrocytes in a clinical case study, as well as a mouse model of ICH. Deletion of the MMP-9 gene significantly ameliorated edema in mouse ICH. And our astrocyte cultures showed that hemoglobin potently and rapidly upregulates MMP-9 in these cells, consistent with the in vivo findings. Taken together, the combination of clinical, in vivo and cell culture evidence suggest that MMP-9 is an important mediator of brain edema in ICH. An important point to be discussed here involves the opposite results obtained by Zhang and coworkers recently, where hemorrhagic severity and mortality was increased in MMP-9 KO mice compared to WT mice (Tang et al, 2004). In that study, bacterial collagenase was injected into mouse striatum to damage collagen and laminin substrates in the microvascular basal lamina, thus causing hemorrhage. The advantage of the collagenase model is that it is highly reproducible, with basically spherical hematoma developing over 3 to 4 h. Furthermore, it replicates the primary vascular trauma and rupture that occurs in clinical ICH. However, the caveat with this model is the presence of bacterial collagenase, which may enhance inflammatory cascades and modify ICH pathology in subtle ways. In our present study, we simply infused homologous blood into the mouse striatum. The drawback here is that lesions tend to be more variable, and there is no primary vascular wall damage. But the advantage is that we do not have potentially deleterious agents other than blood in the model system. In contrast to the Tang et al study, we find that MMP-9 gene KO was beneficial by significantly ameliorating brain edema. Although we cannot unequivocally determine the reasons for these different results, a critical factor may ultimately involve the ICH model used. The finding that deletion of MMP-9 reduced edema in our mouse ICH models may not be surprising per se. After all, we have previously shown that BBB damage was reduced in MMP-9 KO mice after cerebral ischemia (Asahi et al, 2001a, b). What was somewhat unexpected however, was that it did not seem to matter whether we deleted MMP-9 from the infused blood or the recipient brain. After ICH, a whole host of activated blood components and cytokines extravasate into brain parenchyma. For MMPs, this may be a reasonable source for the pathogenesis of edema, since activated leukocytes can produce large amounts of this protease (Gidday et al, 2005; Justicia et al, 2003). Matrix metalloproteinase-9 levels are low in normal brain but relatively higher in blood anyway. So when blood-borne MMP-9 diffuses into brain after ICH, neurovascular proteolysis may ensue with the resulting effect of BBB leakage and spread of edema beyond the primary confines of the initial hemorrhagic lesion. Additionally, our data suggest that brain is also an important source of MMP-9 in ICH. Perihematomal astrocytes are MMP-9-positive and deletion of only brain MMP-9 also reduced edema. A limitation here is that we cannot determine whether blood or brain MMP-9 plays a more quantitatively important role. We were also unable to detect any additive effect of brain versus blood MMP-9 deletion. It is likely that there are close interactions and signaling feedback loops between blood and tissue as the ICH evolves. Nevertheless, this overall link between MMP-9 and hemorrhagic edema is consistent with emerging clinical findings. In humans, increased expression of plasma MMP-9 within the first 24 h of ICH has been reported (Alvarez-Sabin et al, 2004). Moreover, plasma MMP-9 measured at arrival among ICH patients has been shown to be correlated with peri-hematomal edema volume measured on CT scans, as well as progressive hematoma enlargement over the next 48 h (Abilleira et al, 2003). Taken together, our clinical, animal and cell culture data suggest a significant role for astrocytic MMP-9. However, there are several caveats to keep in mind. First, we acknowledge that our in vivo mouse model does not fully replicate the clinical condition of ICH. Translating these experimental findings into a clinical context should be performed with caution. A second limitation here is that we cannot unequivocally define the cellular mechanisms involved. It has been suggested that both intracellular (presumably cytotoxic) and extracellular (presumably vasogenic) edema occurs after ICH. Which component comprises the rate-limiting step here is unknown because we measure edema using a relatively gross end point, that is, total brain water content. Nevertheless, it is generally accepted that the wet/dry weight assay is most reflective of overall edema in animal models. Furthermore, it is acknowledged that MMP-9 signals can also be detected in neurons, endothelium, and microglia as well. To examine cell cultures in all of these would be outside the scope of our study. We only focus on astrocytes but mechanisms of response in other cells remain to be fully dissected. Thirdly, although our pharmacologic data in cell culture suggest that oxidative stress from hemoglobin may be the trigger of MMP-9 in astrocytes, we do not directly measure free radical formation nor do we have evidence for this pathway in vivo. However, others have shown that oxidative stress 465

7 466 is involved in ICH (Peeling et al, 2001a, b), so our cell culture data is at least consistent with these other findings. A fourth caveat involves the question of timing. How rapidly does MMP-9 go up, and how does this compare with the time course of edema progression? In the clinical case, MMP-9 was detected within 6 h, whereas our mouse model data is limited to 48 h measurements. In animal models of cerebral ischemia and trauma, MMP-9 can be rapidly elevated within just a few hours. Clinical edema may peak by 24 to 48 h. Although there is no direct proof, the timing suggests that MMPs may precede the development of edema. A fifth question is related to the significance of edema in our admittedly artificial mouse model. Are the rather small changes in water content, in the range of 2% to 3%, of any biologic significance? A review of the literature shows that the wet/dry brain weight assay of water content typically yields numbers in this range for rodent models, so our data are consistent with known data. Even with massive brain swelling in rodents, wet/dry weight assays reveal water content elevations of only a few percent. But how do these changes affect neuronal cell death per se? Others have shown that neuronal apoptosis can be reduced by MMP inhibitors (Gu et al, 2002, 2005), so there is relevance to parenchymal damage. Of course, the potential confound here then is that we may not be able to say whether reductions in edema are directly caused by MMP-9 gene KO or whether this is a secondary effect of decreased neuronal injury. Ultimately, future studies will have to carefully examine how these improvements in edema relate to clinical outcomes. A sixth caveat is that our study is only focused on MMP-9, whereas others have shown that many others within the large MMP family may be dysregulated after ICH (Power et al, 2003). Further studies are needed to dissect the network effects of these other proteases when a particular member is deleted or blocked. And finally, we acknowledge that our focus here on acute stages of brain injury may be oversimplified. Whereas neurovascular proteolysis via MMPs may be damaging in the acute phase, emerging data now suggest that during delayed phases of stroke, MMPs may mediate potentially beneficial pathways of neurogenesis and plasticity (Lee et al, 2006; Zhao et al, 2006). How MMPs may be involved in the resolution and recovery phase after ICH needs to be carefully examined. In conclusion, our data suggest that although blood itself may be an obvious source of damaging proteases, endogenous astrocytic MMP-9 responses that are triggered by oxidative stress after ICH may also play a significant role in the pathogenesis of edema. Further studies are warranted to test whether specific blockade of MMPs or blunting of astrocytic or other parenchymal reactions to injury, perhaps via antioxidants, may offer new ways of ameliorating acute edema after ICH. Acknowledgements We will always be grateful to Dr Robert Senior of Washington University St Louis for kindly allowing us to use his MMP-9 mice and reagents for so many years. We thank Dr Arantxa Ortega-Aznar for help with the neuropathology. References Abilleira S, Montaner J, Molina CA, Monasterio J, Castillo J, Alvarez-Sabin J (2003) Matrix metalloproteinase-9 concentration after spontaneous intracerebral hemorrhage. J Neurosurg 99:65 70 Alvarez-Sabin J, Delgado P, Abilleira S, Molina CA, Arenillas J, Ribo M, Santamaria E, Quintana M, Monasterio J, Montaner J (2004) Temporal profile of mnatrix metalloproteinases and their inhibitors after spontaneous intracerebral hemorrhage: relationship to clinical and radiological outcome. Stroke 35: Aoki T, Sumii T, Mori T, Wang X, Lo EH (2002) Blood brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury: mechanical versus embolic focal ischemia in spontaneously hypertensive rats. Stroke 33: Arai K, Lee SR, Lo EH (2003) Essential role for ERK MAP kinase in MMP-9 regulation in rat cortical astrocytes. Glia 43: Ariesen MJ, Claus SP, Rinkel GJ, Algra A (2003) Risk factors for intracerebral hemorrhage in the general population: a systematic review. Stroke 34: Asahi M, Asahi K, Jung JC, del Zoppo GJ, Fini ME, Lo EH (2000) Role of matrix metalloproteinase 9 in focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94. J Cereb Blood Flow Metab 20: Asahi M, Sumii T, Fini ME, Itohara S, Lo EH (2001a) Matrix metalloproteinase 2 gene knockout has no effect on acute brain injury after focal ischemia. NeuroReport 12: Asahi M, Wang X, Mori T, Sumii T, Jung J, Moskowitz MA, Fini ME, Lo EH (2001b) Effects of matrix metalloproteinase 9 gene knockout on the proteolysis of blood brain barrier and white matter components after cerebral ischemia. J Neurosci 21: Castillo J, Davalos A, Alvarez-Sabin J, Pumar JM, Leira R, Silva Y, Montaner J, Kase CS (2002) Molecular signatures of brain injury after intracerebral hemorrhage. Neurology 58:624 9 Clark AW, Krekoski CA, Bou S, Chapman KR, Edwards RE (1997) Increased gelatinase A (MMP-2) and gelatinase- B (MMP-9) activities in human brain after focal ischemia. Neurosci Lett 238:53 6 del Zoppo GJ, Mabuchi T (2003) Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab 23: Felberg RA, Grotta JC, Shirzadi AL, Strong R, Narayana P, Hill-Felberg SJ, Aronowski J (2002) Cell death in experimental intracerebral hemorrhage: the black hole model of hemorrhagic damage. Ann Neurol 51: Fisher KE, Fei Q, Laird ER, Stock JL, Allen MR, Sahagan BG, Strick CA (2002) Engineering autoactivating forms of matrix metalloproteinase-9 and expression of the active enzyme in cultured cells and transgenic mouse brain. Biochemistry 41:

8 Fujimura M, Gasche Y, Morita-Fujimura Y, Massengale J, Kawase M, Chan PH (1999) Early appearance of activated matrix metalloproteinase 9 and blood brain barrier disruption in mice after focal cerebral ischemia and reperfusion. Brain Res 842: Gasche Y, Copin JC, Sugawara T, Fujimura M, Chan PH (2001) Matrix metalloproteinase inhibition prevents oxidative stress-associated blood brain barrier disruption after transient focal cerebral ischemia. J Cereb Blood Flow Metab 21: Gasche Y, Fujimura Y, Copin J, Kawase M, Masengale J, Chan PH (1999) Early appearance of activated MMP-9 after focal cerebral ischemia in mice. J Cereb Blood Flow Metab 19: Gidday JM, Gasche YG, Copin JC, Shah AR, Perez RS, Shapiro SD, Chan PH, Park TS (2005) Leukocytederived matrix metalloproteinase-9 mediates blood brain barrier breakdown and is pro-inflammatory after transient focal cerebral ischemia. Am J Physiol 289: H Gingrich MB, Traynelis SF (2000) Serine proteases and brain damage. Trends Neurosci 23: Gong C, Boullis N, Qian J, Tuner DE, Hoff JT, Keep RF (2001) Intracerebral hemorrhage induced neuronal death. Neurosurgery 48: Gu Z, Cui J, Brown S, Fridman R, Mobashery S, Strongin AY, Lipton SA (2005) A highly specific inhibitor of matrix metalloproteinase-9 rescues laminin from proteolysis and neurons from apoptosis in transient focal cerebral ischemia. J Neurosci 25: Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC, Lipton SA (2002) S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science 297: Heo JH, Lucero J, Abumiya T, Koizol JA, Copeland BR, del Zoppo GJ (1999) Matrix metalloproteinases increase very early during experimental focal cerebral ischemia. J Cereb Blood Flow Metab 19: Hoff JT, Xi G (2003) Brain edema from intracerebral hemorrhage. Acta Neurochir 86(Suppl):11 5 Hua Y, Xi G, Keep RF, Hoff JT (2000) Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg 92: Jiang X, Namura S, Nagata I (2001) Matrix metalloproteinase inhibitor KB-R7785 attenuates brain damage resulting from permanent focal cerebral ischemia in mice. Neurosci Lett 305:41 4 Justicia C, Panes J, Sole S, Cervera A, Deulofeu R, Chamorro A, Planas AM (2003) Neutrophil infiltration increases matrix metalloproteinase-9 in the ischemic brain after occlusion/reperfusion of the middle cerebral artery in rats. J Cereb Blood Flow Metab 23: Kitaoka T, Hua Y, Xi G, Hoff JT, Keep RF (2002) Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage. Stroke 33: Kobari M, Gotoh F, Tomita M, Tanahashi N, Shinohara T, Terayama Y, Mihara B (1988) Bilateral hemispheric reduction of cerebral blood volume and blood flow immediately after experimental cerebral hemorrhage in cats. Stroke 19:991 6 Lee KR, Colon GP, Betz AL, Keep RF, Kim S, Hoff JT (1996) Edema from intracerebral hemorrhage: the role of thrombin. J Neurosurg 84:91 6 Lee S, Lo EH (2004) Induction of caspase-mediated cell death by matrix metalloproteinase after hypoxia-reoxygenation in human brain microvscular endothelial cells. J Cereb Blood Flow Metab 24:720 7 Lee SR, Kim HY, Rogowska J, Zhao BQ, Bhide P, Parent JM, Lo EH (2006) Involvement of matrix metalloproteinase in neuroblast cell migration from the subventricular zone after stroke. J Neurosci 26: Lee SR, Tsuji K, Lee S, Lo EH (2004) Role of matrix metalloproteinases in delayed neuronal damage after transient global cerebral ischemia. J Neurosci 24:671 8 Lo EH, Wang X, Cuzner ML (2002) Extracellular proteolysis in brain injury and inflammation: role for plasminogen activators and matrix metalloproteinases. J Neurosci Res 69:1 9 Lyden PD, Zivin JA (1993) Hemorrhagic transformation after cerebral ischemia: mechanisms and incidence. Cerebrovasc Brain Metab Rev 5:1 16 Matsushita K, Wang X, Meng W, Asahi M, Asahi K, Moskowitz MA, Lo EH (2000) Evidence for apoptosis after intracerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab 20: Matz PG, Fujimura M, Chan PH (2000) Subarachnoid hemolysate produces DNA fragmentation in a pattern similar to apoptosis in mouse brain. Brain Res 858: Mun-Bryce S, Rosenberg GA (1998) Matrix metalloproteinases in cerebrovascular disease. J Cereb Blood Flow Metab 18: Noble LJ, Donovan F, Igarashi T, Goussev S, Werb Z (2002) Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events. J Neurosci 22: Peeling J, Del Bigio MR, Corbett D, Green AR, Jackson DM (2001a) Efficacy of disodium 4-[(tert-butylimino)- methyl]benzene-1,3-disulfonate N-oxide(NXY-059), a free radical trapping agent, in a rat model of hemorrhagic stroke. Neuropharmacology 40:433 9 Peeling J, Yan HJ, Corbett D, Xue M, Del Bigio MR (2001b) Effect of FK-506 on inflammation and behavioral outcome followingintracerebral hemorrhage in rat. Exp Neurol 167:341 7 Power C, Henry S, Del Bigio MR, Larsen PH, Corbett D, Imai Y, Yong VW, Peeling J (2003) Intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases. Ann Neurol 53: Romanic AM, White RF, Arleth AJ, Ohlstein EH, Barone FC (1998) Matrix metalloproteinase expression increases after cerebral focal ischemia in rats. Stroke 29: Rosand J, Eskey C, Chang Y, Gonzalez RG, Greenberg SM, Koroshetz WJ (2002) Dynamic single section CT demonstrates reduceed cerebral blood flow in acute intracerebral hemorrhage. Cerebrovasc Dis 14: Rosenberg GA (2002) Matrix metalloproteinases in neuroinflammation. Glia 39: Rosenberg GA, Cunningham LA, Wallace J, Aleander S, Estrada EY, Grossetete M, Razhagi A, Miller K, Gearing A (2001) Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures. Brain Res 893: Rosenberg GA, Estrada EY, Dencoff JE (1998) Matrix metalloproteinases and TIMPs are associated with blood brain barrier opening after reperfusion in rat brain. Stroke 29: Rosenberg GA, Navratil M, Barone F, Feuerstein G (1996) Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J Cereb Blood Flow Metab 16:360 6 Sumii T, Lo EH (2002) Involvement of matrix metalloproteinase in thrombolysis-associated hemorrhagic 467

9 468 transformation after embolic focal ischemia in rats. Stroke 33:831 6 Tang J, Liu J, Zhou C, Alexander JS, Nanda A, Granger DN, Zhang JH (2004) MMP-9 deficiency enhances collagenase-induced intracerebral hemorrhage and brain injury in mutant mice. J Cereb Blood Flow Metab 24: Wang J, Rogove AD, Tsirka AE, Tsirka SE (2003) Protective role of tuftsin fragment 1-3 in an animal model of intracerebral hemorrhage. Ann Neurol 54: Wang J, Tsirka SE (2005a) Neuroprotection by inhibition of matrix metalloproteinases in a mouse model of intracerebral hemorrhage. Brain 128: Wang J, Tsirka SE (2005b) Tuftsin fragment 1 3 is beneficial when delivered after the induction of intracerebral hemorrhage. Stroke 36:613 8 Wang X, Asahi M, Lo EH (1999) Tissue plasminogen activator amplifies hemoglobin induced neurotoxicity in rat neuronal cultures. Neurosci Lett 274:79 82 Wang X, Jung J, Asahi M, Chwang W, Russo L, Moskowitz MA, Dixon CE, Fini ME, Lo EH (2000) Effects of matrix metalloproteinase 9 gene knockout on morphological and motor outcomes after traumatic brain injury. J Neurosci 20: Wang X, Mori T, Sumii T, Lo EH (2002) Hemoglobininduced cytotoxicity on rat cerebral cortical neurons: caspase activation and oxidative stress. Stroke 33: Wells JE, Hurlbert RJ, Fehlings MG, Yong VW (2003) Neuroprotection by minocycline facilitates significant recovery from spinal cord injury in mice. Brain 126: Xi G, Wagner KR, Keep RF, Hua Y, de Courten-Myers GM, Broderick JP, Brott TG, Hoff JT (1998) Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke 29: Yang G, Betz AL, Chenevert TL, Brunberg JA, Hoff JT (1994) Experimental intracerebral hemorrhage: relationship between brain edema, blood flow, and blood brain barrier permeability in rats. J Neurosurg 81: Yong VW, Power C, Forsyth P, Edwards DR (2001) Metalloproteinases in biology and pathologyof the nervous system. Nature Rev Neurosci 2: Zazulia AR, Diringer MN, Videen TO, Adams RE, Yundt K, Aiyagari V, Grubb RL, Powers WJ (2001) Hypoperfusion without ischemia surrounding acute intracerebral hemorrhage. J Cereb Blood Flow Metab 21: Zhao BQ, Wang S, Kim HY, Storrie H, Rosen BR, Mooney DJ, Wang X, Lo EH (2006) Role of matrix metalloproteinases in delayed cortical responses after stroke. Nature Med 12:441 5