Otoprotective properties of 6α-methylprednisolone-loaded nanoparticles against cisplatin: In vitro and in vivo correlation.

6α-Methylprednisolone-loaded surfactant-free nanoparticles have been developed to palliate cisplatin ototoxicity. Nanoparticles were based on two different amphiphilic pseudo-block copolymers obtained by free radical polymerization and based on N-vinyl pyrrolidone and a methacrylic derivative of α-tocopheryl succinate or α-tocopherol. Copolymers formed spherical nanoparticles by nanoprecipitation in aqueous media that were able to encapsulate 6α-methylprednisolone in their inner core. The obtained nanovehicles were tested in vitro using HEI-OC1 cells and in vivo in a murine model. Unloaded nanoparticles were not able to significantly reduce the cisplatin ototoxicity. Loaded nanoparticles reduced cisplatin-ototoxicity in vitro being more active those based on the methacrylic derivative of vitamin E, due to their higher encapsulation efficiency. This formulation was able to protect hair cells in the base of the cochlea, having a positive effect in the highest frequencies tested in a murine model. A good correlation between the in vitro and the in vivo experiments was found. FROM THE CLINICAL EDITOR: Cisplatin is a commonly used chemotherapeutic agent against many cancers clinically. However, one of the significant side-effects remains ototoxicity. Here, the authors presented their data on using 6α-methylprednisolone-loaded nanoparticles in the reduction of ototoxicity in in-vitro and in-vivo experiments. Early promising results should enable further refinement of adopting this new approach in future experiments.

Hemangiopericytoma of the parapharyngeal space: a diagnostic challenge.

Parapharyngeal space tumors are known for having a difficult approach, misleading diagnosis and for representing a treatment challenge. Hemangiopericytomas account for less than 1% of all vascular neoplasms and 3% of all soft tissue sarcomas. Only 14 cases have been reported in the worldwide literature in this location. We present a case of a 44-year-old male who was referred for evaluation. A CT scan and MRI showed a large parapharyngeal mass of a possible salivary gland origin. The patient underwent a lateral cervicotomy associated with a transparotid-transmandibular approach, obtaining a vimentin-positive immunostaining tumor defining the diagnosis. The accurate management and prognosis of this type of neoplasm are provided by the definite diagnosis obtained by a correct histopathologic assessment. A high clinical suspicion is essential.

Reactive oxygen species in apoptosis induced by cisplatin: review of physiopathological mechanisms in animal models.

Cisplatin is a highly effective chemotherapeutic agent but displays significant ototoxic side effects. The most prominent change seen in the cochlea after cisplatin administration consists of loss of outer hair cells. Several mechanisms are believed to mediate cisplatin-induced apoptosis: binding of cisplatin to guanine bases on DNA and the formation of inter- and intra-strand chain cross-linking, generation of reactive oxygen species (ROS) with increased lipid peroxidation and Ca(2+) influx and, finally, inflammation mediated by cisplatin. The aim of the present review is to analyze the role of ROS in the mechanisms causing cisplatin-mediated apoptosis in the inner ear and the contribution of the different pathways involved, emphasizing the main strategies to blockade events leading to apoptosis of cochlear cells.

[Radiological diagnostic of the non-pathological conditions of the petrous apex]. / Diagnóstico radiológico de las condiciones no patológicas del ápex petroso.

Many patients with otological symptoms are remitted to the otolaryngology outpatient clinics every day. These patients commonly undergo imaging studies, generally magnetic resonance imaging (MRI). In some cases, a positive unilateral result is found in the form of a potentially pathological signal that can be observed in the petrous apex region. We present the cases of 6 patients (aged between 26 and 62 years) with asymmetric bone marrow distribution or trapped mucous fluid secretions in the petrous apex, collected over a 6-year period. Diagnosis was made with the use of CT scans and MRI. All of the patients were referred for skull base surgery. In all cases a non-pathologic asymmetry was diagnosed in the petrous apex. Certain non-pathologic conditions of the petrous apex must be treated expectantly without any surgery.

[Designing a new tool for hearing exploration]. / Diseño de una nueva herramienta para la exploración auditiva.

INTRODUCTION: Many presbycusic patients have difficulty in understanding certain words. This could be justified because certain sounds in Spanish are more difficult to perceive, particularly the sounds with energy in the high frequencies. We propose to use a sentence as a tool to check this theory. MATERIALS AND METHOD: All the Spanish sounds were analyzed, measuring the degree of acoustic energy in all the frequencies. The conclusions drawn from the comparison of the results allowed the design of the tool that is proposed here. RESULTS: We established a gradient of perception difficulty, occlusive consonants being the least perceptible, followed by fricative, and finally all those segments with harmony and a clear formant structure. The Spanish sentence "Ana vio ese coche rojizo fino" is proposed as the tool for this study. This sentence has some as it comprises certain peculiarities that makes it particularly useful for this purpose. It will allow us to check whether understanding deteriorates as we move from beginning to end, helping evaluate the importance of high frequencies for intelligibility. CONCLUSIONS: A positive result could help in the design of amplification systems to improve speech intelligibility. In addition, the exploratory tool could allow neuro-acoustic exploration, useful in the central auditory pathology studies.

Does the serological study for viral infection in autoimmune inner ear disease make sense?

Viral infections of the labyrinth have been considered a major source of auditory and vestibular system pathology. However, the involvement of virus in the development of immune reactions responsible for immunomediated inner ear disease has not been studied enough. Following viral infection, an effector immune response, humoral (B cell) and/or cytotoxic (T cell) is directed against a virus and it might cross-react with self-protein or autoantigen, evoking an autoimmune response. Since clinically it can be very difficult to establish a viral etiology for such disorders, serologic studies can be used to confirm the suspected diagnosis. Patients affected by immunomediated inner ear disease that had presented an upper respiratory tract infection underwent an immunologic workup study including microbiological study. After the application of this diagnostic protocol, only one patient, that was subsequently diagnosed with Cogan's syndrome, showed a positive serological test for viral infection. On the basis of the low efficacy of serological testing and due to the lack of evidence, we do not recommend to carry out serologic studies for viral infection.

Adverse effects of glucocorticoid therapy for inner ear disorders.

BACKGROUND: Because of their anti-inflammatory effects and suppression of the immune system, glucocorticoids have been widely used in otolaryngologic disorders and perioperative conditions. OBJECTIVE: The objective of the present study was to determine the incidence of adverse effects after the administration of glucocorticosteroids in patients affected by diverse inner ear disorders. METHODS: One hundred and sixty-three patients affected by sudden sensorineural hearing loss, 39 with progressive sensorineural hearing loss and 16 with fluctuating sensorineural hearing loss were subjected to glucocorticosteroid therapy with 6-methylprednisolone at a starting dose of 1 mg/kg body weight per day; this therapy was tapered during the next 21-28 days. In 20 patients with profound hearing loss (>70 dB), 3 boluses of prednisolone-21-hydrogen-succinate (500 mg per day) were administered. After receiving the boluses, these patients continued with the oral 6-methylprednisolone scheme. RESULTS: Mild adverse effects were observed in 16 patients (7.01%). Only 2 patients (0.9%) with sudden sensorineural hearing loss showed severe complications: peptic ulcer and avascular necrosis of the femoral heads. CONCLUSIONS: The low percentage of severe adverse effects observed in the present study validates the use of corticosteroids for the treatment of inner ear disorders although we should not underestimate these rare complications.

[Environmental scanning electron microscopy for biofilm detection in tonsils]. / Microscopia electrónica de barrido ambiental para la detección de biopelículas en las amígdalas.

INTRODUCTION AND OBJECTIVE: To describe an environmental scanning electron microscopic method for the study of biofilms in clinical samples. A comparison with standard scanning electron microscopy is performed. PATIENTS AND METHOD: Nine patients with a past history of recurrent tonsillitis underwent tonsillectomy. Samples from each patient were obtained for both conventional and environmental scanning electron microscopy. The tonsils removed from 2 patients with sleep apnoea syndrome were used as controls. RESULTS: Eight of nine tonsils had biofilms on their surface. Scanning electron microscopy showed accumulations of bacteria covered by fibrillar structures resulting from the sample dehydration process. Environmental scanning electron microscopy provided a view of bacteria embedded in a homogeneous, amorphous substance that was preserved during the examination. CONCLUSIONS: Environmental scanning electron microscopy permits the imaging of wet systems at different degrees of dehydration. It therefore allows researchers to observe biofilms in their natural hydrated state.

[Ultra-structural study of the lateral portion of the auditory sensorial organ using a decalcification-free method]. / Estudio ultraestructural de la porción lateral del órgano sensorial auditivo mediante un método sin descalcificación.

OBJECTIVE: To explain the development of a new personal technique to study the spiral ligament and stria vascularis in Guinea pig cochleae by obtaining sample tissue without decalcification and to assess its validity for electron microscopy analysis. MATERIAL AND METHOD: Samples were taken from five female Guinea pigs weighing 200-250 g and were fixed in glutaraldehyde and paraformaldehyde for analysis of the spiral ligament and stria vascularis ultrastructure by transmission electron microscopy. RESULTS: All of the ultrastructure components in the spiral ligament and stria vascularis could be examined without the need for decalcification. CONCLUSIONS: Our method to obtain and analyze samples of cochlea side wall is valid, easy and faster.

[Intervention of spiral ligament fibrocytes in the metabolic regulation of the inner ear]. / Intervención de los fibrocitos del ligamento espiral en la regulación metabólica del oído interno.

Maintenance of the K(+) gradient between endolymph and perilymph is essential for normal hearing and depends primarily on the activity of the stria vascularis. Abundant Na-K-ATPase in marginal strial cells provides a pumping mechanism for preserving the K(+) level of the endolymph and consequently, the endocochlear potential. Fibrocytes in the lateral wall of the cochlea supply K(+) to the strial pump, via gap junctions, by recycling back into the stria the ions that efflux from the scala media during auditory transduction. The lateral wall of the cochlea encloses five types of fibrocytes, differentiated by their location, structural features and content of enzymes mediating or energizing ion transport. The disruption of the gap junction bonds by connexin mutations and other pathologies leads to an interruption of K(+) recirculation pathways. The expression of cochlin and otoraplin, proteins that participate in structural or regulatory functions in the inner ear, suggests more diversity and complexity of the mesenchymal tissues than envisioned previously. The presence of otospiralin, a novel protein found in fibrocytes of spiral limbus, spiral ligament and subepithelial regions of the vestibule, represent a critical finding since that protein has been shown to be essential for the survival of the hair cells and supporting cells of the inner ear. A more profound knowledge and understanding of the function of inner ear fibrocytes will provide a new and promising aetiopathogenic approach to the treatment of inner ear disorders.

Azathioprine reduces the risk of audiometric relapse in immune-mediated hearing loss. / La azatioprina reduce el riesgo de recaída audiométrica en hipoacusia inmunomediada.

INTRODUCTION: Current schemes for treatment of immune-mediated hearing loss with sporadic short-course, low-dose corticosteroids, are insufficient. METHODS: To determine the role of azathioprine in the control of auditory impairment, a longitudinal, observational, descriptive study was performed with 20 patients treated with azathioprine (1.5-2.5mg/kg/day into two doses) for 1year. The loss of 10dB on two consecutive frequencies or 15dB on an isolated frequency was considered as relapse. RESULTS: The mean age of the patients was 52.50years (95%CI: 46.91-58.17), half were women. Bilateral affectation was 65%. 75% had organ specific disease and 25% had systemic autoimmune disease. The difference between baseline PTA (46.49dB; DS18.90) and PTA at 12months (45.47dB; DS18.88) did not reach statistical significance (P=.799). There was a moderate positive correlation between female sex and the presence of systemic disease (R=.577). By applying Student's t for paired data, a significant difference (P=.042) was obtained between the PTA in frequencies up to 1000 Hz (PTA125-1000Hz). The relative incidence rate of relapse per year was .52 relapses/year (95%CI: .19-1.14]). The median time to audiometric relapse-free was 9.70months (DS1.03). CONCLUSIONS: Azathioprine maintains the hearing threshold, decreases the risk of relapse, and slows down the rate at which patients relapse, altering the course of immune-mediated inner ear disease.

Analysis of audiometric relapse-free survival in patients with immune-mediated hearing loss exclusively treated with corticosteroids. / Análisis de supervivencia libre de recaída audiométrica en pacientes con hipoacusia inmunomediada tratados exclusivamente con corticoides.

OBJECTIVE: To describe the results in terms of audiometric relapse-free survival and relapse rate in immunomediated hearing loss patients treated exclusively with corticosteroids. METHOD: Retrospective study of patients with audiometric relapses, monitored from 1995 to 2014, in two centres of the Community of Madrid. RESULTS: We evaluated 31 patients with a mean age of 48.52 years (14.67 SD), of which 61.3% were women. Most hearing loss was fluctuating (48.4%). Only 16.1% of patients had systemic autoimmune disease. There is a moderate positive correlation between the sex variable and the systemic involvement variable (Spearman's correlation coefficient=0.356): specifically, between being female and systemic disease. The relative incidence rate of relapse in the first year was 2.01 relapses/year with a 95% CI (1.32 to 2.92). The mean survival time of the event (audiometric relapse) was 5.25 months (SD 0.756). With multivariate analysis, the only variable that achieved statistical significance was age, with a hazard ratio of 1.032 (95% CI; 1.001-1.063, P=.043). CONCLUSIONS: Immune-mediated disease of the inner ear is a chronic disease with relapses. Half of the patients with immunomediated hearing loss treated exclusively with corticosteroids relapse before 6 months of follow-up. In addition, if a patient has not relapsed, they are more likely to relapse as each year passes. Analysis of the of audiometric relapse- free survival will enable the effect of future treatments to be compared and their capacity to reduce the rhythm of relapses.

pH-sensitive polymeric nanoparticles with antioxidant and anti-inflammatory properties against cisplatin-induced hearing loss.

Polymeric nanoparticles (NPs) based on smart synthetic amphiphilic copolymers are used to transport and controlled release dexamethasone in the inner ear to protect against the ototoxic effect of cisplatin. The NPs were based on a mixture of two pseudo-block polymer drugs obtained by free radical polymerization: poly(VI-co-HEI) and poly(VP-co-MVE) or poly(VP-co-MTOS), being VI 1-vinylimidazole, VP N-vinylpyrrolidone, and HEI, MVE and MTOS the methacrylic derivatives of ibuprofen, α-tocopherol and α-tocopheryl succinate, respectively. The NPs were obtained by nanoprecipitation with appropriate hydrodynamic properties, and isoelectric points that matched the pH of inflamed tissue. The NPs were tested both in vitro (using HEI-OC1 cells) and in vivo (using a murine model) with good results. Although the concentration of dexamethasone administered in the NPs is around two orders of magnitude lower that the conventional treatment for intratympanic administration, the NPs protected from the cytotoxic effect of cisplatin when the combination of the appropriate properties in terms of size, zeta potential, encapsulation efficiency and isoelectric point were achieved. To the best of our knowledge this is the first time that pH sensitive NPs are used to protect from cisplatin-induced hearing loss by intratympanic administration.

Evolution of middle ear changes after permanent eustachian tube blockage.

OBJECTIVE: To develop a valid animal model for otitis media with effusion (OME). DESIGN: Forty specific pathogen-free Wistar rats underwent a procedure based on the permanent obstruction of pharyngeal eustachian tube by means of electrocoagulation without any manipulation. SETTING: Ear Research Group, Department of Otorhinolaryngology, Puerta de Hierro Hospital, Universidad Autonoma de Madrid, Madrid, Spain. MAIN OUTCOME MEASURES: The assessment of OME by otoscopy and tympanometry. The rats were humanely killed at 15 and 90 days, and temporal bones were obtained and processed for histopathologic study. RESULTS: The histopathologic study of the temporal bones demonstrated the occurrence of chronic effusion and mucosal changes owing to mucoperiosteal enlargement. CONCLUSIONS: Comparison with other experimental models was made. Our animal model was consistent and reproducible and resembled human OME.

Morphological sequence of Plastipore extrusion in experimental otitis media.

OBJECTIVES: Plastipore prostheses are still used by many surgeons, although the functional results are controversial. The aim of this study was the morphological analysis of Plastipore material performance in the middle ear of rats, with special attention to extrusion. METHODS: Twenty-four Wistar rats were given implants made of commercially available Plastipore and assigned to 3 groups: group A, with implantation in a healthy middle ear; group B, with implantation and cauterization of the nasopharyngeal orifice of the eustachian tube (hypoventilation); and group C, with implantation, cauterization of the eustachian tube, and bacterial inoculation with Pseudomonas aeruginosa. RESULTS: The pathological study showed in nearly all cases the disintegration of the biomaterial. Adhesion between the biomaterial and bone could be seen in 1 rat from group C (hypoventilation and infection). In group C, the Plastipore was in contact with the tympanic membrane in 1 case and was extruding in 2 animals. Different phases of extrusion were defined. No extrusion was observed in the other groups. CONCLUSIONS: The sequential stages of Plastipore extrusion are demonstrated. Infection seems to be the most important factor in Plastipore extrusion in our model.

Tissular changes induced by Pseudomonas aeruginosa in an otitis media rat model with tubal obstruction.

CONCLUSIONS: This is a suitable model for the study of different features of middle ear inflammation. This model allows manipulations inside the middle ear while preserving relevant structures such as the tympanic membrane, and provides a useful model for the study of interactions between bacterial infection and eustachian tube dysfunction. OBJECTIVES: Analysis of early and late histological features in an experimental model of Pseudomonas aeruginosa middle ear inoculation in the rat designed for the study of middle ear procedures. MATERIALS AND METHODS: Thirty Wistar rats were inoculated with Pseudomonas aeruginosa in the tympanic bulla followed by the cauterization of the eustachian tube. Culturing of middle ear effusion was carried out at 7 days follow-up and at sacrifice. Processing of the temporal bones for light microscopy was performed at 7, 14, 30 and 60 days. RESULTS: Early cultures were positive in most cases, thus proving that middle ear inflammation was due to the presence of inoculated Pseudomona aeruginosa. Mucoperiosteal inflammatory changes similar to those observed in human middle ear infection were seen. Acute inflammatory cell infiltration was seen at 7 and 14 days, gradually decreasing to chronic inflammatory changes with fibroplasia at 60 days. Bone resorption was observed at 7 and 14 days, changing to a bony deposition at 30 and 60 days.

[Autoimmune hearing loss: improving diagnostic performance]. / Sordera autoinmunitaria: mejorando el rendimiento de su diagnóstico.

OBJECTIVES: Due to the lack of specific serological markers for the diagnosis of immune-mediated hearing loss, an exhaustive immunologic work-up study for patients is usually performed. The aim of the present study is to find the most cost-effective laboratory tests used for the diagnosis of this entity. PATIENTS AND METHOD: Comparative study between 2 groups of patients with a high suspicion of suffering diverse clinical forms of immune-mediated hearing loss, subjected to different serologic testing designs: the classical immunologic work-up study (125 patients) in comparison with a more restricted examination analysis (57 patients), based on a high risk profile recently reported. RESULTS: Diagnostic efficiency was similar. CONCLUSIONS: Since financial resources are limited, ANA and immunophenotype of PBL (peripheral blood lymphocytes) are recommended for the evaluation of a patient with suspected immune-mediated hearing loss.

[Immunomediated inner ear disease: diagnostic validation by means of a systematic analysis of the scientific literature]. / Enfermedad inmunomediada del oído interno: validación diagnóstica mediante un análisis sistemático de la literatura científica.

INTRODUCTION: Numerous tests have been developed to help in the diagnosis of the immunomediated inner ear disease (IMIED), although their usefulness is still the subject of some dispute. MATERIAL AND METHOD: Studies of cohorts of patients with suspected IMIED have been reviewed. A number of different serological tests were carried out and their response to immunosuppressive therapy was noted. RESULTS: After reviewing 790 articles, the studies analyzed present great heterogeneity in the clinical characteristics of the patients recruited (sudden deafness, fluctuating and rapidly progressive hearing loss, Menière's disease), the inclusion and exclusion criteria, the delay between the diagnosis and the start of medical treatment or the response criteria. CONCLUSIONS: The diagnosis of IMIED is based on clinical presentation and response to corticosteroids. At present no test represents the gold standard in the diagnosis of IMIED. Serologic tests can support the diagnosis, but a methodologically more rigorous approach is needed to identify the true clinical importance of these tests for daily practice.

Polymeric nanoparticles loaded with dexamethasone or α-tocopheryl succinate to prevent cisplatin-induced ototoxicity.

The aim of this work is the development of highly protective agents to be administered locally within the middle ear to avoid cisplatin-induced ototoxicity, which affects to 100% of the clinical patients at ultra-high concentrations (16mg/kg). The protective agents are based on polymeric nanoparticles loaded with dexamethasone or α-tocopheryl succinate as anti-inflammarory and anti-apoptotic molecules. Dexamethasone and α-tocopheryl succinate are poorly soluble in water and present severe side effects when systemic administered during long periods of time. Their incorporation in the hydrophobic core of nanoparticles with the appropriate hydrodynamic properties provides the desired effects in vitro (lower cisplatin-induced toxicity, decreasing of caspase 3/7 activity, and lower IL-1ß release) and in vivo (reducing the hearing loss at the local level). The local administration of the nanoparticles by bullostomy provides an adequate dose of drug without systemic interference with the chemotherapeutic effect of cisplatin. STATEMENT OF SIGNIFICANCE: 100% of the cancer patients receiving ultra-high doses of CDDP (16mg/kg) suffer severe hearing loss, being a limiting factor in antineoplastic treatments. In this paper we describe the application of polymeric nanoparticles loaded with dexamethasone or α-tocopheryl succinate to palliate the cisplatin ototoxicity derived from chemotherapy treatment. These new nanoparticles, that encapsulate, transport, and deliver dexamethasone or α-tocopheryl succinate in the middle ear, are able to partially prevent ototoxicity derived from high doses of CDDP. This is an interdisciplinary study in which in vitro and in vivo experiments are described and extensively discussed. The importance of the results opens an excellent opportunity to the translation to the clinic.

Ageing evokes an intrinsic pro-apoptotic signalling pathway in rat cochlea.

CONCLUSIONS: These findings support the hypothesis that age-related apoptosis evokes an intrinsic pathway of pro-apoptotic signalling within the rat cochlea. OBJECTIVES: The aim of this study was to explore the effects of ageing on cochlear apoptosis in rats, as well as the different signalling pathways involved. MATERIALS AND METHODS: Female Sprague Dawley rats of different ages were used (mean age 7.72+/-1.93 months, n=100). Luciferase assays were used to determine the different caspase activities and ATP levels in rat cochlear protein extracts. Protein and gene expression was examined by Western blotting and real-time RT-PCR assays, respectively. RESULTS: Caspase-3/7 activity, as well as caspase-3 gene expression, were statistically higher in early-mature rats (EM, 276,139+/-13 669 RLUs (relative light units), p<0.001; and 390+/-50 arbitrary units, p=0.0017, respectively) or in aged-mature rats (AM, 371,020+/-26,457, p<0.0001; and 1510+/-90, p<0.0001, respectively) than younger rats (YR 147,129+/-8485 and 0.14+/-0.004, respectively). An increased caspase-9 activity with ageing was also observed (YR 49,932+/-2046 RLUs vs EM 260,890+/-5939, p<0.0001 or AM 118,241+/-12,423, p<0.0001). Caspase-8 activity was not affected significantly by age. Bax protein expression also increased by age (YR 38,200+/-1790 arbitrary units vs EM 76,549+/-5450, p<0.05), in contrast to Bcl-xL protein expression (YR 27,000+/-5000 arbitrary units vs EM 10,200+/-5000, p<0.005).