Anna Rosell

Increased Brain Expression of Matrix Metalloproteinase-9 After Ischemic and Hemorrhagic Human Stroke

Background and Purpose— Abnormal expression of some matrix metalloproteinases (MMP) has shown to play a deleterious role in brain injury in experimental models of cerebral ischemia. We aimed to investigate MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in brain parenchyma in both ischemic and hemorrhagic strokes. Methods— Postmortem fresh brain tissue from 6 ischemic and 8 hemorrhagic stroke patients was obtained within the first 6 hours after death. Finally, 78 brain tissue samples from different areas (infarct, peri-infarct, perihematoma and contralateral hemisphere) were studied. To quantify gelatinase content we performed gelatin zymograms that were confirmed by Western Blot Analysis, immunohistochemistry to localize MMP source, and in situ zymography to detect gelatinase activity. Results— Among ischemic cases, gelatin zymography showed increased MMP-9 content in infarct core although peri-infarct tissue presented also higher levels than contralateral hemisphere (P<0.0001 and P=0.042, respectively). Within infarct core, MMP-9 was mainly located around blood vessels, associated to neutrophil infiltration and activated microglial cells. In peri-infarct areas the major source of MMP-9 were microglial cells. Tissue around intracranial hemorrhage also displayed higher MMP-9 levels than contralateral hemisphere (P=0.008) in close relationship with glial cells. MMP-2 was constitutively expressed and remained invariable in different brain areas. Conclusions— Our results demonstrate in situ higher levels of MMP-9 in human brain tissue after ischemic and hemorrhagic stroke, suggesting a contribution of MMP-9 to ischemic brain injury and perihematoma edema.

MMP-9–Positive Neutrophil Infiltration Is Associated to Blood–Brain Barrier Breakdown and Basal Lamina Type IV Collagen Degradation During Hemorrhagic Transformation After Human Ischemic Stroke

Background and Purpose— An abnormal expression of some matrix metalloproteinases (MMPs) is related with hemorrhagic transformation events after stroke. Our aim was to investigate MMP-2 and MMP-9 in the ischemic brain and its relation with blood–brain barrier breakdown after hemorrhagic transformation in human stroke. Methods— We assessed 5 cases of fatal ischemic strokes with hemorrhagic complications; brain samples were obtained from infarct, hemorrhagic, and contralateral tissue. MMP-9 and MMP-2 content was analyzed by zymography and immunohistochemistry was performed to localize MMP-9 and to assess collagen IV integrity in the basal lamina. Laser capture microdissection was performed to isolate blood–brain barrier vessels to study these MMPs. Results— Overall, MMP-9 levels were higher both in hemorrhagic and nonhemorrhagic infarcted tissue compared to contralateral areas (P<0.0001 and P<0.05). Moreover, levels of the cleaved MMP-9 85kDa-form were significantly elevated in the hemorrhagic compared to nonhemorrhagic and contralateral areas (P=0.033 and P<0.0001). No changes were found for MMP-2 content. Immunostaining revealed a strong MMP-9–positive neutrophil infiltration surrounding brain microvessels associated with severe basal lamina type IV collagen degradation and blood extravasation. Microdissection confirmed that content of MMP-9 was similarly high in microvessel endothelium from hemorrhagic and infarcted areas compared to contralateral hemisphere vessels (P<0.05), pointing to neutrophils surrounding dissected microvessels as the main source of MMP-9 in hemorrhagic areas. Conclusions— Our results show a strong neutrophil infiltration in the infarcted and hemorrhagic areas with local high MMP-9 content closely related to basal lamina collagen IV degradation and blood–brain barrier breakdown. Microvessel and inflammatory MMP-9 response are associated with hemorrhagic complications after stroke.

Etiologic Diagnosis of Ischemic Stroke Subtypes With Plasma Biomarkers

Background and Purpose— Because there is no biologic marker offering precise information about stroke etiology, many patients receive a diagnosis of undetermined stroke even after all available diagnostic tests are done, precluding correct treatment. Methods— To examine the diagnostic value of a panel of biochemical markers to differentiate stroke etiologies, consecutive acute stroke patients were prospectively evaluated. Brain computed tomography, ultrasonography, cardiac evaluations, and other tests were done to identify an etiologic diagnosis according to TOAST classification. Blood samples were drawn on Emergency Department arrival (<24 hours) to test selected biomarkers: C-reactive protein, D-dimer, soluble receptor for advanced glycation end products, matrix metalloproteinase-9, S-100b, brain natriuretic peptide (BNP), neurotrophin-3, caspase-3, chimerin, and secretagogin (assayed by ELISA). Results— Of 707 ischemic stroke patients included, 36.6% were cardioembolic, 21.4% atherothrombotic, 18.1% lacunar, and 23.9% of undetermined origin. High levels of BNP, soluble receptor for advanced glycation end products, and D-dimer (P<0.0001) were observed in patients with cardioembolic stroke. Independent predictors (odds ratios with CIs are given) of cardioembolic stroke were as follows: atrial fibrillation 15.3 (8.4–27.7, P<0.001); other embolic cardiopathies 14.7 (4.7–46, P<0.001); total anterior circulation infarction 4 (2.3–6.8, P<0.001); BNP >76 pg/mL 2.3 (1.4–3.7, P=0.001); and D-dimer >0.96 &mgr;g/mL 2.2 (1.4–3.7, P=0.001). Even among patients with transient symptoms (n=155), a high BNP level identified cardioembolic etiology (6.7, 2.4–18.9; P<0.001). A model combining clinical and biochemical data had a sensitivity of 66.5% and a specificity of 91.3% for predicting cardioembolism. Conclusions— Using a combination of biomarkers may be a feasible strategy to improve the diagnosis of cardioembolic stroke in the acute phase, thus rapidly guiding other diagnostic tests and accelerating the start of optimal secondary prevention.

Extension of the Thrombolytic Time Window With Minocycline in Experimental Stroke

Background and Purpose— Thrombolysis with tPA is the only FDA-approved therapy for acute ischemic stroke. But its widespread application remains limited by narrow treatment time windows and the related risks of cerebral hemorrhage. In this study, we ask whether minocycline can prevent tPA-associated cerebral hemorrhage and extend the reperfusion window in an experimental stroke model in rats. Methods— Spontaneously hypertensive rats were subjected to embolic focal ischemia using homologous clots and treated with: saline at 1 hour; early tPA at 1 hour, delayed tPA at 6 hours; minocycline at 4 hours; combined minocycline at 4 hours plus tPA at 6 hours. Infarct volumes and hemorrhagic transformation were quantified at 24 hours. Gelatin zymography was used to measure blood levels of circulating matrix metalloproteinase-9 (MMP-9). Results— Early 1-hour thrombolysis restored perfusion and reduced infarction. Late 6-hour tPA did not decrease infarction but instead worsened hemorrhagic conversion. Combining minocycline with delayed 6-hour tPA decreased plasma MMP-9 levels, reduced infarction, and ameliorated brain hemorrhage. Blood levels of MMP-9 were also significantly correlated with volumes of infarction and hemorrhage. Conclusion— Combination therapy with minocycline may extend tPA treatment time windows in ischemic stroke.

Multiphasic roles for matrix metalloproteinases after stroke.

Matrix metalloproteinases (MMPs) comprise a family of zinc endopeptidases that play major roles in the physiology and pathology of the mammalian central nervous system (CNS). These proteinases are evolutionarily conserved as modulators of extracellular matrix during CNS development. After acute tissue injury such as that which occurs after stroke, MMPs become dysregulated and subsequently mediate acute neurovascular disruption and parenchymal destruction. Data from gene knockout models and pharmacologic experiments suggest that MMPs may be attractive therapeutic targets for stroke. However, emerging data now also suggest that some aspects of MMP activity during the delayed neuroinflammatory response may contribute to remodelling and stroke recovery. Ultimately, a more nuanced approach to modifying the MMP response after stroke may be needed in order to optimize inhibition during acute stages of injury without interfering with beneficial endogenous mechanisms of neurovascular remodelling.

Intra-Arterial Bone Marrow Mononuclear Cells in Ischemic Stroke A Pilot Clinical Trial

Background and Purpose— Bone marrow mononuclear cell (BM-MNC) intra-arterial transplantation improves recovery in experimental models of ischemic stroke. We aimed to assess the safety, feasibility, and biological effects of autologous BM-MNC transplantation in patients with stroke. Methods— A single-blind (outcomes assessor) controlled Phase I/II trial was conducted in patients with middle cerebral artery stroke. Autologous BM-MNCs were injected intra-arterially between 5 and 9 days after stroke. Follow-up was done for up to 6 months and blood samples were collected for biological markers. The primary outcome was safety and feasibility of the procedure. The secondary outcome was improvement in neurological function. Results— Ten cases (BM-MNC-treated) and 10 control subjects (BM-MNC-nontreated) were consecutively included. Mean National Institutes of Health Stroke Scale before the procedure was 15.6. Mean BM-MNCs injected were 1.59×108. There was no death, stroke recurrence, or tumor formation during follow-up, although 2 cases had an isolate partial seizure at 3 months. After transplantation, higher plasma levels of beta nerve growth factor (&bgr;-nerve growth factor) were found compared with control subjects (P=0.02). There were no significant differences in neurological function at 180 days. A trend to positive correlation between number of CD34+ cells injected and Barthel Index was found (r=0.56, P=0.09). Conclusions— Intra-arterial BM-MNC transplantation in subacute ischemic stroke is feasible and seems to be safe. Larger randomized trials are needed to confirm the safety and elucidate the efficacy of BM-MNC transplantation. Clinical Trial Registration-URL— www.clinicaltrials.gov. Unique identifier: NCT00761982.

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

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.

A Matrix Metalloproteinase Protein Array Reveals a Strong Relation Between MMP-9 and MMP-13 With Diffusion-Weighted Image Lesion Increase in Human Stroke

Background and Purpose— Matrix metalloproteinases (MMPs) are involved in tissue destruction produced by the neuroinflammatory response that follows ischemic stroke. In the present study we use an MMP array to investigate the blood levels of several MMPs in stroke patients and its relation with brain tissue damage and neurological outcome. Methods— Twenty-four patients with middle cerebral artery occlusion who received thrombolytic therapy were included. Blood samples were drawn before tissue plasminogen activator treatment and an MMP array (multiplex enzyme-linked immunosorbent assay [ELISA]) was performed including gelatinases (MMP-2 and MMP-9), collagenases (MMP-1, MMP-8, and MMP-13), stromelysines (MMP-3 and MMP-10), and MMP endogen inhibitors (TIMP-1 and TIMP-2). To assess tissue lesion a serial multimodal MRI study was performed (pretreatment and at 24 hours). Results— Neither initial diffusion lesion nor hypoperfused volume was associated with metalloproteinase expression within the first 3 hours after stroke onset. Nevertheless, a strong correlation was found between MMP-9 and MMP-13 with diffusion-weighted image (DWI) lesion expansion (r=0.54, P=0.05 and r=0.60, P=0.017, respectively). Baseline levels of both MMP-9 (OR, 14;95% CI, 1.5 to 131; P=0.019) and MMP-13 (OR, 73; 95% CI, 3.9 to 1388; P=0.004) were independent predictors of final increase in brain infarct volume at 24 hours. Conclusions— Our results demonstrate that within the neuroinflammatory response, high levels of MMP-9 and MMP-13 are involved in DWI lesion growth despite thrombolytic therapy, suggesting its ultra-early role in brain injury.

Metalloproteinase and stroke infarct size: role for anti-inflammatory treatment?

Deregulation of matrix metalloproteinases (MMPs), the largest class of human proteases, has been implicated in brain damage in both animal and human studies. Some MMPs are elevated after stroke (both in plasma and in brain tissue), and their expression is enhanced by t‐PA during thrombolysis related to hemorrhagic transformation events. Although the exact cellular source of MMPs remains unknown, brain endothelium, astrocytes, neurons, and inflammatory‐activated cells, such as neutrophils, may release MMP‐2, MMP‐3, MMP‐8, MMP‐9, MMP‐10, and/or MMP‐13. Neurovascular perturbations occurring after stroke lead to blood–brain barrier leakage, edema, hemorrhage, leukocyte infiltration, and progressive inflammatory reactions to brain injury over hours or even days after the initial stroke. Synthesized MMP inhibitors and several compounds used for stroke secondary prevention, such as anti‐inflammatory drugs, might decrease MMPs and improve the acute treatment of human brain ischemia without compromising the beneficial effects of matrix plasticity during stroke recovery.

Increased intranuclear matrix metalloproteinase activity in neurons interferes with oxidative DNA repair in focal cerebral ischemia

J. Neurochem. (2010) 112, 134–149.