TY - JOUR A1 - Kraft, Peter A1 - Drechsler, Christiane A1 - Gunreben, Ignaz A1 - Nieswandt, Bernhard A1 - Stoll, Guido A1 - Heuschmann, Peter Ulrich A1 - Kleinschnitz, Christoph T1 - Von Willebrand Factor Regulation in Patients with Acute and Chronic Cerebrovascular Disease: A Pilot, Case-Control Study JF - PLoS ONE N2 - Background and Purpose In animal models, von Willebrand factor (VWF) is involved in thrombus formation and propagation of ischemic stroke. However, the pathophysiological relevance of this molecule in humans, and its potential use as a biomarker for the risk and severity of ischemic stroke remains unclear. This study had two aims: to identify predictors of altered VWF levels and to examine whether VWF levels differ between acute cerebrovascular events and chronic cerebrovascular disease (CCD). Methods A case–control study was undertaken between 2010 and 2013 at our University clinic. In total, 116 patients with acute ischemic stroke (AIS) or transitory ischemic attack (TIA), 117 patients with CCD, and 104 healthy volunteers (HV) were included. Blood was taken at days 0, 1, and 3 in patients with AIS or TIA, and once in CCD patients and HV. VWF serum levels were measured and correlated with demographic and clinical parameters by multivariate linear regression and ANOVA. Results Patients with CCD (158±46%) had significantly higher VWF levels than HV (113±36%, P<0.001), but lower levels than AIS/TIA patients (200±95%, P<0.001). Age, sex, and stroke severity influenced VWF levels (P<0.05). Conclusions VWF levels differed across disease subtypes and patient characteristics. Our study confirms increased VWF levels as a risk factor for cerebrovascular disease and, moreover, suggests that it may represent a potential biomarker for stroke severity, warranting further investigation. KW - cerebrovascular diseases KW - sex addiction KW - biomarkers KW - ischemic stroke KW - blood KW - stroke KW - platelets KW - demography Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119588 SN - 1932-6203 VL - 9 IS - 6 ER - TY - JOUR A1 - Kraft, Peter A1 - Schwarz, Tobias A1 - Meijers, Joost C. M. A1 - Stoll, Guido A1 - Kleinschnitz, Christoph T1 - Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) Deficient Mice Are Susceptible to Intracerebral Thrombosis and Ischemic Stroke N2 - Background: Thrombus formation is a key step in the pathophysiology of acute ischemic stroke and results from the activation of the coagulation cascade. Thrombin plays a central role in this coagulation system and contributes to thrombus stability via activation of thrombin-activatable fibrinolysis inhibitor (TAFIa). TAFIa counteracts endogenous fibrinolysis at different stages and elevated TAFI levels are a risk factor for thrombotic events including ischemic stroke. Although substantial in vitro data on the influence of TAFI on the coagulation-fibrinolysis-system exist, investigations on the consequences of TAFI inhibition in animal models of cerebral ischemia are still lacking. In the present study we analyzed stroke development and post stroke functional outcome in TAFI-/- mice. Methodology/Principal Findings: TAFI-/- mice and wild-type controls were subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 hours, functional outcome scores were assessed and infarct volumes weremeasured from 2,3,5-Triphenyltetrazoliumchloride (TTC)-stained brain slices. Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus formation within the infarcted brain areas was analyzed by immunoblot. Infarct volumes and functional outcomes did not significantly differ between TAFI-/- mice and controls (p.0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. TAFI deficiency also had no influence on intracerebral fibrin(ogen) formation after tMCAO. Conclusion: Our study shows that TAFI does not play a major role for thrombus formation and neuronal degeneration after ischemic brain challenge. KW - Thrombus Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68519 ER - TY - JOUR A1 - Ruck, Tobias A1 - Bittner, Stefan A1 - Afzali, Ali Maisam A1 - Göbel, Kerstin A1 - Glumm, Sarah A1 - Kraft, Peter A1 - Sommer, Claudia A1 - Kleinschnitz, Christoph A1 - Preusse, Corinna A1 - Stenzel, Werner A1 - Wiendl, Heinz A1 - Meuth, Sven G. T1 - The NKG2D-IL-15 signaling pathway contributes to T-cell mediated pathology in inflammatory myopathies JF - Oncotarget N2 - NKG2D is an activating receptor on T cells, which has been implicated in the pathogenesis of autoimmune diseases. T cells are critically involved in idiopathic inflammatory myopathies (IIM) and have been proposed as specific therapeutic targets. However, the mechanisms underlying T cell-mediated progressive muscle destruction in IIM remain to be elucidated. We here determined the involvement of the NKG2D - IL-15 signaling pathway. Primary human myoblasts expressed NKG2D ligands, which were further upregulated upon inflammatory stimuli. In parallel, shedding of the soluble NKG2D ligand MICA (sMICA) decreased upon inflammation potentially diminishing inhibition of NKG2D signaling. Membrane-related expression of IL-15 by myoblasts induced differentiation of naive CD8\(^+\) T cells into highly activated, cytotoxic \(CD8^+NKG2D^{high}\) T cells demonstrating NKG2D-dependent lysis of myoblasts in vitro. \(CD8^+NKG2D^{high}\) T cell frequencies were increased in the peripheral blood of polymyositis (PM) patients and correlated with serum creatinine kinase concentrations, while serum sMICA levels were not significantly changed. In muscle biopsy specimens from PM patients expression of the NKG2D ligand MICA/B was upregulated, IL-15 was expressed by muscle cells, CD68\(^+\) macrophages as well as CD4\(^+\) T cells, and \(CD8^+NKG2D^+\) cells were frequently detected within inflammatory infiltrates arguing for a local signaling circuit in the inflammatory muscle milieu. In conclusion, the NKG2D - IL-15 signaling pathway contributes to progressive muscle destruction in IIM potentially opening new therapeutic avenues. KW - MIC ligands KW - pathology section KW - T cell activation KW - idiopathic inflammatory myopathies KW - polymyositis KW - IL-15 KW - NKG2D KW - receptor KW - expression KW - lymphokine-activated killer KW - human muscle-cells KW - multiple sclerosis KW - celiac disease KW - tumor immunity KW - NKG2D ligands KW - cutting edge Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136047 VL - 6 IS - 41 ER - TY - JOUR A1 - Pham, Mirko A1 - Helluy, Xavier A1 - Kleinschnitz, Christoph A1 - Kraft, Peter A1 - Bartsch, Andreas J. A1 - Jakob, Peter A1 - Nieswandt, Bernhard A1 - Bendszus, Martin A1 - Guido, Stoll T1 - Sustained Reperfusion after Blockade of Glycoprotein-Receptor-Ib in Focal Cerebral Ischemia: An MRI Study at 17.6 Tesla JF - PLoS ONE N2 - Background: Inhibition of early platelet adhesion by blockade of glycoprotein-IB (GPIb) protects mice from ischemic stroke. To elucidate underlying mechanisms in-vivo, infarct development was followed by ultra-high field MRI at 17.6 Tesla. Methods: Cerebral infarction was induced by transient-middle-cerebral-artery-occlusion (tMCAO) for 1 hour in C57/BL6 control mice (N = 10) and mice treated with 100 mg Fab-fragments of the GPIb blocking antibody p0p/B 1 h after tMCAO (N = 10). To control for the effect of reperfusion, additional mice underwent permanent occlusion and received anti-GPIb treatment (N = 6; pMCAO) or remained without treatment (N = 3; pMCAO). MRI 2 h and 24 h after MCAO measured cerebral-blood-flow (CBF) by continuous arterial-spin labelling, the apparent-diffusion-coefficient (ADC), quantitative-T2 and T2-weighted imaging. All images were registered to a standard mouse brain MRI atlas and statistically analysed voxel-wise, and by cortico-subcortical ROI analysis. Results: Anti-GPIb treatment led to a relative increase of postischemic CBF vs. controls in the cortical territory of the MCA (2 h: 44.2 +/- 6.9 ml/100g/min versus 24 h: 60.5 +/- 8.4; p = 0.0012, F((1,18)) = 14.63) after tMCAO. Subcortical CBF 2 h after tMCAO was higher in anti-GPIb treated animals (45.3 +/- 5.9 vs. controls: 33.6 +/- 4.3; p = 0.04). In both regions, CBF findings were clearly related to a lower probability of infarction (Cortex/Subcortex of treated group: 35%/65% vs. controls: 95%/100%) and improved quantitative-T2 and ADC. After pMCAO, anti-GPIb treated mice developed similar infarcts preceded by severe irreversible hypoperfusion as controls after tMCAO indicating dependency of stroke protection on reperfusion. Conclusion: Blockade of platelet adhesion by anti-GPIb-Fab-fragments results in substantially improved CBF early during reperfusion. This finding was in exact spatial correspondence with the prevention of cerebral infarction and indicates in-vivo an increased patency of the microcirculation. Thus, progression of infarction during early ischemia and reperfusion can be mitigated by anti-platelet treatment. KW - Von-Willebrand-factor KW - Experimental stroke KW - Magnetic-resonance KW - Arterial water KW - Brain KW - Perfusion KW - Mice KW - Inflammation KW - Coefficient KW - mechanisms Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142608 VL - 6 IS - 4 ER - TY - JOUR A1 - Kraft, Peter A1 - Drechsler, Christiane A1 - Gunreben, Ignaz A1 - Heuschmann, Peter Ulrich A1 - Kleinschnitz, Christoph T1 - Regulation of Blood Coagulation Factors XI and XII in Patients with Acute and Chronic Cerebrovascular Disease: A Case-Control Study JF - Cerebrovascular Diseases N2 - Background: Animal models have implicated an integral role for coagulation factors XI (FXI) and XII (FXII) in thrombus formation and propagation of ischemic stroke (IS). However, it is unknown if these molecules contribute to IS pathophysiology in humans, and might be of use as biomarkers for IS risk and severity. This study aimed to identify predictors of altered FXI and FXII levels and to determine whether there are differences in the levels of these coagulation factors between acute cerebrovascular events and chronic cerebrovascular disease (CCD). Methods: In this case-control study, 116 patients with acute ischemic stroke (AIS) or transitory ischemic attack (TIA), 117 patients with CCD, and 104 healthy volunteers (HVs) were enrolled between 2010 and 2013 at our University hospital. Blood sampling was undertaken once in the CCD and HV groups and on days 0, 1, and 3 after stroke onset in patients with AIS or TIA. Correlations between serum FXI and FXII levels and demographic and clinical parameters were tested by linear regression and analysis of variance. Results: The mean age of AIS/TIA patients was 70 ± 12. Baseline clinical severity measured with NIHSS and Barthel Index was 4.8 ± 6.0 and 74 ± 30, respectively. More than half of the patients had an AIS (58%). FXI levels were significantly correlated with different leukocyte subsets (p < 0.05). In contrast, FXII serum levels showed no significant correlation (p > 0.1). Neither FXI nor FXII levels correlated with CRP (p > 0.2). FXII levels were significantly higher in patients with CCD compared with those with AIS/TIA (mean ± SD 106 ± 26% vs. 97 ± 24%; univariate analysis: p < 0.05); these differences did not reach significance in multivariate analysis adjusted for sex and age. FXI levels did not differ significantly between study groups. Sex and age were significantly associated with FXI and/or FXII levels in patients with AIS/TIA (p < 0.05). In contrast, no statistical significant influence was found for treatment modality (thrombolysis or not), pre-treatment with platelet inhibitors, and severity of stroke. Conclusions: In this study, there was no differential regulation of FXI and FXII levels between disease subtypes but biomarker levels were associated with patient and clinical characteristics. FXI and FXII levels might be no valid biomarker for predicting stroke risk. KW - biomarker KW - factor XI KW - factor XII KW - ischemic stroke KW - chronic cerebrovascular disease Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199076 SN - 1015-9770 SN - 1421-9786 N1 - This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. VL - 38 IS - 5 ER - TY - JOUR A1 - Heuschmann, Peter U. A1 - Montellano, Felipe A. A1 - Ungethüm, Kathrin A1 - Rücker, Viktoria A1 - Wiedmann, Silke A1 - Mackenrodt, Daniel A1 - Quilitzsch, Anika A1 - Ludwig, Timo A1 - Kraft, Peter A1 - Albert, Judith A1 - Morbach, Caroline A1 - Frantz, Stefan A1 - Störk, Stefan A1 - Haeusler, Karl Georg A1 - Kleinschnitz, Christoph T1 - Prevalence and determinants of systolic and diastolic cardiac dysfunction and heart failure in acute ischemic stroke patients: The SICFAIL study JF - ESC Heart Failure N2 - Aims Ischaemic stroke (IS) might induce alterations of cardiac function. Prospective data on frequency of cardiac dysfunction and heart failure (HF) after IS are lacking. We assessed prevalence and determinants of diastolic dysfunction (DD), systolic dysfunction (SD), and HF in patients with acute IS. Methods and results The Stroke‐Induced Cardiac FAILure in mice and men (SICFAIL) study is a prospective, hospital‐based cohort study. Patients with IS underwent a comprehensive assessment of cardiac function in the acute phase (median 4 days after IS) including clinical examination, standardized transthoracic echocardiography by expert sonographers, and determination of blood‐based biomarkers. Information on demographics, lifestyle, risk factors, symptoms suggestive of HF, and medical history was collected by a standardized personal interview. Applying current guidelines, cardiac dysfunction was classified based on echocardiographic criteria into SD (left ventricular ejection fraction < 52% in men or <54% in women) and DD (≥3 signs of DD in patients without SD). Clinically overt HF was classified into HF with reduced, mid‐range, or preserved ejection fraction. Between January 2014 and February 2017, 696 IS patients were enrolled. Of them, patients with sufficient echocardiographic data on SD were included in the analyses {n = 644 patients [median age 71 years (interquartile range 60–78), 61.5% male]}. In these patients, full assessment of DD was feasible in 549 patients without SD (94%). Prevalence of cardiac dysfunction and HF was as follows: SD 9.6% [95% confidence interval (CI) 7.6–12.2%]; DD in patients without SD 23.3% (95% CI 20.0–27.0%); and clinically overt HF 5.4% (95% CI 3.9–7.5%) with subcategories of HF with preserved ejection fraction 4.35%, HF with mid‐range ejection fraction 0.31%, and HF with reduced ejection fraction 0.78%. In multivariable analysis, SD and fulfilment of HF criteria were associated with history of coronary heart disease [SD: odds ratio (OR) 3.87, 95% CI 1.93–7.75, P = 0.0001; HF: OR 2.29, 95% CI 1.04–5.05, P = 0.0406] and high‐sensitive troponin T at baseline (SD: OR 1.78, 95% CI 1.31–2.42, P = 0.0003; HF: OR 1.66, 95% CI 1.17–2.33, P = 0.004); DD was associated with older age (OR 1.08, 95% CI 1.05–1.11, P < 0.0001) and treated hypertension vs. no hypertension (OR 2.84, 95% CI 1.23–6.54, P = 0.0405). Conclusions A substantial proportion of the study population exhibited subclinical and clinical cardiac dysfunction. SICFAIL provides reliable data on prevalence and determinants of SD, DD, and clinically overt HF in patients with acute IS according to current guidelines, enabling further clarification of its aetiological and prognostic role. KW - Stroke KW - Heart failure KW - Cardiac dysfunction| Brain natriuretic peptide KW - Troponin Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225656 VL - 8 IS - 2 ER - TY - JOUR A1 - Kleinschnitz, Christoph A1 - Grund, Henrike A1 - Wingler, Kirstin A1 - Armitage, Melanie E. A1 - Jones, Emma A1 - Mittal, Manish A1 - Barit, David A1 - Schwarz, Tobias A1 - Geis, Christian A1 - Kraft, Peter A1 - Barthel, Konstanze A1 - Schuhmann, Michael K. A1 - Herrmann, Alexander M. A1 - Meuth, Sven G. A1 - Stoll, Guido A1 - Meurer, Sabine A1 - Schrewe, Anja A1 - Becker, Lore A1 - Gailus-Durner, Valerie A1 - Fuchs, Helmut A1 - Klopstock, Thomas A1 - de Angelis, Martin Hrabe A1 - Jandeleit-Dahm, Karin A1 - Shah, Ajay M. A1 - Weissmann, Norbert A1 - Schmidt, Harald H. H. W. T1 - Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration N2 - Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox42/2) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox42/2 mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy. KW - Schlaganfall Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68416 ER - TY - JOUR A1 - Kraft, Peter A1 - De Meyer, Simon F. A1 - Kleinschnitz, Christoph T1 - Next-Generation Antithrombotics in Ischemic Stroke: Preclinical Perspective on ‘Bleeding-Free Antithrombosis’ JF - Journal of Cerebral Blood Flow and Metabolism N2 - The present antithrombotic drugs used to treat or prevent ischemic stroke have significant limitations: either they show only moderate efficacy (platelet inhibitors), or they significantly increase the risk for hemorrhages (thrombolytics, anticoagulants). Although most strokes are caused by thrombotic or embolic vessel occlusions, the pathophysiological role of platelets and coagulation is largely unclear. The introduction of novel transgenic mouse models and specific coagulation inhibitors facilitated a detailed analysis of molecular pathways mediating thrombus formation in models of acute ischemic stroke. Prevention of early platelet adhesion to the damaged vessel wall by blocking platelet surface receptors glycoprotein Ib alpha (GPIbα) or glycoprotein VI (GPVI) protects from stroke without provoking bleeding complications. In addition, downstream signaling of GPIbα and GPVI has a key role in platelet calcium homeostasis and activation. Finally, the intrinsic coagulation cascade, activated by coagulation factor XII (FXII), has only recently been identified as another important mediator of thrombosis in cerebrovascular disease, thereby disproving established concepts. This review summarizes the latest insights into the pathophysiology of thrombus formation in the ischemic brain. Potential clinical merits of novel platelet inhibitors and anticoagulants as powerful and safe tools to combat ischemic stroke are discussed. KW - von Willebrand factor KW - platelets KW - glycoprotein Ib KW - FXII KW - coagulation KW - Stim Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126538 VL - 32 IS - 10 ER - TY - JOUR A1 - Schuhmann, Michael K. A1 - Gunreben, Ignaz A1 - Kleinschnitz, Christoph A1 - Kraft, Peter T1 - Immunohistochemical Analysis of Cerebral Thrombi Retrieved by Mechanical Thrombectomy from Patients with Acute Ischemic Stroke JF - International Journal of Molecular Sciences N2 - Mechanical thrombectomy is a novel treatment option for patients with acute ischemic stroke (AIS). Only a few studies have previously suggested strategies to categorize retrieved clots according to their histologic composition. However, these reports did not analyze potential biomarkers that are of importance in stroke-related inflammation. We therefore histopathologically investigated 37 intracerebral thrombi mechanically retrieved from patients with AIS, and focused on the composition of immune cells and platelets. We also conducted correlation analyses of distinctive morphologic patterns (erythrocytic, serpentine, layered, red, white, mixed appearance) with clinical parameters. Most T cells and monocytes were detected in erythrocytic and red clots, in which the distribution of these cells was random. In contrast, von Willebrand factor (vWF)-positive areas co-localized with regions of fibrin and collagen. While clots with huge amounts of vWF seem to be associated with a high National Institute of Health Stroke Scale score at admission, histologic findings could not predict the clinical outcome at discharge. In summary, we provide the first histologic description of mechanically retrieved intracerebral thrombi regarding biomarkers relevant for inflammation in ischemic stroke. KW - thrombus formation KW - immune cells KW - lymphocytes KW - mechanical thrombectomy KW - ischemic stroke KW - inflammation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166206 VL - 17 IS - 3 ER - TY - JOUR A1 - Kraft, Peter A1 - Schuhmann, Michael K. A1 - Garz, Cornelia A1 - Jandke, Solveig A1 - Urlaub, Daniela A1 - Mencl, Stine A1 - Zernecke, Alma A1 - Heinze, Hans-Jochen A1 - Carare, Roxana O. A1 - Kleinschnitz, Christoph A1 - Schreiber, Stefanie T1 - Hypercholesterolemia induced cerebral small vessel disease JF - PLoS ONE N2 - Background While hypercholesterolemia plays a causative role for the development of ischemic stroke in large vessels, its significance for cerebral small vessel disease (CSVD) remains unclear. We thus aimed to understand the detailed relationship between hypercholesterolemia and CSVD using the well described Ldlr\(^{−/-}\) mouse model. Methods We used Ldlr\(^{−/-}\) mice (n = 16) and wild-type (WT) mice (n = 15) at the age of 6 and 12 months. Ldlr\(^{−/-}\) mice develop high plasma cholesterol levels following a high fat diet. We analyzed cerebral capillaries and arterioles for intravascular erythrocyte accumulations, thrombotic vessel occlusions, blood-brain barrier (BBB) dysfunction and microbleeds. Results We found a significant increase in the number of erythrocyte stases in 6 months old Ldlr\(^{−/-}\) mice compared to all other groups (P < 0.05). Ldlr\(^{−/-}\) animals aged 12 months showed the highest number of thrombotic occlusions while in WT animals hardly any occlusions could be observed (P < 0.001). Compared to WT mice, Ldlr\(^{−/-}\) mice did not display significant gray matter BBB breakdown. Microhemorrhages were observed in one Ldlr\(^{−/-}\) mouse that was 6 months old. Results did not differ when considering subcortical and cortical regions. Conclusions In Ldlr\(^{−/-}\) mice, hypercholesterolemia is related to a thrombotic CSVD phenotype, which is different from hypertension-related CSVD that associates with a hemorrhagic CSVD phenotype. Our data demonstrate a relationship between hypercholesterolemia and the development of CSVD. Ldlr\(^{−/-}\) mice appear to be an adequate animal model for research into CSVD. KW - hypercholesterolemia KW - cerebral small vessel disease KW - mouse model KW - histology Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170493 VL - 12 IS - 8 ER -