Radiation-Induced Brain Injury : Age Dependency of Neurocognitive Dysfunction Following Radiotherapy

Cranial radiotherapy is a known risk factor for neurocognitive impairment in cancer survivors. Although radiation-induced cognitive dysfunction is observed in patients of all ages, children seem to be more vulnerable than adults to suffering age-related deficits in neurocognitive skills. So far, the underlying mechanisms by which IR negatively influences brain functions as well as the reasons for the profound age dependency are still insufficiently known. We performed a comprehensive Pubmed-based literature search to identify original research articles that reported on age dependency of neurocognitive dysfunction following cranial IR exposure. Numerous clinical trials in childhood cancer survivors indicate that the severity of radiation-induced cognitive dysfunction is clearly dependent on age at IR exposure. These clinical findings were related to the current state of experimental research providing important insights into the age dependency of radiationinduced brain injury and the development of neurocognitive impairment. Research in pre-clinical rodent models demonstrates age-dependent effects of IR exposure on hippocampal neurogenesis, radiation-induced neurovascular damage and neuroinflammation

Measuring out-of-field dose to the hippocampus in common radiotherapy indications

Background The high susceptibility of the hippocampus region to radiation injury is likely the causal factor of neurocognitive dysfunctions after exposure to ionizing radiation. Repetitive exposures with even low doses have been shown to impact adult neurogenesis and induce neuroinfammation. We address the question whether the out-offeld doses during radiotherapy of common tumour entities may pose a risk for the neuronal stem cell compartment in the hippocampus. Methods The dose to the hippocampus was determined for a single fraction according to diferent treatment plans for the selected tumor entities: Point dose measurements were performed in an anthropomorphic Alderson phantom and the out-of-feld dose to the hippocampus was measured using thermoluminescence dosimeters. Results For carcinomas in the head and neck region the dose exposure to the hippocampal region for a single fraction ranged from to 37.4 to 154.8 mGy. The hippocampal dose was clearly diferent for naso-, oro- and hypopharynx, with maximal values for nasopharynx carcinoma. In contrast, hippocampal dose levels for breast and prostate cancer ranged between 2.7 and 4.1 mGy, and therefore signifcantly exceeded the background irradiation level. Conclusion The mean dose to hippocampus for treatment of carcinomas in the head and neck region is high enough to reduce neurocognitive functions. In addition, care must be taken regarding the out of feld doses. The mean dose is mainly related to scattering efects, as is confrmed by the data from breast or prostate treatments, with a very diferent geometrical set-up but similar dosimetric results

Assessment of DNA damage by 53PB1 and pKu70 detection in peripheral blood lymphocytes by immunofluorescence and high-resolution transmission electron microscopy

Purpose 53BP1 foci detection in peripheral blood lymphocytes (PBLs) by immunofluorescence microscopy (IFM) is a sensitive and quantifiable DNA double-strand break (DSB) marker. In addition, high-resolution transmission electron microscopy (TEM) with immunogold labeling of 53BP1 and DSB-bound phosphorylated Ku70 (pKu70) can be used to determine the progression of the DNA repair process. To establish this TEM method in the PBLs of patients with cancer, we analyzed and characterized whether different modes of irradiation influence the formation of DSBs, and whether accompanying chemotherapy influences DSB formation. Methods We obtained 86 blood samples before and 0.1, 0.5, and 24 h after irradiation from patients (n = 9) with head and neck or rectal cancers receiving radiotherapy (RT; n = 4) or radiochemotherapy (RCT; n = 5). 53BP1 foci were quantified by IFM. In addition, TEM was used to quantify gold-labelled pKu70 dimers and 53BP1 clusters within euchromatin and heterochromatin of PBLs. Results IFM analyses showed that during radiation therapy, persistent 53BP1 foci in PBLs accumulated with increasing numbers of administered RT fractions. This 53BP1 foci accumulation was not influenced by the irradiation technique applied (3D conformal radiotherapy versus intensity-modulated radiotherapy), dose intensity per fraction, number of irradiation fields, or isodose volume. However, more 53BP1 foci were detected in PBLs of patients treated with accompanying chemotherapy. TEM analyses showed that DSBs, indicated by pKu70, were present for longer periods in PBLs of RCT patients than in PBLs of RT only patients. Moreover, not every residual 53BP1 focus was equivalent to a remaining DSB, since pKu70 was not present at every damage site. Persistent 53BP1 clusters, visualized by TEM, without colocalizing pKu70 likely indicate chromatin alterations after repair completion or, possibly, defective repair. Conclusion IFM 53BP1 foci analyses alone are not adequate to determine individual repair capacity after irradiation of PBLs, as a DSB may be indicated by a 53BP1 focus but not every 53BP1 focus represents a DSB

Region-Specific Effects of Fractionated Low-Dose Versus Single-Dose Radiation on Hippocampal Neurogenesis and Neuroinflammation

Background: Despite technical advances in hippocampus-sparing radiotherapy, radiationinduced injury to neural stem cell compartments may affect neurocognitive functions. In pre-clinical mouse models with fractionated low-dose radiation (FLDR) and single-dose radiation (SDR), the accurate response to radiation-induced injury was analyzed in different hippocampal subregions. Methods: Adult and juvenile C57BL/6NCrl mice were exposed to FLDR (20 × 0.1 Gy, daily exposure from Monday to Friday for 4 weeks) or SDR (1 × 2 Gy). In addition, 72 h after the last exposure, neuroglia (astrocytes and microglia) and neuroprogenitor cells were characterized and quantified in the hippocampal cornu ammonis (CA) and dentate gyrus (DG) by immunofluorescence studies. Results: After analyzing different hippocampal subregions, it was observed that radiation responses varied between non-neurogenic CA, with no detectable inflammatory alterations, and neurogenic DG, characterized by impaired neurogenesis and subsequent neuroinflammation. Age-dependent differences in radiosensitivity appeared to depend on the varying proliferative potential of neural stem cell niches. Using the same overall dose for FLDR and SDR (2 Gy), both the cumulative dose over time and also the single dose fraction have decisive impacts on hippocampal damage. Conclusion: Region-specific effects of radiation-induced hippocampal injury relies primarily on cell deaths of proliferating neuroprogenitors. Dose per fraction defines the extent of neuronal injury, and subsequently activated microglia and reactive astrocytes modulate dynamic processes of neuroinflammation. Thus, limiting both cumulative doses and dose fractions to hippocampal DG is an important issue of clinical radiotherapy to preserve neurocognitive functions

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment

Pneumotonometrische Vermessung des biomechanischen Profils in Augen mit Keratokonus und nach refraktiver Chirurgie

Hintergrund: Mithilfe der pneumotonometrischen Vermessung der Hornhaut durch den Ocular Response Analyzer (ORA) können biomechanische Eigenschaften der Hornhaut erfasst werden. Nach einem kurzen Luftstoß in das Auge misst ein Laser die Lichtreflexion bei Applanation der Hornhaut und erstellt anhand dieser Daten eine WaveForm-Kurve. Hierdurch können Erkrankungen wie der Keratokonus, eine nicht entzündliche Veränderung der Hornhaut, die im Verlauf zu einer kegelförmigen Vorwölbung mit Ausdünnung der Hornhaut führt, diagnostiziert werden. In dieser Arbeit wird die WaveForm-Kurve der Hornhaut bei Gesunden, bei Patienten mit Keratokonus und bei Patienten mit Zustand nach LASIK-Behandlung untersucht. Es wird analysiert, ob die Patienten anhand der WaveForm-Parameter in eine Kontrollgruppe und die Gruppen „Keratokonus Grad 1“ (KK1), „Subklinischer Keratokonus“ (SKK) und „Zustand nach LASIK-Behandlung“ (LASIK) eingeteilt werden können. Dies würde erlauben Augen mit Keratokonus möglichst früh erkennen zu können. Durch die Veränderung der Hornhaut beim Keratokonus kann es im Verlauf zu einer Destabilisierung des Tränenfilms kommen. Ein weiteres Ziel der Arbeit ist daher zu untersuchen, ob das Messergebnis des ORA durch die Gabe von befeuchtenden Augentropfen vor der Messung beeinflusst wird. Dies könnte sowohl die allgemeine Qualität der Messung als auch die korrekte Erkennung des Keratokonus beeinflussen. Patienten und Methoden: In der ersten Studie wurden die Untersuchungsergebnisse je eines Auges von 180 Patienten aus dem Homburger Keratokonus Zentrum retrospektiv ausgewertet. Die Patienten wurden zunächst anhand des tomographischen Befundes der Pentacam-Untersuchung, die als Goldstandard-Untersuchung der Vorderabschnitts-Tomographie des Auges genutzt wird, sowie anhand der Angaben aus ihrer Patientenakte in die Gruppen „Kontrollgruppe“ und „KK1“ eingeteilt. Die Gruppe „SKK“ wurde von Patienten gebildet, die an einem Auge an Keratokonus Grad 1 erkrankt sind und an deren Partnerauge diagnostisch noch kein Keratokonus festgestellt werden konnte. Da der Keratokonus im Verlauf meist beide Augen betrifft, ist bei diesen Partneraugen eine subklinische Form des Keratokonus anzunehmen. Die Patienten der Gruppe „LASIK“ wurden durch Daten des Refraktiven Zentrums der Klinik für Augenheilkunde ermittelt. Nach der Gruppeneinteilung durch die Pentacam und der Daten aus den Patientenakten wurde untersucht, welcher Grad der Übereinstimmung in der Einteilung der Patienten anhand der Ergebnisse des ORA erreicht werden kann. Bei den Gruppenvergleichen wurden mittels Mann-Whitney-U Test die WaveForm-Parameter ausgewählt, die sich signifikant zwischen den Gruppen unterscheiden. Diese signifikanten WaveForm-Parameter wurden jeweils in eine logistische Regressionsanalyse eingeschlossen, anhand derer verschiedene Modelle und ROC-Kurven entwickelt wurden, um die untersuchten Augen in die verschiedenen Gruppen einzuteilen. In der zweiten Studie wurden prospektiv bei 43 Patienten, davon 20 Patienten mit gesichertem Keratokonus und 23 gesunde Probanden, Messungen am ORA vor und nach Benutzung von befeuchtenden Augentropfen durchgeführt. Die Daten vor und nach Benutzung der Augentropfen wurden mittels Wilcoxon-Vorzeichen-Rang-Test auf signifikante Veränderungen überprüft. Ergebnisse: Zur Differenzierung der Patienten in die einzelnen Gruppen wurden bei jedem Gruppenvergleich die signifikant unterschiedlichen WaveForm-Parameter ausgewählt und in eine logistische Regression eingeschlossen. Hierdurch konnte jeweils ein Modell erstellt werden, welches die Patienten mit einer hohen Übereinstimmung in die nach der Pentacam eingeteilten Gruppen einteilte. Es zeigte sich, dass von den signifikanten WaveForm-Parametern in jedes Modell unterschiedliche Parameter eingeschlossen wurden. Die Parameter dslope1 und alphf wurden jedoch in 4 von 6 Modellen verwendet. Gesunde Augen und Augen mit Hornhautauffälligkeiten wurden in 76,7% der Fälle in die gleiche Gruppe wie durch die Pentacam eingeteilt. Die Area under the Curve (AUC) betrug 0,83 in der Receiver Operating Characteristic (ROC) Kurvenanalyse. In mehreren Vergleichen zwischen den Gruppen „LASIK“, „KK1“, „SKK“ und einer zusammengeführten Gruppe aus „KK1“ und „SKK“ lag die richtige Klassifizierungswahrscheinlichkeit jeweils bei über 84% und die AUC jeden Modells bei über 0,9 in der ROC-Kurvenanalyse. Bei der Unterscheidung zwischen „KK1“ und „SKK“ konnten nur 66,7% der Augen richtig klassifiziert werden mit einer AUC von 0,721 in der ROC-Kurvenanalyse. Bei der Untersuchung, ob befeuchtende Augentropfen das Messergebnis des ORA verbessern, zeigte sich, dass sich bei Patienten mit Keratokonus die Parameter p1area (<0,022), h1 (<0,011) und alphf (<0,03) signifikant änderten. Dies bedeutet, dass der erste Peak der WaveForm-Kurve höher wurde und mehr Licht reflektiert wurde. Der Parameter alphf sank nach Anwendung der Tropfen. Das heißt, durch die Augentropfen konnte das Rauschen zwischen den Peaks reduziert werden. Eine signifikante Verbesserung des WaveForm-Scores, der die Qualität der ORA-Messung beschreibt, konnte nicht erreicht werden. Eine signifikante Veränderung des Keratokonus Match Index (KMI) gelang ebenfalls nicht. Schlussfolgerung: Diese Arbeit konnte zeigen, dass mithilfe des ORA eine gute Differenzierung zwischen gesunden Augen und Augen mit verschiedenen Hornhautauffälligkeiten gelingt. Eine Unterscheidung zwischen gesunden Augen und Augen mit Keratokonus, sowie eine Unterscheidung zwischen Augen nach LASIK-Behandlung und Keratokonus konnte mittels der erstellten Modelle suffizient erreicht werden. Eine zuverlässige Unterscheidung zwischen Keratokonus Grad 1 und Subklinischer Keratokonus gelang nicht. Durch die Benutzung von befeuchtenden Augentropfen konnte keine signifikante Verbesserung der ORA-Messergebnisse erzielt werden. Ebenso ergab sich keine Veränderung des KMI, was darauf hindeutet, dass die befeuchtenden Augentropfen lediglich einen Einfluss auf einzelne Parameter haben, das Gesamtergebnis der Messung jedoch nicht verändern. Durch die Ergebnisse dieser Arbeit lässt sich zusammenfassend feststellen, dass die WaveForm-Parameter des ORA trotz Zusammenführung von unterschiedlichen Parametern und optimierter Auswertungsmodelle nicht zuverlässig zur alleinigen frühzeitigen Keratokonusdiagnostik genutzt werden können.Purpose: With the help of the pneumotonometric measurement of the cornea by the Ocular Response Analyzer (ORA), biomechanical properties of the cornea can be obtained. After a short blast of air into the eye, a laser measures the light reflection when the cornea is applanated and uses this data to create a waveform curve. This can be used to diagnose conditions such as keratoconus, a non-inflammatory change in the cornea that progressively leads to a cone-shaped protrusion with thinning of the cornea. In this paper, the WaveForm curve of the cornea is investigated in healthy individuals, in patients with keratoconus and in patients with a condition after LASIK treatment. It is analysed whether the patients can be divided into a control group and the groups "keratoconus grade 1" (KK1), "subclinical keratoconus" (SKK) and "condition after LASIK treatment" (LASIK) on the basis of the WaveForm parameters. This would allow eyes with keratoconus to be recognised as early as possible. The corneal alterations can lead to a destabilization of the tear film over the course of the disease. Another aim of the study is therefore to investigate whether the ORA measurement result is influenced by the administration of moisturizing eye drops before the measurement. This may affect the overall quality of measurement as well as the effectiveness in the detection of keratoconus. Patients and methods: In the first study, the examination results of one eye of each of 180 patients from the Homburg Keratoconus Centre were retrospectively analysed. The patients were first divided into the groups "control group" and "KK1" on the basis of both the tomographic findings of the Pentacam examination, which is used as the gold standard examination of the anterior segment tomography of the eye, and the information from their patient file. The "SKK" group was formed by patients who had keratoconus grade 1 in one eye and whose partner eye had not yet been diagnosed with keratoconus. Since keratoconus usually affects both eyes in the course of its development, a subclinical form of keratoconus can be assumed in these partner eyes. The patients in the "LASIK" group were identified based on data from the Refractive Centre of Ophthalmology of Saarland. After the classification on the basis of Pentacam and the data from the patient records, it was investigated which degree of agreement could be achieved in the classification of the patients based on the results of the ORA. In the group comparisons, the WaveForm parameters that differed significantly between the groups were selected using the Mann-Whitney-U test. These significant WaveForm parameters were each included in a logistic regression analysis, which was used to develop different models and ROC curves to classify the eyes studied into the different groups. In the second study, 43 patients, including 20 patients with confirmed keratoconus and 23 healthy subjects, were prospectively measured for ORA before and after use of moisturizing eye drops. The data before and after use of the eye drops were checked for significant changes using the Wilcoxon signed-rank test. Results: To classify patients based on ORA into the individual groups, the significantly different WaveForm parameters were selected for each group comparison and included in a logistic regression. This made it possible to create a model that divided the patients with a high degree of agreement into the groups classified according to the Pentacam. It was shown that of the significant WaveForm parameters, different parameters were included in each model. However, the parameters dslope1 and alphf were used in 4 of 6 models. The ORA-classification of healthy eyes versus eyes with corneal abnormalities showed agreement with the Pentacam-classification in 76.7% of cases. The Area under the curve (AUC) was 0.83 in the Receiver Operating Characteristic (ROC) curve analysis. In several comparisons between the groups "LASIK", "KK1", "SKK" and a merged group of "KK1" and "SKK", the agreement was over 84% and the AUC of each model was over 0.9 in the ROC curve analysis. When distinguishing between "KK1" and "SKK", only 66.7% of the eyes could be correctly classified with an AUC of 0.721 in the ROC curve analysis. When investigating whether moisturizing eye drops improve the quality of measurement of the ORA, it was found that the parameters p1area (<0.022), h1 (<0.011) and alphf (<0.03) changed significantly in patients with keratoconus. This means that the first peak of the WaveForm curve became higher and more light was reflected. The parameter alphf decreased after application of the drops. This means that the eye drops reduced the noise between the peaks. There was no significant improvement in the WaveForm score, which describes the overall quality of the ORA measurement. There was also no significant change in the Keratoconus Match Index (KMI). Conclusion: This study showed that the ORA is able to differentiate between healthy eyes and eyes with different corneal abnormalities. The created models allowed a differentiation between healthy eyes and eyes with keratoconus, as well as a differentiation between eyes after LASIK treatment and keratoconus. A reliable classification into keratoconus grade 1 and subclinical keratoconus could not be achieved. The use of moisturizing eye drops did not significantly improve the ORA measurement results. Similarly, there was no change in the KMI, indicating that the moisturizing eye drops only influence individual parameters, but do not change the overall result of the measurement. In summary, the results of the present work show that the WaveForm parameters of the ORA cannot be used for reliable early keratoconus diagnosis on their own, even when combining different parameters and using optimized statistical models

Prognostischer Wert der zerebralen computertomografischen Perfusionsuntersuchung bei Patienten mit akutem Schlaganfall

Hintergrund: Bei einem akuten ischämischen Schlaganfall ist eine frühe Bildgebung wichtig, um therapierelevante Informationen über die Ursache, Lokalisation und Größe des Schlaganfalls zu erhalten. Es ist von Interesse, minderdurchblutetes Gewebe (Penumbra) von bereits irreversibel geschädigtem Gewebe (Infarktkern) zu unterscheiden, da die reversibel geschädigte Penumbra durch frühzeitige therapeutische Maßnahmen erhalten werden kann. Eine Nativ-CT-Untersuchung kann in der Akutsituation den Infarktkern und die Penumbra nicht ausreichend beurteilen. Bei einer computertomografischen Perfusionsuntersuchung (PCT-Untersuchung) dagegen können durch verschiedene Computertechniken und Berechnungen funktionelle Perfusionsparameter dargestellt werden, anhand derer zu einem frühen Zeitpunkt Angaben zu einem minderperfundierten oder infarzierten Gewebe gemacht werden können. Im Rahmen dieser Arbeit soll untersucht werden, inwiefern diese Mehrinformation durch die PCT-Untersuchung in der Akutsituation eines ischämischen Schlaganfalls Rückschlüsse zulässt, die von prognostischem Wert sind. Es wird analysiert, ob durch die PCT-Untersuchung die finale Infarktgröße und die Penumbra abgeschätzt werden können und ob es einen bestimmten Perfusionsparameter gibt, der in besonderem Maße mit diesen zusammenhängt. Zusätzlich werden die Ergebnisse der PCT-Untersuchung mit dem neurologischen Befund und dem Kollateralstatus der Patienten, sowie dem Zeitfenster des Symptombeginns bis zur Therapie in Bezug gesetzt. Patienten und Methoden: In diese Studie konnten 57 Patienten aus der Datenbank der Klinik für Diagnostische und Interventionelle Neuroradiologie der Universitätsklinik Homburg im Zeitraum von Dezember 2015 bis einschließlich November 2017 eingeschlossen werden. Um den prognostischen Wert der zerebralen PCT-Untersuchung zu beurteilen, wurden die Daten der Perfusionsuntersuchung mit weiteren patientenrelevanten Parametern aus der Patientenakte und dem Volumen der Infarktdemarkierung in einer CT-Verlaufskontrolle 24 Stunden später verglichen. Zur Auswertung der Daten der PCT-Untersuchung wurde als Postprocessing-Software der Perfusion Mismatch Analyzer der Forschungsgruppe ASIST Japan genutzt. Um Zusammenhänge zwischen den verschiedenen Parametern herzustellen wurde je nach Messniveau eine Rangkorrelation nach Spearman oder eine einfache Regressionsanalyse, gefolgt von einer multiplen Regressionsanalyse mit vorwärts Selektion der Variablen, durchgeführt. Ergebnisse: Mittels Regressionsanalyse konnte gezeigt werden, dass es einen signifikanten Zusammenhang zwischen den Volumen der CBF- und CBV-Perfusionsparameterkarte der PCT-Perfusionsuntersuchung und dem finalen Infarktvolumen gibt. Je größer das minderperfundierte bzw. infarzierte Areal der PCT-Perfusionsparameterkarten desto größer stellt sich das Infarktareal 24 Stunden später in der CT-Verlaufskontrolle dar. Eine multiple Regressionsanalyse im Anschluss ergab, dass die CBF-Perfusionsparameterkarte den stärksten Zusammenhang zum finalen Infarktvolumen zeigte. Jedoch war der Unterschied in der Effektstärke nach Cohen bei der einfachen Regressionsanalyse zwischen der CBF-Perfusionsparameterkarte mit 0,51 und der CBV-Perfusionsparameterkarte mit 0,5 gering, sodass in unserer Arbeit sowohl das Volumen der CBF- als auch der CBV-Perfusionsparameterkarte einen starken Zusammenhang zum finalen Infarktvolumen zeigt und somit eine Vorhersage möglich macht. Auch die Perfusionsparameter, vor allem der CBF- und CBV-Perfusionsparameterkarten, zeigen einen statistisch signifikanten Zusammenhang zum finalen Infarktvolumen. Einen besonders starken Zusammenhang zeigen der Parameter CBV sSVD und die Ratio CBV dSVD(). Ebenfalls konnte gezeigt werden, dass anhand der Daten der PCT-Untersuchung mittels Volumen-Mismatch-Berechnung eine Penumbra berechnet werden konnte, mit deren Hilfe ein potentiell reperfundierbares Gewebe abgeschätzt werden kann. In unserer Arbeit zeigte ein „CBF-CBV-Volumen-Mismatch“ einen deutlich stärkeren statistischen Zusammenhang mit dem reperfundierten Gewebe und dem finalen Infarktvolumen als ein „MTT-CBV-Volumen-Mismatch“. Bei der Korrelation der Ergebnisse der CT-Untersuchungen mit dem neurologischen Befund zeigte sich, dass je schlechter der neurologische Status war, desto größer präsentierten sich die auffälligen Areale in der CBF-, CBV-Perfusionsparameterkarte und das finalen Infarktvolumen in der CT-Verlaufskontrolle. Eine Aussagekraft über die Prognosekraft des PCT in Abhängigkeit einer Therapie konnte nicht zufriedenstellend geklärt werden. Bei der Korrelation der Ergebnisse der CT-Untersuchungen mit dem Kollateralstatus zeigte sich, dass je größer sich das minderperfundierten bzw. infarzierten Areale in der Untersuchung zeigte, desto schlechter war der Kollateralstatus des Patienten. Schlussfolgerung: Zusammenfassend zeigt diese Arbeit, dass die zerebrale computertomografische Perfusionsuntersuchung bei Patienten mit akutem Schlaganfall einen hohen prognostischen Aussagewert hinsichtlich des finalen Infarktvolumen und der Penumbra hat. Es zeigten sich positive Zusammenhänge sowohl zwischen der PCT-Untersuchung und dem neurologischen Status als auch zwischen der PCT-Untersuchung und dem Kollateralstatus der Patienten. Im Vergleich zu einer Nativ-CT-Untersuchung hat man durch die PCT-Untersuchung in der Frühphase eines Schlaganfalls einen deutlich größeren Informationszugewinn. Für eine mögliche Therapieentscheidung kann dies von großer Bedeutung sein.Background: In acute ischaemic stroke, early imaging is important to obtain therapy-relevant information about the cause, localisation and size of the stroke. It is of interest to distinguish low perfused tissue (penumbra) from already irreversibly damaged tissue (infarct core), because the reversibly damaged penumbra can be preserved by early therapeutic measures. A native CT scan cannot adequately assess the infarct core and penumbra in the acute situation. A computed tomographic perfusion scan (CT perfusion), on the other hand, can use various computer techniques and calculations to display functional perfusion parameters, which can be used to provide information about underperfused or infarcted tissue at an early stage. The aim of this study is to investigate the extent to which this additional information provided by CT perfusion in the acute situation of an ischaemic stroke allows conclusions to be drawn that are of prognostic value. It will be analysed whether the final infarct size and the penumbra can be estimated by the CT perfusion and whether there is a specific perfusion parameter that is particularly related to these. In addition, the results of the CT perfusion will be related to the neurological findings and collateral status of the patients, as well as the time window of symptom onset to therapy. Patients and methods: This study was able to include 57 patients from the database of the Department of Diagnostic and Interventional Neuroradiology at Homburg University Hospital from December 2015 to November 2017 inclusive. In order to assess the prognostic value of the cerebral CT perfusion, the data of the perfusion examination were compared with further patient-relevant parameters from the patient file and the volume of the infarct demarcation in a CT follow-up 24 hours later. To analyse the data of the CT perfusion, the Perfusion Mismatch Analyzer of the research group ASIST Japan was used as post-processing software. To establish correlations between the different parameters, a rank correlation according to Spearman or a simple regression analysis, followed by a multiple regression analysis with forward selection of variables, was performed depending on the level of measurement. Results: Regression analysis showed that there is a significant correlation between the volumes of the CBF and CBV perfusion parameter maps of the CT perfusion and the final infarct volume. The larger the inferiorly perfused or infarcted area of the CT perfusion parameter maps, the larger the infarct area 24 hours later in the CT follow-up. A subsequent multiple regression analysis showed that the CBF perfusion parameter map had the strongest association with final infarct volume. However, the difference in effect size according to Cohen in the simple regression analysis between the CBF perfusion parameter map with 0,51 and the CBV perfusion parameter map with 0,5 was small, so that in our work both the volume of the CBF and the CBV perfusion parameter map showed a strong correlation to the final infarct volume and thus made prediction possible. The perfusion parameters, especially the CBF and CBV perfusion parameter maps, also show a statistically significant relationship to the final infarct volume. The parameter CBV sSVD and the ratio CBV dSVD() show a particularly strong correlation. It was also shown that a penumbra could be calculated based on the data of the CT perfusion using volume mismatch calculation, which can be used to estimate a potentially reperfusable tissue. In our work, a "CBF-CBV volume mismatch" showed a significantly stronger statistical correlation with reperfused tissue and final infarct volume than a "MTT-CBV volume mismatch". The correlation of the results of the CT examinations with the neurological findings showed that the worse the neurological status, the larger the abnormal areas in the CBF and CBV perfusion parameter map and the final infarct volume in the CT follow-up. The prognostic value of CT perfusion in relation to therapy could not be satisfactorily clarified. The correlation of the results of the CT examinations with the collateral status showed that the larger the inferiorly perfused or infarcted areas in the examination, the worse the collateral status of the patient. Conclusion: In summary, this work shows that cerebral computed tomographic perfusion examination in patients with acute stroke has a high prognostic value with regard to final infarct volume and penumbra. Positive correlations were shown between CT perfusion and neurological status as well as between CT perfusion and collateral status of patients. Compared to a native CT scan, the CT perfusion provides significantly more information in the early phase of a stroke. This can be of great importance for a possible therapy decision

Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin

Background: Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. Methods: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. Results: Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. Conclusions: The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns

Cultured Human Foreskin as a Model System for Evaluating Ionizing Radiation-Induced Skin Injury

Purpose: Precise molecular and cellular mechanisms of radiation-induced dermatitis are incompletely understood. Histone variant H2A.J is associated with cellular senescence and modulates senescence-associated secretory phenotype (SASP) after DNA-damaging insults, such as ionizing radiation (IR). Using ex vivo irradiated cultured foreskin, H2A.J was analyzed as a biomarker of radiation-induced senescence, potentially initiating the inflammatory cascade of radiation-induced skin injury. Methods: Human foreskin explants were collected from young donors, irradiated ex vivo with 10 Gy, and cultured in air-liquid interphase for up to 72 h. At different time-points after ex vivo IR exposure, the foreskin epidermis was analyzed for proliferation and senescence markers by immunofluorescence and immunohistochemical staining of sectioned tissue. Secretion of cytokines was measured in supernatants by ELISA. Using our mouse model with fractionated in vivo irradiation, H2A.J expression was analyzed in epidermal stem/progenitor cell populations localized in different regions of murine hair follicles (HF). Results: Non-vascularized foreskin explants preserved their tissue homeostasis up to 72 h (even after IR exposure), but already nonirradiated foreskin epithelium expressed high levels of H2A.J in all epidermal layers and secreted high amounts of cytokines. Unexpectedly, no further increase in H2A.J expression and no obvious upregulation of cytokine secretion was observed in the foreskin epidermis after ex vivo IR. Undifferentiated keratinocytes in murine HF regions, by contrast, revealed low H2A.J expression in non-irradiated skin and significant radiation-induced H2A.J upregulations at different time-points after IR exposure. Based on its staining characteristics, we presume that H2A.J may have previously underestimated the importance of the epigenetic regulation of keratinocyte maturation. Conclusions: Cultured foreskin characterized by highly keratinized epithelium and specific immunological features is not an appropriate model for studying H2A.J-associated tissue reactions during radiation-induced dermatitis