Establishment of an optimized guinea pig model of cisplatin-induced ototoxicity

BackgroundCisplatin is among the most effective antineoplastic agents and has revolutionized the treatment of many cancer diseases. However, one of its serious side effects is a progressive and irreversible hearing loss, occurring in both adults and children. For the development of otoprotective therapies that prevent this side effect, cisplatin-induced hearing loss animal models are indispensable. Due to the high toxicity of cisplatin, the establishment of such animal models is a difficult and time-consuming task. Here we introduce the detailed protocol of a sophisticated guinea pig model with a sufficient and permanent hearing loss induced by cisplatin. This manuscript is intended to provide guidance in the development of future cisplatin guinea pig models which may reduce the mortality rate of the animals and help to gain more reproducible results.MethodsPigmented and unpigmented guineapigs were treated with an intravenous single application of 8 mg/kg cisplatin under general anesthesia. An extensive and long-term intensive care protocol consisting of scheduled application of fluids, antiemetics, analgesics, glucose and supportive feeding among others, was used to ensure wellbeing of the animals. Hearing tests were performed prior to and 5 days after cisplatin application. Animals were then euthanized.ResultsThe ABR audiometry 5 days after cisplatin application revealed a hearing threshold ranging from 70 dB to 90 dB in the frequencies from 1 kHz to 32 kHz respectively.All animals presented a good health condition despite the treatment with cisplatin.DiscussionThe introduced care protocol in this manuscript is intended to serve as a guidance for the establishment of a stable guinea pig model for short- and long-term investigation regarding the inner ear and its protection in the frame work of cisplatin-induced damage

The nucleotide composition of microsatellites impacts both replication fidelity and mismatch repair in human colorectal cells

Microsatellite instability is a key mechanism of colon carcinogenesis. We have previously studied mutations within a (CA)13 microsatellite using an enhanced green fluorescent protein (EGFP)-based reporter assay that allows the distinction of replication errors and mismatch repair (MMR) activity. Here we utilize this assay to compare mutations of mono- and dinucleotide repeats in human colorectal cells. HCT116 and HCT116+chr3 cells were stably transfected with EGFP-based plasmids harboring A10, G10, G16, (CA)13 and (CA)26 repeats. EGFP-positive mutant fractions were quantitated by flow cytometry, mutation rates were calculated and the mutant spectrum was analyzed by cycle sequencing. EGFP fluorescence pattern changed with the microsatellite's nucleotide sequence and cell type and clonal variations were observed in mononucleotide repeats. Replication errors (as calculated in HCT116) at A10 repeats were 5–10-fold higher than in G10, G16 were 30-fold higher than G10 and (CA)26 were 10-fold higher than (CA)13. The mutation rates in hMLH1-proficient HCT116+chr3 were 30–230-fold lower than in HCT116. MMR was more efficient in G16 than in A10 clones leading to a higher stability of poly-G tracts. Mutation spectra revealed predominantly 1-unit deletions in A10, (CA)13 and G10 and 2-unit deletions or 1-unit insertion in (CA)26. These findings indicate that both replication fidelity and MMR are affected by the microsatellite's nucleotide composition

Optimizing glucocorticoid-therapy of the inner ear

Obwohl Hörstörungen sehr häufig sind und es viele Studien gibt, die sich mit diesem Thema beschäftigten, existiert kaum hochqualitative Evidenz für die pharmakologische Therapie des akuten Hörverlustes. Nichtsdestotrotz werden Glucocorticoide - aufgrund vieler kleiner Studien die positive Ergebnisse erbrachten - bei einigen Innenohr-Erkrankungen wie zum Beispiel beim Hörsturz, bei autoimmun bedingten Hörverlusten oder beim Lärmtrauma als Erstlinientherapie verwendet. In den letzten Jahren konzentrierte sich die Forschung auf die topische Glucocorticoid Therapie, da diese Applikationsart zu wesentlich höheren Innenohrkonzentrationen als die systemische Gabe führt. Zur selben Zeit wurden selektive Glucocorticoid Rezeptor Modulatoren als vielversprechender neuer Therapieansatz für entzündliche Erkrankungen entwickelt. Vor diesem Hintergrund war das Ziel dieser Dissertation daher, die Therapie des Innenohres sowohl durch eine Optimierung der lokalen Wirkstoff-Applikation, als auch durch die Evaluation von selektiven Glucocorticoid Rezeptor Modulatoren zu verbessern. Um dieses Ziel zu erfüllen, wurden verschiedene Poloxamer407 Hydrogele in vitro getestet. In diesen Versuchen kristallisierte sich ein 20% Poloxamer407 Hydrogel mit 30% Triamcinolon-Acetonid als vielversprechendster Kandidat für weitere in vivo Versuche heraus. Die intratympanale Applikation dieses Hydrogels resultierte in therapeutisch relevanten Glucocorticoid Spiegeln im Innenohr, welche für zehn Tage messbar waren. In weiterer Folge verglichen wir die otoprotektive Wirkung von intraoperativ verabreichten Triamcinolon-Acetonid-, Dexamethason- und Kontroll- Hydrogelen in einem Meerschweinchen Modell für die Cochlea Implantation. Da diese Studie keinen otoprotektiven Effekt der Hydrogele zeigte, wurde das Protokoll adaptiert und das Glucocorticoid Hydrogel in einer Nachfolgestudie bereits sieben Tage beziehungsweise einen Tag vor der Cochlea Implantation verabreicht. In dieser experimentellen Serie zeigte sich eine statistisch signifikante Verringerung der postoperativen Hörverluste, wenn das Dexamethason Hydrogel einen Tag vor der Cochlea Implantation appliziert wurde. Um selektive Glucocorticoid Rezeptor Modulatoren auf ihre Effekte auf das Innenohr zu untersuchen, wählten wir Compound A als Modellsubstanz. Während die intraperitoneale Injektion von Compound A keine negativen Effekte auf das Hören hatte, kam es nach intratympanaler Applikation zu einem signifikanten Hörverlust. Daher wurde in weiterer Folge nur die systemische Gabe von Compound A in einem Lärmtrauma-Modell auf potentielle otoprotektive Effekte untersucht. In dieser experimentellen Serie zeigten sich keine otoprotektiven Eigenschaften dieses selektiven Glucocorticoid Rezeptor Modulators. Zusammenfassend zeigt diese Dissertation, dass die intratympanale Applikation von Triamcinolon-Acetonid in einem Poloxamer407 Hydrogel in hohen Innenohrkonzentrationen des Glucocorticoids resultiert und dass die präoperative Gabe von Glucocorticoid Hydrogelen durch die Elektrodeninsertion bedingte Hörverluste im Rahmen der Cochlea Implantation reduzieren kann. Zusätzlich wurden keine otoprotektiven Effekte des selektiven Glucocorticoid Rezeptor Modulators Compound A, dessen lokale Gabe sogar zu ototoxischen Effekten führte, gefunden. In Zusammenschau liefern diese Ergebnisse eine gute Basis für weitere translationale und klinische Studien, die dazu dienen könnten die Therapie von Innenohr Erkrankungen weiter zu verbessern.Even though hearing impairments are very common and various studies addressing this issue have been performed, there is only limited high quality evidence supporting the pharmacologic treatment of acute hearing loss. Nevertheless, based on a large number of small studies that reported protective effects, glucocorticoids serve as first line therapy for inner ear conditions like sudden sensorineural hearing loss, auto- immune hearing loss or noise induced hearing loss. In recent years, research focused on the optimization of topical glucocorticoid therapy as this approach results in significantly higher inner ear drug levels than systemic administration. At the same time, selective glucocorticoid receptor modulators emerged as a promising class of drugs for the treatment of inflammatory conditions. Given this background, the aim of this thesis was to attempt an improvement of inner ear therapy by the optimization of topical drug administration as well as by the evaluation of selective glucocorticoid receptor modulators. To approach this aim, we evaluated thermoreversible poloxamer407 hydrogels of different concentrations in vitro. These studies resulted in the selection of a 20% poloxamer407 hydrogel containing 30% triamcinolone-acetonide as most promising candidate for further in vivo studies. In the guinea pig model, intratympanic application of the triamcinolone-acetonide hydrogel caused therapeutically relevant glucocorticoid levels in perilymph for a minimum of 10 days. We then compared the otoprotective effects of intraoperative triamcinolone-acetonide, dexamethasone and control hydrogel application in a guinea pig model of cochlear implantation. This study did not show otoprotection and we therefore adapted the protocol, applying the glucocorticoid hydrogel seven days and one day prior to cochlear implantation in a follow up study. In this experimental series we observed a statistically significant reduction of postoperative hearing threshold shifts if a dexamethasone hydrogel was applied one day before surgery. To evaluate selective glucocorticoid receptor modulators for their effects on the inner ear, we chose compound A as model molecule. While the intraperitoneal application of compound A did not impede hearing, its intratympanic application caused significant hearing loss. Therefore, only intraperitoneal compound A administration was further evaluated for potential ototoxic effects in a noise trauma model. In this experimental series we were unable to show otoprotective effects of the selective glucocorticoid receptor modulator. In summary, this thesis shows that intratympanic application of triamcinolone- acetonide in poloxamer407 hydrogels results in high perilymph concentrations of the glucocorticoid and that preoperative glucocorticoid application reduces hearing threshold shifts caused by cochlear implantation. Additionally, no otoprotective effects of the selective glucocorticoid receptor modulator compound A were found. To the contrary, intratympanic application of this drug resulted in ototoxic effects. Together these results represent a sound basis for future translational and clinical studies addressing potential improvements of inner ear therapy.submitted by Dr. Clemens HonederZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2016OeBB(VLID)192331

Fixed and adaptive beamforming improves speech perception in noise in cochlear implant recipients equipped with the MED-EL SONNET audio processor

Objective: To determine the impact of the fixed and adaptive beamforming technology of the new MED-EL SONNET cochlear implant audio processor on speech perception in noise. Methods: The study cohort comprises 18 postlingually deafened adult cochlear implant recipients with at least six months of experience. Speech reception thresholds were measured with the Oldenburg Sentence Test in continuous, speech-shaped noise. Target sentences were presented in front of the listener, with noise sources placed at -135° and 135°, respectively. Outcome measures were the differences in speech reception threshold using omnidirectional, fixed and adaptive beamformer microphone settings. Results: The use of directional microphones significantly improved speech reception thresholds: fixed beamformer vs. omnidirectional: 4.3 dB (95%-CI [3.15.5]), p<0.0001adaptive beamformer vs. omnidirectional: 6.1 dB (95%-CI [4.97.3]), p<0.0001and adaptive beamformer vs. fixed beamformer: 1.8 dB (95%-CI [0.73.0]), p = 0.001. Conclusion: This study confirms the previously reported improvements in speech perception in noise of the fixed beamformer microphone setting and is the first to report significant improvements in speech perception in noise when applying the adaptive beamformer microphone settings of the SONNET audio processor. Cochlear implant users may be able to benefit from improved hearing performance especially in difficult listening situations

Fixed and adaptive beamforming improves speech perception in noise in cochlear implant recipients equipped with the MED-EL SONNET audio processor

Objective To determine the impact of the fixed and adaptive beamforming technology of the new MED-EL SONNET cochlear implant audio processor on speech perception in noise. Methods The study cohort comprises 18 postlingually deafened adult cochlear implant recipients with at least six months of experience. Speech reception thresholds were measured with the Oldenburg Sentence Test in continuous, speech-shaped noise. Target sentences were presented in front of the listener, with noise sources placed at -135 and 135, respectively. Outcome measures were the differences in speech reception threshold using omnidirectional, fixed and adaptive beamformer microphone settings. Results The use of directional microphones significantly improved speech reception thresholds: fixed beamformer vs. omnidirectional: 4.3 dB (95%-CI [3.1; 5.5]), p<0.0001; adaptive beamformer vs. omnidirectional: 6.1 dB (95%-CI [4.9; 7.3]), p<0.0001; and adaptive beamformer vs. fixed beamformer: 1.8 dB (95%-CI [0.7; 3.0]), p = 0.001. Conclusion This study confirms the previously reported improvements in speech perception in noise of the fixed beamformer microphone setting and is the first to report significant improvements in speech perception in noise when applying the adaptive beamformer microphone settings of the SONNET audio processor. Cochlear implant users may be able to benefit from improved hearing performance especially in difficult listening situations.(VLID)479101

Sustained release of triamcinolone acetonide from an intratympanically applied hydrogel designed for the delivery of high glucocorticoid doses

The pharmacokinetic properties and tolerability of a triamcinolone acetonide poloxamer 407 hydrogel for intratympanic application were investigated in a guinea pig model. Evaluation of in vivo release kinetics showed very high initial perilymph drug levels, with clinically relevant levels present for a minimum of 10 days. Assessment of auditory brainstem response thresholds showed a minimal, delayed and transient threshold shift, which was apparent on day 3 and resolved by day 10. No relevant histological changes of the middle and inner ear structures were noted, and hair cell counts showed no significant differences between treated and untreated ears. Thus, the triamcinolone-acetonide-loaded poloxamer 407 hydrogel is an effective vehicle for sustained high-dose inner ear glucocorticoid delivery