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Objective This study was designed to investigate the indirect neuroprotective properties of recombinant human erythropoietin (rhEPO) pretreatment in a rat model of transient middle cerebral artery occlusion (MCAO). Methods One hundred and ten male Wistar rats were randomly assigned to four groups receiving either 5,000 IU/kg rhEPO intravenously or saline 15 minutes prior to MCAO and bilateral craniectomy or sham craniectomy. Bilateral craniectomy aimed at elimination of the space-consuming effect of postischemic edema. Diagnostic workup included neurological examination, assessment of infarct size and cerebral edema by magnetic resonance imaging, wet–dry technique, and quantification of hemispheric and local cerebral blood flow (CBF) by flat-panel volumetric computed tomography. Results In the absence of craniectomy, EPO pretreatment led to a significant reduction in infarct volume (34.83 ± 9.84% vs. 25.28 ± 7.03%; p = 0.022) and midline shift (0.114 ± 0.023 cm vs. 0.083 ± 0.027 cm; p = 0.013). We observed a significant increase in regional CBF in cortical areas of the ischemic infarct (72.29 ± 24.00% vs. 105.53 ± 33.10%; p = 0.043) but not the whole hemispheres. Infarct size-independent parameters could not demonstrate a statistically significant reduction in cerebral edema with EPO treatment. Conclusions Single-dose pretreatment with rhEPO 5,000 IU/kg significantly reduces ischemic lesion volume and increases local CBF in penumbral areas of ischemia 24 h after transient MCAO in rats. Data suggest indirect neuroprotection from edema and the resultant pressure-reducing and blood flow-increasing effects mediated by EPO.

Great advances have been made in our understanding of Takotsubo syndrome in the past decade, but the aetiology of the condition remains incompletely understood. The most established theory, that catecholamine-mediated myocardial stunning is provoked by emotional or physiological stress, is supported by the presence of supraphysiological levels of plasma catecholamines in patients with Takotsubo syndrome. For this reason, the hyperexcitability of the autonomic nervous system under conditions of physical and emotional stress is often assessed in these patients. Observational studies have indicated that a predisposing influence of chronic or traumatic stress, anxiodepressive disorders, and maladaptive personality traits are linked to the pathogenesis of Takotsubo syndrome. Chronic stress can influence autonomic function through dysregulation of the hypothalamic-pituitary-adrenal axis and contribute to the development of cardiovascular disorders. In this Perspectives article, we discuss the current knowledge of the psychoneuroendocrinological and psychosocial mechanisms underlying the pathophysiology of Takotsubo syndrome.

Background Computer-assisted planimetry is widely used in experimental stroke research to assess the size of the ischemic lesion or hemispheric volume. New method Only insufficient data exist on the training required to achieve sufficient reliability in planimetry. Therefore, planimetry was performed over 15 months by two blinded raters who were initially inexperienced in the method. For inter-rater reliability, the hemispheric and lesional volume of 227 male Wistar Unilever rats subjected to middle cerebral artery occlusion were determined in diffusion- and T2-weighted sequences. For the intra-rater agreement, one investigator assessed the hemispheric and lesional volume in 87 T2-weighted sequences twice within a six-week interval. The correlation was calculated using Krippendorff’s alpha and Bland-Altman plots illustrated the agreement. Results Inter-rater agreement increased during the first seven weeks and remained at high values (Krippendorff’s alpha > 0.88). For intra-rater agreement, Krippendorff’s alpha was 0.84 for hemispheric and 0.85 for lesional volume. The Bland-Altman plot indicated solid agreement between raters in the absence of systematic errors. Comparison with existing methods Simplified geometrical models or automated methods for planimetry can be used to determine lesional volume, but both approaches are inappropriate to assess hemispheric volume. Conclusion Computer-assisted planimetry can be an appropriate method to determine hemispheric or ischemic lesion volume in rodents but requires a sufficiently long learning period of approximately two months. Even an experienced investigator can generate data with serious variation. Inter- and intra-rater-dependent bias should be considered during the design and performance of respective studies.

It is well known that recanalization of thrombosed cerebral sinuses occurs early but without marked influence on the long-term outcome and on final venous infarct volume on magnetic resonance imaging. To better understand the possible microvascular mechanisms behind these clinical observations, we evaluated the sequels of subacute superior sagittal sinus (SSS) thrombosis in rats using micro- and nano-CT imaging of the same specimen to provide large volume and high resolution CT image data respectively. SSS thrombosis was induced in 11 animals which were euthanized after 6 h (n = 4) or 6 weeks (n = 7). Eight sham-operated rats served as controls. After infusion of contrast into the vasculature of the brains, these were isolated and scanned using micro-, nano-, and synchrotron-based micro-CT ((8 {mu}m{sup 3}), (900 nm){sup 3}, and (1.9 {mu}m{sup 3}) voxel sizes). The cross-sectional area of the superior sagittal sinus, microvessels and cortical veins were quantified. Tissue sections were stained against VEGF antigen. Immunohistochemistry was confirmed using quantitative rtPCR. SSS thrombosis led to a congestion of the bridging veins after 6 h. After 6 weeks, a network of small vessels surrounding the occluded SSS was present with concurrent return towards the diameter of the draining bridging veinsmore » of controls. This microvascular network connected to cortical veins as demonstrated by nano- and synchrotron-based micro-CT. Also the volume fraction and number of cortical veins increased significantly. Immunohistochemistry in the region of the microsvascular network demonstrated a strong immunoreactivity against VEGF, confirmed by rtPCR. The sequel of subacute SSS thrombosis induced a network of microvessels ('venogenesis') draining the bridging veins. Also the volume fraction of cortical veins increased significantly.« less

Background Early recanalization of large cerebral vessels in ischemic stroke is associated with improved clinical outcome, however persisting hypoperfusion leads to poor clinical recovery despite large vessel recanalization. Limited experimental sonothrombolysis studies have shown that addition of microbubbles during treatment can improve microvascular patency. We aimed to determine the effect of two different microbubble formulations on microvascular patency in a rat stroke model. Methods We tested BR38 and SonoVue® microbubble-enhanced sonothrombolysis in Wistar rats submitted to 90-minute filament occlusion of the middle cerebral artery. Rats were randomized to treatment (n = 6/group): control, rt-PA, or rt-PA+3-MHz ultrasound insonation with BR38 or SonoVue® at full or 1/3 dose. Treatment duration was 60 minutes, beginning after withdrawal of the filament, and sacrifice was immediately after treatment. Vascular volumes were evaluated with microcomputed tomography. Results Total vascular volume of the ipsilateral hemisphere was reduced in control and rt-PA groups (p0.1). Conclusions Microbubble-enhanced sonothrombolysis improves microvascular patency. This effect is not dose- or microbubble formulation-dependent suggesting a class effect of microbubbles promoting microvascular reopening. This study demonstrates that microbubble-enhanced sonothrombolysis may be a therapeutic strategy for patients with persistent hypoperfusion of the ischemic territory.

Objectives Postoperative cognitive decline (POCD) has a perceivable influence on daily living and is noticed more often by close relatives than by patients themselves 3 months after aortic valve replacement. This study aimed to elucidate the longitudinal course of the subjective awareness of POCD. Design Follow-up of a prospective observational study. Setting A single cardiothoracic center in Germany. Participants The study included 108 patients scheduled for elective aortic valve replacement surgery and 85 close relatives of the patients. Interventions In addition to conducting a neuropsychologic examination, the authors previously interviewed 82 patients with a Cognitive Failure Questionnaire for self-assessment (s-CFQ), and 62 relatives with the Cognitive Failure Questionnaire for others (f-CFQ) before and 3 months after surgery. Up until 12 months after surgery, the authors continuously interviewed additional patients (baseline and 3 months after surgery), thereby enlarging the original sample, and included the entire group (108 patients, 85 relatives) for the 12-month follow-up. Results The analysis showed that relatives (p = 0.026) and patients experienced patients’ cognitive decline 3 months after surgery (p = 0.009). All changes still were observed in questions related to memory and attention. After 1 year, the s-CFQ no longer differed between baseline and postoperative assessment. Mean scores in the f-CFQ still were above baseline, barely missing statistical significance (p = 0.051). In patients with “change to worse” in the f-CFQ at 1-year follow-up, declining cognitive results in nonverbal learning (p = 0.021) could be observed 3 months postoperatively. Only a decrease in 3-month f-CFQ correlated with a decline in specific neuropsychologic tests 3 months after surgery. Conclusions Contrary to the authors’ previous results, the impact of POCD on daily living functions also was recognized by the patients themselves. The long-term influence and the associations between subjective deficits and psychometric cognitive measures seemed to be assessed more reliably by close relatives.

The aetiology of takotsubo cardiomyopathy (TTC) is still poorlyunderstood. The most established theory of catecholamine-mediatedmyocardial stunning being provoked by a stressful event is supportedby supraphysiological levels of plasma catecholamines in TTC patients[1]. TTC predominantly occurs in postmenopausal women, and severalstudies suggest a predisposing role of psychiatric or personality traitsin its pathogenesis [2,3].In our recent study, female TTC patients were screened in detail forpsychosocialattributes,butourfindingslendsupportforneithernotice-able psychiatric or personality characteristics nor for an increasedoccurrence of social or work hassles. However, TTC patients showed ablunted cortisol stress response (CSR) compared with cardiac-healthycontrols and high incidences of stressful or traumatic life-events com-pared with healthy controls and non-ST-segment elevation myocardialinfarction (NSTEMI) patients [4].It issuggested thatcertainpersonalitytraits,suchaslocusof control(LOC), largely determine how people cope with stress. Internal LOCrefers to the conviction that outcomes of live events are determinedby one's own behaviour and choices, and is associated with a more ac-tive search for solutions to reduce stress. To the contrary, external LOCimpliesthatoutcomeisdeterminedbyluck,chance,orpowerfulothers.It is associated with unfavourable stress management strategies, leadsto higher stress levels, and might predispose to somatic illness [5].To our knowledge, LOC has not been investigated in TTC patients sofar, and only the research from Hefner and colleagues focuses on stressmanagement in these patients. In their retrospective study [6], the re-searchers compared 31 female TTC patients with 104 healthy womenfrom a normative control sample of the stress-coping questionnaire(SVF-120) and found TTC patients to use some positive strategies,such as deemphasising, less often than controls. In their second paper[7], the authors did not report differences in stress management whenthese TTC patients were compared with 30 females with a history ofacute coronary syndrome (ACS).The present hypothesis-generating study aims to identify internaland external LOC as a possible predisposing personality trait and its re-lationship to stress management in TTC patients for the first time, andcontrasts the results to those of NSTEMI patients and healthy controls.Data were collected from 19 female TTC patients diagnosed atKerckhoff Heart and Thorax Center, Bad Nauheim, Germany. Twentywomen with a history of NSTEMI and 20 cardiac-healthy female volun-teers served as controls. The groups were matched by age (mean age60.5 ± 9.2), and the TTC and NSTEMI patients were additionallymatched by their index event date in relation to study inclusion(mean months 18.4 ± 8.5). All participants gave written, informedconsent and have recently attended our previous study. For extensiveinformationaboutmethodological,clinicalandsociodemographicchar-acteristicspleaserefertothispublication[4].Thelocalethicscommitteeapproved the study.We used the German IPC questionnaire [8] to determine internal(I-Scale) and two dimensions of external LOC (P-scale = powerfulothers, C-scale = chance) which measure one's belief in ‘who’ has thecontroland‘howmuch’controlonehasoverone'slife.Theshortversionof the stress-coping questionnaire (SVF-78, [9]) investigates stressmanagement. It comprises 78 items divided into 13 subscales ofeffective or maladaptive strategies of coping with stress (Table 1).Behaviours defined in the subscales 2–5and8–10 are considered as

Objective Alterations in cortisol awakening and stress responses (CAR, CSR) are sensitive markers for the basal activity and responsiveness of the hypothalamus–pituitary–adrenal axis (HPAA) in psychopathological conditions. We investigated whether patients with takotsubo cardiomyopathy (TTC) differ in these markers when compared with non-ST-segment elevation myocardial infarction (NSTEMI) patients and healthy controls. Methods 19 female TTC patients were compared with 20 female NSTEMI patients and with 20 healthy women, matched by age and index event date. Salivary sampling indicated cortisol release, questionnaires assessed personality, life events, chronic stress and psychiatric symptoms. Results The groups did not differ relevantly in their basal HPAA activity, psychiatric or personality profiles. Despite increased heart rates in response to stress (median difference (MD diff )=3.5, p=0.002) and higher nervousness scores (MD diff =−3.0, p=0.024), TTC patients revealed a blunted CSR with a medium effect compared to the controls (MD diff =−3.2 nmol/L, p=0.022, r=0.36); even when controlled for prestress cortisol differences (p=0.044, r=0.33). In comparison with NSTEMI patients, no significant differences in CSR (MD diff =−1.9 nmol/L, p=0.127, r=0.25) or nervousness (MD diff =2.0, p=0.107) can be observed. Stressful life events, for example, traumatic experiences, occurred more often in TTC (42%) than in NSTEMI patients and controls (both 10%, p=0.031). Conclusions In this small exploratory trial, a trend for a blunted CSR and high incidences of stressful life events were observed in TTC patients. If these results can be confirmed in larger studies, chronic stress and the inhibitory influence of cortisol on catecholamine release might be significant for the pathogenesis of TTC.

Flat-panel volumetric computed tomography (fpVCT) is a non-invasive approach to three-dimensional small animal imaging. The capability of volumetric scanning and a high resolution in time and space enables whole organ perfusion studies. We aimed to assess feasibility and validity of fpVCT in cerebral perfusion measurement with impaired hemodynamics by evaluation of three well-established rat stroke models for temporary and permanent middle cerebral artery occlusion (MCAO). Male Wistar rats were randomly assigned to temporary (group I: suture model) and permanent (group II: suture model; III: macrosphere model) MCAO and to a control group. Perfusion scans with respect to cerebral blood flow (CBF) and volume (CBV) were performed 24h post intervention by fpVCT, using a Gantry rotation time of 1s and a total scanning time of 30s. Postmortem analysis included infarct-size calculation by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Infarct volumes did not differ significantly throughout intervention groups. After permanent MCAO, CBF significantly decreased in subcortical regions to 78.2% (group II, p=0.005) and 79.9% (group III, p=0.012) and in total hemisphere to 77.4% (group II, p=0.010) and 82.0% (group III, p=0.049). CBF was less impaired with temporary vessel occlusion. CBV measurement revealed no significant differences. Results demonstrate feasibility of cerebral perfusion quantification in rats with the fpVCT, which can be a useful tool for non-invasive dynamic imaging of cerebral perfusion in rodent stroke models. In addition to methodological advantages, CBF data confirm the macrosphere model as a useful alternative to the suture model for permanent experimental MCAO.

In monitoring of recanalization and in sonothrombolysis, contrast-enhanced ultrasound (CEUS) is applied in extended time protocols. As extended use may increase the probability of unwanted effects, careful safety evaluation is required. We investigated the safety profile and beneficial effects of CEUS in a reperfusion model. Wistar rats were subjected to filament occlusion of the right middle cerebral artery (MCA). Reperfusion was established after 90 minutes, followed by recombinant tissue-type plasminogen activator (rt-PA) treatment and randomization to additional CEUS (contrast agent: SonoVue; 60 minutes). Blinded outcome evaluation consisted of magnetic resonance imaging (MRI), neurologic assessment, and histology and, in separate experiments, quantitative 3D nano-computed tomography (CT) angiography (900 nm3 voxel size). Nano-CT revealed severely compromised microcirculation in untreated animals after MCA reperfusion. The rt-PA partially improved hemispheric perfusion. Impairment was completely reversed in animals receiving rt-PA and CEUS. This combination was more effective than treatment with either CEUS without rt-PA or rt-PA and ultrasound or ultrasound alone. In MRI experiments, CEUS and rt-PA treatment resulted in a significantly reduced ischemic lesion volume and edema formation. No unwanted effects were detected on MRI, histology, and intracranial temperature assessment. This study shows that CEUS and rt-PA is safe in the situation of reperfusion and displays beneficial effects on the level of the microvasculature.

Thrombosis of the cerebral veins and sinus are common causes of stroke. Animal models help us to understand the underlying pathophysiology of this condition. Therefore, the purpose of our study was to evaluate a well-established model for sinus sagittalis (SSS) thrombosis using micro- and nanocomputed tomography (CT) imaging. SSS thrombosis was performed in four rats. After contrast perfusion, brains were isolated and scanned using micro-CT at (8 µm)³ voxel size to generate 3D images of the cerebral vasculature. For more detailed information on vascular perfusion territories, nano-CT imaging was performed to investigate the boundary layer of contrast-enhanced vessels and the occluded veins. The venous and arterial vascular volume fraction and gray scale measurements were obtained in the SSS thrombosis group and compared to controls. The significance of differences in vascular volume fraction and gray scale measurements was tested with analysis of variance. Results were complemented with histology. Micro-CT proved to accurately visualize and differentiate vascular occlusion territories performed in the SSS thrombosis model. Moreover, 3D micro-CT provided quantitative information on arterial and venous vascular volume fraction. Micro-CT imaging enables a total 3D visualization of complications (ventricle rupture) in the SSS thrombosis model. We established gray scale measurements by which focal cerebral ischemia could be radiographically categorized (p

Background Cerebral air microembolization (CAM) is a frequent side effect of diagnostic or therapeutic interventions. Besides reduction of the amount of bubbles, filter systems in the clinical setting may also lead to a dispersion of large gas bubbles and therefore to an increase of the gas–liquid-endothelium interface. We evaluated the production and application of different strictly defined bubble diameters in a rat model of CAM and assessed functional outcome and infarct volumes in relation to the bubble diameter. Methods Gas emboli of defined number and diameter were injected into the carotid artery of rats. Group I (n = 7) received 1800 air bubbles with a diameter of 45 μm, group II (n = 7) 40 bubbles of 160 μm, controls (n = 6) saline without gas bubbles; group I and II yielded the same total injection volume of air with 86 nl. Functional outcome was assessed at baseline, after 4 h and 24 h following cerebral MR imaging and infarct size calculation. Results Computer-aided evaluation of bubble diameters showed high constancy (group I: 45.83 μm ± 2.79; group II: 159 μm ± 1.26). Animals in group I and II suffered cerebral ischemia and clinical deterioration without significant difference. Infarct sizes did not differ significantly between the two groups (p = 0.931 u-test). Conclusions We present further development of a new method, which allows reliable and controlled CAM with different bubble diameters, producing neurological deficits due to unilateral cerebral damage. Our findings could not display a strong dependency of stroke frequency and severity on bubble diameter.