Optische Analyse mitochondrialer Funktion und Heterogenität in kultivierten hippocampalen Astrozyten

Diese Dissertation beschreibt die Untersuchung mitochondrialer Heterogenität in kultivierten Astrozyten des neonatalen Rattenhippocampus. Im Rahmen der Neurophysiologie und Pathologie erfuhren sowohl die Rolle von Astrozyten als auch jene zerebraler Mitochondrien während der vergangenen zwei Jahrzehnte eine umfassende Neubewertung. So wurde die Wichtigkeit von Astrozyten als Garanten neuronalen Überlebens und als Modulatoren neuronaler Kommunikation sowie des neuronalen Metabolismuses entdeckt. Die Pathogenese einer Reihe neurodegenerativer Erkrankungen, z.B. der Parkinsonerkrankung oder verschiedener demenzieller Syndrome, konnte auf den Defekt einer funktionierenden Atmungskette zurückgeführt werden. Dies rückte den Einfluss von Mitochondrien auf den zellulären Energieaushalt, die Kalziumhomöostase und Apoptose in diversen Zelltypen in den Forschungsfokus. Selbst intrazellulär scheinen Mitochondrien keine uniform agierenden Organellen darzustellen. Diese Arbeit kombiniert erstmals für diesen Zelltyp hochauflösende konventionelle und Zweiphotonenemissionslasermikroskopie mit der Möglichkeit ratiometrischer Analyse der JC-1 Fluoreszenz, um quantitativ wie qualitativ Aussagen über zelluläre Lage, Form, Transmembranpotenzial (m) sowie Ca2+ Homöostase unter physiologischen wie pathophysiologischen Bedingungen treffen zu können. Es zeigte sich, dass Mitochondrien strategisch um das endoplasmatische Retikulum (ER) gruppiert sind. Periphere Mitochondrien wurden dabei mithilfe des Bildsplitters Dual-View für stärker polarisiert befunden. Mitochondrien unterschieden sich ebenso bezüglich ihrer Reaktion auf metabolische Herausforderungen, die durch die Applikation von Zyanid, FCCP und anderen Chemikalien simuliert wurden. Regelmäßige m Schwankungen erschienen dabei zunehmend eine natürliche Charakteristik von Mitochondrien mit funktionierender Atmungskette und intrazellulärem Ca2+ Haushalt zu sein, wie durch Experimente mit Dantrolen und 2-APB als Blockern der Ryanodin- und IP3-Rezeptoren des ER sowie durch entsprechende Versuche mit dem Ca2+ Indikator Fluo-3, welche eine Unterdrückung sonst bestehender Ca2+ Wellen ergaben, gezeigt werden konnte. Zudem wurden hier erstmalig raumzeitliche Frequenzmuster intrazellulärer Synchronisation und Kinetik dieser m Schwankungen in Echtzeit analysiert, indem der potenziometrische Fluoreszenzfarbstoff JC-1, welcher zwei Emissionswellenlängen hat, verwendet wurde. Eine Korrelationsanalyse des m mit Verteilung und Form von Mitochondrien vervollständigte diese Dissertation.Zusammenfassend verdeutlichen diese Ergebnisse die interaktive Rolle von Mitochondrien im astrozytären Metabolismus und zeitgleich ihre autonome wie auch variable Aktivität selbst innerhalb einer Zelle

Effect of Contrast Agent Dose Reduction on Vascular Enhancement and Image Quality in Thoracoabdominal Dynamic 3-Dimensional Magnetic Resonance Angiography: A Systematic Intraindividual Analysis in Pigs

OBJECTIVE: High spatial and temporal resolution contrast-enhanced magnetic resonance angiography (MRA) with gadolinium-based contrast agents (GBCAs) at standard dose offers both detailed anatomic information on both arterial and venous vessels and hemodynamic characteristics. Several preclinical and clinical dynamic 3-dimensional (3D) MRA studies that focused on arterial vessels only proposed that high image quality may also be achieved with significantly reduced GBCA doses, calling into question the need to use standard doses. A systematic analysis of GBCA doses and resulting image quality for both arteries and veins has not yet been performed. The purpose of this study was therefore to systematically analyze dose-dependent vascular enhancements in dynamic 3D-MRA of the thoracoabdominal vasculature at 1.5 T in an animal model to determine the optimal contrast agent protocol for optimized vascular assessment. MATERIALS AND METHODS: The vascular enhancement in thoracoabdominal dynamic 3D-MRA (time-resolved angiography with interleaved stochastic trajectories, TWIST at 1.5 T) was interindividually and intraindividually compared in 5 anesthetized Göttingen minipigs using gadobutrol at the standard dose (0.1 mmol/kg body weight, ie, 0.1 mL/kg) and at reduced doses (0.08, 0.06, 0.04, 0.02 mmol/kg) in a randomized order. All injections were performed at 2 mL/s followed by 20 mL saline. Images were quantitatively analyzed, measuring signal intensities in 5 regions that covered the passage of the GBCA through the body at different representative stages of circulation (pulmonary, arterial, and venous system). The evaluation of GBCA dose-dependent signal intensity changes in the different vascular regions was performed by linear regression analysis.The qualitative image analysis of dynamic 3D-MRA by 3 independent radiologists included the visibility of 25 arterial and venous vessel segments at different stages of GBCA passage. Possible quality losses were statistically tested by comparing image quality ratings at the reduced dose with that of the standard dose using Friedman test followed by Dunn post hoc test for multiple comparison. Significance was stated at P < 0.05. RESULTS: Quantitative analysis revealed shorter time-to-peak intervals and bolus durations in line with decreasing GBCA dose and volume in all vessels. Although the peak signal was almost independent of the administered GBCA dose at the level of the pulmonary trunk, a linear signal decrease in the abdominal aorta ( r2 = 0.96), the renal arteries ( r2 = 0.99), the inferior vena cava ( r2 = 0.99), and the portal vein ( r2 = 0.97) was observed. Cumulative analysis of arterial segments revealed significantly lower image quality at doses below 40% of the standard dose, whereas in venous segments, significantly lower image quality was observed at doses below 60% of the standard dose. CONCLUSIONS: In dynamic 3D-MRA at 1.5 T, dose reduction leads to a signal loss that is most pronounced in the venous system and results in significantly lower image quality according to the dose and vessels of interest. Careful dose reduction is thus required according to the specific diagnostic needs. For dynamic 3D-MRA of the arterial and venous system, GBCA doses of at least 60% of the standard dose up to the full dose are preferable, whereas 40% of the standard dose seems feasible if only the arterial system is to be imaged

A pilot study of magnetic resonance fingerprinting in Parkinson's disease

Parkinson's disease (PD) affects more than six million people, but reliable MRI biomarkers with which to diagnose patients have not been established. Magnetic resonance fingerprinting (MRF) is a recent quantitative technique that can provide relaxometric maps from a single sequence. The purpose of this study is to assess the potential of MRF to identify PD in patients and their disease severity, as well as to evaluate comfort during MRF. Twenty-five PD patients and 25 matching controls underwent 3 T MRI, including an axial 2D spoiled gradient echo MRF sequence. T1 and T2 maps were generated by voxel-wise matching the measured MRF signal to a precomputed dictionary. All participants also received standard inversion recovery T1 and multi-echo T2 mapping. An ROI-based analysis of relaxation times was performed. Differences between patients and controls as well as techniques were determined by logistic regression, Spearman correlation and t-test. Patients were asked to estimate the subjective comfort of the MRF sequence. Both MRF-based T1 and T2 mapping discriminated patients from controls: T1 relaxation times differed most in cortical grey matter (PD 1337 ± 38 vs. control 1386 ± 37 ms; mean ± SD; P =.0001) and, in combination with normal-appearing white matter, enabled correct discrimination in 85.7% of cases (sensitivity 83.3%; specificity 88.0%; receiver-operating characteristic [ROC]) area under the curve [AUC] 0.87), while for T2 mapping the left putamen was the strongest classifier (40.54 ± 6.28 vs. 34.17 ± 4.96 ms; P =.0001), enabling differentiation of groups in 84.0% of all cases (sensitivity 80.0%; specificity 88.0%; ROC AUC 0.87). Relaxation time differences were not associated with disease severity. Standard mapping techniques generated significantly different relaxation time values and identified other structures as different between groups other than MRF. Twenty-three out of 25 PD patients preferred the MRF examination instead of a standard MRI. MRF-based mapping can identify PD patients with good comfort but needs further assessment regarding disease severity identification and its potential for comparability with standard mapping technique results