Johann Otto Pelz

A novel quantification of blood-brain barrier damage and histochemical typing after embolic stroke in rats.

Treatment strategies in acute ischemic stroke are still limited. Considering numerous translation failures, research is tending to a preferred use of human-like animal models, and a more-complex perspective of tissue salvaging involving endothelial, glial and neuronal components according to the neurovascular unit (NVU) concept. During ischemia, blood-brain barrier (BBB) alterations lead to brain edema and hemorrhagic transformation affecting NVU components. The present study aims on a novel quantification method of BBB damage and affected tissue following experimental cerebral ischemia, closely to the human condition. Wistar rats underwent embolic middle cerebral artery occlusion, followed by an intravenous application of fluorescein isothiocyanate (FITC)-tagged albumin (≈70kDa) and/or biotinylated rat IgG (≈150kDa) as BBB permeability markers. Both fluorescent agents revealed similar leakage and allow quantification of BBB permeability by fluorescence microscopy, and after immunohistochemical conversion into a permanent diaminobenzidine label at light-microscopical level. The following markers were identified for sufficient detection of NVU components: Rat endothelial cell antigen-1 (RECA) and laminin for vessels, Lycopersicon esculentum and Griffonia simplicifolia agglutinin for vessels and microglial subpopulations, ionized calcium binding adaptor molecule 1 (Iba1), CD68 and CD11b for macrophages, activated microglia, monocytes and neutrophils, S100β for astroglia, as well as NeuN and HuC/D for neurons. This is the first report confirming the usefulness of simultaneously applied FITC-albumin and biotinylated rat IgG as BBB permeability markers in experimental stroke, and, specifying antibodies and lectins for multiple fluorescence labeling of NVU components. Newly elaborated protocols might facilitate a more-complex outcome measurement in drug development for cerebral ischemia.

Interrelations between blood-brain barrier permeability and matrix metalloproteinases are differently affected by tissue plasminogen activator and hyperoxia in a rat model of embolic stroke

BackgroundIn ischemic stroke, blood-brain barrier (BBB) regulations, typically involving matrix metalloproteinases (MMPs) and inhibitors (TIMPs) as mediators, became interesting since tissue plasminogen activator (tPA)-related BBB breakdown with risk of secondary hemorrhage was considered to involve these mediators too. Despite high clinical relevance, detailed interactions are purely understood. After a pilot study addressing hyperoxia as potential neuroprotective co-treatment to tPA, we analyzed interrelations between BBB permeability (BBB-P), MMPs and TIMPs.FindingsRats underwent embolic middle cerebral artery occlusion (eMCAO) and treatment with normobaric (NBO) or hyperbaric oxygen (HBO), tPA, tPA+HBO, or no treatment. BBB-P was assessed by intravenously applied FITC-albumin at 4 or 24 hours. MMP-2/-9 and TIMP-1/-2 serum levels were determined at 5 or 25 hours. Time point-corrected partial correlations were used to explore interrelations of BBB-P in ischemic regions (extra-/intravasal FITC-albumin ratio) and related serum markers. BBB-P correlated positively with MMP-2 and MMP-9 in controls, whereas hyperoxia led to an inverse association, most pronounced for HBO/MMP-9 (r = -0.606; P < 0.05). As expected, positive coefficients were observed after treatment with tPA. Co-treatment with HBO attenuated and in part reversed this effect, but to a lower degree than HBO alone. Amongst MMPs and TIMPs, significant associations shifted from MMP-9 to -2 when comparing treatment with HBO/tPA and tPA+HBO. TIMPs were significantly interrelated after tPA, tPA+HBO, and interestingly, HBO alone.ConclusionsHBO was found to reverse the positively directed interrelation of BBB-P and MMPs after eMCAO, but this effect failed to sustain in the expected amount when HBO and tPA were given simultaneously.

Early outcome and blood-brain barrier integrity after co-administered thrombolysis and hyperbaric oxygenation in experimental stroke

BackgroundAfter promising results in experimental stroke, normobaric (NBO) or hyperbaric oxygenation (HBO) have recently been discussed as co-medication with tissue plasminogen activator (tPA) for improving outcome. This study assessed the interactions of hyperoxia and tPA, focusing on survival, early functional outcome and blood-brain barrier (BBB) integrity following experimental stroke.MethodsRats (n = 109) underwent embolic middle cerebral artery occlusion or sham surgery. Animals were assigned to: Control, NBO (60-minute pure oxygen), HBO (60-minute pure oxygen at 2.4 absolute atmospheres), tPA, or HBO+tPA. Functional impairment was assessed at 4 and 24 hours using Menzies score, followed by intravenous application of FITC-albumin as a BBB permeability marker, which was allowed to circulate for 1 hour. Further, blood sampling was performed at 5 and 25 hours for MMP-2, MMP-9, TIMP-1 and TIMP-2 concentration.ResultsMortality rates did not differ significantly between groups, whereas functional improvement was found for NBO, tPA and HBO+tPA. NBO and HBO tended to stabilize BBB and to reduce MMP-2. tPA tended to increase BBB permeability with corresponding MMP and TIMP elevation. Co-administered HBO failed to attenuate these early deleterious effects, independent of functional improvement.ConclusionsThe long-term consequences of simultaneously applied tPA and both NBO and HBO need to be addressed by further studies to identify therapeutic potencies in acute stroke, and to avoid unfavorable courses following combined treatment.

Endothelial barrier antigen-immunoreactivity is conversely associated with blood-brain barrier dysfunction after embolic stroke in rats

While the concept of the Neurovascular Unit (NVU) is increasingly considered for exploring mechanisms of tissue damage in ischemic stroke, immunohistochemical analyses are of interest to specifically visualize constituents like the endothelium. Changes in immunoreactivity have also been discussed to reflect functional aspects, e.g., the integrity of the blood-brain barrier (BBB). This study aimed to characterize the endothelial barrier antigen (EBA) as addressed by the antibody SMI-71 in a rat model of embolic stroke, considering FITC-albumin as BBB leakage marker and serum levels of BBB-associated matrix metalloproteinases (MMPs) to explore its functional significance. Five and 25 h after ischemia onset, regions with decreased BBB integrity exhibited a reduction in number and area of EBA-immunopositive vessels, while the stained area per vessel was not affected. Surprisingly, EBA content of remaining vessels tended to be increased in areas of BBB dysfunction. Analyses addressing this interrelation resulted in a significant and inverse correlation between the vessels’ EBA content and degree of BBB permeability. In conclusion, these data provide evidence for a functional relationship between EBA-immunoreactivity and BBB dysfunction in experimental ischemic stroke. Further studies are required to explore the underlying mechanisms of altered EBA-immunoreactivity, which might help to identify novel neuroprotective strategies.

Failure to confirm benefit of acetyl- dl -leucine in degenerative cerebellar ataxia: a case series

Recently, Strupp and co-workers [1] demonstrated that acetyl-DL-leucine (Tanganil ; Pierre Fabre Médicament, Boulogne, France) may lead to substantial symptomatic improvement in various forms of degenerative cerebellar ataxia (DCA) differing widely in symptom duration, severity and etiology. Medication was given for about 7 days, and 12 of their 13 patients benefited, while no side effects were reported [1]. However, endpoints were assessed unblinded, rendering objective evaluation of treatment response difficult. Here, we report our observations in a series of patients suffering from DCA who were treated with acetyl-DL-leucine. Pharmacological treatment was combined with physioand occupational therapy as both are important non-pharmaceutical components of the treatment of DCA [2, 3]. The study was conducted as a series of individual treatment efforts and confirms to the 1964 Declaration of Helsinki and its later amendments. After they had given informed consent for the off-label use of acetyl-DL-leucine, 10 patients with DCA (Table 1) were treated in-hospital with 5 g acetyl-DL-leucine once daily for a total duration of 7 days. Furthermore, each patient received altogether five sessions of physiotherapy at 45 min and five sessions of occupational therapy at 30 min, individually matched to the patient’s symptoms and including dedicated gait and balance training. Video-recorded measurements at baseline (off-drug) and on day 7 of active treatment (on-drug) were based on the Scale for the Assessment and Rating of Ataxia (SARA) [5, 6]. SARA scores were assessed from the video recordings by three investigators who were blinded with regard to the time point of video recording. Additionally, patients were asked about their subjective improvement on medication, and possible side effects during treatment were evaluated. Statistical analyses were performed with SPSS version 20.0 (IBM Corporation; New York, NY, USA). A p value \0.05 was considered as statistically significant. There was excellent interrater agreement for assessment of SARA scores from video recordings with an intraclass correlation (absolute mode) between investigators of C0.97 (p\ 0.001). Mean and median SARA scores were similar between baseline and at day 7 of treatment with acetyl-DLleucine (Wilcoxon signed-rank test; p = 0.17, respectively, p = 0.38; Table 2). During in-hospital treatment, no side effects were observed. Although 7 of 10 patients reported subjective amelioration of cerebellar symptoms, we failed to detect any significant improvement as measured by SARA in assessments blinded to treatment status to reduce rater bias [7]. As physiotherapy was shown to be beneficial for individuals with DCA [2, 3], it is unlikely to have obscured any positive effect of acetyl-DL-leucine. Notably, (pre-)clinical trials studied acetyl-DL-leucine only in vestibular diseases [8, 9], so its potential mode of action in DCA remains speculative. One limitation of our case series is that, in contrast to Strupp and co-workers [1], patients in our case series received the liquid formulation of acetyl-DL-leucine orally, because Tanganil tablets were no longer available in Germany. Since both, the solid and liquid formulation contain neither stabilizers nor any active pharmaceutical & Joseph Classen joseph.classen@medizin.uni-leipzig.de

Sonographic evaluation of the vagus nerves: Protocol, reference values, and side-to-side differences: Sonographic Reference Values for VNs

Reported sonographic reference values for the vagus nerves (VNs) vary greatly. We aimed to generate reference values in a large cohort and examine intrarater, interrater, and across‐ultrasound systems agreement.

Evaluation Of freehand high‐resolution 3‐dimensional ultrasound of the median nerve

Introduction: In this study we evaluated freehand 3D ultrasound (3DUS) of the median nerve in comparison to 2D ultrasound (2DUS) and assessed the influence of tilting the transversal plane on cross‐sectional area (CSA) measurement. Methods: Two examiners investigated the median nerves of 22 healthy subjects over a distance of 20 cm using 3DUS. Image quality and CSA were assessed at random points within the virtual 3D volume and compared with 2DUS. Results: Image quality within the virtual 3D volume was good/sufficient/poor in 53.0%/40.2%/6.8% (examiner 1) and 21.6%/69.6%/8.8% (examiner 2), respectively. CSA measurements with 3DUS were smaller than with 2DUS (–12% and –17%; Wilcoxon test, P < 0.001). Interrater agreement for 3DUS and intermethod agreement between 2DUS and 3DUS were moderate. Stepwise tilting of the transversal plane increased CSA significantly. Conclusion: Freehand 3DUS of the median nerve over 20 cm is feasible and may help overcome some of the limitations and pitfalls of 2DUS. Muscle Nerve 55: 206–212, 2017

Evaluation of freehand high‐resolution 3D ultrasound of the median nerve

Introduction: In this study we evaluated freehand 3D ultrasound (3DUS) of the median nerve in comparison to 2D ultrasound (2DUS) and assessed the influence of tilting the transversal plane on cross‐sectional area (CSA) measurement. Methods: Two examiners investigated the median nerves of 22 healthy subjects over a distance of 20 cm using 3DUS. Image quality and CSA were assessed at random points within the virtual 3D volume and compared with 2DUS. Results: Image quality within the virtual 3D volume was good/sufficient/poor in 53.0%/40.2%/6.8% (examiner 1) and 21.6%/69.6%/8.8% (examiner 2), respectively. CSA measurements with 3DUS were smaller than with 2DUS (–12% and –17%; Wilcoxon test, P < 0.001). Interrater agreement for 3DUS and intermethod agreement between 2DUS and 3DUS were moderate. Stepwise tilting of the transversal plane increased CSA significantly. Conclusion: Freehand 3DUS of the median nerve over 20 cm is feasible and may help overcome some of the limitations and pitfalls of 2DUS. Muscle Nerve 55: 206–212, 2017

Evaluation of Freehand B-Mode and Power-Mode 3D Ultrasound for Visualisation and Grading of Internal Carotid Artery Stenosis

Background Currently, colour-coded duplex sonography (2D-CDS) is clinical standard for detection and grading of internal carotid artery stenosis (ICAS). However, unlike angiographic imaging modalities, 2D-CDS assesses ICAS by its hemodynamic effects rather than luminal changes. Aim of this study was to evaluate freehand 3D ultrasound (3DUS) for direct visualisation and quantification of ICAS. Methods Thirty-seven patients with 43 ICAS were examined with 2D-CDS as reference standard and with freehand B-mode respectively power-mode 3DUS. Stenotic value of 3D reconstructed ICAS was calculated as distal diameter respectively distal cross-sectional area (CSA) reduction percentage and compared with 2D-CDS. Results There was a trend but no significant difference in successful 3D reconstruction of ICAS between B-mode and power mode (examiner 1 {Ex1} 81% versus 93%, examiner 2 {Ex2} 84% versus 88%). Inter-rater agreement was best for power-mode 3DUS and assessment of stenotic value as distal CSA reduction percentage (intraclass correlation coefficient {ICC} 0.90) followed by power-mode 3DUS and distal diameter reduction percentage (ICC 0.81). Inter-rater agreement was poor for B-mode 3DUS (ICC, distal CSA reduction 0.36, distal diameter reduction 0.51). Intra-rater agreement for power-mode 3DUS was good for both measuring methods (ICC, distal CSA reduction 0.88 {Ex1} and 0.78 {Ex2}; ICC, distal diameter reduction 0.83 {Ex1} and 0.76 {Ex2}). In comparison to 2D-CDS inter-method agreement was good and clearly better for power-mode 3DUS (ICC, distal diameter reduction percentage: Ex1 0.85, Ex2 0.78; distal CSA reduction percentage: Ex1 0.63, Ex2 0.57) than for B-mode 3DUS (ICC, distal diameter reduction percentage: Ex1 0.40, Ex2 0.52; distal CSA reduction percentage: Ex1 0.15, Ex2 0.51). Conclusions Non-invasive power-mode 3DUS is superior to B-mode 3DUS for imaging and quantification of ICAS. Thereby, further studies are warranted which should now compare power-mode 3DUS with the angiographic gold standard imaging modalities for quantification of ICAS, i.e. with CTA or CE-MRA.

Autonomic reactions and peri-interventional alterations in body weight as potential supplementary outcome parameters for thromboembolic stroke in rats

BackgroundSince several neuroprotectives failed to reproduce promising preclinical results under clinical conditions, efforts emerged to implement clinically relevant endpoints in animal stroke studies. Thereby, insufficient attention was given on autonomic reactions due to experimental stroke, although clinical trials reported on high functional and prognostic impact. This study focused on autonomic consequences and body weight changes in a translational relevant stroke model and investigated interrelations to different outcome measurements.MethodsForty-eight rats underwent thromboembolic middle cerebral artery occlusion (MCAO) while recording heart rate (HR) and mean arterial pressure (MAP). After assessing early functional impairment (Menzies score), animals were assigned to control procedure or potentially neuroprotective treatment with normobaric (NBO) or hyperbaric oxygen (HBO). Four or 24 hours after ischemia onset, functional impairment was re-assessed and FITC-albumin administered intravenously obtaining leakage-related blood–brain barrier (BBB) impairment. Body weight was documented prior to MCAO and 4 or 24 hours after ischemia onset.ResultsDuring MCAO, HR was found to increase significantly while MAP decreased. The amount of changes in HR was positively correlated with early functional impairment (P = 0.001): Severely affected animals provided an increase of 15.2 compared to 0.8 beats/minute in rats with low impairment (P = 0.048). Regarding body weight, a decrease of 9.4% within 24 hours after MCAO occurred, but treatment-specific alterations showed no significant correlations with respective functional or BBB impairment.ConclusionsFuture studies should routinely include autonomic parameters to allow inter-group comparisons and better understanding of autonomic reactions due to experimental stroke. Prospectively, autonomic consequences might represent a useful outcome parameter enhancing the methodological spectrum of preclinical stroke studies.