Saskia Grudzenski

Effect of Simvastatin on MMPs and TIMPs in Human Brain Endothelial Cells and Experimental Stroke

Clinical studies demonstrated favorable effects of statins in stroke beyond lipid-lowering effects. In acute stroke, the disruption of the blood–brain barrier (BBB) is mediated by matrix metalloproteinases (MMPs). A modified MMP metabolism may account for the beneficial effects of statins. Cultured human brain microvascular endothelial cells (BMECs) were pretreated with simvastatin and subjected to oxygen glucose deprivation (OGD). Gene expression and protein secretion of MMP-2 and MMP-9 and the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were measured by quantitative real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Simvastatin significantly dampened the expression but not secretion of MMP-2 under OGD. MMP-9 synthesis rate was low and unaffected by simvastatin treatment, while the gene expression and protein secretion of TIMP-1 and TIMP-2 were both strongly induced. Our results provide evidence for a positive effect of simvastatin on the MMP metabolism in human BMECs and experimental stroke mainly by means of the increased expression and secretion of TIMP-1 and TIMP-2.

Temporal Profile of Matrix Metalloproteinases and Their Inhibitors in a Human Endothelial Cell Culture Model of Cerebral Ischemia

Background: Matrix metalloproteinases (MMPs) are key players in proteolytic blood-brain barrier (BBB) disruption during ischemic stroke, leading to vascular edema, hemorrhagic transformation and infiltration by leukocytes. Their effect is dampened by the endogenous tissue inhibitors of metalloproteinases (TIMPs). The respective cellular source of specific MMPs and TIMPs during BBB breakdown is still under investigation. Methods: We analyzed the MMP and TIMP release of human brain microvascular endothelial cells (BMECs) under oxygen glucose deprivation (OGD). Cultured human BMECs (the hCMEC/D3 cell line) were subjected to OGD (6, 12, 18 and 24 h). Gene expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were serially measured by quantitative real time-PCR and compared to ELISA-detected cell culture medium levels. Results: OGD induced a significant and long-lasting increase in MMP-2 gene expression, reaching a plateau after 12 h. Medium protein levels of MMP-2 were correspondingly elevated at 12 h of OGD. The MMP-9 synthesis rate was detectable at very low levels and remained unaffected by OGD. TIMP-1 gene expression and secretion declined under OGD, whereas both expression and secretion of TIMP-2 remained stable. Contrary to the respective gene expression rate, medium levels of MMP-2, TIMP-1 and TIMP-2 started a simultaneous decline after 12 h of OGD. This is most likely due to an impaired synthesis and enhanced consumption rate under OGD. Conclusions: The objective of our study was to determine the contribution of human BMECs to the MMP metabolism under in vitro OGD conditions simulating ischemic stroke. Our results suggest that human BMECs switch to a proinflammatory state by means of an enhanced production of MMP-2, attenuated release of TIMP-1, and unaffected production of TIMP-2. Thus, human BMECs might participate in the MMP-mediated BBB breakdown during ischemic stroke. However, our data does not support human BMECs to be a source of MMP-9.

Chemical shift sodium imaging in a mouse model of thromboembolic stroke at 9.4 T

To estimate changes in the 23Na density and in the 23Na relaxation time T2* in the anatomically small murine brain after stroke.

Thrombolysis in Experimental Cerebral Amyloid Angiopathy and the Risk of Secondary Intracerebral Hemorrhage

Background and Purpose— Intracerebral hemorrhage (ICH) is the most adverse event of thrombolysis in ischemic stroke. Cerebral amyloid angiopathy increases the risk for spontaneous lobar ICH. Although thrombolysis may be performed in cerebral amyloid angiopathy–affected patients, there is still little knowledge available on the risk for secondary ICH. Methods— We investigated the effect of recombinant tissue-type plasminogen activator on experimental ischemic stroke in APP23 transgenic mice (n=18) and wild-type littermates (n=15). Focal ischemic stroke was induced in 26-month-old mice by temporal middle cerebral artery occlusion (filament model), followed by treatment with 10 mg/kg recombinant tissue-type plasminogen activator. Twenty-four hours later, a functional score was assessed and the mice were euthanized for histological analysis. ICH was classified as grades 1 to 3 depending on severity. Results— The groups did not differ regarding mortality (P=0.67) and functional deficit (P=0.18). Compared with wild-type mice, the APP23 genotype was associated with a higher appearance for ICH in the infarct area (P=0.05). ICH severity grades 2 and 3 correlated significantly with infarct size (P=0.004 and 0.008, respectively). Conclusions— The APP23 genotype was not associated with increased mortality or worse functional outcome. Our results suggest an increased risk for ICH in the cerebral amyloid angiopathy–affected brain; however, no ICH was observed outside the ischemic area.

The effect of adipose tissue-derived stem cells in a middle cerebral artery occlusion stroke model depends on their engraftment rate

BackgroundIn the field of experimental stem cell therapy, intra-arterial (IA) delivery yields the best results concerning, for example, migrated cell number at the targeted site. However, IA application also appears to be associated with increased mortality rates and infarction. Since many rodent studies systemically apply 1 × 106 cells, this could also be a consequence of engrafted cell number. The aim of this study was therefore to investigate the effect of different doses of adipose tissue-derived stem cells (ASCs) on engraftment rates and stroke outcome measured in vivo using 9.4-T high-field magnetic resonance imaging (MRI).MethodsMale Wistar rats (n = 43) underwent a middle cerebral artery occlusion (MCAo) for 45 or 90 min, followed by IA delivery of either saline or 1 × 106, 3 × 105, or 5 × 104 ASCs pre-labelled with very small superparamagnetic iron oxide particles (VSOPs). MRI (9.4-T) analysis was performed 48 h and 9 days post-MCAo. Lesion volumes were assessed by analysis of T2-weighted images and cell signal tracking showing cell engraftment and active cell migration by an improved T2*-analysis.ResultsThe ASC-derived signal intensity increased in the affected hemisphere 48 h post MCAo with injected cell number (p < 0.05). The analysis of stroke volumes revealed an increased infarction after injection of 1 × 106 ASCs compared to controls or application of 5 × 104 ASCs (p < 0.05). At 9 days post-MCAo, injection of 3 × 105 ASCs resulted in reduced infarct volumes (p < 0.05). Correspondingly, MRI analysis revealed no changes in cell numbers between both MRI examinations but showed active ASC migration to the site of infarction.ConclusionOur results confirm that IA injection is an efficient way of targeting damaged brain tissue but its usefulness strongly depends on the right dose of delivered stem cells since this factor has a strong influence on migration rate and infarct volume, with better results for doses below 1 × 106 cells. Future challenges will include the determination of therapeutic doses for best cellular engraftment and stroke outcome.

Efficacy of Alteplase in a Mouse Model of Acute Ischemic Stroke A Retrospective Pooled Analysis

Background and Purpose— The debate over the fact that experimental drugs proposed for the treatment of stroke fail in the translation to the clinical situation has attracted considerable attention in the literature. In this context, we present a retrospective pooled analysis of a large data set from preclinical studies, to examine the effects of early versus late administration of intravenous recombinant tissue-type plasminogen activator. Methods— We collected data from 26 individual studies from 9 international centers (13 researchers; 716 animals) that compared recombinant tissue-type plasminogen activator with controls, in a unique mouse model of thromboembolic stroke induced by an in situ injection of thrombin into the middle cerebral artery. Studies were classified into early (<3 hours) versus late (≥3 hours) drug administration. Final infarct volumes, assessed by histology or magnetic resonance imaging, were compared in each study, and the absolute differences were pooled in a random-effect meta-analysis. The influence of time of administration was tested. Results— When compared with saline controls, early recombinant tissue-type plasminogen activator administration was associated with a significant benefit (absolute difference, −6.63 mm3; 95% confidence interval, −9.08 to −4.17; I2=76%), whereas late recombinant tissue-type plasminogen activator treatment showed a deleterious effect (+5.06 mm3; 95% confidence interval, +2.78 to +7.34; I2=42%; Pint<0.00001). Results remained unchanged after subgroup analyses. Conclusions— Our results provide the basis needed for the design of future preclinical studies on recanalization therapies using this model of thromboembolic stroke in mice. The power analysis reveals that a multicenter trial would require 123 animals per group instead of 40 for a single-center trial.

Development of cerebral microbleeds in the APP23-transgenic mouse model of cerebral amyloid angiopathy - a 9.4 Tesla MRI study

Background: Cerebral amyloid angiopathy (CAA) is characterized by extracellular deposition of amyloid β (Aβ) around cerebral arteries and capillaries and leads to an increased risk for vascular dementia, spontaneous lobar hemorrhage, convexal subarachnoid hemorrhage, and transient focal neurological episodes, which might be an indicator of imminent spontaneous intracerebral hemorrhage. In CAA cerebral microbleeds (cMBs) with a cortical/juxtacortical distribution are frequently observed in standard magnetic resonance imaging (MRI). In vivo MRI of transgenic mouse models of CAA may serve as a useful tool to investigate translational aspects of the disease. Materials and Methods: APP23-transgenic mice demonstrate cerebrovascular Aβ deposition with subsequent neuropathological changes characteristic for CAA. We performed a 9.4 Tesla high field MRI study using T2, T2* and time of flight-magnetic resonance angiograpy (TOF-MRA) sequences in APP23-transgenic mice and wildtype (wt) littermates at the age of 8, 12, 16, 20 and 24 months, respectively. Numbers, size, and location of cMBs are reported. Results: T2* imaging demonstrated cMBs (diameter 50–300 μm) located in the neocortex and, to a lesser degree, in the thalamus. cMBs were detected at the earliest at 16 months of age. Numbers increased exponentially with age, with 2.5 ± 2 (median ± interquartilrange) at 16 months, 15 ± 6 at 20 months, and 31.5 ± 17 at 24 months of age, respectively. Conclusion: We report the temporal and spatial development of cMBs in the aging APP23-transgenic mouse model which develops characteristic pathological patterns known from human CAA. We expect this mouse model to serve as a useful tool to non-invasively monitor mid- and longterm translational aspects of CAA and to investigate experimental therapeutic strategies in longitudinal studies.

Thromboembolic stroke in C57BL/6 mice monitored by 9.4 T MRI using a 1H cryo probe

BackgroundA new thromboembolic animal model showed beneficial effects of t-PA with an infarct volume reduction of 36.8% in swiss mice. Because knock-out animal experiments for stroke frequently used C57BL76 mice we evaluated t-PA effects in this mouse strain and measured infarct volume and vascular recanalisation in-vivo by using high-field 9.4 T MRI and a 1H surface cryo coil.MethodsClot formation was triggered by microinjection of murine thrombin into the right middle cerebral artery (MCA). Animals (n = 28) were treated with 10 mg/kg, 5 mg/kg or no tissue plasminogen activator (t-PA) 40 min after MCA occlusion. For MR-imaging a Bruker 9.4 T animal system with a 1H surface cryo probe was used and a T2-weighted RARE sequence, a diffusion weighted multishot EPI sequence and a 3D flow-compensated gradient echo TOF angiography were performed.ResultsThe infarct volume in animals treated with t-PA was significantly reduced (0.67 ± 1.38 mm3 for 10 mg/kg and 10.9 ± 8.79 mm3 for 5 mg/kg vs. 19.76 ± 2.72 mm3 ; p < 0.001) compared to untreated mice. An additional group was reperfused with t-PA inside the MRI. Already ten minutes after beginning of t-PA treatment, reperfusion flow was re-established in the right MCA. However, signal intensity was lower than in the contralateral MCA. This reduction in cerebral blood flow was attenuated during the first 60 minutes after reperfusion. 24 h after MCA occlusion and reperfusion, no difference in signal intensity of the contralateral and ipsilateral MCAs was observed.ConclusionsWe confirm a t-Pa effect using this stroke model in the C57BL76 mouse strain and demonstrate a chronological sequence MRI imaging after t-PA using a 1H surface cryo coil in a 9.4 T MRI. This setting will allow testing of new thrombolytic strategies for stroke treatment in-vivo in C57BL76 knock-out mice.

Statin Therapy and the Development of Cerebral Amyloid Angiopathy--A Rodent in Vivo Approach.

Background: Cerebral amyloid angiopathy (CAA) is characterized by vascular deposition of amyloid β (Aβ) with a higher incidence of cerebral microbleeds (cMBs) and spontaneous hemorrhage. Since statins are known for their benefit in vascular disease we tested for the effect on CAA. Methods: APP23-transgenic mice received atorvastatin-supplemented food starting at the age of eight months (n = 13), 12 months (n = 7), and 16 months (n = 6), respectively. Controls (n = 16) received standard food only. At 24 months of age cMBs were determined with T2*-weighted 9.4T magnetic resonance imaging and graded by size. Results: Control mice displayed an average of 35 ± 18.5 cMBs (mean ± standard deviation), compared to 29.3 ± 9.8 in mice with eight months (p = 0.49), 24.9 ± 21.3 with 12 months (p = 0.26), and 27.8 ± 15.4 with 16 months of atorvastatin treatment (p = 0.27). In combined analysis treated mice showed lower absolute numbers (27.4 ± 15.6, p = 0.16) compared to controls and also after adjustment for cMB size (p = 0.13). Conclusion: Despite to a non-significant trend towards fewer cMBs our results failed to provide evidence for beneficial effects of long-term atorvastatin treatment in the APP23-transgenic mouse model of CAA. A higher risk for bleeding complications was not observed.