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In dieser experimentellen Studie wurde der Einfluss einer moderaten Hypothermie nach Induktion einer epiduralen, extraaxialen Raumforderung auf das neurologische Outcome, auf histopathologische Veränderungen und mittels bildgebender Methoden untersucht. Der Hauptaugenmerk wurde dabei eindeutig auf die neurologischen Verlaufsuntersuchungen mit Hilfe einer neuropsychologischen Testbatterie gelegt.Damit konnte in etwa die Hauptphase der klinischen Rekonvaleszens nach Trauma abgedeckt werden.Zudem hatten die meisten experimentellen Arbeiten bereits nach wesentlich kürzeren Zeiträumen ihre Nachuntersuchungen abgeschlossen.Die Gesamtmortalität betrug bei den normotherm behandelten Tieren 55% und bei den hypotherm behandelten Tieren 45%. Der Unterschied betrug damit nur 10% und war nicht signifikant. Betrachtet man aber die Mortalitätsraten differenzierter, so zeigt sich bezüglich der rein schädigungsbedingten Mortalität als Folge von schweren neurologischen Defiziten wie Hemiparese, Inaktivität und damit verbundenen dramatischen Gewichtsverlust eine Mortalität von 5% für die Hypothermiegruppe und 30% in der Normothermiegruppe. Dies findet seine Bestätigung auch in anderen experimentellen Untersuchungen. Für die Anwendung von Hypothermie bei Schädel – Hirn –Traumen und zerebralen Ischämien in klinischen Studien ist die Datenlage bisher noch widersprüchlich. Die bisher größte Multicenterstudie in den USA von 1994 -1998 musste bei 392 Patienten mit SHT abgebrochen werden, nachdem kein therapeutischer Effekt unter Hypothermie festzustellen war (Clifton et al., 2001¹). Nähere Analysen zeigten jedoch eine Verbesserung des Outcomes bei Patienten unter 45 Jahren welche bei Aufnahme bereits hypothermen Bedingungen ausgesetzt waren. Damit stellt sich natürlich die Frage nach dem optimalen Zeitfenster für den Beginn einer hypothermen Behandlung. Als therapeutische Konsequenz erscheint damit unter Umständen ein sofortiger Beginn der Hypothermiebehandlung mit Eintreffen des Notarztes als wirkungsvoller. Zusätzlich konnten wiederum neueste Untersuchungen bei Patienten mit zerebraler Ischämie nach Herz- und Kreislaufstillstand einen protektiven Effekt einer moderater Hypothermie auf das neurologische Outcome aufzeigen (Bernard et al., 2002; Holzer et al., 2002).In unserer Studie sollte aber auf keinen Fall der nur geringe Unterschied in der Gesamtmortalität mit 55 % in der normothermen und 45 % in der hypothermen Gruppe vernachlässigt werden. Die Annäherung der Gesamtmortalität war hierbei auf eine deutlich erhöhte Rate systemischer oder lokaler Infektionen unter den hypothermen Tieren zurückzuführen.In klinischen Studien mehren sich allerdings die Hinweise auf eine durch Hypothermie bedingte Immunsuppression und damit verbundenen erhöhten Infektionsneigung. So konnten erhöhte Pneumonieraten (Schwab et al., 1998; 2001 ; Shiozaki et al., 2001) aber auch ein vermehrtes Auftreten von Meningitiden (Shiozaki et al.,2001) beobachtet werden. Shiozaki konnte zudem signifikant erhöhte Raten von Leuko- und Thrombozytopenien sowie Elektrolytentgleisungen im hypothermen Kollektiv finden (Shiozaki et al., 2001). Schwab fand in einer eigens zur Überprüfung der Nebenwirkungen von Hypothermie bei Patienten mit zerebraler Ischämie aufgelegten Studie erhöhte Raten an Pneumonien (48%), Thrombozytopenien (70%) und Bradykardien (62%) (Schwab et al.,2001). Prospektive Studien von Patienten mit kolorektalen Eingriffen wiesen ebenso unter milder Hypothermie signifikant vermehrt Wundheilungsstörungen (Kurz et al., 1996) und eine geringere Lymphozytenaktivität auf (Beilin et al., 1998). Angewandt auf unsere Studie zeigte sich ebenfalls eine erhöhte Rate von Wundheilungsstörungen unter Hypothermie, ohne dabeijedoch zu einer Beeinflussung der Ergebnisse in den neuropsychologischen Testreihen zu führen.Abschließend kann festgehalten werden, dass in dieser Studie die Induktion einer moderaten Hypothermie nach epiduraler, extraaxialer Raumforderung, zu einer Verbesserung neurologischer Defizite und damit zu einer Besserung der Lebensqualität jener Versuchstiere führte, die den Beobachtungszeitraum überlebten. Eine Verringerung der Gesamtmortalität konnte nicht erreicht werden.
Auditorische Hirnstammimplantate (ABI stellen die einzige Option der Hörrehabilitation bei bilateraler retrocochleärer Ertaubung dar. Die Implantate sind insbesondere in ihrer größten Nutzergruppe - Neurofibromatose Typ 2 Patienten - für ihr sehr variables Hörergebnis bekannt.
Die Evozierbarkeit und die Qualität der intraoperativ abgeleiteten elektrisch evozierten auditorischen Hirnstammantworten wird als möglicher Einflussfaktor auf das Outcome diskutiert. Bisher gelten weder für die Frage des Einsatzes an sich, noch für die Methodik oder die Analyse und Bewertung der EABR in der ABI-Chirurgie einheitliche Konzepte. Ziel dieser Studie ist die detaillierte Analyse der intraoperativ registrierten EABR während ABI-Implantation bei NF2-Patienten.
Zudem stellt Beurteilung der Hörfunktion mit ABI bei NF2-Patienten stellt aufgrund oftmals begleitender Symptomatik der Grunderkrankung eine besondere Herausforderung dar. Sprachtests allein spiegeln die Hörfunktion in dieser Patientengruppe nicht immer umfassend wider. Die in dieser Studie angewendete Würzburger Skala für Implantat-Hören soll dieser Problematik gerecht werden, indem Ergebnisse eines etablierten Sprachtests mit der klinischen Kommunikationsfähigkeit kombiniert werden.
Zusammenfassung der Hauptergebnisse:
Nach intraoperativer Stimulation mittels ABI zeigten sich EABR-Antworten mit null bis 3 Vertex-positiven Peaks (P1, P2, P3), welche in dieser Kohorte im Mittel nach 0,42 ms (P1), 1,43 ms (P2) bzw. 2,40 ms (P3) auftraten. Eine 2-Peak Wellenform war in dieser Studie die am häufigsten beobachtete Morphologie (78,8%). Bei der Stimulation unterschiedlicher Elektrodenkontakte zeigten sich Unterschiede in der EABR-Wellenmorphologie. Alle Antworten konnten in eine der fünf Kategorien der Würzburger EABR-Klassifikation eingeordnet werden.
Für die Latenz von P2 konnte eine statistisch signifikante Korrelation mit der Tumorausdehnung nach Hannover Klassifikation gezeigt werden.
Die Einstufung des Hörergebnisses mit ABI in NF2 nach Ergebnis im MTP-Test und nach Kommunikationsfähigkeit im Alltag unterschied sich in 7 von 22 Fällen (31,2%) um eine Kategorie. Bei der Einordnung in die Würzburger Skala für Implantat-Hören zeigte sich nach Diskussion der divergenten Fälle in 2 Fällen die Kategorisierung zugunsten des Ergebnisses im MTP-Test und in 5 Fällen zugunsten des Ergebnisses der Kommunikationsfähigkeit im Alltag.
Nützliches Hören mit ABI konnte in 95,5% der Patienten gezeigt werden, davon erzielten 68,2% Sprachverständnis.
Die Auslösbarkeit reproduzierbarer intraoperativer EABRs konnte in 95,5% Hörvermögen hervorsagen.
The numbers of monocytes and macrophages in the walls of cerebral blood vessels were counted on perfusion-fixed frozen brain sections (16 JLffi) of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHR-SP), normotensive Wistar-Kyoto (WKY) rats, and young (16-week-old) and old (2-year-old) normotensive Sprague-Dawley rats (SD-l6w and SD-2y, respectively) using monoclonal antiborlies against rat macrophages (ED2). The staining was visualized with fluoresceinlabeled second antiborlies. The ED2-specific staining in brain sections was restricted to macrophages in a perivascular location. The number of perivascular cells per square millimeter of high-power field was significantly greater in SHR-SP (8.6 ± 2.1; n = 4) and SHR (6. 7 ± 0.9; n = 6) than in normotensive WKY (4.0 ± 0.5; n = 6; p <0.01). The number of perivascular macrophages was also greater in SD-2y (7.5 ± 2.7; n = 9) than in SD-l6w (2.9 ± 1.8; n = 8; p < 0.01). No ED2 staining was found in the resident microglia or in the endothelial cells, which were identified by double staining with rhodamine-labeled anti-factor VIII-related antigen antiborlies. The results suggest that the stroke risk factors hypertension and advanced age are associated with increased subendothelial accumulation of monocytes and macrophages. This accumulation could increase the tendency for the endothelium to convert from an anticoagulant to a procoagulant surface in response to mediators released from these subendothelial cells.
The effect of 6-chloro-2,3,4,5-tetrahydro-3-methyi-1-H-3-benzazepine (SKF 86466), a selectlve nonimldazoline alpha-2 adrenoceptor antagonlst, on hippocampal re1ease of norepinephrine and dopamlne in conscious rats was lnvestigated by /n vlvo mlcrodialysis and high-pressure liquid chromatography. Additionally, extracellular concentrations of hippocampal dopamine (DA) and norepinephrtne (NE), durtng Infusion of selective monoamine uptake Inhibitors, were determined in freely moving rats. The basal concentration of NE in the dialysate was 4.9 ± 0.3 pg/20 pl. lntravenous admlnistratlon of 5 or 10 mgJkg of SKF 86466 was associated wlth a transierlt inc:rease (30 min) of 2-fold (12 ± 1 pg/20 ,d; p < .05) and 8-fold (39 ± 3 pg/20 pl; p < .05), respectlvely, in dlalysate NE, whereas a 1-mgfkg dose had no effect. DA was not detected in basal dlalysates, but after the adminlstratlon of 5 or 10 mgJkg of SKF 86466, 3.9 ± 0.4 and 6.4 ± 0.6 pg/20 pl, respectlvely, was present in the dialysates. The rnaxlmum increase in dialysate DA was reached 60 to 90 min after SKF 86466. The DA was not derived from plasma because plasma NE was elevated after the 5 mgJkg dose of SKF 86466 whereas no plasma DA was detected. ln order to determlne whether DA was present in noradrenergic nerve termlnals, the dopamine ß-hydroxylase Inhibitor SKF 1 02698 was administered (50 mgJkg i.p.). The Inhibitor decreased dialysate NE but DA was stin not detected in the dialysate. When SKF 86466 (5 mgJkg t.v.) was adminlstered 4 hr after SKF 102698, DA appeared in the dialysate but there was no lncrease in dialysate NE. Administration through the dialysis probe of the DA uptake Inhibitor, GBR-12909 (0.1 and 1 pM), dose-dependently lnaeased DA Ieveis to 5.7 ± 1.2 and 9.6 ± 2.8 pg/20 pl, respectively. GBR-12909 had no effect on hippocampal NE. Desipramine (5 and 10 pM) lncreased dose-dependently dialysate NE and lncreased DA concentrations to detectable Ieveis (2.7 ± 0.5 and 3.5 ± 0.7 pg/20 ,d, respectively). These results suggest that the a/pha-2 adrenoceptors modulate both NE and DA release in the rat hlppocampus and that DA detected in the hlppocampal dialysate might be released from dopaminergic neurons.
In dieser Arbeit wurde gezeigt, dass aus uniparentalen, embryonalen
Stammzellen mit fehlender maternal geprägter Genexpression (AG-Zellen)
differenzierte neuronale Progenitorzellen (pNPCs) eine ähnliche neuronale
Kapazität wie wildtypische Progenitorzellen haben. Sie bilden nach
histomorphologischen Kriterien in vitro adulte Neurone mit Ausbildung eines
synaptischen Netzwerks. In elektrophysiologischen PatchClamp-
Untersuchungen wurde gezeigt, dass diese Zellen, ähnlich dem wildtypischen
Pendant, spannungsabhängige Natrium- und Kaliumkanälen besitzen, ein
negatives Membranpotential haben und bei Stimulation mit repetitiven
Aktionspotentialen reagieren. Nach Transplantation in einem Schädel-Hirn-
Trauma-Modell konnten nach drei Monaten in vivo Donorzellen mit neuraler
Morphologie und der Expression von jungen, neuronalen und glialen Proteinen
gefunden werden. Die Teratombildung ist im Vergleich zum Wildtyp unverändert,
eine maligne Entartung mit invasivem Wachstum oder ausgedehnter
Metastasierung konnte nicht gefunden werden. Aus AG-Zellen generierte
neuronale Progenitorzellen sind ein starkes Instrument, um neuronale
genomische Prägung zu untersuchen. Außerdem könnte die regenerative
Kapazität für eine patientenspezifische Zellersatztherapie genutzt werden.
Die Ableitung Akustisch evozierter Potentiale (AEP) durch intraoperatives Monitoring wird regelhaft bei der Operation von Vestibularisschwannomen mit dem Ziel des Hörerhaltes durchgeführt.
Trotz AEP-Erhalt am Ende der Operation wurden Fälle mit postoperativer Taubheit beobachtet. Bisher ist es unklar, ob es sich um falsch positive AEP-Befunde oder Fälle von sekundärer Taubheit handelt.
Diese Pilotstudie, bei der zu definierten Zeitpunkten postoperativ AEP-Messungen durchgeführt wurden, zeigt erhebliche Veränderungen der AEP-Befunde im postoperativen Verlauf. Es fanden sich Patienten mit verbesserten AEP-Befunden, aber auch verschlechterten AEP bis zum vollständigen Verlust aller AEP-Komponenten.
Ob ein sekundärer Hörverlust durch frühzeitiges Erkennen von AEP-Veränderungen verhindert werden kann, wird Inhalt von weiteren Studien sein.
This article reviews experimental and clinical data on the use of magnesium as a neuroprotective agent in various conditions of cerebral ischemia. Whereas magnesium has shown neuroprotective properties in animal models of global and focal cerebral ischemia, this effect could not be reproduced in a large human stroke trial. These conflicting results may be explained by the timing of treatment. While treatment can be started before or early after ischemia in experimental studies, there is an inevitable delay of treatment in human stroke. Magnesium administration to women at risk for preterm birth has been investigated in several randomized controlled trials and was found to reduce the risk of neurological deficits for the premature infant. Postnatal administration of magnesium to babies after perinatal asphyxia has been studied in a number of controlled clinical trials. The results are promising but the trials have, so far, been underpowered. In aneurysmal subarachnoid hemorrhage (SAH), cerebral ischemia arises with the onset of delayed cerebral vasospasm several days after aneurysm rupture. Similar to perinatal asphyxia in impending preterm delivery, treatment can be started prior to ischemia. The results of clinical trials are conflicting. Several clinical trials did not show an additive effect of magnesium with nimodipine, another calcium antagonist which is routinely administered to SAH patients in many centers. Other trials found a protective effect after magnesium therapy. Thus, it may still be a promising substance in the treatment of secondary cerebral ischemia after aneurysmal SAH. Future prospects of magnesium therapy are discussed.
This article reviews experimental and clinical data on the use of various neuroprotective agents and therapeutic measures after aneurysmal subarachnoid hemorrhage (SAH). While calcium antagonists have been used in the past and are still part of the standard treatment regimen in most departments involved in the treatment of SAH, other classes of drugs and various other methods have been tested for their potential to inhibit delayed ischemia after SAH. This article reviews the literature about clinical studies about the efficacy of various neuroprotective agents and methods including statins, steroids and Endothelin-antagonists and other - alternative - methods like cisternal lavage, intrathecal drug delivery and hypercapnia, offering future perspectives for the treatment of this hazardous disease.
Einleitung: SSEP sind etabliert, um Patienten intraoperativ zu überwachen, wenn sie sich Operationen im zerebrovaskulären System unterziehen. Das EEG ist eine weitere Methode zur neurophysiologischen Überwachung. In dieser Studie wurde die Wertigkeit des simultanen Ableitens von SSEP und EEG Signalen untersucht. Methode: Dreizehn Patienten (7 Frauen, 6 Männer, mittleres Alter 53.5 Jahre), welche sich dem Clipping eines intrakraniellen Aneurysma unterzogen, wurden eingeschlossen. Die SSEP Latenz 1 (Lat1), Latenz 2 (Lat2) und Amplitude (Amp) wurden kontinuierlich gemessen. Verminderung der Amplitude > 50% oder Verlängerungen der Latenzen > 10% gegenüber den Ausgangswerten wurden als signifikante Ereignisse bewertet. Das EEG wurde mittels einer subduralen Grid-Elektrode gemessen. Alpha % (Al%), Alpha-Delta-Ratio (ADR) und Total Power (TP) wurden ausgewertet. Resultate: Circa 9000 Einzelwerte wurden analysiert. Statistisch signifikante Korrelationen traten zwischen Al% und Amp (K=0.5) auf. Dabei zeigten sich die Veränderungen im EEG (Al%) 6 Minuten vor Ereignissen im SSEP (Amp). Statistisch signifikante Korrelationen traten ebenfalls zwischen Al% und Amp-Ereignissen (K=-0.4) auf. In 6/7 Patienten traten die Al%-Änderungen 7 Minuten vor den Amp-Änderungen auf. Noch stärkere Beziehungen ergaben sich zwischen Lat2 und allen EEG Modalitäten, jedoch reichte die Gesamtzahl der Datenpunkte nicht aus, um statistische Signifikanzen herzuleiten. Schlussfolgerung: Dies ist die erste Beschreibung von signifikanten Beziehungen zwischen quantitativem SSEP und EEG während zerebrovaskulären Operationen. Das quantitative EEG hat das Potenzial, frühe ischämische Ereignisse eher zu detektieren als dies mit SSEP möglich ist.
Ziel der Arbeit war es, dichte Neuronenkulturen in kleinere Untereinheiten zu unterteilen, welche durch ihre Neuriten miteinander in Kontakt standen. Zu diesem Zweck wurden auf MEAs zellbindende Kreisareale mittels Mikrostempeltechnik auf die zellabweisende Schicht aus Polyethylenglykol übertragen. Dudurch wurde gewährleistet, dass scharf abgegrenzte Neuronenareale für mehrere Wochen auf dem MEA wuchsen und nach Ausbildung der neuritischen Verbindungen untereinander, die elektrische Aktivität zwischen den Kreiskammern gemessen werden könnte. Das sollte Auskunft über Informationsausbreitung in Neuralnetzen geben und die Theorien über Synchronität und Synfirechains prüfbar machen.
Aneurysmal subarachnoid hemorrhage (aSAH) remains a disease with high mortality and morbidity. Since treating vasospasm has not inevitably led to an improvement in outcome, the actual emphasis is on finding neuroprotective therapies in the early phase following aSAH to prevent secondary brain injury in the later phase of disease. Within the early phase, neuroinflammation, thromboinflammation, disturbances in brain metabolism and early neuroprotective therapies directed against delayed cerebral ischemia (DCI) came into focus. Herein, the role of neuroinflammation, thromboinflammation and metabolism in aSAH is depicted. Potential neuroprotective strategies regarding neuroinflammation target microglia activation, metalloproteases, autophagy and the pathway via Toll-like receptor 4 (TLR4), high mobility group box 1 (HMGB1), NF-κB and finally the release of cytokines like TNFα or IL-1. Following the link to thromboinflammation, potential neuroprotective therapies try to target microthrombus formation, platelets and platelet receptors as well as clot clearance and immune cell infiltration. Potential neuroprotective strategies regarding metabolism try to re-balance the mismatch of energy need and supply following aSAH, for example, in restoring fuel to the TCA cycle or bypassing distinct energy pathways. Overall, this review addresses current neuroprotective strategies in aSAH, hopefully leading to future translational therapy options to prevent secondary brain injury.
In einer vorangegangenen Phase-1-Studie wurde beobachtet, dass bei Patienten mit schwerer aneurysmatischer SAB, die CBF durch intermittierende kontrollierte Hyperkapnie erhöht werden kann. Zudem zeigte sich in dieser vorherigen Studie nach dem Zurücksetzen der mechanischen Beatmung auf die Ausgangsparameter eine langsame und asymptotische Rückkehr der CBF zu den Ausgangsniveaus ohne einen negativen Rebound-Effekt. Diese Beobachtung legte nahe, dass eine längere Dauer der Hyperkapnie den CBF-erhöhenden Effekt verlängern kann.
Die vorliegende Studie wurde als Dosisoptimierungsstudie geplant, um den Zeitpunkt zu bestimmen, zu dem der CBF ein Maximum erreicht, und unter der Annahme, dass nach diesem Maximum Puffermechanismen in Blut und Liquor zu Anpassungsmechanismen führen können, die nach dem Abbruch zu einem negativen Rebound-Effekt führen.
Es ergab sich eine "Netto" - Optimaldauer der Hyperkapnieintervention von 45 Minuten.
The expression of intercellular adhesion molecule 1 (ICAM-1) was studied in rat focal ischemic cortex. A significant increase in ICAM-1 mRNA expression in the ischemic cortex over Ievels in contralateral (nonischemic) site was observed by means of Northern blot analysis following either permanent or temporary occlusion with reperfusion of the middle cerebral artery (PMCAO or MCAO with reperfusion) in spontaneously hypertensive rats. In the ischemic cortex, Ievels of ICAM-1 mRNA increased significantly at 3 h (2.6-fold, n = 3, P < 0.05), peaked at 6 to 12 h (6.0-fold, P < 0.01) and remained elevated up to 5 days (2.5-fold, P < 0.05) after PMCAO. The profile of ICAM-1 mRNA expression in the ischemic cortex following MCAO with reperfusion was similar to that following PMCAO, except that ICAM-1 mRNA was significantly increased as early as 1 h (6.3-fold, n = 3, P < 0.05) and then gradually reached a peak at 12 h (12-fold, P < 0.01) after reperfusion. ICAM-1 mRNA expression in ischemic cortex following PMCAO was significantly greater in hypertensive rats than in two normotensive rat strains. Immunostaining using anti-ICAM-1 antiborlies indicated that upregulated ICAM-1 expressionwas localized to endotheIial cells of intraparenchymal blood vessels in the ischemic but not contralateral cortex. The data suggest that an upregulation of ICAM-1 mRNA and protein on brain capillary endothelium may play an important rote in leukocyte migration into ischemic brain tissue.
The present study was performed to qua ntify the distribution of a peptide neurotransmitter after microinjection into the medial preoptic area (POM), using a technique suitable for conscious animal preparations. The results indicate that only 50-ni volumes of injected tracer were sufficiently localized with 77 ± 9% recovery in the POM. Injections of higher volumes resulted in an increasing spread of tracer into distant anatomical regions and structures, including the needle tract and cerebral ventricles. The amount of tracer localized in the POM decreased to 38±4% (200 nl) (P < 0.05) and 41 ±8% (500 nl) (P <0.05), respectively. The data suggest that the volume of injection is critical for intraparenchymal injections into structures of a diameter of I mm or less, such as the POM and should not exceed 50 nl in conscious animal preparations.
Thyrotropin-releasing hormonewas shown to exert potent ventilatory effects after centrat administration. These data, however, were derived from studies using anesthetized animal preparations. Since TRH elicits strong arousal reactions, the observed ventilatory effects of TRH under anesthesia may have been due to nonspecific reduction in the anesthetic state of the animals. In order to clarify the extent to which the reversal of anesthesia may change ventilatory parameters after TRH application, we investigated the effect of TRH on Ventilation rate, relative tidal volume, relative respiratory minute volume, CO\(_2\) production CO\(_2\) consumption, and locomotor activity in the conscious, unrestrained rat. Intracerebroventricular application of TRH induced a dose-dependent, sustained increase in ventilation rate, relative tidal volume, and relative respiratory minute volume of maximally 128%, 890%, and 235%, respectively. In addition, CO\(_2\) production and O\(_2\) consumption were elevated by 4.6 and 11.7 fold, whiJe no significant changes in locomotor activity were observed. The results suggest that TRH stimulates ventilation by a mechanism independent of its analeptic properties.
The present study was performed in order to evaluate the effects of the selective 02- adrenoceptor antagonist 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine (SK&F 86466) on dermorphin-induced analgesia, respiratory depression and inhibition of locomotor activity in the conscious rat. Intracerebroventricular (icv) administration of dermorphin (3 nmol/rat) decreased respiration rate and relative ventilatory minute volume maximally by 38 % and 50 % of baseline respectively. SK&F 86466 dose-dependently reversed the dermorphin-induced depression of ventilatory parameters, while SK&F 86466 exerted no effect on dermorphin-induced analgesia or depression of locomotor activity due to catalepsia. It appears, therefore, that a 2-adrenoceptors selectively interact with Jl2-opioid-receptor mediated effects, such as respiratory depression, but are not involved in the modulation of Jl,-opioid-related effects, such as supraspinal analgesia and depression of locomotor activity.
Background
Hypophosphatasia (HPP) is a rare, inherited metabolic disorder caused by loss-of-function mutations in the ALPL gene that encodes the tissue-nonspecific alkaline phosphatase TNAP (ORPHA 436). Its clinical presentation is highly heterogeneous with a remarkably wide-ranging severity. HPP affects patients of all ages. In children HPP-related musculoskeletal symptoms may mimic rheumatologic conditions and diagnosis is often difficult and delayed. To improve the understanding of HPP in children and in order to shorten the diagnostic time span in the future we studied the natural history of the disease in our large cohort of pediatric patients. This single centre retrospective chart review included longitudinal data from 50 patients with HPP diagnosed and followed at the University Children's Hospital Wuerzburg, Germany over the last 25 years.
Results
The cohort comprises 4 (8%) perinatal, 17 (34%) infantile and 29 (58%) childhood onset HPP patients. Two patients were deceased at the time of data collection. Diagnosis was based on available characteristic clinical symptoms (in 88%), low alkaline phosphatase (AP) activity (in 96%), accumulating substrates of AP (in 58%) and X-ray findings (in 48%). Genetic analysis was performed in 48 patients (31 compound heterozygous, 15 heterozygous, 2 homozygous mutations per patient), allowing investigations on genotype-phenotype correlations. Based on anamnestic data, median age at first clinical symptoms was 3.5 months (min. 0, max. 107), while median time to diagnosis was 13 months (min. 0, max. 103). Common symptoms included: impairment of motor skills (78%), impairment of mineralization (72%), premature loss of teeth (64%), musculoskeletal pain and craniosynostosis (each 64%) and failure to thrive (62%). Up to now 20 patients started medical treatment with Asfotase alfa.
Conclusions
Reported findings support the clinical perception of HPP being a chronic multi-systemic disease with often delayed diagnosis. Our natural history information provides detailed insights into the prevalence of different symptoms, which can help to improve and shorten diagnostics and thereby lead to an optimised medical care, especially with promising therapeutic options such as enzyme-replacement-therapy with Asfotase alfa in mind.
Non-small cell lung cancer, ovarian cancer, and pancreatic cancer all present with high morbidity and mortality. Systemic chemotherapies have historically been the cornerstone of standard of care (SOC) regimens for many cancers, but are associated with systemic toxicity. Multimodal treatment combinations can help improve patient outcomes; however, implementation is limited by additive toxicities and potential drug–drug interactions. As such, there is a high unmet need to develop additional therapies to enhance the efficacy of SOC treatments without increasing toxicity. Tumor Treating Fields (TTFields) are electric fields that exert physical forces to disrupt cellular processes critical for cancer cell viability and tumor progression. The therapy is locoregional and is delivered noninvasively to the tumor site via a portable medical device that consists of field generator and arrays that are placed on the patient’s skin. As a noninvasive treatment modality, TTFields therapy-related adverse events mainly consist of localized skin reactions, which are manageable with effective acute and prophylactic treatments. TTFields selectively target cancer cells through a multi-mechanistic approach without affecting healthy cells and tissues. Therefore, the application of TTFields therapy concomitant with other cancer treatments may lead to enhanced efficacy, with low risk of further systemic toxicity. In this review, we explore TTFields therapy concomitant with taxanes in both preclinical and clinical settings. The summarized data suggest that TTFields therapy concomitant with taxanes may be beneficial in the treatment of certain cancers.
Background
Early inflammatory processes may play an important role in the development of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Experimental studies suggest that anti-inflammatory and membrane-stabilizing drugs might have beneficial effects, although the underlying mechanisms are not fully understood. The aim of this study was to investigate the effect of early treatment with methylprednisolone and minocycline on cerebral perfusion and EBI after experimental SAH.
Methods
Male Sprague-Dawley rats were subjected to SAH using the endovascular filament model. 30 minutes after SAH, they were randomly assigned to receive an intravenous injection of methylprednisolone (16mg/kg body weight, n=10), minocycline (45mg/kg body weight, n=10) or saline (n=11). Mean arterial blood pressure (MABP), intracranial pressure (ICP) and local cerebral blood flow (LCBF) over both hemispheres were recorded continuously for three hours following SAH. Neurological assessment was performed after 24 hours. Hippocampal damage was analyzed by immunohistochemical staining (caspase 3).
Results
Treatment with methylprednisolone or minocycline did not result in a significant improvement of MABP, ICP or LCBF. Animals of both treatment groups showed a non-significant trend to better neurological recovery compared to animals of the control group. Mortality was reduced and hippocampal damage significantly attenuated in both methylprednisolone and minocycline treated animals.
Conclusion
The results of this study suggest that inflammatory processes may play an important role in the pathophysiology of EBI after SAH. Early treatment with the anti-inflammatory drugs methylprednisolone or minocycline in the acute phase of SAH has the potential to reduce brain damage and exert a neuroprotective effect.
Frühe entzündliche Vorgänge scheinen eine große Rolle in der Entstehung der globalen Hirnschädigung in der Frühphase nach einer Subarachnoidalblutung (SAB) zu spielen.
Ziel der vorliegenden Arbeit war es den Effekt der anti-inflammatorischen Medikamente Methylprednisolon und Minozyklin auf die Gehirndurchblutung und frühe Hirnschädigung nach SAB zu untersuchen.
Hierzu wurde ein randomisiertes und kontrolliertes Tierexperiment durchgeführt. Mit Hilfe des endovaskulären Perforationsmodells wurde bei männlichen Sprague-Dawley-Ratten eine SAB ausgelöst. Den Tieren wurde 30 Minuten nach Auftreten der SAB Methylprednisolon, Minozyklin oder Kochsalzlösung intraperitoneal verabreicht.
Sowohl Methylprednisolon als auch Minozyklin verminderten den Anteil Caspase 3 positiver Zellen in immunhistochemischen Färbungen der Hippocampie der Versuchstiere signifikant. In Bezug auf die klinische Untersuchung, den intrakraniellen Druck und die Hirndurchblutung der Ratten ergaben sich keine signifikanten Unterschiede zwischen den Versuchsgruppen. Die Ergebnisse suggerieren, dass Methylprednisolon und Minozyklin den akuten Zellschaden nach SAB reduzieren. Daher könnten sich beide Mittel als geeignet für die Therapie der „Early Brain Injury“ nach SAB erweisen. Weitere Studien zum besseren Verständnis der zugrunde liegenden Wirkmechanismen von Methylprednisolon und Minozyklin auf die Frühphase der SAB sind nötig.
Die zervikale spondylotische Myelopathie kann zu schweren neurologischen Funktionsstörungen führen. Vor allem durch die Störung der Feinmotorik und des Gangbilds kann es zu einer Beeinträchtigung im alltäglichen Leben kommen. Eine operative Dekompression des Rückenmarks kann ein Fortschreiten der neurologischen Symptome verhindern oder abmildern.
Ziel dieser Studie war die Erfassung unterschiedlicher neurologischer Dysfunktionen bei zervikaler Myelopathie präoperativ sowie die Evaluierung der funktionellen Veränderung nach operativer Dekompression. Hierbei wurden verschiedene - objektive und subjektive - Messmethoden angewandt. Die Erfassung der feinmotorischen Dysfunktion lag dabei im Vordergrund, da diese in der Literatur bis jetzt nur unzureichend untersucht und beschrieben wurden. Daneben wurde die Regeneration der kernspintomographisch gemessenen Schädigung des Myelons nach operativer Dekompression untersucht.
In dieser Studie wurde ein digitales Graphiktablett zur quantitativen und objektiven Erfassung der feinmotorischen Dysfunktion bei zervikaler Myelopathie benutzt. Eine Beeinträchtigung der Feinmotorik wurde vor allem bei komplexen Schreibaufgaben festgestellt, welche am ehesten Schreibaufgaben im alltäglichen Leben entsprechen. Im Einklang mit früheren Studien wurde auch in dieser Studie eine Störung des Gangbilds bei Patienten mit zervikaler Myelopathie festgestellt.
Die Dauer der Symptome bis zur Operation konnte als einziger signifikanter Einflussfaktor für den Schweregrad der feinmotorischen Dysfunktion ausgemacht werden. Das Geschlecht, der BMI, der Beruf und Sport hatten in dieser Analyse weder einen Einfluss auf den klinischen noch auf den neuroradiologischen Schweregrad der zervikalen Myelopathie.
Nach operativer Dekompression verbesserten sich die feinmotorische Funktion sowie das Gangbild der Patienten. Auch die subjektiven Scores zur Erfassung der neurologischen Funktion (European Myelopathy Score, Nurick-Score) sowie die Schmerzen (Numerische Rating-Skala) verbesserten sich postoperativ. Zudem nahm die Länge des pathologischen Signals im MRT ab.
Faktoren, die einen Trend zur Korrelation mit den postoperativen Veränderungen der Funktion zeigten, waren das Alter der Patienten und die Symptomdauer. In dieser Studie konnte kein Einfluss des BMIs, der Anzahl der operierten Höhen oder der Art des Operationszugangs festgestellt werden.
Obwohl die Anzahl der untersuchten Patienten begrenzt war, gestaltete sich die Gruppe heterogen (Alter, Geschlecht, BMI, Dauer der Symptome). So konnte ein möglichst breites Spektrum an Patienten mit zervikaler Myelopathie untersucht werden.
Die wichtigste Limitation dieser Studie war die hohe drop-out Rate in den postoperativen Verlaufsuntersuchungen. Daher konnten die Ergebnisse der Längsschnittanalyse nur an einer kleinen Patientengruppe ermittelt werden und bei der Untersuchung von Einflussfaktoren für das postoperative Ergebnis nur Tendenzen erkannt werden. Ob diese Faktoren einen prognostischen Wert für das postoperative Ergebnis haben, müsste in weiteren Studien an einem größeren Kollektiv untersucht werden.
Temporary hypercapnia has been shown to increase cerebral blood flow (CBF) and might be used as a therapeutical tool in patients with severe subarachnoid hemorrhage (SAH). It was the aim of this study was to investigate the optimum duration of hypercapnia. This point is assumed to be the time at which buffer systems become active, cause an adaptation to changes of the arterial partial pressure of carbon dioxide (PaCO2) and annihilate a possible therapeutic effect. In this prospective interventional study in a neurosurgical ICU the arterial partial pressure of carbon dioxide (PaCO\(_2\)) was increased to a target range of 55 mmHg for 120 min by modification of the respiratory minute volume (RMV) one time a day between day 4 and 14 in 12 mechanically ventilated poor-grade SAH-patients. Arterial blood gases were measured every 15 min. CBF and brain tissue oxygen saturation (StiO\(_2\)) were the primary and secondary end points. Intracranial pressure (ICP) was controlled by an external ventricular drainage. Under continuous hypercapnia (PaCO\(_2\) of 53.17 ± 5.07), CBF was significantly elevated between 15 and 120 min after the start of hypercapnia. During the course of the trial intervention, cardiac output also increased significantly. To assess the direct effect of hypercapnia on brain perfusion, the increase of CBF was corrected by the parallel increase of cardiac output. The maximum direct CBF enhancing effect of hypercapnia of 32% was noted at 45 min after the start of hypercapnia. Thereafter, the CBF enhancing slowly declined. No relevant adverse effects were observed. CBF and StiO\(_2\) reproducibly increased by controlled hypercapnia in all patients. After 45 min, the curve of CBF enhancement showed an inflection point when corrected by cardiac output. It is concluded that 45 min might be the optimum duration for a therapeutic use and may provide an optimal balance between the benefits of hypercapnia and risks of a negative rebound effect after return to normal ventilation parameters.
Based on recent findings that show that depletion of factor XII (FXII) leads to better posttraumatic neurological recovery, we studied the effect of FXII-deficiency on post-traumatic cognitive and behavioral outcomes in female and male mice. In agreement with our previous findings, neurological deficits on day 7 after weight-drop traumatic brain injury (TBI) were significantly reduced in FXII\(^{−/−}\) mice compared to wild type (WT) mice. Also, glycoprotein Ib (GPIb)-positive platelet aggregates were more frequent in brain microvasculature of WT than FXII\(^{−/−}\) mice 3 months after TBI. Six weeks after TBI, memory for novel object was significantly reduced in both female and male WT but not in FXII\(^{−/−}\) mice compared to sham-operated mice. In the setting of automated home-cage monitoring of socially housed mice in IntelliCages, female WT mice but not FXII\(^{−/−}\) mice showed decreased exploration and reacted negatively to reward extinction one month after TBI. Since neuroendocrine stress after TBI might contribute to trauma-induced cognitive dysfunction and negative emotional contrast reactions, we measured peripheral corticosterone levels and the ration of heart, lung, and spleen weight to bodyweight. Three months after TBI, plasma corticosterone levels were significantly suppressed in both female and male WT but not in FXII\(^{−/−}\) mice, while the relative heart weight increased in males but not in females of both phenotypes when compared to sham-operated mice. Our results indicate that FXII deficiency is associated with efficient post-traumatic behavioral and neuroendocrine recovery.
Introduction
A novel neurostimulation system allows steering current in horizontal directions by combining segmented leads and multiple independent current control. The aim of this study was to evaluate directional DBS effects on parkinsonian motor features and adverse effects of subthalamic neurostimulation.
Methods
Seven PD patients implanted with the novel directional DBS system for bilateral subthalamic DBS underwent an extended monopolar review session during the first postoperative week, in which current thresholds were determined for rigidity control and stimulation-induced adverse effects using either directional or ring-mode settings.
Results
Effect or adverse effect thresholds were modified by directional settings for each of the 14 STN leads. Magnitude of change varied markedly between leads, as did orientation of optimal horizontal current steering.
Conclusion
Directional current steering through chronically implanted segmented electrodes is feasible, alters adverse effect and efficacy thresholds in a highly individual manner, and expands the therapeutic window in a monopolar review as compared to ring-mode DBS.
Thromboxanes are abundantly present in the rat brain but their possible physiological functions in the brain are not known. The prostaglandin endoperoxide analogue U-46619 is a selective agonist of TxA2 receptors in many peripheral tissues. In the present study the ·central cardiovascular and ventilatory effects of U-46619 were investigated in rats. In conscious spontaneously hypertensive rats (SHR) U-46619 (1-100 nmol/kg i.c.v.) induced a strong dose-related increase in blood pressure but had no significant effect on heart rate. In conscious normotensive rats (NR) neither blood pressure nor heart rate was significantly affected. Furthermore, U-46619 (0.1-100 nmol/kg i.c.v.) had no significant effect on blood pressure, heart rate or ventilation in urethane-anaesthetised NR . The results demonstrate an increased sensitivity of SHR to TxA2.
Introduction
Structural plasticity with synapse formation and elimination is a key component of memory capacity and may be critical for functional recovery after brain injury. Here we describe in detail two surgical techniques to create a cranial window in mice and show crucial points in the procedure for long-term repeated in vivo imaging of synaptic structural plasticity in the mouse neocortex.
Methods
Transgenic Thy1-YFP(H) mice expressing yellow-fluorescent protein (YFP) in layer-5 pyramidal neurons were prepared under anesthesia for in vivo imaging of dendritic spines in the parietal cortex either with an open-skull glass or thinned skull window. After a recovery period of 14 days, imaging sessions of 45–60 min in duration were started under fluothane anesthesia. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate fixed to metal base. The animals were set under a two-photon microscope with multifocal scanhead splitter (TriMScope, LaVision BioTec) and the Ti-sapphire laser was tuned to the optimal excitation wavelength for YFP (890 nm). Images were acquired by using a 20×, 0.95 NA, water-immersion objective (Olympus) in imaging depth of 100–200 μm from the pial surface. Two-dimensional projections of three-dimensional image stacks containing dendritic segments of interest were saved for further analysis. At the end of the last imaging session, the mice were decapitated and the brains removed for histological analysis.
Results
Repeated in vivo imaging of dendritic spines of the layer-5 pyramidal neurons was successful using both open-skull glass and thinned skull windows. Both window techniques were associated with low phototoxicity after repeated sessions of imaging.
Conclusions
Repeated imaging of dendritic spines in vivo allows monitoring of long-term structural dynamics of synapses. When carefully controlled for influence of repeated anesthesia and phototoxicity, the method will be suitable to study changes in synaptic structural plasticity after brain injury.
ln the present study the effects of thyrotropin releasing hormone (TRH) and its stable analogue, CG3703, on cardiac output (thermodilution, Cardiomax) and regional blood flow (BF; directional pulsed Doppler technique) were investigated in hypovolemic hypotension in the rat. In urethan-anesthetized rats TRH (0.5 or 2 mg/ kg ia) or CG3703 (0.05 or 0.5 mg/kg ia) reversed the bleeding (27% of the blood volume)-induced decreases in mean arterial ...
The effects of i.c.v. administered dermorphin, a highly selective \(\mu\)-opioid agonist, on cardiac function and renal, mesenteric and hindquarter blood ftow were studied in conscious rats. Core temperature, blood gases, arterial plasma levels of norepinephrine, epinephrine, dopamine, 3,4-dihydroxyphenylalanine and dihydroxyphenylacetic acid (DOPAC) also were examined. Cardiac output was rneasured using a thermodilution technique and regional blood ftows using directional pulsed Doppler velocimetry. Dermorphin, at doses of 0.1-100 nmol/kg, increased blood pressure and hindquarter blood flow, renal and mesenteric resistance, and core temperature. Higher doses (1-5 \(\mu\)mol/kg) caused respiratory depression, acidosis, and shock despite profaund sympatho-adrenomedullary stimulation. Circulating Ieveis of catecholamines were significantly increased at the dermorphin doses of 0.1-1 00 nmol/kg. At the 100 nmol/kg dose, plasma levels of epinephrine, norepinephrine, the dopamine metabellte dihydroxyphenylacetic acid and the catecholamine precursor 3,4,-dihydroxyphenylalanine were increased by 2-15-fold. The data indicate that mu opioid receptor Stimulation exerts potent effects on cardiorespiratory functions, activates the sympathoadrenomedullary system and produces a pattem of blood flow changes consistent with the stress-induced •detense· response (skeletal muscle vasodilation and splanchnic vasoconstriction). Excessive mu opioid receptor Stimulation Ieads to shock due to respiratory and hemodynamic collapse.
In addition to the endocrine effects, the thyrotropin releasing hormone (TRH) is known to induce dose-dependent increases in blood pressure and heart rate after intracerebroventricular (i.c.v.) administration in urethane-anaesthetised rats (1, 2). The a~ of the present study was to investigate whether TRH has similar effects in conscious rats of various strains i.e. spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY) and Wistar (NR) rats.
N-Acetyl-leukotriene E\(_4\) administered to conscious freely moving rats produced a dose-dependent vasoconstriction in the mesenteric vessels which led to profound reduction of blood flow to the gut. Renal and hindquarter blood flow and vascular resistance were not affected even by high doses of N-acetyl-leukotriene E\(_4\) . N-Acetyl-leukotriene E\(_4\) was 10-fold more potent than the thromboxane analog U-46619 and 1000-fold more potent than prostaglandin F\(_{2a}\) but 2-5-fold less potent than leukotriene D\(_4\)/E\(_4\) to induce mesenteric vasoconstriction. These data indicatc that N-acetylleukotriene E\(_4\) is a biologically active metabolite of peptide leukotrienes, and might play a role in cardiovascular derangements mediated by leukotrienes.
Tbe mechanisms mediating the etl'ects ofthyrotropin-releasing hormone (TRH) on the cardiovascular system were studied in the conscious rat. Intracerebroventricolar (i.c. v.) injection of TRH (8 pmol-80 nmollkg) induced dose-dependent lncreases in mean arterial pressure, heart rate, and cardiac index. Rindquarter blood Oow increased due to vasodilation, while an lncrease in renal and mesenteric vascular resistance caused a decrease in blood Oow in the respective organs. The plasma Ievels of norepinephrine a~d epinephrine were increased by TRH, while there was no change in plasma renin activity or vasopressin. Tbe cardiovascular actions of i.c. v. TRH were not in.fluenced by blockade of the renin-angiotensin system or vasopressin receptors. Tbe ganglion blocker chlorisondamine and the a 1- aod al-adrenoreceptor antagooist phentolamlne (2 mg/kg i.v.) abolished the increase in blood pressure and mesenteric vasoconstriction after i.c. v. TRH. Propranolol (2 mg/kg i. v.) blocked the TRH-ioduced increase in cardiac index, heart rate, and hindquarter blood flow. The hindquarter vasodllatlon lnduced by TRH was also blocked by the selective ß1-adrenocept9r antagonist ICI 188,551 (1 or 2 mg/kg i.v.), while tbe ,8,-adrenoceptor blocker practolol (10 mg/kg i.v.) had no eft'ect on the hindquarter vasodiJation produced by TRH but totally blocked the increase in cardiac Index. In adrenal demedullated rats, the systemic hemodynamic eft'ects ofi.c. v. TRH were dimlnished along with the decrease in renal blood flow and lncrease in renal vascular resistance; however, the iocrease in hfndquarter blood flow was attenuated only in adrenal demedullated rats pretreated with the sympathetlc blocker bretylium. The renal vasoconstriction induced by i.c. v. TRH was not abolished by renal denervation. In sinoaortic debufl'ered rats, the pressor, tachycardic, and mesenteric vasoconstrictor responses to centrally administered TRH were significantly potentiated. Taken together, these data soggest that the putative rieurotransmitter TRH may play a role in central regulation of cardiac functions and organ blood flow distribution through both tbe sympathetic nerves and the adrenal medulla. A pivotal roJe for ß1-adrenoceptors in mediation ofhindquarter vasodilation ls also demonstrated.
Background and Purpose: We reported previously that stroke risk factors prepared the brain stem for the development of ischemia and hemorrhage and induced the production of tumor necrosis factor following an intrathecal injection of Iipopolysaccharide, a prototypic monocyte-activating stimulus. This study evaluates whether blood or brain cells of hypertensive rats produce more proinflammatory and prothrombotic mediators than do blood or brain cells of normotensive rats. MethotJs: Levels of tumor necrosis factor, platelet-activating factor, 6-ketoprostaglandin F1a, and thromboxane B2 in the cerebrospinal fluid and blood of spontaneously hypertensive and normotensive Wistar-Kyoto rats were monitored before and after achallenge with Iipopolysaccharide. Results: Little or no activity from these media tors was found in the cerebrospinal fluid or blood of saline-injected control animals. Intravenous administration of Iipopolysaccharide (0.001, 0.1, and 1.8 mg/kg) produced dose-dependent increases in blood levels of all mediators in hypertensive rats. In normotensive rats the levels were less than in hypertensive rats and were not c1early dose-related. When Iipopolysaccharide was injected intracerebroventricularly, more tumor necrosis factor was measured in the cerebrospinal fluid than in the blood, suggesting local synthesis of this cytokine. Levels of tumor necrosis factor and platelet-activating factor in the cerebrospinal fluid were higher in hypertensive than in normotensive rats. The thromboxane A2/prostacyclin ratio was not aItered significantly between the two rat strains. Conclusions: It is suggested that the higher incidence of brain stem ischemia and hemorrhage after the intrathecal injection oflipopolysaccharide in hypertensive rats than in normotensive rats might be related to the higher levels of the two cytotoxic factors tumor necrosis factor and platelet-activating factor produced in response to such challenge.
Thyrotropin releasing hormone (TRH, I-pyroglutamyl-l-histidyl-l-prolinamide) was the fIrst hypothalamic releasing SUbstance to be isolated, chemically characterized and synthetized /1/. The studies to date have revealed that the thyrotropin release from the pituitary gland is only one of the numerous actions of TRH. In addition to its endocrine actions (TSH and prolactin release) this tripeptide has central nervous system actions totally unrelated to its effects on the hypothalamo-pituitary axis. This review aims to summarize the studies on the central nervous system' actions of TRH with special emphasis on the autonomic pharmacology of this peptide.
The cardiovascular and endocrine activity of three analogs of thyrotropin releasing hor.mone (TRH), 4-nitro-imidazole TRH (4-nitroTRH), 2-trifluoro-methyl-imidazole TRH (2-TFM-TRH) and 4-trifluoromethyl- imidazole TRH (4-TFM-TRH), was compared to TRH in conscious rats. Injection of TRH or the three analogs (1 mg/kg or 5 mg/kg) into the arterial line induced increases in mean arterial pressure, pulse pressure and heart rate and raised plasma prolactin (PRL). None of the analogs were more potent than TRH in inducing cardiovascular changes. The 4-TFM-TRH was significantly less potent than the 2-TFM-TRH in increasing blood pressure, while the nitro-TRH was more potent than the 2-TFM-TRH in producing tachycardia. TRH induced a two-fold increase in PRL at the 5 mg/kg dose, while both the fluorinated analogs elici ted a 4 to 5 fold increase in PRL at the higher dose. The present results suggest that the receptors for TRH-elicited PRL release differ from TRH-receptors involved in its cardiovascular actions.
The acute effect ofT-2 toxemia on local blood flow and vascular resistance in hindquarter. mesenteric. and renal vascular beds was continuously measured by the directional pulsed Doppler technique in conscious, male Sprague-Dawley rats. Intravenous injection ofT-2 toxin (I mg/kg) in the conscious rat reduced blood flow and increased vascular resistance in all blood vessels studied but had no significant effect on mean arterial pressure or heart rate. The blood flow in hindquarters gradually decreased to a minimum of -77 ± 9% (mean ±SE) 6 hr after the toxin injection. The hindquarter vascular resistance concomitantly increased to a maximum value of + 323 ± 69% above thc resistance before toxin administration. Mesenteric and renal blood flow initially increased (slightly) and then gradually decreased. The maximum drop of blood flow, -90 ± 13% and -76 ± 13% for the mesenteric and renal vascular beds, respectively, was achieved 4 hr after T-2 toxin injection and the blood flow values remained low for up to 6 hr. Simultaneously with the impairment of
Opioid peptidesandmultiple opioid receptors are found in brain cardiovascular nuclei, autonomic ganglia, the heart, and blood vessels, and opioids induce potent cardiovascular changes. The role of endogenaus opioids in normal cardiovascular homeostasis is unclear; however, current data suggest opioid involvement in stress.
The effect of the selective \(\mu\)-opioid agonist o-Ala\(^2\)-Me-Phe\(^4\)-Gly-ol'-enkephalin (DAGO), injected into the medial preoptic nucleus of hypothalamus, on cardiac output and regional blood flow was studied in the conscious rat and the effect of DAGO on renal sympathetic nerve activity and renal blood flow was studied in anesthetized rats. In conscious rats, injections of DAGO (1 or 10 nmol) into the preoptic nucleus increased the blood pressure in a dose-related manner. The maximum rises of mean arterial pressure and pulse pressure after the larger dose were +23 ± 5 mmHg (mean ±SEM, P < 0.01) and + 17 ± 3 mmHg(P < 0.01), respectively. A small dose (0.1 nmol) increased heart rate ( +47 ± 13 bpm, P < 0.05); thc 1 nmol dosc produced bradycardia (- 39 ± 11 bpm, P < 0.05), while the 10 nmol dose initially decreased heart rate ( -68 ± 15 bpm (P < 0.01) and then gradually increased heart rate to a maximum of + 74 ± 13 bpm, (P < 0.0 1). A long-lasting increase in cardiac output was also elicited by DAGO, with maximum changes after 1 and 10 nmol of + 14 ± 6% and +22 ± 7% (P < 0.01), respectively. B1ood flow in the hindquarters increascd after DAGO but the mesenteric and renal blood ftow decreased in a dose-related manner. Significant responscs in hindquarter and mesenteric blood fl.ow after DAGO were independent of systemic hemodynamic responses at the dose ofO.l nmol. The vascular resistance in the hindquarters significantly decreased after a small dose of DAGO while the larger doses dose-dependently increased mesenteric and renal vascular resistance. A crucial role of the sympathetic nervous system in the hemodynamic effects of DAGO was demonstrated: (1) by the profound activation of renal sympathetic nerve activity after injections of DAGO (I nmol/100 nl) into the preoptic nucleus, (2) by blockade of the pressor, tachycardic and regional hemodynamic effects of DAGO (I nmol) by the ganglion blocker ch1orisondamine (5 mg/kg i.v.). The results suggest that the pressor effect of DAGO in preoptic nucleus is due primarily to an increase in cardiac output. The differential changes in blood ftow in organs further suggest that the opioid \(\mu\)-receptors in the preoptic nucleus might be involved in the integration of peripheral blood ftow in the hypothalamus during affective behavior.
Cardiovascular Effects of Anatoxin-A in the Conscious Rat. SJREN, A.-L., AND FEUERSTEIN, G. (1990). Toxicol. Appl. Pharmacol. 102,91-100. The effects ofanatoxin-A on mean arterial pressure (MAP), heart rate, cardiac index (CI), and blood flow (BF) in hindquarter (HQ), renal (R). and mesenteric (M) vascular beds were studied after intravenous (iv) and intracerebroventricular (icv) administration in the conscious rat. The pharmacological profile of anatoxin-A was further compared to nicotine administered iv and icv. MAP and heart rate were measured from femoral artery, CI by thermodilution method, and blood flow by Doppler velocimetry. Anatoxin-A and nicotine (30, 100 and 300 1-!g/kg iv) produced an increase in MAP with concomitant bradycardia. The highest doses increased Cl. MBF and RBF decreased due to a vasoconstriction in M and R vasculature. These effects were attenuated by the ganglion blocker chlorisondamine (5 mg/kg, iv). Anatoxin-A ( 100 1-!g/k~ iv) increased plasma epinephrine Ievels by 2- fold with virtually no effect on norepinephrine whereas nicotine ( 100 ~oLg/kg, iv) increased plasma epinephrine and norepinephrine by 20- to 30-fold. Central administration of anatoxin-A and nicotine (30-100 ,ug/kg icv) increased MAP with no effect on heart rate and produced M and R vasoconstriction. In summary, the present study demonstrates that anatoxin-A acts as a nicotinic cholinergic agonist in the c.onscious rat after both systemic and centrat administration. Anatoxin-A and nicotine produced pressor and reno-splanchnic vasoconstrictor responses and at high doses increased cardiac output. These effects were mediated by activation ofthe nicotinic receptors in the adrenal medulla and sympathetic ganglia. However, marked differences were found in the potency ofanatoxin-A versus nicotine to stimulate the sympathoadrenomedullary axis.
The capacity of L-649,923-sodium ( ßS, -yR * )-4-(3-( 4-acetyl-3-hydroxy-2-propylphenoxy)propylthio)-- y-hydroxy-ß-methylbenzene butanoate-to block vascular receptors of leukotriene D\(_4\) ( L TD\(_4\)) was examined in the conscious rat. Hindquarter (HQ), renal, and mesenteric blood flow and vascular resistance were evaluated in the conscious rat chronically equipped with miniaturized Doppler probes for organ blood flow measurement by directional pulsed Doppler technique. In addition, cardiac outpul was measured by thermodilution technique in conscious rats equipped with minithermistors in the ascending aorta. Systemic hemodynamic variables. mean arterial pressure, and heart rate were monitored through femoral catheters. L TD\(_4\) (I or 10 \(\mu\)g/kg) produced a marked dose dependent increase in the mesenteric vascular resistance associated with a marked decrease in blood flow whereas no consistent effects were demonstrated in the renal circulation. L TD\(_4\) • at I \(\mu\)g/kg. increased the HQ blood flow whereas the higher dose of LTD\(_4\) produced a biphasic response: an early increase followed by a decrease in blood flow. Infusion of L TD\(_4\) • 3 \(\mu\)g/kg per min over 10 min decreased cardiac output and increased total peripheral resistance. L-649,923 (10 or 30 mg/kg, i.v.) effectively blocked the L TD4-induced mesenteric constriction and the second I phase of HQ vasoconstriction but did not modify the , LTD\(_4\) induced HQ vasodilation. L-649,923 also effectively attenuated the cardiac effects of LTD\(_4\) infusion. I These studies suggest that L-649,923 could preserve cardiac and vascular functions in pathologic states mediated by cysteinylleukotrienes, such as traumatic or endotoxin shock. Key Words: Leukotriene D4 -Cardiovascular system- Leukotriene antagonist- Mesenteric blood tlow-Renal blood flow-Hindquarter blood flowAnaphylaxis.
Prostag1andin F\(_{2\alpha}\) (PGF\(_{2\alpha}\)) is one of the most common metabo1ites of arachidonic acid (M) in rat brain. When administered intracerebroventricularly (i.c.v.) to rats, both AA and PGFal exert dose-related hypertensive, tachycardic and hyperthermic effects. Metabolie alterations in the endogenaus formation of some prostaglandins in the brain-stem of spontaneously hypertensive rats (SHR) have been reported. Therefore the central effects of AA and PGF \(_{2\alpha}\) on blood pressure, heart rate and body temperature were studied both in SHR and nonootensive Wistar rats (NR) under urethane-anaesthesia. The hypertensive effect of AA i.c.v. (0.01-100 \(\mu\)g/rat) was larger in magni tude in SHR than in NR, but there was no significant difference in the M-induced changes of heart rate and body temperature between the groups. Pretreatment of NR wi th soditm1 :meclofenamate (1 mg/rat i.c.v.) antagonised the central effects of M indicating that these effects are not due to M itself but to its conversion to prostaglandins. Unlike the effects of AA, the central hypertensive, tachycardic and hyperthennic responses to PGF\(_{2\alpha}\) (0.5-50 l-lg/rat i.c.v .) were significantly attenuated in SHR. The present results obtained with M are conpatible with the previous assumption that the synthesis of prostaglandins in the brain of SHR might differ from that in NR. The results also demonstrate that the central effects of PGF\(_{2\alpha}\) are reduced in SHR.
Prostaglandin E2 (PGE2) increased the blood pressure, heart rate and body temperature, when administered at the doses ofO.OOI-IO,ug into the lateral cerebral ventricle (i.c.v.) of the urethane-anesthetised rat. The highest dose of 10 ,ug/rat induced a strong initial hypotensive effect. lntravenously (i.v.), PGE2 at the doses of 0.01-10 ,ug/rat caused a biphasic blood pressure response with dose-related initial decreases followed by slight increases in blood pressure. The heart rate and body temperature were slightly increased by i.v. administrations of PGE2 . The highest i.v. dose of 10 ,ug/rat initially decreased also the heart rate. Central pretreatment with indomethacin ( I mg/rat i.c.v.) partly antagonised all of the recorded central effects of PGE2 , while sodium meclofenamate (I mg/rat i.c. v.) abolished the hypertensive response to i.c. v. administered PGE2 but failed to significantly affect the PGE2-induced rises of heart rate and body temperature. The results support the previous suggestions that PGE2 may participate in the central cardiovascular and thermoregulatory contro!. The results also suggest that indomethacin and sodium meclofenamate antagonize the effects of exogenous prostaglandins. Since sodium meclofenamate, unlike indomethacin, affected preferentially the hypertensive response to centrally administered PGE2 , there may be differences in the sites and/or modes of action between these drugs.
Prostacyclin (PGI2) induced a dose-dependent decrease in blood pressure with slight increases in heart rate and body temperature, when administered at the doses of 0.1-100 ~g into the lateral cerebral ventricle (i.c.v.) of the urethane-anaesthetised rat. When the same doses were administered intravenously, both the blood pressure and heart rate decreased. Central pretreatment wib~ sodiurn meclofenamate (1 mg/rat i.c.v.) antagonised the central hypotensive effect of PGI2 but i.c.v. pretreatrnent of the rats with indomethacin (1 mg/rat) failed to affect the PGI 2-induced hypotension. Central pretreatment with two histamine H2-receptor antagonists, cimetidine (500 ~g/rat i.c.v.) or metiamide (488 ~g/rat i.c.v.), antagonised the blood pressure lowering effect of 0.1 ~g dose of PGI2 but failed to affect the hypotension induced by higher PGI2 doses. Therefore the main central hypotensive effect of PGI2 seems not to be associated with the stimulation of histamine H2 -receptors in the brain. The hypotensive effect of i.c.v. administered PGI2 appears to be due to an action upon the central nervous system rather than to a leakage into the peripheral circulation. This assurnption is supported by the fact that sodiurn meclofenamate i.c.v. antagonised the effect of PGI 2. In addition, the chronotropic response to i.c.v. PGI2 was opposite to that induced by intravenous administration. The results also suggest that there may be differences in the mode of action between sodiurn meclofenamate and indomethacin.
Prostaglandin D2 (PGD2) is the most common prostaglandin type of tile rat brain. Recently a neurornodulator role for PGD2 has been suggested. In the present work the central cardiovascular and thermal effects of PGDz were studied in urethane-anaesthetised rats. Mlen adrndnistered at the doses of 0.001-10 ~g/rat into the lateral cerebral ventricle(i.c.v.), PGD2 slightly increased the blood pressure, heart rate and body ternpera~ ure. The highest dose caused also an initial hypotensive effect. Upon lntravenous injections PGD2 (0.1-10 ~g/rat) initially decreased and then weakly increased the blood pressure but had only negligible effects on heart rate and body temperature. Central pretreatment with sodium meclofenamate or indomethacin (1 mg/rat i.c.v.) antagonised effectively all the recorded central effects of PGD2. The central cardiovascular and thermal effects of PGD2 were much weaker than those obtained earlier with other prostaglandins, such as PGF2alpha and PGE2.. Therefore, in spite of its abundance in the brain PGD2 may not be very important for the central cardiovascular and thermal regulation in the rat.
Background and Purpose: We earlier reported that risk factors for stroke prepare brain stem tissue for a modified Shwartzman reaction, incIuding the development of ischemia and hemorrhage and the production of tumor necrosis factor-a, after a provocative dose of lipopolysaccharide. In the present study, we sought to determine whether blood and central nervous system cells of rats with the stroke risk factor of advanced age produce more proinflammatory and prothrombotic media tors than do those of young rats of the same strain. Methods: Levels of tumor necrosis factor-a and platelet activating factor in the cerebrospinal fluid and tumor necrosis factor-a in the serum of 2-year-old and 16-week-old Sprague-Dawley rats were monitored before and after challenge with lipopolysaccharide. Results: No consistent tumor necrosis factor-a activity was found in the cerebrospinal fluid or blood of control animals. Intravenous administration of lipopolysaccharide (1.8 mg/kg) increased serum tumor necrosis factor-a levels but had no effect on tumor necrosis factor-a in the cerebrospinal fluid. Serum tumor necrosis factor-a increased much more in aged rats than in young rats. When lipopolysaccharide was injected intracerebroventricularly, tumor necrosis factor-a activity in cerebrospinal fluid increased significantly more in old rats than in young rats. Baseline levels of platelet activating factor in cerebrospinal fluid were significantly higher in old rats than in young rats, and the levels increased to a greater degree in aged rats on stimulation. Conclusions: Rats with the stroke risk factor of advanced age respond to lipopolysaccharide with a more exuberant production of tumor necrosis factor-a and platelet activating factor than young rats of the same strain. These findings are consistent with our working hypothesis that perivascular cells are capable of exaggerated signaling of endothelium through cytokines such as tumor necrosis factor-a in animals with stroke risk factors. The effect of such signaling might be to prepare the endothelium of the local vascular segment for thrombosis or hemorrhage in accord with the local Shwartzman reaction paradigm.
We used a sensitive silver degeneration staining method to study the effects of insertion of microdialysis probes in rat dorsal hippocampus and neocortex. Nine animals were sacrificed 24 h, 3 days or 7 days after implantation of dialysis tubing. Although mild neuronal cell death and small petechial hemorrhages were seen in elose proximity to the implantation site, the striking finding was the presence of degenerating axons both adjacent to the implantation site and in remote sites such as the corpus callosum and contralateral hippocampus. The observed changes could alter brain function near or remote from the implantation site and should be considered in analysis of dialysis experiments.
Das Glioblastom ist mit einem Anteil von 20% an allen hirneigenen Tumoren der häufigste und bösartigste primäre intrakranielle Tumor. Trotz multimodalem Therapiekonzept, das operative Resektion, Strahlen- und Chemotherapie verbindet, haben Patienten eine Prognose von im Mittel nur 14,6 Monaten. Sein aggressives Wachstum zieht eine Vaskularisierung nach sich, die jedoch nicht in ausreichendem Maße die Sauerstoffversorgung des Tumorgewebes sicherstellen kann. Studien mit Messelektroden zeigten einen deutlich reduzierten Sauerstoffpartialdruck im Tumor im Vergleich zum umliegenden Hirngewebe. Dieses hypoxische Milieu löst genetische Alterationen und adaptive Veränderungen der Proteinexpression aus, die eine Selektion besonders aggressiver Tumorzellen bewirkt. Für eine bessere prognostische Einschätzung sowie als zukünftige therapeutische Ziele zur Erhöhung der Response auf Chemo- und Strahlentherapie ist es von großem Interesse, solche Faktoren als mögliche Hypoxiemarker aufzufinden. In dieser Arbeit wurden die Proteine HIF-1α, CAIX, VEGF, EPO und NDRG1 auf mRNA- und Proteinebene in den Glioblastomzelllinien GaMG, U251 und U373 auf eine Veränderung ihrer Expression unter hypoxischen Bedingungen untersucht. Ausmaß (5% O2, 1% O2 und 0,1% O2) und Dauer (1 h, 6 h und 24 h) der Hypoxie wurde variiert. Anschließend wurde über 24 h und 48 h eine Reoxygenierung durchgeführt. Auch wurden Expressionsuntersuchungen an Gewebeproben von Normalhirnen, Astrozytomen WHO Grad II und Glioblastomen vorgenommen. Die Verwendbarkeit von GAPDH als Marker für diese Analysen wurde durch Experimente sichergestellt, die dessen mRNA und das Protein als nicht durch Hypoxie oder Malignisierung reguliert nachwiesen. Wir bestätigten die Rolle von HIF-1α als Mediator der hypoxischen Zellantwort. Während die mRNA konstant blieb, wurde das Protein unter hypoxischen Bedingungen hochreguliert. Dies zeigte auch, dass unser experimentelles Setting funktionierte. NDRG1, CA-IX sowie EPO wurden unter Hypoxie sowohl auf mRNA-, als auch Proteinebene hochreguliert und blieben unter Reooxygenierung stabil. In Glioblastomen waren diese Gene auf mRNA-Ebene bedeutend stärker exprimiert als in niedriggradigen Astrozytomen. HIF-1α, NDRG1, CA-IX sowie EPO können also in humanen Glioblastomzellen als Hypoxiemarker dienen und möglicherweise auch eine prognostische und therapeutische Bedeutung haben.
Medulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma.
Ten thiosemicarbazone ligands obtained by condensation of pyridine-2-carbaldehyde, quinoline-2-carbaldehyde, 2-acetylpyridine, 2-acetylquinoline, or corresponding 2-pyridyl ketones with thiosemicarbazides RNHC(S)NHNH\(_{2}\) and R=CH\(_{3}\), C\(_{6}\)H\(_{5}\) were prepared in good yield. The reaction of [PdCl\(_{2}\)(cod)] with cod=1,5-cyclooctadiene or K\(_{2}\)[PtCl\(_{4}\)] resulted in a total of 17 Pd(II) and Pt(II) complexes isolated in excellent purity, as demonstrated by \(^{1}\)H, \(^{13}\)C, and, where applicable, \(^{195\)Pt NMR spectroscopy combined with CHNS analysis. The cytotoxicity of the title compounds was studied on four human glioblastoma cell lines (GaMG, U87, U138, and U343). The most active compound, with a Pd(II) metal centre, a 2-quinolinyl ring, and methyl groups on both the proximal C and distal N atoms exhibited an EC\(_{50}\) value of 2.1 μM on the GaMG cell lines, thus being slightly more active than cisplatin (EC\(_{50}\) 3.4 μM) and significantly more potent than temozolomide (EC\(_{50}\) 67.1 μM). Surprisingly, the EC\(_{50}\) values were inversely correlated with the lipophilicity, as determined with the “shake-flask method”, and decreased with the length of the alkyl substituents (C\(_{1}\)>C\(_{8}\)>C\(_{10}\)). Correlation with the different structural motifs showed that for the most promising anticancer activity, a maximum of two aromatic rings (either quinolinyl or pyridyl plus phenyl) combined with one methyl group are favoured and the Pd(II) complexes are slightly more potent than their Pt(II) analogues.
Glioblastoma (GBM) sind bösartige hirneigene Tumore, deren schlechte Prognose einer innovativen Therapie bedarf. Aus diesem Grund wurde ein neuer Therapieansatz entwickelt, der auf einer lokalen Ultraschall-vermittelten Zytostatika Applikation beruht. Hierfür wurden stabile Microbubbles (MB) bestehend aus Phospholipiden synthetisiert. Es konnte gezeigt werden, dass MB als auch fokussierter Ultraschall niedriger Intensität (LIFU) keinen negativen Einfluss auf GBM-Zellen hat. MB hingegen konnten mittels LIFU destruiert werden, wodurch das in den MB eingeschlossene Chemotherapeutikum freigesetzt werden kann. Es wurden verschiedene Platin(II)- und Palladium(II)-Komplexe auf GBM Zellen getestet. Zur Beladung der MB wurde Doxorubicin (Dox) verwendet. Es konnte eine Beladungseffizienz der MB mit Dox von 52 % erreicht werden, auch eine Aufreinigung dieser mittel Ionenaustausch-Chromatographie und Dialyse war erfolgreich. Die Austestung der mit Dox beladenen MB (MBDox) erfolgte auf GBM-Zellen in 2D- und 3D-Zelkulturmodellen. Dabei zeigte sich, dass die Behandlung mit MBDox und LIFU für 48 h eine zytotoxische Wirkung hatte, die sich signifikant von der Behandlung mit MBDox ohne LIFU unterschied. Zur Austestung der MBDox in 3D-Zellkulturmodellen wurden zwei Scaffold-Systeme eingesetzt. Es zeigte sich in den Versuchen, dass MBDox mit LIFU im Vergleich zu MBDox ohne LIFU Applikation einen zytotoxischen Effekt auf GBM-Zellen haben. Somit konnte die Wirksamkeit der Zytostatika Applikation mittels MB und LIFU in 2D- und 3D-Zellkulturmodellen erfolgreich etabliert werden. Als weiterer Schritt wurden zwei 3D in vitro Modelle erarbeitet. Dabei wurden zunächst organotypische hippocampale Slice Kulturen (organotypic hippocampal brain slice cultures, OHSC) aus der Maus hergestellt und anschließend mit fluoreszent-markierten Mikrotumoren aus GBM-Zelllinien, Primärzellen (PZ) und aus Patienten generierten GBM-Organoiden hergestellt. Diese GBM-Modelle wurden mit Tumor Treating Fields (TTFields) behandelt. Dabei war eine Abnahme der Tumorgröße von Mikrotumoren aus GBM-Zellen und PZ unter TTFields-Behandlung für 72 h messbar. Als weiteres in vitro Modell wurden humane Tumorschnitte aus intraoperativ entferntem GBM-Patientenmaterial hergestellt. Die Schnitte wiesen ein heterogenes Ansprechen nach 72 h TTFields-Applikation auf. Dies spiegelt die Heterogenität des GBM sehr gut wider und bestärkt die Eignung des Modelles zur Untersuchung von neuen Therapieansätzen zur Behandlung von GBM.
Gegenstand dieser Doktorarbeit war die Beschreibung des Urokinaseplaminaktivators uPA im C6-Sphäroidmodell der Ratte und dessen Lokalisation in Bezug auf den Primärtumor. Das hierbei verwendete Tiermodell basiert auf der C6-Tumorzellreihe, welche durch Transfektion von Rattengliomzellen mit dem Vaskularisierungsfaktor VEGF entwickelt wurde. Die gesteigerte Expression von VEGF resultiert in einer stärkeren Vaskularisierung und einer erhöhten Wachstumsrate des Tumors. Im Vorfeld der Tumorimplantation konnte die Expression von uPA durch die C6-Tumorzellen mittels reverser RNA-Transkription und Polymerasekettenreaktion nachgewiesen werden. In vitro gelang der Nachweis von uPA im C6-Sphäroiden mittels Fluoreszenz-Färbung. Im Rahmen des Tierversuches wurden aus den Tumorzellen ca. 300µm große Sphäroide hergestellt, welche den Ratten in den Kortex des linken Frontallappens implantiert wurden und dort solide Hirntumoren bildeten. Die Versuchstiere wurden anschließend in zwei Gruppen aufgeteilt. Der Positivgruppe wurde täglich über einen Zeitraum von 19 bzw. 21 Tagen der Proteasehemmer WX-UK1 in die Bauchhöhle injiziert, die Kontrollgruppe erhielt ein Placebo. Nach Ablauf des Behandlungszeitraumes konnte an den explantierten Gehirnen mittels histochemischer Peroxidasefärbung die Protease uPA im Tumorgewebe nachgewiesen werden. Die Konzentration von uPA war besonders im invasionsaktiven Bereich des Tumors erhöht. Dieser entspricht der Randzone des soliden Tumors, sowie den distanzierten Zellnestern im gesunden Hirngewebe, welche als so genannte Invasionszone zusammengefasst werden. Die tragende Rolle von uPA bei der Invasion der Tumorzellen in das gesunde Hirngewebe konnte somit bestätigt werden. Die Messung von erhöhten uPA-Konzentrationen an der Basalmembran von Hirngefäßen korreliert mit Beobachtungen, dass die Tumorzellen entlang von Gefäßen und Plexus migrieren, aber nicht in der Lage sind, in das Gefäßlumen einzudringen. Der Nachweis der erfolgreichen orthotopen Sphäroidimplantation mittels MRT-Bildgebung der Hirntumoren unterstreicht den Vorteil der offenen Implantationstechnik gegenüber der Zellinjektion. Die peritoneale Verabreichung des Proteasehemmers WX-UK1 führte im Rahmen dieser Untersuchungen zu keiner signifikanten Reduktion des Tumorwachstums, welches mittels Volumenmessung im MRT dokumentiert wurde. Des Weiteren konnte keine Minderung der uPA-Konzentration in den Tumoren der Positivgruppe gegenüber der Kontrollgruppe gemessen werden. Neben der fehlenden Biodistribution des Wirkstoffes kommt hierfür auch eine mangelnde Spezifität von WX-UK1 für uPA oder ein alternativer Aktivierungsweg der Proteolyse innerhalb der Tumorzellen in Betracht. Diese Arbeit führt zur Weiterentwicklung des C6-Sphäroidmodells und unterstützt die zukünftige Entwicklung von Wirkstoffen gegen das Tumorwachstum auf Basis der anti-invasiven Therapie.
Hirntumoren werden nach histologischen und molekulargenetischen Gesichtspunkten unterteilt. Neben dem Krankheitsverlauf unterscheiden sich LGA auch genetisch von GBM. Wie die mRNA der Proteine ATF5, KiSS1, RPS27, BRMS1 und TTK in LGA exprimiert ist bzw. sich im Verlauf verändert, war in dieser Form noch nicht in einem Patientenpanel untersucht worden. Ziel dieser Arbeit war es, die mRNA-Expressionsraten zu bestimmen sowie Korrelationen und Kointegrationen mit klinischen Daten zu analysieren. Außerdem wurden Besonderheiten der Verteilung innerhalb des Patientenpanels beschrieben. Quantitative-PCR-Analysen wurden durchgeführt. Die Expressionswerte wurden auf die Expression des Haushaltsgens GAPDH normalisiert. Die resultierenden ∆CTm - bzw. ∆∆CT-Werte sowie die klinischen Patientendaten waren Basis für Kointegrations-, Korrelations-, Expressions-, Ähnlichkeits- und Verlaufsanalysen. KiSS1 schien generell kaum oder nicht in der Vielzahl der Tumoren exprimiert zu sein, Aussagen zur klinischen Korrelation erwiesen sich als schwierig. Für RPS27 konnten tendenziell niedrigere Werte in den Verlaufstumoren im Vergleich zu den LGA gefunden werden. Auch für BRMS1 war in der Mehrzahl der Fälle die mRNA der Vorläufertumoren in Relation zu den Rezidiven stärker exprimiert. ATF5 korrelierte nach Kendall RE und PFS (-0,324; p=0,077) in der Gruppe der IDH1-mutierten LGA, für TTK nach Kendall OS und RE (-0,444; p=0,097) im Gesamtpanel und nach Pearson auch RE und PFS (-0,43; p=0,096) in der Gruppe der IDH1-mutierten LGA.
Einführung und Zielsetzung:
Die intraoperative Ableitung von ABRs (auditory brainstem responses) ist eine Standardmethode für das Monitoring der Hörbahn bei der Operation von Vestibularisschwannomen. Als Fernfeldtechnologie zeigt sich diese Methode jedoch oft limitiert. Ziel dieser Arbeit ist, den zusätzlichen Einsatz einer nicht-invasiven Elektrocochleographie (ECochG) als Nahfeldtechnologie zu evaluieren.
Methoden:
Hierzu erfolgte retrospektive Auswertung und Klassifikation von elektrophysiologischen Monitoring Daten von 69 Patienten, welche zwischen 2010 und 2014 mittels retrosigmoidalen Zugang am Vestibularisschwannom operiert wurden. Die ECochG wurde bei diesen Patienten simultan zu den ABR mit einer ans Trommelfell platzierten Kugelelektrode abgeleitet. Die Patientenselektion für diese Studie erfolgte vor allem nach dem Wunsch des Patienten gehörerhaltend operiert zu werden, unabhängig von Tumorausdehnung (von klein bis Hirnstamm komprimierend) oder der präoperativer Hörqualität. Es erfolgte Korrelation mit der prä- und postoperativen Hörqualität.
Ergebnisse:
Präoperativ zeigen die ABR- und ECochG-Klassen nahezu dieselbe Verteilung und Korrelation mit der präoperativen Hörklasse. Allerdings zeigt, wie initial vermutet, die postoperative ECochG schwächere Korrelation mit der postoperativen Hörqualität, als die ABRs: 25 von 43 Patienten mit postoperativer Taubheit zeigten in der ECochG am Ende der OP immer noch cochleäre Potentiale. Neben der cochleären Funktion kann mit der nicht-invasiven ECochG die Hörbahn analog zum ABR dargestellt werden. Die Identifizierbarkeit besonders der späteren Komponenten (Welle III und V) ist mit der nicht-invasiven Elektrocochleographie mindestens genauso gut möglich wie mit den ABR. Weiter liefert die ECochG signifikant größere Amplituden. Der Vergleich der ABR- mit den ECochG-Klassen liefert stark positive Korrelationen. Dies gilt vor allem für die Klassen 1 bis 3, in denen die Welle V noch vorhanden ist.
Schlussfolgerung:
Die signifikant größeren Amplituden der ECochG erlauben kürzere Messzeiten. Dies bietet intraoperativ Sicherheit für den Fall von Artefakten oder technischen Störungen sowie Vorteile in technisch schwierigen Phasen der OP. Neben der cochleären Funktion kann mit der ECochG die Hörbahn analog zum ABR bis in den Hirnstamm erfasst und überwacht werden. Die ECochG kann die ABR Ableitung nicht gänzlich ersetzen, da ihre Verlässlichkeit bei elektrisch darstellbarer Beeinträchtigung der Hörbahn sinkt. Wann immer eine Welle V vorhanden ist, ist Monitoring mit der nicht-invasiven ECochG genauso gut möglich wie mit ABR.
Analysis of cerebral glucose metabolism following experimental subarachnoid hemorrhage over 7 days
(2023)
Little is known about changes in brain metabolism following SAH, possibly leading towards secondary brain damage. Despite sustained progress in the last decade, analysis of in vivo acquired data still remains challenging. The present interdisciplinary study uses a semi-automated data analysis tool analyzing imaging data independently from the administrated radiotracer. The uptake of 2-[18F]Fluoro-2-deoxy-glucose ([\(^{18}\)F]FDG) was evaluated in different brain regions in 14 male Sprague–Dawley rats, randomized into two groups: (1) SAH induced by the endovascular filament model and (2) sham operated controls. Serial [\(^{18}\)F]FDG-PET measurements were carried out. Quantitative image analysis was performed by uptake ratio using a self-developed MRI-template based data analysis tool. SAH animals showed significantly higher [\(^{18}\)F]FDG accumulation in gray matter, neocortex and olfactory system as compared to animals of the sham group, while white matter and basal forebrain region showed significant reduced tracer accumulation in SAH animals. All significant metabolic changes were visualized from 3 h, over 24 h (day 1), day 4 and day 7 following SAH/sham operation. This [\(^{18}\)F]FDG-PET study provides important insights into glucose metabolism alterations following SAH—for the first time in different brain regions and up to day 7 during course of disease.
In a recent study, we showed in an in vitro murine cerebellar microvascular endothelial cell (cerebEND) model as well as in vivo in rats that Tumor-Treating Fields (TTFields) reversibly open the blood–brain barrier (BBB). This process is facilitated by delocalizing tight junction proteins such as claudin-5 from the membrane to the cytoplasm. In investigating the possibility that the same effects could be observed in human-derived cells, a 3D co-culture model of the BBB was established consisting of primary microvascular brain endothelial cells (HBMVEC) and immortalized pericytes, both of human origin. The TTFields at a frequency of 100 kHz administered for 72 h increased the permeability of our human-derived BBB model. The integrity of the BBB had already recovered 48 h post-TTFields, which is earlier than that observed in cerebEND. The data presented herein validate the previously observed effects of TTFields in murine models. Moreover, due to the fact that human cell-based in vitro models more closely resemble patient-derived entities, our findings are highly relevant for pre-clinical studies.
Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood–brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB’s integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100–300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.
Progressive deterioration of the central nervous system (CNS) is commonly associated with aging. An important component of the neurovasculature is the blood-brain barrier (BBB), majorly made up of endothelial cells joined together by intercellular junctions. The relationship between senescence and changes in the BBB has not yet been thoroughly explored. Moreover, the lack of in vitro models for the study of the mechanisms involved in those changes impede further and more in-depth investigations in the field. For this reason, we herein present an in vitro model of the senescent BBB and an initial attempt to identify senescence-associated alterations within.
Bei einem SHT handelt es sich um eine mechanische Schädigung des Hirngewebes, verursacht durch eine Gewalteinwirkung auf den Kopf. Diese initiale Schädigung des Hirngewebes weitet sich nachfolgend aus. Wirksame Therapien, um diese sekundären Pathomechanismen zu inhibieren, gibt es nicht. Sofern das SHT überlebt wird, ist es eine der häufigsten Ursachen für bleibende Behinderungen. Wichtige Pathomechanismen, welche zur Ausweitung der Hirnschädigung beitragen, sind das posttraumatische Hirnödem und Entzündungsreaktionen. Beides wird durch die Aktivierung des so-genannten Kallikrein-Kinin-Systems begünstigt. In der vorliegenden Arbeit wurde dieses System 1 Stunde nach experimentellem SHT durch die Applikation des C1-Inhibitors gehemmt und am nachfolgenden Tag die Auswirkungen bewertet. Die Ergebnisse zeigen, dass diese Behandlung nach der Hirnverletzung zu einer Reduktion des Hirnödems und der Entzündungsreaktion führt. Die Bildung von Thromben in den Hirngefäßen ist geringer als in Kontrolltieren, vermutlich da der C1-Inhibitor auch die intrinsische Gerinnungs-kaskade hemmt. Insgesamt führt die Behandlung zu kleineren Hirnläsionen als in entsprechenden Kontrolltieren. Hiermit stellt der C1-Inhibitor ein potenzieller Therapieansatz bei SHT dar. Jedoch bleibt es offen, inwiefern sich diese Ergebnisse auf das menschliche SHT übertragen lassen.
Ischemic stroke is one of the leading causes of death worldwide. It damages neurons and other supporting cellular elements in the brain. However, the impairment is not only confined to the region of assault but the surrounding area as well. Besides, it also brings about damage to the blood-brain barrier (BBB) which in turn leads to microvascular failure and edema. Hence, this necessitates an on-going, continuous search for intervention strategies and effective treatment. Of late, the natural sweetener stevioside proved to exhibit neuroprotective effects and therapeutic benefits against cerebral ischemia-induced injury. Its injectable formulation, isosteviol sodium (STVNA) also demonstrated favorable results. Nonetheless, its effects on the BBB have not yet been investigated to date. As such, this present study was designed to assess the effects of STVNA in our in vitro stroke model of the BBB.The integrity and permeability of the BBB are governed and maintained by tight junction proteins (TJPs) such as claudin-5 and occludin. Our data show increased claudin-5 and occludin expression in oxygen and glucose (OGD)-deprived murine brain capillary cerebellar endothelial cells (cerebEND) after STVNa treatment. Likewise, the upregulation of the transmembrane protein integrin-αv was also observed. Finally, cell volume was reduced with the simultaneous administration of STVNA and OGD in cerebEND cells. In neuropathologies such as stroke, the failure of cell volume control is a major feature leading to loss of cells in the penumbra as well as adverse outcomes. Our initial findings, therefore, point to the neuroprotective effects of STVNA at the BBB in vitro, which warrant further investigation for a possible future clinical intervention.
Bislang ist ungeklärt, warum PNPs teils schmerzhaft und teils schmerzlos verlaufen. Die in der vorliegenden Arbeit untersuchte Hypothese lautete, dass ein Ungleichgewicht zwischen pro- und anti-inflammatorischen Zytokinen der unterschiedlichen Schmerzausprägung zugrunde liegt.Es wurden 32 Patienten mit schmerzhafter PNP, 20 Patienten mit schmerzloser PNP und 44 Kontrollpersonen auf die Expression und Produktion ausgewählter pro- und anti-inflammatorischer Zytokine untersucht. Zur Messung der Schmerzhaftigkeit wurden etablierte Schmerzfragebögen verwendet. Zusätzlich wurden nahezu alle Patienten mit der Allgemeinen Depressionsskala befragt. Die Diagnose, Ätiologie, Dauer, klinische Manifestation der PNP sowie die Medikation der Patienten wurde auf standardisierten Erhebungsbögen dokumentiert. Zur Messung der Zytokine wurde morgens Blut in EDTA- und Serummonovetten asserviert und entsprechend der Messmethodik weiterverarbeitet. Die relative Genexpression wurde aus Gesamt-RNA mittels reverser Transkription und quantitativer real-time PCR, die Serumproteine mittels enzyme-linked immunosorbant assay gemessen. Die Patienten mit schmerzhafter PNP hatten in der Mehrzahl Neuropathie-typische Plussymptome und mittelstarke Schmerzen, die eine starke bis sehr starke Behinderung darstellten. Die hier untersuchten Zytokinmuster bei Patienten mit schmerzhafter und schmerzloser PNP zeigten eine Verschiebung zu pro-inflammatorischen Zytokinen bei Patienten mit schmerzhafter PNP. Die Zytokinexpression der Patienten mit schmerzhafter PNP war im Vergleich zu Patienten mit schmerzloser PNP und Kontrollen bezüglich der IL-2 und TNF Expression und Produktion signifikant erhöht. Umgekehrt lagen bei Patienten mit schmerzloser PNP die Produktion und die Expression des IL-4 im Vergleich zu Patienten mit schmerzhafter PNP und Kontrollen höher. Die Expression des IL-10 lag bei Patienten mit schmerzloser PNP ebenfalls höher als bei Patienten mit schmerzloser PNP und Kontrollen, unterschied sich aber auf Proteinebene nicht in den drei Gruppen. Die einleitend gestellte Hypothese, dass der schmerzhafte oder schmerzlose Verlauf einer PNP durch unterschiedliche Zytokinprofile bedingt ist, kann durch die vorliegenden Ergebnisse gestützt werden. In Zusammenschau mit den Daten aus der Grundlagenforschung scheint einem pro-inflammatorischen Zytokinmuster eine entscheidende Rolle an der Entstehung und Aufrechterhaltung neuropathischer Schmerzen zuzukommen. Für TNF sind entsprechende pathophysiologische Wirkungen bekannt. Anti-inflammatorische Zytokine, wie IL-4 und IL-10 zeigten analgetische Wirkungen im Tierversuch. Die Mitwirkung des IL-2 an peripheren Opioid-Rezeptoren lässt eine endogene periphere Analgesie vermuten. Hieraus lassen sich Folgerungen für zukünftige Diagnostik und Therapie neuropathischer Schmerzen ziehen. Durch Erkennung von Zytokin-Imbalancen wären schmerzhafte PNPs früher einer adäquaten Therapie zuzuführen. Durch die Modulation von Zytokinprofilen im Rahmen schmerzhafter PNPs könnten sich zusätzlich therapeutische Möglichkeiten eröffnen.
Trotz jahrelanger, intensiver Forschung ist es bisher nicht gelungen, die Morbidität und
Mortalität der aneurysmatischen Subarachnoidalblutung (SAB) signifikant zu senken. Der Fokus dieser Bemühungen lag in den letzten Jahrzehnten vor allem auf der frühen Erkennung und Behandlung des verzögert, typischerweise zwischen Tag 4 und 14 auftretenden, symptomatischen Vasospasmus´ und somit einer zerebralen Ischämie, die mit einer höheren Rate an klinischen Komplikationen, reduzierter Alltagsaktivität, schlechterer kognitiver Leistungsfähigkeit und einer insgesamt höheren Mortalität einhergeht sowie mit einem insgesamt schlechteren neurologischen Outcome assoziiert ist.
Diese Arbeit beschäftigt sich per Tierexperiment mit der Akutphase der SAB und zweier möglicher Therapiestrategien (Hyper-HES und Hypothermie), die möglichst früh im Verlauf angewendet werden und für eine histologisch sowie neurologisch nachweisbare Verbesserung im Gesamtergebnis sorgen können.
Insgesamt entsprechen sowohl die osmotischwirksame Therapie mit Hydroxyethylstärke als auch die hypotherme Therapie nach unseren Resultaten der primären Zielsetzung der Studie. Die Möglichkeit einer prinzipiell überall verfügbaren, kostengünstigen und gut steuerbaren Frühphasentherapie nach SAB ohne Kompromittierung der initialen Diagnostik und der klinischen Differentialdiagnosen
scheint hier jeweils gegeben zu sein.
Early treatment with glucocorticoids could help reduce both cytotoxic and vasogenic edema, leading to improved clinical outcome after stroke. In our previous study, isosteviol sodium (STVNA) demonstrated neuroprotective effects in an in vitro stroke model, which utilizes oxygen-glucose deprivation (OGD). Herein, we tested the hypothesis that STVNA can activate glucocorticoid receptor (GR) transcriptional activity in brain microvascular endothelial cells (BMECs) as previously published for T cells. STVNA exhibited no effects on transcriptional activation of the glucocorticoid receptor, contrary to previous reports in Jurkat cells. However, similar to dexamethasone, STVNA inhibited inflammatory marker IL-6 as well as granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion. Based on these results, STVNA proves to be beneficial as a possible prevention and treatment modality for brain ischemia-reperfusion injury-induced blood–brain barrier (BBB) dysfunction.
Both nerve injury and complex regional pain syndrome (CRPS) can result in chronic pain. In traumatic neuropathy, the blood nerve barrier (BNB) shielding the nerve is impaired—partly due to dysregulated microRNAs (miRNAs). Upregulation of microRNA-21-5p (miR-21) has previously been documented in neuropathic pain, predominantly due to its proinflammatory features. However, little is known about other functions. Here, we characterized miR-21 in neuropathic pain and its impact on the BNB in a human-murine back translational approach. MiR-21 expression was elevated in plasma of patients with CRPS as well as in nerves of mice after transient and persistent nerve injury. Mice presented with BNB leakage, as well as loss of claudin-1 in both injured and spared nerves. Moreover, the putative miR-21 target RECK was decreased and downstream Mmp9 upregulated, as was Tgfb. In vitro experiments in human epithelial cells confirmed a downregulation of CLDN1 by miR-21 mimics via inhibition of the RECK/MMP9 pathway but not TGFB. Perineurial miR-21 mimic application in mice elicited mechanical hypersensitivity, while local inhibition of miR-21 after nerve injury reversed it. In summary, the data support a novel role for miR-21, independent of prior inflammation, in elicitation of pain and impairment of the BNB via RECK/MMP9.
In einem experimentellen Schädel-Hirn-Trauma-Modell der fokalen Kälteläsion bei der Maus wurde die Effektivität der B1R-Blockade untersucht. Die Ergebnisse dieser Untersuchung dokumentierten auf der Suche nach einer grundlegenden spezifischen Therapie des vasogenen traumatischen Hirnödems die B1R-Blockade als einen potentiellen Ansatz zu Reduktion der sekundären Hirn-schäden. Zum Einen konnte durch die selektive Blockade von B1R mit dem Präparat R-715 nach einer fokalen Kälteläsion im Mausmodell die Hirnschädigung um etwa 75 % gegenüber den Tieren der Kontrollgruppen reduziert werden. Zum Anderen lässt sich nach der B1R-Blockade u. a. eine signifikante Abschwächung des vasogenen Hirnödems um etwa 50 % im Vergleich zu den Tieren der Kontrollgruppen feststellen. Die Reduktion der sekundären Hirnschädigung durch die B1R-Blockade 24 Stunden nach der Läsionsinduktion macht die selektive B1R-Blockade als kausaler Therapie-ansatz eine interessante Behandlungsoption des posttraumatischen vasogenen Hirnödems.
Die Critical-lllness-Polyneuropathie (CIP), eine Erkrankung des peripheren Nervensystems nach einer schweren intensivmedizinisch behandlungspflichtigen Erkrankung, ist bereits seit dem 19. Jahrhundert bekannt und lässt sich nur sehr schwer von einer Critical-Illness- Myopathie, die im gleichen Kontext auftreten kann, unterscheiden. Erschwert wird die Situation dadurch, dass beide Funktionsstörungen kombiniert auftreten können. Auf Grund der Weiterentwicklung in der Medizin ist in den letzten 2 Jahrzehnten eine Häufigkeitssteigerung zu verzeichnen mit der Forderung geeignete Maßnahmen zu finden, die häufig schwerwiegenden Folgeerscheinungen zu mindern. Mit Entwicklung eines bestimmten neurorehabilitativen Behandlungsregimes ergeben sich die Fragen, welche CIP Patienten profitieren, ergeben sich prognoserelevante Faktoren und welche Art und Dauer der Neurorehabilitation vorgehalten werden muss. In der vorliegenden Arbeit wurden retrospektiv die Entlassungsbriefe der Patienten mit einer CIP der Abteilung für neurologisch-neurochirurgisch Frührehabilitation der Jahre 2004-2008 ausgewertet. Die Beurteilung der Fähigkeitsstörungen erfolgte mit dem Frührehabilitations-Barthel-Index nach Schönle, dem Barthel-Index sowie dem FIM und das Handicap wurde mit der 8-stufigen Glasgow-Outcome-Scale bewertet. Zusätzliche Parameter waren das Alter und Geschlecht, die Ursache der CIP, die Zeit von Erkrankungsbeginn bis zur Aufnahme, die Behandlungsdauer, Komplikationen sowie die Entlassungsart. 200 Patienten, 67,5% Männer und 32,5% Frauen konnten ausgewertet werden. Eine Häufung des Erkrankungsbildes fand sich bei Patienten ab dem 60. Lebensjahr. Eine Ursachenpräferenz fand sich nicht, wobei im Wesentlichen kardiale, respiratorische und gastrointestinale Erkrankungen vorgefunden worden waren. Alle Patienten konnten, nach einer durchschnittlichen Behandlungsdauer von 40 Tagen, von der Frührehabilitationsbehandlung profitieren. So konnten 34% in weiterführende rehabilitative Behandlungsstufen und 17,5% der Patienten gebessert nach Hause entlassen werden. Die Sterblichkeit mit 11% muss der besonderen Schwere des Krankheitsbildes angelastet werden. Dies wird durch die Feststellung gestützt, dass die Komplikationsrate mit über 60% während der rehabilitativen Behandlung sehr hoch lag. Hervorzuheben ist, dass sich herausarbeiten ließ, dass sich eine längere Behandlung positiv auf die Behandlungsergebnisse, auch bei schwer Betroffenen auswirkte. Die auslösende Ursache scheint keinen wesentlichen Einfluss auf das outcome zu haben. Männer profitieren bei der Wiedererlangung motorischer Fähigkeiten etwas besser als Frauen. Die älteren Patienten zeigen gute Verbesserungen bei körperlichen Funktionsstörungen. Bezogen auf die kognitiven Fähigkeiten muss aber das Alter als negativer Prädiktor angesehen werden. Der Grad der Behinderung hat einen deutlichen Einfluss auf die Behandlungsdauer und Behandlungsergebnisse. Demgegenüber stellt eine primäre Intensivpflicht einen negativen Prädiktor, auch wenn auch diese Patienten von der Rehabilitation profitieren.
As recently reviewed, 1026 neuroprotective drug candidates in stroke research have all failed on their road towards validation and clinical translation, reasons being quality issues in preclinical research and publication bias. Quality control guidelines for preclinical stroke studies have now been established. However, sufficient understanding of the underlying mechanisms of neuronal death after stroke that could be possibly translated into new therapies is lacking. One exception is the hypothesis that cellular death is mediated by oxidative stress. Oxidative stress is defined as an excess of reactive oxygen species (ROS) derived from different possible enzymatic sources. Among these, NADPH oxidases (NOX1-5) stand out as they represent the only known enzyme family that has no other function than to produce ROS. Based on data from different NOX knockout mouse models in ischemic stroke, the most relevant isoform appears to be NOX4. Here we discuss the state-of-the-art of this target with respect to stroke and open questions that need to be addressed on the path towards clinical translation.
Differences in stem cell marker and osteopontin expression in primary and recurrent glioblastoma
(2022)
Background
Despite of a multimodal approach, recurrences can hardly be prevented in glioblastoma. This may be in part due to so called glioma stem cells. However, there is no established marker to identify these stem cells.
Methods
Paired samples from glioma patients were analyzed by immunohistochemistry for expression of the following stem cell markers: CD133, Musashi, Nanog, Nestin, octamer-binding transcription factor 4 (Oct4), and sex determining region Y-box 2 (Sox2). In addition, the expression of osteopontin (OPN) was investigated. The relative number of positively stained cells was determined. By means of Kaplan–Meier analysis, a possible association with overall survival by marker expression was investigated.
Results
Sixty tissue samples from 30 patients (17 male, 13 female) were available for analysis. For Nestin, Musashi and OPN a significant increase was seen. There was also an increase (not significant) for CD133 and Oct4. Patients with mutated Isocitrate Dehydrogenase-1/2 (IDH-1/2) status had a reduced expression for CD133 and Nestin in their recurrent tumors. Significant correlations were seen for CD133 and Nanog between OPN in the primary and recurrent tumor and between CD133 and Nestin in recurrent tumors. By confocal imaging we could demonstrate a co-expression of CD133 and Nestin within recurrent glioma cells. Patients with high CD133 expression had a worse prognosis (22.6 vs 41.1 months, p = 0.013). A similar trend was seen for elevated Nestin levels (24.9 vs 41.1 months, p = 0.08).
Conclusions
Most of the evaluated markers showed an increased expression in their recurrent tumor. CD133 and Nestin were associated with survival and are candidate markers for further clinical investigation.
Im Rahmen der vorliegenden Arbeit wurden Daten von Patienten ausgewertet, die an einem Vestibularisschwannom erkrankt sind. Dabei wurde der Einfluss einer Facialisparese auf die Lebensqualität untersucht. Die Auswertung der Daten zeigte statistisch signifikante Zusammenhänge zwischen verschiedenen Domänen der Lebensqualität und einer postoperativen Facialisparese.
Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.
Traumatic brain injury (TBI) is the leading cause of death and disability in polytrauma and is often accompanied by concomitant injuries. We conducted a retrospective matched-pair analysis of data from a 10-year period from the multicenter database TraumaRegister DGU\(^®\) to analyze the impact of a concomitant femoral fracture on the outcome of TBI patients. A total of 4508 patients with moderate to critical TBI were included and matched by severity of TBI, American Society of Anesthesiologists (ASA) risk classification, initial Glasgow Coma Scale (GCS), age, and sex. Patients who suffered combined TBI and femoral fracture showed increased mortality and worse outcome at the time of discharge, a higher chance of multi-organ failure, and a rate of neurosurgical intervention. Especially those with moderate TBI showed enhanced in-hospital mortality when presenting with a concomitant femoral fracture (p = 0.037). The choice of fracture treatment (damage control orthopedics vs. early total care) did not impact mortality. In summary, patients with combined TBI and femoral fracture have higher mortality, more in-hospital complications, an increased need for neurosurgical intervention, and inferior outcome compared to patients with TBI solely. More investigations are needed to decipher the pathophysiological consequences of a long-bone fracture on the outcome after TBI.
Introduction: Striatal dopamine depletion disrupts basal ganglia function and causes Parkinson’s disease (PD). The pathophysiology of the dopamine-dependent relationship between basal ganglia signaling and motor control, however, is not fully understood. We obtained simultaneous recordings of local field potentials (LFPs) from the subthalamic nucleus (STN) and electromyograms (EMGs) in patients with PD to investigate the impact of dopaminergic state and movement on long-range beta functional connectivity between basal ganglia and lower motor neurons.
Methods: Eight PD patients were investigated 3 months after implantation of a deep brain stimulation (DBS)-system capable of recording LFPs via chronically-implanted leads (Medtronic, ACTIVA PC+S®). We analyzed STN spectral power and its coherence with EMG in the context of two different movement paradigms (tonic wrist extension vs. alternating wrist extension and flexion) and the effect of levodopa (L-Dopa) intake using an unbiased data-driven approach to determine regions of interest (ROI).
Results: Two ROIs capturing prominent coherence within a grand average coherogram were identified. A trend of a dopamine effect was observed for the first ROI (50–150 ms after movement start) with higher STN-EMG coherence in medicated patients. Concerning the second ROI (300–500 ms after movement start), an interaction effect of L-Dopa medication and movement task was observed with higher coherence in the isometric contraction task compared to alternating movements in the medication ON state, a pattern which was reversed in L-Dopa OFF.
Discussion: L-Dopa medication may normalize functional connectivity between remote structures of the motor system with increased upper beta coherence reflecting a physiological restriction of the amount of information conveyed between remote structures. This may be necessary to maintain simple movements like isometric contraction. Our study adds dynamic properties to the complex interplay between STN spectral beta power and the nucleus’ functional connectivity to remote structures of the motor system as a function of movement and dopaminergic state. This may help to identify markers of neuronal activity relevant for more individualized programming of DBS therapy.
Tyr-o-Arg\(^2\)-Phe-sarcosine\(^4\) (TAPS), a mu-selective tetrapeptide analog of dermorphin, induced sustained antinociception and stimulated ventilatory minute volume (MV) at the doses of 3 to 100 pmol i.c.v. The doses of 30 and 100 pmol i.c.v. induced catalepsy. The effect of TAPS on MV was in negative correlation with the dose and the maximal response was achieved by the lowest (3 pmol) dose (+63 ± 23%, P < .05). Morphine, an agonist at both mu\(_1\) and mu\(_2\) sites, at a dose of 150 nmol i.c.v. (equianalgetic to 100 pmol of TAPS decreased the MV by 30%, due to a decrease in ventilatory tidal volume. The antinociceptive effect of TAPS was antagonized by naloxone and the mu, receptor antagonist, naloxonazine. Naloxonazine also attenuated the catalepsy produced by 1 00 pmol of TAPS i.c. v. and the respiratory Stimulation produced by 3 pmol of TAPS i.c.v. Pretreatment with 30 pmol of TAPS antagonized the respiratory depression induced by the mu opioid agonist dermorphin (changes in MV after dermorphin alone at 1 or 3 nmol were -22 ± 1 0% and -60 ± 9% and, after pretreatment with TAPS, +44 ± 11 % and -18 ± 5%, respectively). After combined pretreatment with naloxonazine and TAPS, 1 nmol of dermorphin had no significant effect on ventilation. In contrast, pretreatment with a low respiratory stimulant dose (10 pmol i.c.v.) of dermorphin did not modify the effect of 1 nmol of dermorphin. ln conclusion, the antinociceptive, cataleptic and respiratory stimulant effects of TAPS appear to be a related to its agonist action at the mu, opioid receptors. TAPS did not induce respiratory depression (a mu\(_2\) opioid effect) but antagonized the respiratory depressant effect of another mu agonist. Thus, in vivo TAPS appears to act as a mu\(_2\) receptor antagonist.
The selective opioid mu receptor agonist dermorphin increased the locomotor activity of rats dose dependently at 1 0 to 1 00 pmol/kg i.c.v. Respiratory rate, relative tidal volume and respiratory minute volume also increased unrelated to changes in locomotor activity. Higher doses, on the other hand, produced catalepsy and respiratory depression. Pretreatment of the rats with the mu,-selective antagonist naloxonazine (10 mg/kg i.v.) blocked the stimulant locomotor and respiratory effects of low doses of dermorphin (1 0--1 00 pmol/kg), but potentiated the respiratory depressant effect of a high dose (1 0 nmol/kg) of dermorphin. The selective benzodiazepine antagonist flumazenil (5 mg/kg), which has been shown previously to antagonize catalepsy and respiratory depression produced by relatively high doses of dermorphin, did not antagonize the respiratory or locomotor stimulant effect of dermorphin. The data suggest that mu\(_1\)-opioid receptors are responsible for the low dose stimulant effects of dermorphin on locomotor activity and respiration whereas mu\(_2\) receptors mediate the respiratory depressant effect of dermorphin.
lnteractions of p-opioid receptors with the benzodiazepine system were studied by examining the modulatory effects of flumazenil (a benzodiazepine antagonist) and alprazolam (a benzodiazepine agonist) on the respiratory effects ofthe opioid peptide dermorphin. Dermorphin, 1-30 nmol administered i.c.v., to conscious, unrestrained rats decreased ventilation rate (VR) and minute volume (MV) dose-dependently. The ventilatory depression was antagonized by naloxone and by the benzodiazepine antagonist flumazenil. The benzodiazepine alprazolam potentiateri the respiratory inhibition of a small (I nmol) dose of dermorphin but antagonized that of a higher dos:~ (3 nmol). The results suggest that the benzodiazepine/GABA receptor complex modulates respiratory depression induced by centrat p-receptor Stimulation in the rat.
The possibility that \(\mu\)Opioid-induced tachycardia and bradycardia could be mediated by different subtypes of the \(\mu\)·receptor was studied in conscious Sprague-Dawley rats. The selective \(\mu\)·receptor agonist dermorphin and its analog, TAPS (Tyr-o-Arg-Phe-sarcosine), a putative \(\mu _1\)-receptor agonist, were given centrally. Tyr-o-Arg-Phe-sarcosine increased the heart rate, the response being inversely correlated to the dose (an increase of 71 ± 22, 49 ± 14 and 30 ± 17 beats/min at doses of 0.3, 3 and 30 pmol, respectively). Dermorphin induced less clear changes in heart rate (maximum increase of 39 ± 14 beats/min at the dose of 1 pmol). Aftertreatment with the Jl 1-selective antagonist naloxonazine (NAZ), TAPS 30 pmol and dennorphin I pmol decreased heart rate by -22 ± 10 and -24 ± 7 bpm, respectively. The bradycardic effect oflarger doses of dennorphin was potentiated by NAZ (from -25 ± 8 to -97 ± 22 bpm) but abolished by the non-selective antagonist naloxone. These data suggest that the high affinity \(\mu _1\)-opioid receptors mediate tachycardic responses and \(\mu _2\)-receptors mediate bradycardic responses.
Thyrotropin releasing hormone (TRH), 10 pmol kg-1 injected in the region of locus coeruleus, caused a rapid (within 1 min) rise of mean arterial pressure in the urethane- naesthetized rat. No clear-cut effects in heart rate or ventilation were observed. When TRH was injected into the lateral ventricle, a dose more than 10-fold higher was required to achieve a comparable rise in arterial pressure. It is concluded that TRH may have a physiological rote in centrat cardiovascular regulation.
Cardioventilator responses were studied in anaesthetized rats after injections of TRH into either the lateral (i.c.v. lat) or the fourth (i.c.v. IV) cerebral ventricles. TRH induced a morerapid hypertensive effect i.c.v. IV than i.c.v. lat. Blocking of the cerebral aqueduct abolished the hypertensive and tachypnoeic effects of TRH i.c.v. lat but not those of TRH i.c.v. IV. It is concluded that TRH increased blood pressure and ventilation rate via brain stem structures close to the fourtli ventricle.
Background
There is growing evidence that endothelial failure and subsequent blood brain barrier (BBB) breakdown initiate cerebral small vessel disease (CSVD) pathology. In spontaneously hypertensive stroke-prone rats (SHRSP) endothelial damage is indicated by intraluminal accumulations of erythrocytes (erythrocyte thrombi) that are not observed with current magnetic resonance imaging techniques. Two-photon microscopy (2 PM) offers the potential for real-time direct detection of the small vasculature. Thus, within this pilot study we investigated the sensitivity of 2 PM to detect erythrocyte thrombi expressing initiating CSVD phenomena in vivo.
Methods
Eight SHRSP and 13 Wistar controls were used for in vivo imaging and subsequent histology with haematoxylin-eosin (HE). For 2 PM, cerebral blood vessels were labeled by fluorescent Dextran (70 kDa) applied intraorbitally. The correlation between vascular erythrocyte thrombi observed by 2 PM and HE-staining was assessed. Artificial surgical damage and parenchymal Dextran distribution were analyzed postmortem.
Results
Dextran was distributed within the small vessel walls and co-localized with IgG.
Artificial surgical damage was comparable between SHRSP and Wistar controls and mainly affected the small vasculature. In fewer than 20% of animals there was correlation between erythrocyte thrombi as observed with 2 PM and histologically with HE.
Conclusions
Contrary to our initial expectations, there was little agreement between intravital 2 PM imaging and histology for the detection of erythrocyte thrombi. Two-photon microscopy is a valuable technique that complements but does not replace the value of conventional histology.
Simple Summary
In glioblastoma, tumor recurrence is inevitable and the prognosis of patients is poor, despite multidisciplinary treatment approaches involving surgical resection, radiotherapy and chemotherapy. Recently, Tumor Treating Fields (TTFields) have been added to the therapeutic set-up. These alternating electric fields are applied to glioblastoma at 200 kHz frequency via arrays placed on the shaved scalp of patients. Patients show varying response to this therapy. Molecular effects of TTFields have been investigated largely in cell cultures and animal models, but not in patient tissue samples. Acquisition of matched treatment-naïve and recurrent patient tissues is a challenge. Therefore, we suggest three reliable patient-derived three-dimensional ex vivo models (primary cells grown as microtumors on murine organotypic hippocampal slices, organoids and tumor slice cultures) which may facilitate prediction of patients’ treatment responses and provide important insights into clinically relevant cellular and molecular alterations under TTFields.
Abstract
Glioblastoma (GBM) displays a wide range of inter- and intra-tumoral heterogeneity contributing to therapeutic resistance and relapse. Although Tumor Treating Fields (TTFields) are effective for the treatment of GBM, there is a lack of ex vivo models to evaluate effects on patients’ tumor biology or to screen patients for treatment efficacy. Thus, we adapted patient-derived three-dimensional tissue culture models to be compatible with TTFields application to tissue culture. Patient-derived primary cells (PDPC) were seeded onto murine organotypic hippocampal slice cultures (OHSC), and microtumor development with and without TTFields at 200 kHz was observed. In addition, organoids were generated from acute material cultured on OHSC and treated with TTFields. Lastly, the effect of TTFields on expression of the Ki67 proliferation marker was evaluated on cultured GBM slices. Microtumors exhibited increased sensitivity towards TTFields compared to monolayer cell cultures. TTFields affected tumor growth and viability, as the size of microtumors and the percentage of Ki67-positive cells decreased after treatment. Nevertheless, variability in the extent of the response was preserved between different patient samples. Therefore, these pre-clinical GBM models could provide snapshots of the tumor to simulate patient treatment response and to investigate molecular mechanisms of response and resistance.
While glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs (peripheral blood mononuclear cells) and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future.
MACC1 - ein prognostischer Blutmarker für das Überleben von Patienten mit Glioblastoma multiforme?
(2023)
Das GBM ist der aggressivste primäre Hirntumor bei Erwachsenen ohne bekannten Tumormarker. Wir haben im Blutplasma zirkulierende mRNA Transkripte von MACC1, einem prognostischen Biomarker für solide Tumoren, auf ihre Korrelation mit dem klinischem Outcome und der Therapieantwort bei GBM-Patienten getestet. MACC1 mRNA Transkripte waren signifikant erhöht bei GBM-Patienten im Vergleich zur Kontrollgruppe. Eine niedrige MACC1 mRNA Transkript-Konzentration clusterte mit anderen prognostisch wertvollen Faktoren, wie z.B. dem IDH1 Mutationsstatus: Patienten mit der IDH1 R132H Mutation in Kombination mit einer niedrigen MACC1 mRNA Transkript-Konzentration wiesen das längste Gesamtüberleben von über 2 Jahren auf, IDH1 wildtyp und eine hohe MACC1 mRNA Transkript-Konzentration führten zum schlechtesten Outcome (medianes Gesamtüberleben 8,1 Monate). Patienten mit IDH1 wildtyp und einer niedriger MACC1 mRNA Transkript-Konzentration waren intermediär (medianes Gesamtüberleben 9,1 Monate). Kein Patient hatte eine IDH1 R132H Mutation und eine hohe MACC1 mRNA Transkript-Konzentration. Patienten mit niedriger MACC1 mRNA Transkript-Konzentration, die die Standardtherapie nach Stupp erhielten, überlebten länger (medianes Gesamtüberleben 22,6 Monate) als Patienten mit einer hohen MACC1 mRNA Transkript-Konzentration (medianes Gesamtüberleben 8,1 Monate). Patienten, die keine Standardtherapie erhielten, zeigten das schlechteste Outcome, unabhängig von der MACC1 mRNA Transkript-Konzentration (niedrig: 6,8 Monate, hoch: 4,4 Monate). Durch das Hinzufügen der MACC1 mRNA TranskriptKonzentrationen zur präoperativen Diagnostik könnte somit die Prognose und das Outcome von GBM Patienten genauer evaluiert werden und so eine genauere Einteilung in Therapie- und Risikogruppen erfolgen
Obwohl es sich bei Vestibularisschwannomen (VS) um benigne Tumoren handelt, können sie die Lebensqualität der betroffenen Patienten deutlich beeinträchtigen. Gerade bei Patienten, die an einer NF 2 leiden und sich daher wiederholt operativen Eingriffen unterziehen müssen, ist es notwendig, eine medikamentöse Therapiealternative anbieten zu können, die ohne die Notwendigkeit einer operativen Intervention auskommt und gleichzeitig schwerwiegenden Folgen der Tumorerkrankung – wie dem drohenden Hörverlust – Einhalt gebietet. Die vorliegende Arbeit hatte zum Ziel, sich dieser medikamentösen Therapiealternative einen Schritt anzunähern, indem molekulare Pathomechanismen, die dem VS zugrunde liegen könnten, untersucht wurden. Im Mittelpunkt standen der Chemokinrezeptor CXCR4, das Tumorsuppressorprotein Merlin und die Metalloprotease ADAM9. Für CXCR4 ließen sich keine Effekte in Bezug auf die Aktivierung der ERK- und AKT-Signalwege erkennen. Auch beeinflusste eine Merlinüberexpression in VS-Zellen die CXCR4- und ADAM9-Proteinexpression nicht. Für ADAM9 zeigte sich eine potenzielle Relevanz für die Pathogenese des VS: Wurde die ADAM9-Konzentration durch einen knock-down reduziert, hatte dies eine verminderte VS-Zellzahl zur Folge. Des Weiteren scheint Integrin α6 ein Substrat von ADAM9 zu sein, das möglicherweise in die Zytoskelettmodifikation durch ADAM9 involviert ist. Somit stellt die ADAM9-Inhibition einen interessanten Angriffspunkt für eine mögliche medikamentöse Behandlung von VS dar. Ferner wurden Cytokine gefunden, die bisher nicht in einen Zusammenhang mit dem VS gebracht worden waren. Vor allem die Bedeutung der Cytokine TIMP-2 und CXCL7 sollte für das VS näher untersucht werden. Somit konnte diese Arbeit weitere Aspekte aufdecken, die für die Pathogenese des VS relevant sein könnten und an die zukünftige Forschung anknüpfen sollte.
Objective
Recently, we described a disintegrin and metalloproteinase 9 (ADAM9) overexpression by Schwann cells of vestibular schwannoma (VS) and suggested that it might be a marker for VS tumor growth and invasiveness. This research note provides additional data utilizing a small cohort of VS primary cultures and tissue samples. We examined whether reconstitution of Merlin expression in VS cells regulates ADAM9 protein expression and performed lentiviral ADAM9 knock down to investigate possible effects on VS cells numbers. Moreover, the co-localization of ADAM9 and Integrins α6 and α2β1, respectively, was examined by immunofluorescence double staining.
Results
ADAM9 expression was not regulated by Merlin in VS. However, ADAM9 knock down led to 58% reduction in cell numbers in VS primary cell cultures (p < 0.0001). While ADAM9 and Integrin α2β1 were co-localized in only 22% (2 of 9) of VS, ADAM9 and Integrin α6 were co-localized in 91% (10 of 11) of VS. Therefore, we provide first observations on possible regulatory functions of ADAM9 expression in VS.
Aufgrund seiner infausten Prognose und des häufigen Auftretens nimmt das GBM unter den Hirntumoren eine besondere Rolle ein. Viele intrazelluläre Signalwege und Tumormarker sind bereits gut erforscht und verstanden. Hierzu gehört auch der epigenetisch determinierte Methylierungsgrad des MGMT-Genpromotors. Die Bestimmung des MGMT-Status gehört bei allen Patienten mittlerweile zur Standarddiagnostik, um den Effekt der Radiochemotherapie auf den Tumor zu prognostizieren. Ist der MGMT-Genpromotor unmethyliert, haben alkylierende Substanzen wie TMZ nur einen geringen Effekt auf die Tumorzellen. Solche Patienten profitieren kaum von der Standardtherapie nach dem Stupp-Schema. Es sind jedoch Fälle aufgetreten, bei denen sich der Methylierungsgrad des MGMT-Genpromotors im Behandlungsverlauf der Patienten verändert hat.
Aufgrund dessen untersuchte ich in meiner Arbeit, ob man Änderungen im MGMT-Genmethylierungsstatus und in der MGMT-Genexpression auf mRNA-und Proteinebene unter Nachahmung der Standardtherapie experimentell auslösen kann. Mit den verwendeten Versuchsansätzen konnte ich in der Zellkultur keine Veränderungen feststellen. Lediglich auf mRNA-Ebene konnte nach 5 Tagen fraktionierter Bestrahlung bei der methylierten Zelllinie U87 eine leichte Steigerung der MGMT-mRNA-Expression verzeichnet werden. Diese Expressionssteigerung stand allerdings nicht im Zusammenhang mit einer Änderung des MGMT-Methylierungsstatus und spiegelte sich auch nicht auf Proteinebene wider.
Dieses Ergebnis lässt weitere Forschungen in die Richtung der therapieinduzierten Änderungen am MGMT-Genpromotor sinnvoll erscheinen, um letztendlich die Therapie am Patienten effektiver und individueller zu gestalten und das mediane Überleben sowie dieLebensqualität unter der Behandlung vor allem für Patienten mit unmethyliertem MGMT-Genpromotor zu verbessern.
Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.
Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.
Inflammation following ischaemic stroke attracts high priority in current research, particularly using human-like models and long-term observation periods considering translational aspects. The present study aimed on the spatio-temporal course of macrophage-like cell accumulation after experimental thromboembolic stroke and addressed microglial and astroglial reactions in the ischaemic border zone. Further, effects of tissue plasminogen activator (tPA) as currently best treatment for stroke and the potentially neuroprotective co-administration of hyperbaric oxygen (HBO) were investigated. Rats underwent middle cerebral artery occlusion and were assigned to control, tPA or tPA+HBO. Twenty-four hours, 7, 14 and 28 days were determined as observation time points. The accumulation of macrophage-like cells was semiquantitatively assessed by CD68 staining in the ischaemic area and ischaemic border zone, and linked to the clinical course. CD11b, ionized calcium binding adaptor molecule 1 (Iba), glial fibrillary acidic protein (GFAP) and Neuronal Nuclei (NeuN) were applied to reveal delayed glial and neuronal alterations. In all groups, the accumulation of macrophage-like cells increased distinctly from 24 hours to 7 days post ischaemia. tPA+HBO tended to decrease macrophage-like cell accumulation at day 14 and 28. Overall, a trend towards an association of increased accumulation and pronounced reduction of the neurological deficit was found. Concerning delayed inflammatory reactions, an activation of microglia and astrocytes with co-occurring neuronal loss was observed on day 28. Thereby, astrogliosis was found circularly in contrast to microglial activation directly in the ischaemic area. This study supports previous data on long-lasting inflammatory processes following experimental stroke, and additionally provides region-specific details on glial reactions. The tendency towards a decreasing macrophage-like cell accumulation after tPA+HBO needs to be discussed critically since neuroprotective properties were recently ascribed to long-term inflammatory processes.
The intercellular adhesion of circulating leukocytes to vascular endothellum ls a prerequisite for leukocyte emigration from the blood to extravascular tlssues. This process is facllltated by adhesion molecules on the surfaces of both the vascular endothelial cells and the leukocytes. The experiments presented here demonstrate for the first time that the leukocyte adhesion receptor, intercellular adhesion molecule-1, is constitutively expressed on cultured cerebromicrovascular endothelial cell lines derived from both spontaneously hypertensive (SHR) rats and normotensive WistarKyoto (WKY) rats. Both cultures contained simliar numbers of cells constitutively expressing this adhesion molecule (31.4% and 29.6%, respectlvely). Adhesion molecule expression was up-regulated by interleukin-1 ß, tumor necrosis factor-a, interferon-y and lipopolysaccharide in a dose- and time-dependent manner. Both cultures exhibited similar maximum levels of adhesion molecule up-regulation to optimal concentrations of all three cytokines. However, SHR endothelial cells were moresensitive to all three cytokines; significantly higher levels of intercellular adhesion molecule-1 expresslon were seen on SHR as opposed to WKY endothelial cells cultured with sub-optimal cytokine concentrations. It was also observed that lipopolysaccharide up-regulated intercellular adhesion molecule-1 expression on SHR endothelial cells to a greater extent than on WKY endothelial cells.
The findings that intercellular adhesion molecule-1 can be up-regulated to a greater degree on SHR endothelial cells may have important implications for in vivo perivascular leukocyte accumulation under hypertensive conditions. These observations indicate a possible mechanism by which hypertension may predispose to the development of disorders such as atherosclerosis and stroke.
Lipopolysaccharidc (LPS)-induced (i.v. or i.c.v., 1.8 mg/kg) release of von Willebrand factor (vWF) ·was examined in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. SHR rats releascd significantly (P < 0.05) more vWF than WKY rats in response to LPS. LPS also inhibited factor VIII procoagulant activity (FVIII: c) which may indicate an increase in thrombin activity. Cultured cerebrovascular endothelial cells (EC) derived from both SHR and WKY rats, as weil as human umbilical vein EC (HUVEC) cultures constitutively released vWF. Treatment with agonists including LPS, thrombin and tumor necrosis factor-a (TNFa) did not affect the in vitro secretion of vWF by cerebrovascular EC cultures but significantly upregulated vWF release by HUVEC cultur~s. Preincubation of cerebrovascular EC cultures with interleukin-1 OL-l) ± TNFa or co-culturing in the presence of LPS-activated syngeneic monocytes had no effect on vWF secretion. The findings demoostrate that conditions of hypertension may affect endothelial cells and make them more responsive to agonist Stimulation and thereby increase secretion of vWF, an important factqr in hemostasis as weil as thrombosis. The capacity of LPS to significantly affect the in vivo secretion of vWF in SHR and WKY rats but not cultured cerebrovascular EC indicates that observed elevations in plasma vWF were not derived from cerebrovascular EC. lt is suggested that hypertension may function as a risk factor for thrombotic stroke by influencing factors involved in coagulation processes, such as vWF and factor VIII : c.