Björn Reuter

Time to treatment with recombinant tissue plasminogen activator and outcome of stroke in clinical practice: retrospective analysis of hospital quality assurance data with comparison with results from randomised clinical trials

Objective To study the time dependent effectiveness of thrombolytic therapy for acute ischaemic stroke in daily clinical practice. Design A retrospective cohort study using data from a large scale, comprehensive population based state-wide stroke registry in Germany. Setting All 148 hospitals involved in acute stroke care in a large state in southwest Germany with 10.4 million inhabitants. Participants Data from 84 439 patients with acute ischaemic stroke were analysed, 10 263 (12%) were treated with thrombolytic therapy and 74 176 (88%) were not treated. Main outcome measures Primary endpoint was the dichotomised score on a modified Rankin scale at discharge (“favourable outcome” score 0 or 1 or “unfavourable outcome” score 2-6) analysed by binary logistic regression. Patients treated with recombinant tissue plasminogen activator (rtPA) were categorised according to time from onset of stroke to treatment. Analogous analyses were conducted for the association between rtPA treatment of stroke and in-hospital mortality. As a co-primary endpoint the chance of a lower modified Rankin scale score at discharge was analysed by ordinal logistic regression analysis (shift analysis). Results After adjustment for characteristics of patients, hospitals, and treatment, rtPA was associated with better outcome in a time dependent pattern. The number needed to treat ranged from 4.5 (within first 1.5 hours after onset; odds ratio 2.49) to 18.0 (up to 4.5 hours; odds ratio 1.26), while mortality did not vary up to 4.5 hours. Patients treated with rtPA beyond 4.5 hours (including mismatch based approaches) showed a significantly better outcome only in dichotomised analysis (odds ratio 1.25, 95% confidence interval 1.01 to 1.55) but the mortality risk was higher (1.45, 1.08 to 1.92). Conclusion The effectiveness of thrombolytic therapy in daily clinical practice might be comparable with the effectiveness shown in randomised clinical trials and pooled analysis. Early treatment was associated with favourable outcome in daily clinical practice, which underlines the importance of speeding up the process for thrombolytic therapy in hospital and before admission to achieve shorter time from door to needle and from onset to treatment for thrombolytic therapy.

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.

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.

Aphemia: an isolated disorder of speech associated with an ischemic lesion of the left precentral gyrus

Sirs, Aphemia, also termed apraxia of speech, cortical anarthria or pure word mutism, is an isolated disorder of coordinated speech articulation that results in severe affection of verbal motor output [8, 9]. This rare and possibly underdiagnosed syndrome was first postulated by Paul Broca in 1861 [1]; however, the definition remained imprecise during the course of history. Especially the differentiation between aphemia and aphasia—the latter of which was shortly after described by Armand Trousseau— as well as dysarthria has been in discussion since then. A 61-year-old right-handed dentist with treated hypertension awoke with acute loss of speech and saliva running out of the right corner of his mouth. He wrote ‘‘stroke’’ on a piece of paper for his wife, who called the ambulance. In the emergency room, he was nearly mute due to grossly distorted motor output, but he had full comprehension of speech, could communicate through gestures and intact, fluent writing (Fig. 1a). Speech was very difficult with strangled vowels, severe phonematic paraphasia and abnormal prosody that was not facilitated by singing, reading or repetition. This and the constant effort to correct himself cumulated in visible frustration, whereas, surprisingly, the patient was able to produce—apparently emotionally triggered—short, but intact and fluently articulated commentaries of the situation (e.g., ‘‘It’s not working!’’, ‘‘That’s terrible!’’). There was a mild right facial palsy without further affection of coordinated buccofacial movement (whistling, etc.) or oropharyngeal sensibility (NIHSS 4). Diffusion-weighted MRI performed shortly after presentation to the emergency room showed an acute ischemic lesion of the left precentral gyrus that was not yet visible on T2-weighted FLAIR images. The lesion extended slightly to the medial part of the premotor cortex (Fig. 2). Ultrasound and MRA demonstrated a highgrade stenosis of the left internal carotid artery (ICA). As extensive stroke workup failed to show an alternative source of embolism, symptomatic ICA stenosis was considered the most likely cause of stroke, and the patient was treated with endarterectomy on day 4. Under speech therapy, the symptoms improved gradually, and speech became more and more fluent after day 3. In the beginning, syntax presented incompletely because of the effort of speech, but became intact by day 3. From day 1, repeated dictations and spontaneous essays (see Fig. 1b) could demonstrate the constantly fluent and intact written language. During observation, dysarthric components never occurred. At 1 week, the patient recovered completely, including the right facial palsy. Still not clearly classified as an articulatory or language disorder, aphemia is today understood as an isolated disorder of the planning of motor articulation of speech. These patients, even when mute, can write correctly and have no difficulty in the production of verbal sequences as long as they do not have to articulate them, distinguishing the disorder from motor aphasia. The presentation of abnormal prosody, ‘‘false starts,’’ self corrections and the occurrence of undisturbed ‘‘insulas’’ in speech can help to differentiate it from dysarthria. Often, right-sided hemiparesis, limb apraxia, mild buccofacial apraxia and central right facial palsy are associated symptoms. The lesion of the precentral gyrus corresponds to recent data of the few similar cases published [2, 5, 7]. These case studies have linked the C. Ottomeyer (&) B. Reuter T. Jager C. Rosmanith M. G. Hennerici K. Szabo Department of Neurology, Universitatsklinikum Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany e-mail: c.ottomeyer@neuro.ma.uni-heidelberg.de

Intravenous thrombolysis for acute ischaemic stroke in the elderly: data from the Baden-Wuerttemberg stroke registry

In Europe intravenous thrombolysis (IVT) for ischaemic stroke is still not approved for patients aged >80 years. However, elderly patients are frequently treated based on individual decision making. In a retrospective observational study a consecutive and prospective stroke registry in southwest Germany was analysed.

Restriction of therapy mainly explains lower thrombolysis rates in reduced stroke service levels

Objective: To assess the influence of preexisting disabilities, age, and stroke service level on standardized IV thrombolysis (IVT) rates in acute ischemic stroke (AIS). Methods: We investigated standardized IVT rates in a retrospective registry-based study in 36,901 patients with AIS from the federal German state Baden-Wuerttemberg over a 5-year period. Patients admitted within 4.5 hours after stroke onset were selected. Factors associated with IVT rates (patient-level factors and stroke service level) were assessed using robust Poisson regression modeling. Interactions between factors were considered to estimate risk-adjusted mortality rates and potential IVT rates by service level (with stroke centers as benchmark). Results: Overall, 10,499 patients (28.5%) received IVT. The IVT rate declined with service level from 44.0% (stroke center) to 13.1% (hospitals without stroke unit [SU]). Especially patients >80 years of age and with preexisting disabilities had a lower chance of being treated with IVT at lower stroke service levels. Interactions between stroke service level and age group, preexisting disabilities, and stroke severity (all p < 0.0001) were observed. High IVT rates seemed not to increase mortality. Estimated potential IVT rates ranged between 41.9% and 44.6% depending on stroke service level. Conclusions: Differences in IVT rates among stroke service levels were mainly explained by differences administering IVT to older patients and patients with preexisting disabilities. This indicates considerable further potential to increase IVT rates. Our findings support guideline recommendations to admit acute stroke patients to SUs.

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.

MMP-2 concentrations in stroke according to etiology: Adjusting for enzyme degradation in stored deep-frozen serum and other methodological pitfalls

Matrix metalloproteinases (MMP) have a prominent role in the pathophysiology of stroke. We investigated potential differences in MMP-2 concentrations with respect to acute stroke etiology. For another MMP family member, MMP-9, significant degradation over time has been found even when stored at -80 °C, so we measured temporal degradation of MMP-2 and adjusted for this and other factors potentially affecting our results. For 264 patients with acute stroke at baseline and a control cohort of 120 subjects, MMP-2 concentrations were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits. For each stroke patient, stroke etiology was categorized as cardioembolic, large vessel or small vessel ischemic stroke, or primary hemorrhage. Stroke patients had significantly lower MMP-2 concentrations than controls (mean ± standard deviation: 175.6 ± 65.6 ng/mL versus 212.0 ± 54.8 ng/mL, p<0.001). However, sample degradation (average sample storage time: 240.0 ± 113.7 days) was considerable, amounting to approximately 15% per year. The full extent of differences in MMP-2 concentrations between stroke of different subtypes only became evident when results were adjusted for enzyme degradation during storage and other methodological pitfalls. Before adjustment, the only significant difference between etiologies was that the cardioembolic stroke group had a significantly higher concentration of MMP-2 than the hemorrhage group. After adjustment for time to analysis and ELISA plate clustering, patients with cardioembolic stroke had significantly higher MMP-2 concentrations in comparison to all other stroke subtypes.

True Effects or Bias? MMP-2 and MMP-9 Serum Concentrations after Acute Stroke.

Background: Average serum matrix metalloproteinase (MMP) concentrations in patients with acute stroke have shown to be varying across studies. Possibly, next to true effects, other factors may influence MMP levels. The aim of this study was to investigate the dynamics of these enzymes in repeated measurements in the acute post-stroke period, in respect to different stroke etiologies, and highlight potential sources for variability. Methods: Serum in 233 patients with acute ischemic or hemorrhagic stroke (stroke cohort; SC) was ascertained within 24 h after onset and then 1, 3 and 7 days thereafter. One hundred five controls (control cohort; Co) were recruited. Multi-variable adjustment was carried out using salient extraneous covariates including stroke etiology, clinical severity and lesion size next to a set of routine laboratory parameters. Results: Unadjusted SC MMP-2 concentrations are significantly lower (SC 165.4, 95% CI 158.5-172.4; Co 203.7 ng/ml, 95% CI 190.7-216.5; p < 0.001) and MMP-9 concentrations significantly higher than in controls (SC 608.5 ng/ml, 95% CI 555.3-661.8; Co 475.6 ng/ml, 95% CI 413.6-537.6; p < 0.001). Adjustment mitigates associations between MMP concentrations and stroke etiology, clinical severity, lesion size or differences in temporal profile shown present without adjustment. Salient covariates absorb much of the effect: age, leukocyte count and albumin concentrations are associated significantly with MMP-2 concentrations; only leukocyte count is significantly associated with MMP-9. Conclusions: Concentrations of MMP-2 and MMP-9 in serum in humans measured after acute stroke are potentially influenced by extraneous covariates rather than being directly associated with characteristics of the underlying stroke.