Biocompatibility studies for novel therapeutic strategies at the tympanic membrane level

Die vorliegende Arbeit beschäftigte sich mit der Anwendbarkeit und dem Nutzen neuartiger Strategien zur Behandlung zweier Ohrpathologien: zum einen der optoakustischen Stimulation des Hörsystems, die zur Behandlung von Hörverlusten eingesetzt werden soll. Zum anderen wurde die Applikation von selbstklebenden Silikonpflastern zum unkomplizierten und direkten Verschluss von Trommelfellperforationen untersucht. Zentraler Applikationsort beider Methoden war dabei das Trommelfell. Das Trommelfell ist ein essenzieller Bestandteil des Hörsystems und spielt eine wichtige Rolle für das Hörvermögen, da es die Schallwellen, die gesammelt durch den äußeren Gehörgang auf die dünne, elastische Membran treffen, aufnimmt und durch diese zum Schwingen gebracht wird, dem ersten Schritt im Hörvorgang. Andererseits fungiert es als Grenzfläche zwischen äußerem Ohr und Mittelohr auch als physiologische Barriere und schützt das Mittelohr vor dem Eindringen von Keimen. Ist das Hörvermögen eines Menschen eingeschränkt, z.B. durch altersbedingte Schwerhörigkeit, kommen Hörsysteme zum Einsatz. Diese dienen der symptomatischen Behandlung des Hörverlustes und sollen somit den Patienten wieder die Teilnahme am Leben ermöglichen. Nachteile der konventionellen Therapie sind allerdings eine geringe frequenzspezifische Auflösung des Eingangssignals mit folgender Differenzierungsstörung trotz ausreichender Verstärkung, geminderter Tragekomfort durch einen Verschluss- und damit verbundenen wiederkehrenden Entzündungen des äußeren Gehörganges usw. Die optoakustische Stimulation bietet eine alternative Stimulationsmethode des Hörsystems mit einer zu erwarteten erhöhten Frequenzauflösung und gleichzeitiger Verbesserung des Tragekomforts, da der Verschluss des Gehörganges bei Nutzen dieser Methode entfallen würde. Dabei werden kurze Laserpulse eingesetzt, um Vibrationen im bestrahlten Gewebe, z.B. dem Trommelfell oder weiteren vibrationsfähigen Komponenten des peripheren Hörsystems, zu erzeugen. Als grundsätzlicher Baustein dieser neuen Stimulationsmethode war ein Ziel dieser Arbeit die Analyse ihrer Biokompatibilität, welche im Tiermodell und in Zellkulturen untersucht wurde. Hierbei konnte am Trommelfell der Maus gezeigt werden, dass für einen gepulsten Laser, mit 532 nm Wellenlänge, ab einer Schwelle von 89 mW mittlerer Laserleistung, Zonen mit apoptotischen und nekrotischen Zellen entstanden, die mit steigender Laserleistung in ihrer Größe zunahmen. Es konnte kein negativer Einfluss auf das Hörvermögen der Tiere durch die Stimulation nachgewiesen werden. In Zellkulturversuchen mit drei humanen Zelllinien konnte gezeigt werden, dass die erhaltenen Schädigungsschwellen je nach Zelllinie deutlich höher waren. Außerdem konnte hier eine reduzierte Zellviabilität nachgewiesen werden, die wiederum abhängig von der Strahlungsintensität war und sich in der Ausprägung zwischen den Zelllinien unterschied. Überdies wurden durch die Bestrahlung regulatorische Prozesse in den Zellen induziert, die sich über Genexpressionsanalysen nachweisen ließen. Das Hörvermögen kann, neben der Altersschwerhörigkeit, auch durch Trommelfellperforationen maßgeblich beeinträchtigt sein. Durch die Perforationen wird die Schallweiterleitung erheblich geschwächt und es entsteht eine Schallleitungsschwerhörigkeit. Des Weiteren können durch die Perforation Keime in das Mittelohr eindringen und dort akute Entzündungen auslösen, die weitreichende Folgen, etwa die Entstehung von chronischen Prozessen und die Bildung von Cholesteatomen, mit sich bringen können. Um eine direkt wirksame Behandlung von Trommelfellperforationen zu ermöglichen, hat die AG um Prof. Dr. Arzt vom Leibniz Institut für Neue Materialien (INM) in Kollaboration mit uns selbsthaftende Silikon-Pflaster zur Abdeckung der Perforationen entwickelt. Diese wurden in zwei verschiedenen Ausführungen, unstrukturiert und mikrostrukturiert, produziert und dienen, neben dem Verschluss, später auch der verbesserten Heilung der verletzten Trommelfelle. Beide Pflaster zeigten sich ex vivo als gut haftend auf dem Trommelfell der Maus. In vivo, im lebenden, narkotisierten Tier, fanden wir signifikant erhöhte Hörschwellen und signifikant reduzierte Distorsionsprodukte otoakustischer Emissionen (DPOAE) nach Induzieren einer Perforation. Das Abdecken der Perforation mit den neuartigen Pflastern erhöhte die DPOAE-Signale signifikant, die Hörschwellen in der Click-ABR (engl. auditory brainstem responses)-Messung wurden hingegen nicht signifikant verbessert. In weiteren Versuchen konnten wir zeigen, dass die Applikation von dickeren Pflastern keine signifikante Verbesserung der DPOAE-Signale mit sich brachte. Das Aufbringen der Pflaster auf das gesunde Trommelfell führte bei der dicken und der dünnen Ausführung zu einer signifikanten Reduzierung der DPOAE-Signale. Zusammenfassend erbrachten beide therapeutische Ansätze vielversprechende Ergebnisse, die zusammen mit weiteren translationalen Untersuchungen, auf einen zukünftigen Einsatz im klinischen Alltag hoffen lassen.The presented thesis investigated the applicability and benefit of two innovative therapeutic strategies for the treatment of ear pathologies: on one side, the optoacoustic stimulation as an alternative stimulation method for the hearing system affected by hearing loss. On the other hand, the application of self-adhesive silicone patches for the simple and direct coverage of tympanic membrane perforations. The central site of application of both methods thereby was the tympanic membrane. The tympanic membrane, or eardrum, plays an essential role for the hearing function, as it collects incoming sound waves, traveling from the outer ear canal and is thereby set into vibrations, the first step of the hearing process. Furthermore, the eardrum forms the interface between the outer ear and the middle ear and functions as a physiological barrier to protect the middle ear from the entering of pathogenic germs. If the hearing function of people is impaired e.g., by presbycusis, age-related hearing loss, hearing aids are needed. Conventionally, patients get supplied with hearing aids that are meant to compensate the hearing loss symptomatically aiming to enable the patients to participate in everyday life. The main drawbacks of conventional hearing aids are the insufficient frequency resolution of the input signal leading to insufficient auditive discrimination of sounds despite sufficient amplification, discomfort wearing them in the occluded ear canal and because of occlusion, recurrent infections. The optoacoustic stimulation offers an alternative stimulation method with an estimated high frequency resolution and a non-occlusive way of application. Thereby, short laser pulses are used to induce vibrations in irradiated tissues e.g., the tympanic membrane or further vibratory components of the peripheral hearing system. As one basic element of this innovative stimulation method, one major goal of the presented work was the investigation of its biocompatibility that was performed in an animal model and in cell culture experiments. The optoacoustic stimulation at the murine tympanic membrane, with our currently used laser parameters, was demonstrated to be safe up to 89 mW. Areas with apoptotic and necrotic cells could be detected starting at 89 mW and increased in their dimensions with rising laser power. We could not identify any negative effect of the stimulation on the hearing function of these animals. In cell culture assays of three human cell lines, our group noticed significantly higher damage thresholds, depending on the irradiated and analyzed cell line. Furthermore, the cell viability was reduced through irradiation with an intensity that was depending on the irradiation power and the respective cell line. Also, regulatory processes were induced by irradiation in the cells, which were investigated by gene expression analysis. Besides age-related hearing loss, eardrum defects play a crucial role in the loss of hearing function as well. Tympanic membrane perforations significantly affect sound transmission, and a conductive hearing loss occurs. Furthermore, pathogenic germs may enter the middle ear through the perforated membrane, predictably leading to acute inflammations that may cause multiple complications up to chronic inflammatory processes and cholesteatoma formation. To allow a very direct and quick therapeutic method for tympanic membrane perforations, in collaboration with the group led by Prof. Dr. Eduard Arzt from the Leibniz Institute for New Materials (INM), self-adhesive silicone patches have been designed. Two different conformations of these patches have been analyzed and presented in this work. The unstructured and the microstructured patches aimed to cover the perforation and to support the healing process in the future. The patches demonstrated ex vivo good adhesive characteristics on the murine tympanic membrane. In vivo, acute eardrum perforations in anesthetized mice induced significantly increased hearing thresholds and significantly reduced distortion product otoacoustic emissions (DPOAE) signals. Covering the perforation with our patches, significantly increased the DPOAE signals, however, the click-ABR thresholds were not significantly affected. In further experiments, we demonstrated that applying patches with higher thicknesses could not improve the DPOAE signals anymore. Additionally, the application of thin and thick patches on the intact tympanic membrane reduced the DPOAE signals significantly. To sum up, both innovative therapeutic strategies at the eardrum level demonstrated significant positive results warranting further translational work that could introduce them into the daily medical practice

Optoacoustically induced auditory brainstem responses in the mouse model enhanced through an absorbing film

Significance: Optoacoustic stimulation offers an alternative stimulation strategy for the hearing organ. To serve as the base for a novel auditory prosthesis, the optoacoustic stimulation must be biocompatible and energy-saving. Aim: Enhancing the efficiency of optoacoustic stimulation while reducing the energy input in a suited animal model. Approach: Optoacoustically induced auditory brainstem responses (oABRs) were recorded after the pulsed laser irradiation of the tympanic membrane (TM) in mice. The results were compared with the ABRs induced through acoustic click stimulation. In addition, self-adhesive absorbing films were applied on the TM before the optoacoustic stimulation to investigate their effect on the resulting ABRs. Results: Using an absorbing film on the TM during optical stimulation led to considerably enhanced oABR wave I amplitude values compared with the stimulation of the bare TM. When using our stimulation strategy, we induced oABR waves in the 50% to 60% range of the acoustical stimulation reached with 80-dB SPL click stimuli. Conclusions: The mouse model can be used for certain developmental work for an optoacoustic auditory prosthesis. Using absorbing films on the TM during optical stimulation considerably enhances oABR wave I amplitude. Optimization of the stimulation strategy could further enhance the efficiency within biocompatibility margins

A Self-Adhesive Elastomeric Wound Scaffold for Sensitive Adhesion to Tissue

Pressure sensitive adhesives based on silicone materials are used particularly for skin adhesion, e.g., the fixation of electrocardiogram (ECG) electrodes or wound dressings. However, adhesion to sensitive tissue structures is not sufficiently addressed due to the risk of damage or rupture. We propose an approach in which a poly-(dimethylsiloxane) (PDMS)-based soft skin adhesive (SSA) acts as cellular scaffold for wound healing. Due to the intrinsically low surface free energy of silicone elastomers, functionalization strategies are needed to promote the attachment and spreading of eukaryotic cells. In the present work, the effect of physical adsorption of three different proteins on the adhesive properties of the soft skin adhesive was investigated. Fibronectin adsorption slightly affects adhesion but significantly improves the cellular interaction of L929 murine fibroblasts with the polymeric surface. Composite films were successfully attached to explanted tympanic membranes. This demonstrates the potential of protein functionalized SSA to act as an adhesive scaffold in delicate biomedical applications

Cochlea-Specific Deletion of Cav1.3 Calcium Channels Arrests Inner Hair Cell Differentiation and Unravels Pitfalls of Conditional Mouse Models

Inner hair cell (IHC) Cav1.3 Ca2+ channels are multifunctional channels mediating Ca2+ influx for exocytosis at ribbon synapses, the generation of Ca2+ action potentials in pre-hearing IHCs and gene expression. IHCs of deaf systemic Cav1.3-deficient (Cav1.3-/-) mice stay immature because they fail to up-regulate voltage- and Ca2+-activated K+ (BK) channels but persistently express small conductance Ca2+-activated K+ (SK2) channels. In pre-hearing wildtype mice, cholinergic neurons from the superior olivary complex (SOC) exert efferent inhibition onto spontaneously active immature IHCs by activating their SK2 channels. Because Cav1.3 plays an important role for survival, health and function of SOC neurons, SK2 channel persistence and lack of BK channels in systemic Cav1.3-/- IHCs may result from malfunctioning neurons of the SOC. Here we analyze cochlea-specific Cav1.3 knockout mice with green fluorescent protein (GFP) switch reporter function, Pax2::cre;Cacna1d-eGFPflex/flexand Pax2::cre;Cacna1d-eGFPflex/-. Profound hearing loss, lack of BK channels and persistence of SK2 channels in Pax2::cre;Cacna1d-eGFPflex/- mice recapitulated the phenotype of systemic Cav1.3-/- mice, indicating that in wildtype mice, regulation of SK2 and BK channel expression is independent of Cav1.3 expression in SOC neurons. In addition, we noticed dose-dependent GFP toxicity leading to death of basal coil IHCs of Pax2::cre;Cacna1d-eGFPflex/flex mice, likely because of high GFP concentration and small repair capacity. This and the slower time course of Pax2-driven Cre recombinase in switching two rather than one Cacna1d-eGFPflex allele lead us to study Pax2::cre;Cacna1d-eGFPflex/- mice. Notably, control Cacna1d-eGFPflex/- IHCs showed a significant reduction in Cav1.3 channel cluster sizes and currents, suggesting that the intronic construct interfered with gene translation or splicing. These pitfalls are likely to be a frequent problem of many genetically modified mice with complex or multiple gene-targeting constructs or fluorescent proteins. Great caution and appropriate controls are therefore required

Resting-State Connectivity of the Left Frontal Cortex to the Default Mode and Dorsal Attention Network Supports Reserve in Mild Cognitive Impairment

Reserve refers to the phenomenon of relatively preserved cognition in disproportion to the extent of neuropathology, e.g., in Alzheimer’s disease. A putative functional neural substrate underlying reserve is global functional connectivity of the left lateral frontal cortex (LFC, Brodmann Area 6/44). Resting-state fMRI-assessed global LFC-connectivity is associated with protective factors (education) and better maintenance of memory in mild cognitive impairment (MCI). Since the LFC is a hub of the fronto-parietal control network that regulates the activity of other networks, the question arises whether LFC-connectivity to specific networks rather than the whole-brain may underlie reserve. We assessed resting-state fMRI in 24 MCI and 16 healthy controls (HC) and in an independent validation sample (23 MCI/32 HC). Seed-based LFC-connectivity to seven major resting-state networks (i.e., fronto-parietal, limbic, dorsal-attention, somatomotor, default-mode, ventral-attention, visual) was computed, reserve was quantified as residualized memory performance after accounting for age and hippocampal atrophy. In both samples of MCI, LFC-activity was anti-correlated with the default-mode network (DMN), but positively correlated with the dorsal-attention network (DAN). Greater education predicted stronger LFC-DMN-connectivity (anti-correlation) and LFC-DAN-connectivity. Stronger LFC-DMN and LFC-DAN-connectivity each predicted higher reserve, consistently in both MCI samples. No associations were detected for LFC-connectivity to other networks. These novel results extend our previous findings on global functional connectivity of the LFC, showing that LFC-connectivity specifically to the DAN and DMN, two core memory networks, enhances reserve in the memory domain in MCI

Cross-sectional seroprevalence surveys of SARS-CoV-2 antibodies in children in Germany, June 2020 to May 2021

The rate of SARS-CoV-2 infections in children remains unclear due to many asymptomatic cases. We present a study of cross-sectional seroprevalence surveys of anti-SARS-CoV-2 IgG in 10,358 children recruited in paediatric hospitals across Germany from June 2020 to May 2021. Seropositivity increased from 2.0% (95% CI 1.6, 2.5) to 10.8% (95% CI 8.7, 12.9) in March 2021 with little change up to May 2021. Rates increased by migrant background (2.8%, 4.4% and 7.8% for no, one and two parents born outside Germany). Children under three were initially 3.6 (95% CI 2.3, 5.7) times more likely to be seropositive with levels equalising later. The ratio of seropositive cases per recalled infection decreased from 8.6 to 2.8. Since seropositivity exceeds the rate of recalled infections considerably, serologic testing may provide a more valid estimate of infections, which is required to assess both the spread and the risk for severe outcomes of SARS-CoV-2 infections

Left frontal hub connectivity delays cognitive impairment in autosomal-dominant and sporadic Alzheimer's disease

Patients with Alzheimer's disease vary in their ability to sustain cognitive abilities in the presence of brain pathology. A major open question is which brain mechanisms may support higher reserve capacity, i.e. relatively high cognitive performance at a given level of Alzheimer's pathology. Higher functional MRI-assessed functional connectivity of a hub in the left frontal cortex is a core candidate brain mechanism underlying reserve as it is associated with education (i.e. a protective factor often associated with higher reserve) and attenuated cognitive impairment in prodromal Alzheimer's disease. However, no study has yet assessed whether such hub connectivity of the left frontal cortex supports reserve throughout the evolution of pathological brain changes in Alzheimer's disease, including the presymptomatic stage when cognitive decline is subtle. To address this research gap, we obtained cross-sectional resting state functional MRI in 74 participants with autosomal dominant Alzheimer's disease, 55 controls from the Dominantly Inherited Alzheimer's Network and 75 amyloid-positive elderly participants, as well as 41 amyloid-negative cognitively normal elderly subjects from the German Center of Neurodegenerative Diseases multicentre study on biomarkers in sporadic Alzheimer's disease. For each participant, global left frontal cortex connectivity was computed as the average resting state functional connectivity between the left frontal cortex (seed) and each voxel in the grey matter. As a marker of disease stage, we applied estimated years from symptom onset in autosomal dominantly inherited Alzheimer's disease and cerebrospinal fluid tau levels in sporadic Alzheimer's disease cases. In both autosomal dominant and sporadic Alzheimer's disease patients, higher levels of left frontal cortex connectivity were correlated with greater education. For autosomal dominant Alzheimer's disease, a significant left frontal cortex connectivity × estimated years of onset interaction was found, indicating slower decline of memory and global cognition at higher levels of connectivity. Similarly, in sporadic amyloid-positive elderly subjects, the effect of tau on cognition was attenuated at higher levels of left frontal cortex connectivity. Polynomial regression analysis showed that the trajectory of cognitive decline was shifted towards a later stage of Alzheimer's disease in patients with higher levels of left frontal cortex connectivity. Together, our findings suggest that higher resilience against the development of cognitive impairment throughout the early stages of Alzheimer's disease is at least partially attributable to higher left frontal cortex-hub connectivity

Analysis of proximal ALOX5 promoter binding proteins by quantitative proteomics

5‐Lipoxygenase (5‐LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5‐LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter‐interacting proteins, obtained by DNA pulldown and label‐free quantitative mass spectrometry. Protein preparations from myeloid and B‐lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel‐like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G‐quadruplex structures. As G‐quadruplexes are implicated in gene regulation, spectroscopic and antibody‐based methods were used to confirm their presence within the GC‐rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G‐quadruplex structures, which may play a functional role in ALOX5 gene regulation

Optoacoustically induced auditory brainstem responses in the mouse model enhanced through an absorbing film

Significance: Optoacoustic stimulation offers an alternative stimulation strategy for the hearing organ. To serve as the base for a novel auditory prosthesis, the optoacoustic stimulation must be biocompatible and energy-saving. Aim: Enhancing the efficiency of optoacoustic stimulation while reducing the energy input in a suited animal model. Approach: Optoacoustically induced auditory brainstem responses (oABRs) were recorded after the pulsed laser irradiation of the tympanic membrane (TM) in mice. The results were compared with the ABRs induced through acoustic click stimulation. In addition, self-adhesive absorbing films were applied on the TM before the optoacoustic stimulation to investigate their effect on the resulting ABRs.Results: Using an absorbing film on the TM during optical stimulation led to considerably enhanced oABR wave I amplitude values compared with the stimulation of the bare TM. When using our stimulation strategy, we induced oABR waves in the 50% to 60% range of the acoustical stimulation reached with 80-dB SPL click stimuli. Conclusions: The mouse model can be used for certain developmental work for an optoacoustic auditory prosthesis. Using absorbing films on the TM during optical stimulation considerably enhances oABR wave I amplitude. Optimization of the stimulation strategy could further enhance the efficiency within biocompatibility margins