Lothar Schilling

Publications of Lothar Schilling

• Differential effects of tumor-platelet interaction in vitro and in vivo in glioblastoma.

  Authors: Marc A Brockmann, Birte Bender, Elena Plaxina, Ingo Nolte, Ralf Erber, Katrin Lamszus, Christoph Groden, Lothar Schilling

  Journal of neuro-oncology. 03/2011; 105(1):45-56.

  An elevated platelet count is considered an independent predictor of short survival in glioblastoma and various other tumor entities. Prothrombotic activity of the tumor microcirculation resulting inAn elevated platelet count is considered an independent predictor of short survival in glioblastoma and various other tumor entities. Prothrombotic activity of the tumor microcirculation resulting in platelet activation and release of cytokines from activated platelets has been suggested to play a role. This study was designed to analyze the effects of platelet-released cytokines on glioblastoma and endothelial cell proliferation and migration in vitro, and the influence of platelet count on glioblastoma growth and angiogenesis in vivo. In cultured human glioblastoma, umbilical cord and cerebral microvascular endothelial cells platelet-released cytokines significantly stimulated proliferation and migration as well as sprouting and formation of capillary-like structures. In vivo, glioblastoma cells were implanted in mice followed by platelet depletion starting 1 or 8 days later. Tumor volume, proliferative index, and vessel density analyzed 14 days after engraftment did not differ between animals with a normal and a low platelet count. Likewise, no effect of platelet depletion over 20 days upon the volume of intracerebrally growing tumors was observed in mice. Additionally, proliferative activity and vessel density determined in tumor samples from patients operated upon glioblastoma did not show any correlation with the patients' preoperative platelet count. Thus, we conclude that distinct proliferation- and chemotaxis-stimulating effects of platelet-derived cytokines can be achieved in vitro, while the platelet count does not exert a major influence on tumor growth and tumor angiogenesis in GBM in vivo.

• CORMs protect endothelial cells during cold preservation, resulting in inhibition of intimal hyperplasia after aorta transplantation in rats.

  Authors: Hui Song, Simone Hoeger, Jan-Luuk Hillebrands, Isabell Mandel, Ralf Loesel, Grietje Beck, Lothar Schilling, Peter Schnuelle, Benito Yard

  Transplant international : official journal of the European Society for Organ Transplantation. 11/2010; 23(11):1144-53.

  Allograft vasculopathy is the leading cause for chronic transplant loss. We investigated if the addition of carbon monoxide releasing molecules (CORMs) to the preservation solution would protect theAllograft vasculopathy is the leading cause for chronic transplant loss. We investigated if the addition of carbon monoxide releasing molecules (CORMs) to the preservation solution would protect the endothelium from cold preservation injury in an aortic transplantation model. In particular, we tested if CORM preserve vascular functioning and limit neo-intima formation following cold preservation (Cp). Abdominal aortas from Lewis or Fisher rats were subjected to Cp in University of Wisconsin (UW) solution to which 50 μm of CORM-3 was added or not. Hereafter, whole mount staining, acetylcholine mediated vasorelaxation (AMV) and aortic transplantation was performed. In vitro CORM-3 protected human umbilical vein endothelial cells from Cp injury and prevented denudation and intercellular gap formation in aortic grafts. Cp resulted in loss of AMV of aorta segments. By contrast, AMV was preserved after the addition of CORM-3 during Cp. Two months after transplantation Cp of aorta grafts resulted in an increased adventitial remodelling and neo-intima formation. This was significantly blunted by CORM-3 in syngeneic recipients. Our study demonstrates that addition of CORM-3 to UW solution prevents endothelial damage, thereby maintaining vascular function directly after cold preservation. Hence, our findings might offer a novel strategy to prevent vascular damage during CP.

• In vivo chlorine-35, sodium-23 and proton magnetic resonance imaging of the rat brain.

  Authors: Stefan Kirsch, Mark Augath, David Seiffge, Lothar Schilling, Lothar R Schad

  NMR in biomedicine. 03/2010; 23(6):592-600.

  In this study we demonstrate the feasibility of combined chlorine-35, sodium-23 and proton magnetic resonance imaging (MRI) at 9.4 Tesla, and present the first in vivo chlorine-35 images obtained byIn this study we demonstrate the feasibility of combined chlorine-35, sodium-23 and proton magnetic resonance imaging (MRI) at 9.4 Tesla, and present the first in vivo chlorine-35 images obtained by means of MRI. With the experimental setup used in this study all measurements could be done in one session without changing the setup or moving the subject. The multinuclear measurement requires a total measurement time of 2 h and provides morphological (protons) and physiological (sodium-23, chlorine-35) information in one scanning session. Chlorine-35, sodium-23 and high resolution proton images were acquired from a phantom, a healthy rat and from a rat displaying a focal cerebral infarction. Compared to the healthy tissue a signal enhancement of a factor of 2.2 +/- 0.2 in the chlorine-35 and a factor of 2.9 +/- 0.6 in the sodium-23 images is observed in the areas of infarction. Exemplary unlocalized measurement of the in vivo longitudinal and transversal relaxation time of chlorine-35 in a healthy rat showed multi-exponential behaviour. A biexponential fit revealed a fast and a slow relaxing component with T(1,a) = (1.7 +/- 0.4) ms, T(1,b) = (25.1 +/- 1.4) ms, amplitudes of A = 0.26 +/- 0.02, (1-A) = 0.74 +/- 0.02 and T(2,a) = (1.3 +/- 0.1) ms, T(2,b) = (11.8 +/- 1.1) ms, A = 0.64 +/- 0.02, (1-A) = 0.36 +/- 0.02. Combined proton, sodium-23 and chlorine-35 MRI may provide a new approach for non-invasive studies of ionic regulatory processes under physiological and pathological conditions in vivo.

• Application of Micro-CT in Small Animal Imaging.

  Authors: Sebastian J Schambach, Simona Bag, Lothar Schilling, Christoph Groden, Marc A Brockmann

  Methods (San Diego, Calif.). 09/2009;

  Over the past decade, the number of publications using micro-computed tomography (muCT) imaging in preclinical in vivo studies has risen exponentially. Higher spatial and temporal resolution are theOver the past decade, the number of publications using micro-computed tomography (muCT) imaging in preclinical in vivo studies has risen exponentially. Higher spatial and temporal resolution are the key technical advancements that have allowed researchers to capture increasingly detailed anatomical images of small animals, and to monitor the progression of disease in small animal models. The purpose of this review is to present the technical aspects of muCT, as well as, current research applications. Our objectives are threefold: to familiarize the reader with the basics of muCT-techniques; to present the type of experimental designs currently used; and to highlight limitations, future directions, in muCT-scanner research applications, and experimental methods. As a first step we present different muCT setups and components, as well as, image contrast generation principles. We then present experimental approaches in order of the evaluated organ system. Finally, we provide a short summary of some of the technical limitations of muCT imaging, and discuss potential future developments in muCT-scanner techniques and experimental setups.

• Vascular Imaging in Small Rodents Using Micro-CT.

  Authors: Sebastian J Schambach, Simona Bag, Christoph Groden, Lothar Schilling, Marc A Brockmann

  Methods (San Diego, Calif.). 09/2009;

  In vivo animal models of neoplasm, stroke, subarachnoid haemorrhage, and other diseases involving alterations in vessel anatomy and diameter, require a fast and easy-to-use imaging tool that capturesIn vivo animal models of neoplasm, stroke, subarachnoid haemorrhage, and other diseases involving alterations in vessel anatomy and diameter, require a fast and easy-to-use imaging tool that captures anatomical structure and biologic function data. Micro-computed tomography angiography (muCTA) offers high spatial and temporal resolution and is suitable to perform this task. However, conducting muCTA in small rodents, especially in mice, requires a high degree of accuracy and precision. This article describes a setup for in vivo muCTA in mice using both a bolus technique with a conventional contrast agent, as well as, angiography with a blood pool contrast agent. Our setup in mice is at isotropic resolutions up to 16 mum with scanning times less than one minute. The described protocol also addresses some of the technical challenges associated with the imaging of vascular structures in mice models.

• Ultrafast High-Resolution In Vivo Volume-CTA of Mice Cerebral Vessels.

  Authors: Sebastian J Schambach, Simona Bag, Volker Steil, Cristina Isaza, Lothar Schilling, Christoph Groden, Marc A Brockmann

  Stroke; a journal of cerebral circulation. 03/2009;

  BACKGROUND AND PURPOSE: Animal models developed in rats and mice have become indispensable in preclinical cerebrovascular research. Points of interest include the investigation of the vascular bedBACKGROUND AND PURPOSE: Animal models developed in rats and mice have become indispensable in preclinical cerebrovascular research. Points of interest include the investigation of the vascular bed and the morphology and function of the arterial, capillary, and venous vessels. Because of their extremely small caliber, in vivo examination of these vessels is extremely difficult. In the present study we have developed a method to provide fast 3D in vivo analysis of cerebral murine vessels using volume computed tomography-angiography (vCTA). METHODS: Using an industrial X-ray inspection system equipped with a multifocus cone beam X-ray source and a 12-bit direct digital flatbed detector, high-speed vCTA (180 degrees rotation in 40 s. at 30 fps) was performed in anesthetized mice. During the scan an iodinated contrast agent was infused via a tail vein. Images were reconstructed using a filtered backprojection algorithm. Image analysis was performed by maximum intensity projection (MIP) and 3D volume reconstruction. RESULTS: All mice tolerated i.v. injection of the iodinated contrast agent well. Smallest achievable voxel size of raw data while scanning the whole neurocranium was 16 microm. Anatomy of cerebral vessels was assessable in all animals, and anatomic differences between mouse strains could easily be detected. Mean vessel diameter was measured in C57BL/6 and BALBc mice. Changes of vessel caliber were assessable by repeated vCTA. CONCLUSIONS: Ultra fast in vivo vCTA of murine cerebral vasculature is feasible at resolutions down to 16 microm. The technique allows the assessment of vessel caliber changes in living mice, thus providing an interesting tool to monitor different features such as vasospasm or vessel patency.

• Clazosentan, an endothelin receptor antagonist, prevents early hypoperfusion during the acute phase of massive experimental subarachnoid hemorrhage: a laser Doppler flowmetry study in rats.

  Authors: Gerrit A Schubert, Lothar Schilling, Claudius Thomé

  Journal of neurosurgery. 01/2009; 109(6):1134-40.

  Object Acute cerebral hypoperfusion and early disturbances in cerebral autoregulation after subarachnoid hemorrhage (SAH) have been demonstrated repeatedly and have been shown to contributeObject Acute cerebral hypoperfusion and early disturbances in cerebral autoregulation after subarachnoid hemorrhage (SAH) have been demonstrated repeatedly and have been shown to contribute significantly to acute and secondary brain injury. Acute vasoconstriction has been identified as a major contributing factor. Although increasing evidence implicates endothelin (ET)-1 in the development of cerebral vasospasm, its role in the acute phase after SAH has not yet been investigated. The purpose of this study was to further determine the role of ET in the first minutes to hours after massive experimental SAH induced by prophylactic treatment with the ET receptor antagonist clazosentan. Methods Subarachnoid hemorrhage was induced in 22 anesthetized rats by injection of 0.5-ml arterial, nonheparinized blood into the cisterna magna over the course of 60 seconds. In addition to monitoring intracranial pressure (ICP) and mean arterial blood pressure, laser Doppler flowmetry (LDF) probes were placed stereotactically over the cranial windows to allow online recording of cerebral blood flow (CBF) starting 30 minutes prior to SAH and continuing for 3 hours after SAH. The control group (Group A, 11 rats) received vehicle saline solution via a femoral catheter before SAH, and a second group (Group B, 11 rats) was treated prophylactically with clazosentan, an ET(A) receptor antagonist. Treatment was started 30 minutes prior to bolus injection (1 mg/kg body weight), immediately followed by a continuous infusion of 1 mg/kg body weight/hr until the end of the experiment. Results Induction of SAH in the rats caused an immediate increase in ICP, which led to an acute decrease in cerebral perfusion pressure (CPP). Perfusion, as measured with LDF, was found to have decreased relative to baseline by 30 +/-20% in the control group and 20 +/-9% in the clazosentan-treated group. Intracranial pressure and CPP recovered comparably in both groups thereafter within minutes. Control animals demonstrated prolonged hypoperfusion with a loss of autoregulation independent of CPP changes, finally approaching 80% of baseline values toward the end of the experiment. The authors observed that clazosentan did not influence peracute CPP-dependent hypoperfusion, but prevented continuous CBF reduction. Laser Doppler flowmetry perfusion readings remained depressed in control animals at 73 +/-19% of baseline in comparison with 106 +/-25% of baseline in clazosentan-treated animals (p = 0.001). Conclusions The first hours after a massive experimental SAH can be characterized by a CPP-independent compromise in cerebral perfusion. Prophylactic treatment with the ET receptor antagonist clazosentan prevented hypoperfusion. It is known that in the first days after SAH, a reduction in CBF correlates clinically to high-grade SAH. Although research currently focuses on delayed vasospasm, administration of vasoactive drugs in the acute phase of SAH may reverse perfusion deficits and improve patient recovery.

• Hypothermia reduces cytotoxic edema and metabolic alterations during the acute phase of massive SAH: a diffusion-weighted imaging and spectroscopy study in rats.

  Authors: Gerrit Alexander Schubert, Sven Poli, Lothar Schilling, Sabine Heiland, Claudius Thomé

  Journal of neurotrauma. 07/2008; 25(7):841-52.

  Acute changes in cerebral perfusion and metabolism after subarachnoid hemorrhage (SAH) have been shown to contribute significantly to acute brain injury. The purpose of this study was to examine theAcute changes in cerebral perfusion and metabolism after subarachnoid hemorrhage (SAH) have been shown to contribute significantly to acute brain injury. The purpose of this study was to examine the effects of moderate hypothermia on the acute changes after massive experimental SAH as evaluated by diffusion-weighted imaging (DWI) and magnetic resonance spectroscopy (MRS). SAH in rats was induced by injection of 0.5 mL of arterial blood. Normothermic animals (NT, n = 10) were kept at 37.0 +/- 0.2 degrees C, while temperature was lowered to 32.0 +/- 0.2 degrees C in the primary hypothermia group (pHT, n = 10) prior to SAH and in the secondary hypothermia group (sHT, n = 10) immediately after SAH. DWI and MRS were performed from 30 min prior up to 3 h after injury. The apparent diffusion coefficient (ADC) was measured in cortical and hippocampal regions of interest (ROIs). MRS included lactate, N-acetyl aspartate (NAA), and creatine in a central voxel. DWI showed a generalized, significant decline in ADC after SAH in NT. Significant change in ADC in pHT was absent, and accelerated recovery for animals in sHT was noted. MRS analysis revealed significant lactate accumulation to 204 +/- 40% from baseline only in NT, while sHT was characterized by a transient, less pronounced increase of lactate (159 +/- 11%) and lactate in pHT did not change significantly (117 +/- 11%). NAA did not change significantly when compared to baseline or between groups for NT, pHT, or sHT. Creatine rose significantly to 166 +/- 27% in NT after the insult, indicating increased metabolic stress which was absent in pHT (106 +/- 8%) and sHT (124 +/- 18%). Hypothermia can ameliorate early development of cytotoxic edema, lactate accumulation, and a general metabolic stress response after SAH, even when started after the insult. Our study indicates that a potentially beneficial influence on metabolism and cerebral perfusion in this crucial phase is practicable and might hold the key to further improve outcome in SAH.

• Hypothermia reduces early hypoperfusion and metabolic alterations during the acute phase of massive subarachnoid hemorrhage: a laser-Doppler-flowmetry and microdialysis study in rats.

  Authors: Gerrit Alexander Schubert, Sven Poli, Aminadav Mendelowitsch, Lothar Schilling, Claudius Thomé

  Journal of neurotrauma. 06/2008; 25(5):539-48.

  Morbidity and mortality of subarachnoid hemorrhage (SAH) are correlated with the severity of the patient's acute neurological deficit. This initial presentation has been attributed to cerebralMorbidity and mortality of subarachnoid hemorrhage (SAH) are correlated with the severity of the patient's acute neurological deficit. This initial presentation has been attributed to cerebral hypoperfusion in the acute phase, and we investigated the impact of moderate hypothermia on the early changes in perfusion and metabolism following massive experimental SAH. SAH was induced in 61 anesthetized rats by rapid injection of 0.5 mL of arterial blood into the cisterna magna. In normothermia (NT), animals were kept at 37 degrees C, while in the primary hypothermia (pHT) group, temperature was lowered to 32 degrees C prior to SAH, and in the secondary hypothermia (sHT) group, cooling was started immediately after SAH. From 30 min prior to 180 min after SAH, Laser-Doppler-flowmetry (LDF) probes allowed online recording of cerebral blood flow (CBF) while parenchymal dialysate was collected by microdialysis probes within the frontoparietal cortex. In NT, the acute phase was characterized by impaired autoregulation and prolonged hypoperfusion. In pHT and sHT, autoregulation was preserved and acute hypoperfusion rapidly improved. SAH also caused a highly significant reduction in glucose in NT only. pHT significantly reduced accumulation of lactate, glutamate, and aspartate. Comparable trends were present for histidine, GABA, and taurine, while glutamine consumption was ameliorated. Early perfusion deficits caused by acute hypoperfusion and disruption of cerebral autoregulation can be ameliorated by hypothermia. Also, the acute phase of experimental SAH is characterized by glucose depletion, lactate accumulation, and release of excitatory amino acids, which can be influenced favorably by hypothermia.

• Angiotensin converting enzyme inhibition for arterial hypertension reduces the risk of recurrence in patients with chronic subdural hematoma possibly by an antiangiogenic mechanism.

  Authors: Ralf Weigel, Axel Hohenstein, Linda Schlickum, Christel Weiss, Lothar Schilling

  Neurosurgery. 11/2007; 61(4):788-92; discussion 792-3.

  OBJECTIVE: Chronic subdural hematoma (CSH) is characterized by pathological vascularization of the parietal membrane. Plasma leakage from immature vessels may be involved in hematoma enlargement andOBJECTIVE: Chronic subdural hematoma (CSH) is characterized by pathological vascularization of the parietal membrane. Plasma leakage from immature vessels may be involved in hematoma enlargement and recurrence. We tested the hypothesis that the antiangiogenic side-effect of angiotensin converting enzyme (ACE)-inhibitor treatment for the control of arterial hypertension reduces the risk of recurrence in CSH. METHODS: We analyzed the data of 438 patients with CSH treated by a standard surgical procedure for hematoma evacuation in our department between 1995 and 2003. Patients with coagulopathies, malignancies, and independent neurological disorders were excluded from this study. Patient records were screened for age, sex, pre- and postoperative Markwalder score, arterial hypertension, medication with ACE-inhibitors, and recurrence of CSH. The rate of ACE-inhibitor treatment in our CSH patients was compared with an age-matched control group treated for herniated lumbar disc at the same time. The concentration of vascular endothelial growth factor was analyzed in hematoma samples and corresponding venous blood in 40 consecutive patients. RESULTS: A total of 310 patients were included in this study. The demographic data of Group A (with ACE-inhibition) and Group B (without ACE-inhibition) were comparable. In Group A, 5% (four out of 81) of the patients experienced recurrence as opposed to 18% (42 out of 229) in Group B (P = 0.00345). A negative correlation was found between the yearly rates of medication with ACE-inhibitors and recurrence (r = -0.8488; P = 0.0044). The rate of ACE-inhibitor treatment was lower in the CSH patients (25%) than in the control group (40%). The VEGF content was significantly lower in the hematoma in patients with ACE-inhibition (mean, 8891 pg/ml; range, 4300-18,300 pg/ml) than in patients without (mean, 22,565 pg/ml; range, 4200-89,650 pg/ml; P = 0.0116). CONCLUSION: Our data suggest that ACE-inhibitor treatment for the control of arterial hypertension lowers the risk of recurrence in patients undergoing operation for CSH and possibly even the development of CSH. This effect might be the result of an antiangiogenic mechanism of ACE-inhibitors.

• A new method for superselective middle cerebral artery infusion in the rat.

  Authors: Johannes Woitzik, Lothar Schilling

  Journal of neurosurgery. 06/2007; 106(5):872-8.

  OBJECT: Selective intraarterial drug delivery is used to achieve enhanced local uptake with reduced systemic side effects. In the present paper the authors describe and characterize a newOBJECT: Selective intraarterial drug delivery is used to achieve enhanced local uptake with reduced systemic side effects. In the present paper the authors describe and characterize a new microcatheter-based model of superselective perfusion of the middle cerebral artery (MCA) in rats combined with blockade of blood flow through the MCA. METHODS: Selectivity of administration was shown by infusion of Evans blue which diffusely stained the MCA territory, indicating an increased permeability of the blood-brain barrier during the blockade of blood flow to the MCA. Perfusion of autologous blood through the microcatheter resulted in a flow rate-related increase in the cerebral blood flow measured by laser Doppler flowmetry. Similarly, infusion of an artificial O2 carrier, Oxycyte, was accompanied by an increase in tissue oxygenation as measured using a Licox sensor. Blockade of blood flow to the MCA with the new microcatheter for an extended period of time resulted in the development of ischemia, which was comparable to that induced by intravascular occlusion using a silicone-coated thread. In a 24-hour MCA occlusion model, selective administration of a low dose of MK-801 (0.3 mg/kg body weight) resulted in a significantly smaller infarct volume than systemic application (339 +/- 53 mm(3) compared with 508 +/- 26 mm(3), p < 0.001). CONCLUSIONS: This new model of superselective MCA infusion is a valuable tool for investigating the effect of selective delivery and enhanced drug uptake into cerebral ischemic tissue. Without constant blockade of blood flow through the MCA it may also be useful for enhanced drug uptake, gene transfer, or application of stem cells in other neuropathological conditions.

• Granulocyte-macrophage colony-stimulating factor-induced vessel growth restores cerebral blood supply after bilateral carotid artery occlusion.

  Authors: Ulf C Schneider, Lothar Schilling, Helmut Schroeck, C Thomas Nebe, Peter Vajkoczy, Johannes Woitzik

  Stroke; a journal of cerebral circulation. 05/2007; 38(4):1320-8.

  BACKGROUND AND PURPOSE: Hemodynamic compromise due to occlusive cerebrovascular disease is associated with an increased stroke risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has beenBACKGROUND AND PURPOSE: Hemodynamic compromise due to occlusive cerebrovascular disease is associated with an increased stroke risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been suggested to stimulate collateral blood vessel growth in various models of hemodynamic compromise. The purpose of this study was to investigate the effects of GM-CSF on cerebral hemodynamics and vessel growth in a rat model of chronically impaired cerebral blood flow (CBF). METHODS: Male Sprague-Dawley rats underwent sequential bilateral carotid artery occlusion (BCO) and were treated with GM-CSF or saline for 6 weeks. Sham-occluded animals served as a control group. Baseline CBF was measured by iodo[(14)C]antipyrine autoradiography, and cerebrovascular reserve capacity was assessed by laser-Doppler flowmetry after application of 20 mg/kg body weight acetazolamide. The capillary density and arterioles immunopositive for alpha-smooth muscle actin were counted on brain sections. The cerebral angioarchitecture was visualized with a latex perfusion technique. RESULTS: Baseline CBF as measured by iodo[(14)C]antipyrine autoradiography was not affected by BCO. The cerebrovascular reserve capacity, however, was significantly impaired 1 week after BCO. CBF and cerebrovascular reserve capacity recovered completely in GM-CSF-treated animals but not in solvent-treated animals. Histologic analysis of the hippocampus revealed integrity of the hypoxia-vulnerable neurons in all animals. The capillary density showed a very mild increase in GM-CSF-treated animals. However, the number of intraparenchymal and leptomeningeal arterioles was significantly higher in GM-CSF-treated animals than in both other groups. CONCLUSIONS: Long-term GM-CSF treatment in a BCO model in rats leads to restoration of impaired cerebral hemodynamics and accompanies structural changes in the resistance-vessel network.

• Comparison of different intravascular thread occlusion models for experimental stroke in rats.

  Authors: Johannes Woitzik, Ulf C Schneider, Claudius Thomé, Helmut Schroeck, Lothar Schilling

  Journal of neuroscience methods. 04/2006; 151(2):224-31.

  For the induction of ischemic strokes of varying sizes in rats, different types of threads were used to occlude the middle cerebral artery (MCA) in combination with or without the posteriorFor the induction of ischemic strokes of varying sizes in rats, different types of threads were used to occlude the middle cerebral artery (MCA) in combination with or without the posterior communicating artery (PCOM) and the common carotid artery (CCA). During vessel occlusion brain tissue partial oxygen pressure (ptiO2) and regional cerebral blood flow (rCBF) were monitored using a combined ptiO2/laser Doppler flow probe. Following neurological assessment animals were sacrificed at 3, 8 and 24 h and the necrotic volume was measured on serial coronary slices. In another experimental group, rCBF was measured 1h post-insult with the iodo[14C]antipyrine method. Animals with selective MCA occlusion showed less reduction of ptiO2 and rCBF and smaller infarcts when compared with animals with combined occlusion of the MCA, CCA and PCOM. Both groups, selective MCA occlusion and combined occlusion of the MCA, CCA and PCOM, demonstrated a high reproducibility and low variability of stroke size. Relative growth of stroke size within 24 h was higher in animals with selective MCA occlusion. Therefore, the selective MCAO model may be advantageous for studies of neuroprotective strategies.

• Early administration of a second-generation perfluorochemical decreases ischemic brain damage in a model of permanent middle cerebral artery occlusion in the rat.

  Authors: Johannes Woitzik, Nina Weinzierl, Lothar Schilling

  Neurological research. 07/2005; 27(5):509-15.

  OBJECTIVES: Perfluorochemicals (PFCs) may exert a neuroprotective function in the early phase of ischemia by improving the oxygen supply to the endangered tissue. We have, therefore, investigated theOBJECTIVES: Perfluorochemicals (PFCs) may exert a neuroprotective function in the early phase of ischemia by improving the oxygen supply to the endangered tissue. We have, therefore, investigated the effect of Oxycyte, a second-generation perfluorocarbon solution, on the extent of early ischemic brain damage in a model of permanent focal cerebral ischemia. METHODS: Eight hours of permanent focal cerebral ischemia was induced in isoflurane anesthetized male Sprague-Dawley rats by unilateral middle cerebral artery (MCA) thread occlusion under the control of laser Doppler flowmetry (LDF). Animals were assigned to one of the following treatment groups: nO2-NaCl and hO2-NaCl-NaCl (0.9%, 1 ml/100 g i.v.) and nO2-Oxycyte and hO2-Oxycyte-Oxycyte (1 ml/100 g i.v.). The injection of NaCl or Oxycyte was performed immediately after MCA occlusion. After injection, breathing was changed to pure oxygen in groups hO2-NaCl and hO2-Oxycyte while animals in groups nO2-NaCl and nO2-Oxycyte were allowed to breathe air. The necrotic volume was calculated from serial coronal sections stained with silver-nitrate. In addition, nitrotyrosine production was studied by immunohistochemistry. RESULTS: Upon MCA occlusion, animals showed a reduction of cerebral blood flow of approximately 80% of the LDF signal in all groups. Hemodynamic and metabolic parameters were not affected by the infusion of Oxycyte. The total infarct volume was reduced in hO2-Oxycyte animals [group nO2-NaCl: 341+/-31 mm3 (mean+/-SD), group hO2-NaCl: 351+/-33 mm3, group nO2-Oxycyte: 354+/-24 mm3, and group hO2-Oxycyte: 300+/-29 mm3, p < 0.05 versus all other groups]. Moreover, hO2-Oxycyte animals showed lesser intensity of nitrotyrosine staining when compared with hO2-NaCl animals. DISCUSSION: These results suggest that Oxycyte administered immediately after the onset of vascular occlusion may exert neuroprotective effects in the early phase of brain ischemia.

• Cerebrovascular characterization of clazosentan, the first nonpeptide endothelin receptor antagonist clinically effective for the treatment of cerebral vasospasm. Part I: inhibitory effect on endothelin(A) receptor-mediated contraction.

  Authors: Hartmut Vatter, Michael Zimmermann, Veronika Tesanovic, Andreas Raabe, Lothar Schilling, Volker Seifert

  Journal of neurosurgery. 07/2005; 102(6):1101-7.

  OBJECT: The central role of endothelin (ET)-1 in the development of cerebral vasospasm after subarachnoid hemorrhage is indicated by the successful treatment of this vasospasm in several animalOBJECT: The central role of endothelin (ET)-1 in the development of cerebral vasospasm after subarachnoid hemorrhage is indicated by the successful treatment of this vasospasm in several animal models by using selective ET(A) receptor antagonists. Clazosentan is a selective ET(A) receptor antagonist that provides for the first time clinical proof that ET-1 is involved in the pathogenesis of cerebral vasospasm. The aim of the present investigation was, therefore, to define the pharmacological properties of clazosentan that affect ET(A) receptor-mediated contraction in the cerebrovasculature. METHODS: Isometric force measurements were performed in rat basilar artery (BA) ring segments with (E+) and without (E-) endothelial function. Concentration effect curves (CECs) were constructed by cumulative application of ET-1 or big ET-1 in the absence or presence of clazosentan (10(-9), 10(-8), and 10(-7) M). The inhibitory potency of clazosentan was determined by the value of the affinity constant (pA2). The CECs for contraction induced by ET-1 and big ET-1 were shifted to the right in the presence of clazosentan in a parallel dose-dependent manner, which indicates competitive antagonism. The pA2 values for ET-1 were 7.8 (E+) and 8.6 (E-) and the corresponding values for big ET-1 were 8.6 (E+) and 8.3 (E-). CONCLUSIONS: The present data characterize clazosentan as a potent competitive antagonist of ET(A) receptor-mediated constriction of the cerebrovasculature by ET-1 and its precursor big ET-1. These functional data may also be used to define an in vitro profile of an ET receptor antagonist with a high probability of clinical efficacy.

• Cerebrovascular characterization of clazosentan, the first nonpeptide endothelin receptor antagonist shown to be clinically effective for the treatment of cerebral vasospasm. Part II: effect on endothelin(B) receptor-mediated relaxation.

  Authors: Hartmut Vatter, Michael Zimmermann, Veronika Tesanovic, Andreas Raabe, Volker Seifert, Lothar Schilling

  Journal of neurosurgery. 07/2005; 102(6):1108-14.

  OBJECT: The disturbed balance between nitric oxide and endothelin (ET)-1 in the cerebrovasculature seems to play a major role in the development of cerebral vasospasm after subarachnoid hemorrhage.OBJECT: The disturbed balance between nitric oxide and endothelin (ET)-1 in the cerebrovasculature seems to play a major role in the development of cerebral vasospasm after subarachnoid hemorrhage. Endothelin-1 represents the contractile part in this balance. In addition to the prevailing ET(A) receptor-dependent contractile effect, ET-1 also has ET(B) receptor-mediated vasodilatory attributes. The aim of the present study was to define the actual selectivity of clazosentan, the first putative highly ET(A) receptor-selective antagonist clinically proven to be effective in the treatment of vasospasm in the cerebrovasculature. METHODS: Rat basilar artery ring segments with endothelial function were used for the measurement of isometric force. Concentration effect curves were constructed by cumulative application of sarafotoxin S6c, ET-1, or big ET-1 in the presence or absence of clazosentan (10(-9) to 10(-6) M) after a precontraction was induced by prostaglandin F2alpha. The inhibition by clazosentan was estimated by the value of the affinity constant (pA2). The relaxation induced by sarafotoxin S6c, ET-1, and big ET-1 was inhibited in a competitive manner by clazosentan, yielding pA2 values of 7.1, 6.7, and 6.5, respectively. The selectivity to the ET(A) receptor in the cerebrovascular system was approximately two logarithmic units. CONCLUSIONS: The present investigation shows a competitive inhibition of ET(B) receptor-mediated relaxation in cerebral vessels by clazosentan in therapeutically relevant concentrations. Thus, additional clinical trials should be undertaken to evaluate clazosentan concentrations in cerebrospinal fluid. Furthermore, the present data may be taken to describe the pharmacological properties for an ET receptor antagonist specifically tailored for the treatment of pathological conditions of impaired cerebral blood flow.

• Increased mRNA expression of VEGF within the hematoma and imbalance of angiopoietin-1 and -2 mRNA within the neomembranes of chronic subdural hematoma.

  Authors: Axel Hohenstein, Ralf Erber, Lothar Schilling, Ralf Weigel

  Journal of neurotrauma. 06/2005; 22(5):518-28.

  The aim of the study was to determine the source of vascular endothelial growth factor (VEGF) in hematoma fluid of patients suffering from chronic subdural hematoma (CSH) and to identify the level ofThe aim of the study was to determine the source of vascular endothelial growth factor (VEGF) in hematoma fluid of patients suffering from chronic subdural hematoma (CSH) and to identify the level of gene expression of the pro-angiogenic factors angiopoietin 1 (ANG-1) and ANG-2 in hematoma membranes. Samples of venous blood, hematoma fluid, and outer membrane were obtained during surgery for CSH. The numbers of mononuclear cells were determined in hematoma fluid and in venous blood samples taken from 11 patients. The concentration of VEGF was measured by ELISA technique in hematoma fluid and in plasma. RT-PCR methodology was used to study the expression of different mRNA species in 11 patients. The mRNA species analyzed include VEGF and its receptors, VEGFR-1 and VEGFR-2, and ANG-1, ANG-2 and their receptor, Tie-2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as housekeeping gene and was used for semiquantitative analysis. The VEGF concentration was several hundred fold higher in the hematoma fluid than in corresponding plasma samples. A significant correlation was found between the number of neutrophils and the VEGF content in the hematoma fluid. The expression levels of VEGF, mainly VEGF165 and VEGF121 mRNA were highest in cells obtained from the hematoma fluid. In membrane samples, mRNA encoding for VEGF and its receptors was only inconsistently detected while the mRNA species encoding for ANG-1, ANG-2, and Tie-2 were found throughout all samples. The mean ratio of ANG-1/ANG-2 mRNA expression was 0.48 as opposed to 1.9 in a normal human brain tissue sample. The results suggest that the hematoma cells are the primary source of VEGF. A marked increase in the expression of ANG-2 mRNA over ANG-1 mRNA demonstrates a pro-angiogenic pattern in the hematoma membranes. Persistent activation of the ANG/Tie-2 system in addition to high levels of VEGF may keep the vasculature in a destabilized condition and may account for the continuous formation of new and immature blood vessels resulting in massive plasma extravasation and repeated bleeding episodes. Thus, the present study provides new evidence in favor of pro-angiogenic mechanisms playing an important role in the pathophysiology of CSH.

• Hypothermia as a neuroprotective strategy in subarachnoid hemorrhage: a pathophysiological review focusing on the acute phase.

  Authors: Claudius Thomé, Gerrit A Schubert, Lothar Schilling

  Neurological research. 05/2005; 27(3):229-37.

  Aneurysmal subarachnoid hemorrhage (SAH) remains a very prevalent challenge in neurosurgery associated with a high morbidity and mortality due to the lack of specific treatment modalities. TheAneurysmal subarachnoid hemorrhage (SAH) remains a very prevalent challenge in neurosurgery associated with a high morbidity and mortality due to the lack of specific treatment modalities. The prognosis of SAH patients depends primarily on three factors: (i) the severity of the initial bleed, (ii) the endovascular or neurosurgical procedure to occlude the aneurysm and (iii) the occurrence of late sequelae, namely delayed ischemic neurological deficits due to cerebral vasospasm. While neurosurgeons and interventionalists have put significant efforts in minimizing periprocedural complications and a multitude of investigators have been devoted to the research on chronic vasospasm, the acute phase of SAH has not been studied in comparable detail.In various experimental studies during the past decade, hypothermia has been shown to reduce neuronal damage after ischemia, traumatic brain injury and other cerebrovascular diseases. Clinically, only some of these encouraging results could be reproduced. This review analyses results of studies on the effects of hypothermia on SAH with special respect to the acute phase in an experimental setting. Based on the available data, some considerations for the application of mild to moderate hypothermia in patients with subarachnoid hemorrhage are given.

• Effects of chronic endothelin ET(A) receptor blockade on blood pressure and vascular formation of cyclic guanosine-3',5'-monophosphate in spontaneously hypertensive rats.

  Authors: Michael Kirchengast, Klaus Witte, Kerstin Stolpe, Lothar Schilling, Pavel I Nedvetsky, Harald H H W Schmidt, Björn Lemmer

  Arzneimittel-Forschung. 02/2005; 55(9):498-504.

  Endothelin (ET) mediates vasoconstriction in intact arterial blood vessels with functional endothelium via stimulation of ET(A) receptors, while ET(B) receptor stimulation leads to vasodilation viaEndothelin (ET) mediates vasoconstriction in intact arterial blood vessels with functional endothelium via stimulation of ET(A) receptors, while ET(B) receptor stimulation leads to vasodilation via nitric oxide (NO) release and formation of cyclic guanosine-3',5'-monophosphate (cGMP). In spontaneously hypertensive rats (SHR) the cGMP-forming NO-receptor guanylyl cyclase (sGC) is downregulated. It is unclear whether ET contributes to the hypertensive phenotype of SHR, and whether this involves the disturbed cGMP signaling. The selective ETA receptor antagonist darusentan (CAS 171714-84-4), given orally via drinking water (10 mg kg(-1) d(-1)) for 12 weeks, significantly lowered systolic blood pressure of SHR as determined by radiotelemetry. Neither impaired endothelium-dependent relaxation to acetylcholine was restored nor sGC expression and activity affected when compared to control SHR. While these findings show a role for ETA receptors in blood pressure regulation in genetically elevated blood pressure, downregulation of sGC expression and cGMP-mediated vasorelaxant response in SHR were shown to be independent of ETA receptors. The findings suggest distinct mechanisms of gene expression affecting ET and cGMP mediated vasomotor functions.

• Time course of fluorescence intensity and protein expression in HeLa cells stably transfected with hrGFP.

  Authors: Petra Kirsch, Mathias Hafner, Hanswalter Zentgraf, Lothar Schilling

  Molecules and cells. 07/2003; 15(3):341-8.

  The green fluorescent protein (GFP), obtained from Aequorea victoria, is widely used in transfection experiments to study the dynamics and characteristics of gene expression in a non-invasive manner.The green fluorescent protein (GFP), obtained from Aequorea victoria, is widely used in transfection experiments to study the dynamics and characteristics of gene expression in a non-invasive manner. However, generation of cell lines displaying long-term expression of the GFP protein has repeatedly been reported unsuccessful. In the present study a different marker protein, humanized GFP (hrGFP) obtained from a sea pansy, Renilla reniformis, was used for transfection, and the time course of protein expression was studied by live cell fluorescence microscopy, PCR technology, and immunoblotting. HeLa cells were transfected with a plasmid, phrGFP-IRES-Neo containing the neomycin cassette and the hrGFP gene together with an internal ribosome entry site (IRES) sequence under the control of a CMV promotor. Cells were selected for three weeks after transfection using G418. During this time, hrGFP fluorescence was highest around day 4 and decreased thereafter eventually disappearing until the end of the selection period. In parallel, the proportion of cells staining positive for propidium iodide (i.e. dead cells) increased steadily. Three weeks after transfection non-fluorescent clones were present throughout. A couple of clones were subcultured for another week and used for further analyses. PCR methodology revealed the insertion of the hrGFP into the HeLa genome and the presence of mRNA species coding for hrGFP following reverse transcription. However, immunoblot analysis failed to show expression of the protein. These observations suggest a down-regulation of hrGFP expression at both transcriptional and post-transcriptional levels similar to results previously obtained with GFP. Thus, use of hrGFP does not appear to offer an advantage over GFP in transfection experiments aiming at permanent expression of the marker protein.