Experimental & Translational Stroke Medicine - Latest Articles

Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke

R. Christian Crumrine — 2012-05-16T00:00:00Z

Background: Tissue type plasminogen activator is the only approved thrombolytic agent for the treatmentof ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomaticand asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patientprognosis and increase patient participation in thrombolytic treatment. A novel direct-actingthrombolytic agent, Delta(K2-K5) plasmin, promising an improved safety profile was examinedfor safety in the snare ligature model of stroke in the rat. Methods: Male spontaneously hypertensive rats were subjected to 6 hours middle cerebral arteryocclusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosedwith saline, vehicle, Delta(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissuetypeplasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60minutes. The rats were assessed for Bleeding Score, infarct volume, modified Bederson scoreand general behavioral score. In a parallel study, temporal progression of infarct volume wasdetermined. In an in vitro study, whole blood clots from humans, canines and rats wereexposed to Delta(K2-K5). Clot lysis was monitored by absorbance at 280 nm. Results: The main focus of this study was intracranial hemorrhage safety. Delta(K2-K5) plasmintreatment at the highest dose caused no more intracranial hemorrhage than the lowest dose ofrecombinant tissue type plasminogen activator, but showed at least a 5-fold superior safetymargin. Secondary results include: temporal infarct volume progression shows that thegreatest expansion of infarct volume occurs within 2-3 hours of middle cerebral arteryocclusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6hours ischemia with reflow. Delta(K2-K5) plasmin tended to reduce infarct volume and improvebehavior compared to controls. In vitro data suggests that Delta(K2-K5) plasmin is equallyeffective at lysing clots from humans, canines and rats. Conclusions: The superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Delta(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agenta good candidate for clinical evaluation in the treatment of acute ischemic stroke.

ATP-binding cassette transporters in immortalised human brain microvascular endothelial cells in normal and hypoxic conditions

Christian Lindner — 2012-05-03T00:00:00Z

Background: Rapid reperfusion following ischemia is the most effective therapy in stroke therapy.However, the success may be compromised by ischemia & reperfusion (I/R) injury and at thehuman blood-brain barrier (BBB), therefore the effects on transendothelial transport are ofspecial interest. Current studies suggest the ATP-binding cassette (ABC) transporters to beregulated upon ischemic stroke in a way that impedes the effects of drug therapy. Theimmortalised human brain microvascular endothelial cell line hCMEC/day provides most ofthe unique properties of the BBB with respect to transport and might be a reliable in vitromodel to study transendothelial transport after I/R. Methods: We exposed hCMEC/D3 cells to 24 hours of hypoxia alone and to hypoxia followed by 60min of reoxygenisation as a in vitro model for I/R. Western blot showed mild upregulation ofhypoxia inducible factor (HIF-1alpha) after hypoxia alone and RNA lysates were analysed with awell-established real-time RT-PCR-based TaqMan low-density array detecting 47 of 48known human ABC transporters. Results: No significant increases of ABC mRNA expression levels were detected neither in hypoxicnor in I/R samples. However, slight decrease of ABCC1 in hypoxic and I/R samples and ofABCA10 and ABCD3 in I/R samples was observed. Conclusion: Our data suggests that hCMEC/D3 cell line and - at the moment - in vitro models in generalare a poor basis for stroke research but may be enhanced by co-culturing more cells of theneurovascular unit inducing an overall ischemic response at the BBB.

Brain edema formation correlates with perfusion deficit during the first six hours after experimental subarachnoid hemorrhage in rats

Thomas Westermaier — 2012-05-02T00:00:00Z

Background: Severe brain edema is observed in a number of patients suffering from subarachnoidhemorrhage (SAH). Little is known about its pathogenesis and time-course in the first hoursafter SAH. This study was performed to investigate the development of brain edema and itscorrelation with brain perfusion after experimental SAH. Methods: Male Sprague-Dawley rats, randomly assigned to one of six groups (n = 8), were subjected toSAH using the endovascular filament model or underwent a sham operation. Animals weresacrificed 15, 30, 60, 180 or 360 minutes after SAH. Intracranial pressure (ICP), meanarterial blood pressure (MABP), cerebral perfusion pressure (CPP) and bilateral local cerebralblood flow (LCBF) were continuously measured. Brain water content (BWC) was determinedby the wet/dry-weight method. Results: After SAH, CPP and LCBF rapidly decreased. The decline of LCBF markedly exceeded thedecline of CPP and persisted until the end of the observation period. BWC continuouslyincreased. A significant correlation was observed between the BWC and the extent of theperfusion deficit in animals sacrificed after 180 and 360 minutes. Conclusions: The significant correlation with the perfusion deficit after SAH suggests that the developmentof brain edema is related to the extent of ischemia and acute vasoconstriction in the firsthours after SAH.

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

Dominik Michalski — 2012-04-17T00:00:00Z

Background: Since several neuroprotectives failed to reproduce promising preclinical results under clinicalconditions, efforts emerged to implement clinically relevant endpoints in animal strokestudies. Thereby, insufficient attention was given on autonomic reactions due to experimentalstroke, although clinical trials reported on high functional and prognostic impact. This studyfocused on autonomic consequences and body weight changes in a translational relevantstroke model and investigated interrelations to different outcome measurements. Methods: Forty-eight rats underwent thromboembolic middle cerebral artery occlusion (MCAO) whilerecording heart rate (HR) and mean arterial pressure (MAP). After assessing early functionalimpairment (Menzies score), animals were assigned to control procedure or potentiallyneuroprotective treatment with normobaric (NBO) or hyperbaric oxygen (HBO). Four or 24hours after ischemia onset, functional impairment was re-assessed and FITC-albuminadministered intravenously obtaining leakage-related blood-brain barrier (BBB) impairment.Body weight was documented prior to MCAO and 4 or 24 hours after ischemia onset. Results: During MCAO, HR was found to increase significantly while MAP decreased. The amountof 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 ratswith low impairment (P = 0.048). Regarding body weight, a decrease of 9.4% within 24hours after MCAO occurred, but treatment-specific alterations showed no significantcorrelations with respective functional or BBB impairment. Conclusions: Future studies should routinely include autonomic parameters to allow inter-groupcomparisons and better understanding of autonomic reactions due to experimental stroke.Prospectively, autonomic consequences might represent a useful outcome parameterenhancing the methodological spectrum of preclinical stroke studies.

Focal brain trauma in the cryogenic lesion model in mice

Furat Raslan — 2012-04-05T00:00:00Z

The method to induce unilateral cryogenic lesions was first described in 1958 by Klatzo. We describe here an adaptation of this model that allows reliable measurement of lesion volume and vasogenic edema by 2, 3, 5-triphenyltetrazolium chloride (TTC) -staining and Evans blue extravasation in mice. A copper or aluminium cylinder with a tip diameter of 2.5 mm is cooled with liquid nitrogen (-196degreesC) and placed on the exposed skull bone over the parietal cortex (stereotaxic coordinates from bregma: 1.5 mm posterior, 1.5 mm lateral). The tip diameter and the contact time between the tip and the parietal skull determine the extent of cryolesion. Due to an early damage of the blood brain barrier (BBB), the cryogenic cortical injury is characterized by vasogenic edema, marked brain swelling, and inflammation. The lesion grows during the first 24 hours, a process involving complex interactions between endothelial cells, immune cells, cerebral blood flow, and the intracranial pressure. These contribute substantially to the damage from the initial injury. The major advantage of the cryogenic lesion model is the circumscribed and highly reproducible lesion size and location.

Impact of anesthesia on pathophysiology and mortality following subarachnoid hemorrhage in rats

Konstantin Hockel — 2012-03-13T00:00:00Z

Background: Anesthesia is indispensable for in vivo research but has the intrinsic potential to alter study results. The aim of the current study was to investigate the impact of three common anesthesia protocols on physiological parameters and outcome following the most common experimental model for subarachnoid hemorrhage (SAH), endovascular perforation. Methods: Sprague-Dawley rats (n = 38) were randomly assigned to (1) chloral hydrate, (2) isoflurane or (3) midazolam/medetomidine/fentanyl (MMF) anesthesia. Arterial blood gases, intracranial pressure (ICP), mean arterial blood pressure (MAP), cerebral perfusion pressure (CPP), and regional cerebral blood flow (rCBF) were monitored before and for 3 hours after SAH. Brain water content, mortality and rate of secondary bleeding were also evaluated. Results: Under baseline conditions isoflurane anesthesia resulted in deterioration of respiratory parameters (arterial pCO2 and pO2) and increased brain water content. After SAH, isoflurane and chloral hydrate were associated with reduced MAP, incomplete recovery of post-hemorrhagic rCBF (23 ± 13% and 87 ± 18% of baseline, respectively) and a high anesthesia-related mortality (17 and 50%, respectively). Anesthesia with MMF provided stable hemodynamics (MAP between 100-110 mmHg), high post-hemorrhagic rCBF values, and a high rate of re-bleedings (> 50%), a phenomenon often observed after SAH in humans. Conclusion: Based on these findings we recommend anesthesia with MMF for the endovascular perforation model of SAH.

Intracortical injection of endothelin-1 induces cortical infarcts in mice: effect of neuronal expression of an adenosine transporter

Hanifi Soylu — 2012-03-12T00:00:00Z

Background: Activation of adenosine A1 receptors has neuroprotective effects in animal stroke models. Adenosine levels are regulated by nucleoside transporters. In vitro studies showed that neuron-specific expression of human equilibrative nucleoside transporter 1 (hENT1) decreases extracellular adenosine levels and adenosine A1 receptor activity. In this study, we tested the effect of hENT1 expression on cortical infarct size following intracerebral injection of the vasoconstrictor endothelin-1 (ET-1) or saline. Methods: Mice underwent stereotaxic intracortical injection of ET-1 (1 μl; 400 pmol) or saline (1 μl). Some mice received the adenosine receptor antagonist caffeine (25 mg/kg, intraperitoneal) 30 minutes prior to ET-1. Perfusion and T2-weighted magnetic resonance imaging (MRI) were used to measure cerebral blood flow (CBF) and subsequent infarct size, respectively. Results: ET-1 reduced CBF at the injection site to 7.3 ± 1.3% (n = 12) in hENT1 transgenic (Tg) and 12.5 ± 2.0% (n = 13) in wild type (Wt) mice. At 48 hours following ET-1 injection, CBF was partially restored to 35.8 ± 4.5% in Tg and to 45.2 ± 6.3% in Wt mice; infarct sizes were significantly greater in Tg (9 ± 1.1 mm3) than Wt (5.4 ± 0.8 mm3) mice. Saline-treated Tg and Wt mice had modest decreases in CBF and infarcts were less than 1 mm3. For mice treated with caffeine, CBF values and infarct sizes were not significantly different between Tg and Wt mice. Conclusions: ET-1 produced greater ischemic injury in hENT1 Tg than in Wt mice. This genotype difference was not observed in mice that had received caffeine. These data indicate that hENT1 Tg mice have reduced ischemia-evoked increases in adenosine receptor activity compared to Wt mice.

Mild hypothermia of 34degreesC reduces side effects of rt-PA treatment after thromboembolic stroke in rats

Bernd Kallmunzer — 2012-03-07T00:00:00Z

Background: Hypothermia is neuroprotective in experimental stroke and may extend the so far limited therapeutic time window for thrombolysis. Therefore, hypothermia of 34°C and its effects on delayed thrombolysis including reperfusion-associated injury were investigated in a model of thromboembolic stroke (TE). Methods: Male Wistar rats (n = 48) were subjected to TE. The following treatment groups were investigated: control group - normothermia (37°C); thrombolysis group - rt-PA 90 min after TE; hypothermia by 34°C applied 1.5 to 5 hours after TE; combination therapy- hypothermia and rt-PA. After 24 hours infarct size, brain edema and neuroscore were assessed. Protein markers for inflammation and adhesion, gelatinase activity, and blood brain barrier (BBB) disruption were determined. MRI-measurements investigated infarct evolution and blood flow parameters. Results: The infarct volume and brain swelling were smaller in the hypothermia group compared to the other groups (p < 0.05 to p < 0.01). Thrombolysis resulted in larger infarct and brain swelling than all others. Hypothermia in combination with thrombolysis reduced these parameters compared to thrombolysis (p < 0.05). Moreover, the neuroscore improved in the hypothermia group compared to control and thrombolysis. Animals of the combination therapy performed better than after thrombolysis alone (p < 0.05). Lower serum concentration of sICAM-1, and TIMP-1 were shown for hypothermia and combination therapy. Gelatinase activity was decreased by hypothermia in both groups. Conclusions: Therapeutic hypothermia reduced side-effects of rt-PA associated treatment and reperfusion in our model of TE.

Report on the 3'rd scientific meeting of the "Verein zur Foerderung des Wissenschaftlichen Nachwuchses in der Neurologie" (NEUROWIND e.V.) held in Motzen, Germany, Nov. 4'th - Nov. 6'th, 2011

Christoph Kleinschnitz — 2012-02-23T00:00:00Z

From November 4th- 6th 2011, the 3rd NEUROWIND e.V. meeting was held in Motzen, Brandenburg, Germany. Like in the previous years, the meeting provided an excellent platform for scientific exchange and the presentation of innovative projects for young colleagues in the fields of neurovascular research, neuroinflammation and neurodegeneration. As kick-off to the scientific sessions, Reinhard Hohlfeld, Head of the Institute for Clinical Neuroimmunology in Munich, gave an illustrious overview on the many fascinations of neuroimmunologic research. A particular highlight on the second day of the meeting was the award of the 1'st NEUROWIND e.V. prize for young academics in the field of experimental neurology. This award is posted for young colleagues under the age of 35 with a significant achievement in the field of neurovascular research, neuroinflammation or neurodegeneration and comprises an amount of 20.000 Euro, founded by Merck Serono GmbH, Darmstadt. Germany. The first prize was awarded to Ivana Nikic from Martin Kerschensteiner's group in Munich for her brilliant work on a reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis, published in Nature Medicine in 2011. This first prize award ceremony was a great incentive for the next call for proposals now upcoming in 2012.

An experimental Protocol for mimicking Pathomechanisms of Traumatic Brain Injury in Mice

Christiane Albert-Weissenberger — 2012-02-02T00:00:00Z

Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events. In order to examine injury processes associated with TBI, a number of rodent models to induce brain trauma have been described. However, none of these models covers the entire spectrum of events that might occur in TBI. Here we provide a thorough methodological description of a straightforward closed head weight drop mouse model to assess brain injuries close to the clinical conditions of human TBI.