Use of scanning electron microscopy in the cochlea of guinea pigs / Microscopia eletrônica de varredura em cócleas de cobaias

Abstract Introduction: The use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the preparation of this material is a difficult and delicate task. In an attempt to simplify the handling of these materials, two agents, toluidine blue and ethylenediamine tetra-acetic acid were tested, in addition to the elimination of osmium tetroxide during the preparation of albino guinea pig cochleae. We also tested the applicability of these methodologies in an ototoxicity protocol. Objective: To verify the quality of the images obtained with and without the use of ethylenediamine tetra-acetic acid, toluidine blue and osmium tetroxide in the preparation of cochleae of albino guinea pigs for the scanning electron microscopy. Methods: Three groups of cochleae were used. In Group 1, 10 cochleae were prepared with the usual methodology, dissecting the optical capsule without decalcification and using osmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediamine tetra-acetic acid, injecting toluidine blue in the endolymphatic space to facilitate the identification of the organ of Corti. In Group 3, we used 4 cochleae of guinea pigs that received 3 doses of cisplatin (7.5 mg/kg, D1-D5-D6), two prepared according to the methodology used in Group 1 and two with that used in Group 2. Scanning electron microscopy images were obtained from the organ of Corti region of the basal turn of each cochlea. Results: The organ of Corti was more easily identified with the use of toluidine blue. The dissection of the cochlea was more accurate in the decalcified cochleae. The quality of the images and the preservation of the organ of Corti obtained with the two methodologies were similar. Conclusion: The proposed modifications resulted in images of similar quality as those observed using the traditional methodology.

Resumo Introdução: O emprego da microscopia eletrônica no estudo da orelha interna permitiu observar detalhes minuciosos das células ciliadas especialmente em estudos de ototoxicidade. Entretanto, o preparo desse material é trabalhoso e delicado. Para simplificar a manipulação desses materiais, testou-se o uso de dois agentes, azul de toluidina e ácido etilenodiamino tetra-acético, além da retirada do tetróxido de ósmio na preparação de cócleas de cobaias albinas. Testamos também a aplicabilidade dessas metodologias em um protocolo de ototoxicidade. Objetivo: Verificar a qualidade das imagens obtidas com e sem o uso de ácido etilenodiamino tetra-acético, azul de toluidina e tetróxido de ósmio na preparação de cócleas de cobaias albinas para a microscopia eletrônica de varredura. Método: Foram utilizados três grupos de cócleas. No Grupo 1 preparou-se 10 cócleas com a metodologia usual, dissecando a cápsula ótica sem descalcificac¸ão e utilizando tetróxido de ósmio como pós-fixador. No Grupo 2 preparamos 10 cócleas descalcificadas com ácido etilenodiamino tetra-acético, injetando azul de toluidina no espac¸o endolinfático para facilitar a identificação do órgão de Corti. No Grupo 3 utilizamos 4 cócleas de cobaias que receberam 3 doses de cisplatina (7,5 mg/kg, D1-D5-D6), duas preparadas com a metodologia do Grupo 1 e duas com a do Grupo 2. Foram obtidas imagens da microscopia eletrônica de varredura da região do órgão de Corti do giro basal de cada cóclea. Resultados: O órgão de Corti foi mais facilmente identificado com o azul de touidina. A dissecção da cóclea foi mais precisa nas cócleas descalcificadas A qualidade das imagens e a preservac¸ão do órgão de Corti obtidas com as duas metodologias foi similar. Conclusão: As modificações propostas resultaram em imagens de qualidade similar as observadas com o uso da metodologia tradicional.

Internacionalização, ciência e saúde: a medicina regenerativa global e os mercados paralelos / Internationalization, science and health: global regenerative medicine and the parallel markets

A medicina regenerativa implica em uma mudança de paradigma, a regeneração do organismo ao nível celular ou tecidual – um assunto contemporâneo controverso e de difícil estandardização. O artigo apresenta um resumo das tendências científicas, econômicas, sociais e de regulamentação global nessa área, analisadas em relação a dilemas teóricos relevantes em antropologia médica e sociologia da ciência e da saúde. Em especial, aqueles que tratam da construção de um ‘aparato coletivo de sentido’ para as novas entidades biológicas e ontológicas, a formação da cidadania biológica e a governança pela incerteza. Apresentam-se, também, evidências empíricas sobre um fenômeno chave para a governança e a regulamentação, qual seja a instalação de uma nova demanda transnacional em pesquisa e saúde através de mercados paralelos de óvulos e de terapias celulares em experimentação. Utilizam-se dados qualitativos coletados para uma pesquisa mais abrangente, resenhas jornalísticas e entrevistas com lideranças internacionais. Conclui-se com uma reflexão sobre a importância da governança internacional em ensaios clínicos e dos caminhos a serem explorados, visando uma harmonização da diversidade de práticas normativas.

Regenerative medicine involves a paradigm change due to organism regeneration at cellular and tissue level – a controversial contemporary issue and difficult to regulate. This article presents a summary of the main scientific, economic, social and regulatory global trends, analyzed according to relevant theoretical dilemmas in medical anthropology and in the sociology of science and health. This is especially true of the construction of a ‘collective frame of reference’ on the new biological and ontological entities, the shaping of biological citizenship, and governance through uncertainty. Empirical evidence is also presented on a key aspect in regulation and governance, namely the emergence of a new transnational demand in health research through the establishment of parallel markets for ova and experimental cellular therapies. Qualitative data collected for a broader research paper is analyzed, as well as journal reviews and information gathered during interviews with international leaders. The paper concludes with a discussion on the importance on international governance of clinical trials and on further exploration, towards a multilevel harmonization of a diversity of normative practices.

Efeito da oxigenação hiperbárica em lesão ototóxica produzida pela amicacina em cobaias / The effects of hyperbaric oxygen therapy upon ototoxic injuries produced by amikacin in guinea pigs

A oxigenação hiperbárica têm favorecido a prevenção e o tratamento de afecções auditivas como a ototoxicidade. OBJETIVO: Estudar os efeitos da oxigenação hiperbárica em lesão ototóxica promovida pela amicacina. Forma de estudo: Experimental. MÉTODO: Avaliados aspectos funcionais de 12 cobaias albinas por meio das emissões otoacústicas produtos de distorção e do potencial evocado auditivo de tronco encefálico, antes e após o uso de amicacina (600 mg/kg/dia) e das sessões com oxigenação hiperbárica (2 ATA, 60 minutos). Aspectos morfológicos foram avaliados por meio de microscopia eletrônica de varredura. Grupos de estudo com três animais: grupo 1 - solução salina + oxigenação hiperbárica; grupo 2 - amicacina 8 dias; grupo 3 - amicacina + 7 dias de repouso e grupo 4 - amicacina + oxigenação hiperbárica. RESULTADOS: Grupo 1 apresentou preservação da funcionalidade e da morfologia durante todo experimento. Grupo 2 demonstrou, ao final do experimento, lesões estatisticamente significantes das células ciliadas com alterações funcionais. Grupos 3 e 4 apresentaram alterações estatisticamente significantes dos aspectos funcionais e morfológicos após o uso da amicacina, mantendo estas alterações após os procedimentos propostos. CONCLUSÃO: A oxigenação hiperbárica não promoveu alterações na morfologia das células ciliadas da cóclea e aos limiares eletrofisiológicos das cobaias submetidas à amicacina.

Hyperbaric oxygen therapy (HBOT) has enhanced the prevention and treatment of auditory ailments such as ototoxicity. OBJECTIVE: To study the effects of HBOT upon ototoxic injuries produced by amikacin. METHOD: This experimental study included 12 albino guinea pigs, whose auditory function was assessed through distortion product otoacoustic emissions (DPOAEs) and brainstem auditory evoked potentials (BAEPs) before and after the administration of amikacin (600 mg/kg/day) and HBOT sessions (2 ATA, 60 minutes). Morphological features were analyzed through scanning electron microscopy. Subjects were divided into four groups, as follows: group 1 - saline solution + HBOT; group 2 - amikacin for 8 days; group 3 - amikacin + seven days of rest; and group 4 - amikacin + HBOT. RESULTS: Group 1 subjects had preserved function and morphology throughout the experiment; Group 2 subjects had statistically significant levels of hair cell injury and functional impairment; Subjects on groups 3 and 4 had statistically significant functional and morphological impairment after the administration of amikacin, which were still present after the proposed procedures had been carried out. CONCLUSION: Hyperbaric oxygen therapy did not change the cochlear hair cell morphology or the electro-physiological thresholds of the guinea pigs given amikacin.

Hair cell loss and regeneration in the chick cochlea after treatment with gentamicin

Hair cells can be damaged by countless agents, among them aminoglycoside antibiotics. In the cheek cochlea, the hair cell loss can be recovered by regeneration. The objectives of the present investigation were to estudy the time progression of injury caused by gentamicin and the regeneration process in chick hair cells. Gentamicin was administered in a single subcutaneous dose of 125 or 250 mg/kg to two groups of 3-day old chicks. The cochleae were processed for analysis by scanning electron microscopy on the 1st, 3rd, 5th and 20th day after injection The cellular sequence of degeneration and regeneration was studied. On the 20th day, most it he damaged cochlear area showed regenerated hair and suport cells. Stereocilia and microvilli were observed on the apical surface of the regenerated hair cells.

Hairy cell leukemia: a histo-cytochemical and ultra-structural study

We studied five patients with hairy cell leukemia (HCL) diagnosed within the last ten years at the Department of Hematology of Universidade Federal de Sao Paulo - Escola Paulista de Medicina. Our purpose was to analyze the value of transmission electron microscopy (TEM) by comparing this method with the conventional ones. At diagnosis, patients presented weight loss, spleen enlargement and hairy cells (HC) in peripheral blood and bone marrow slides. HC was characterized by morphology and tartrate test resistance in the acid phosphatase reaction (TRAP). At the evaluation time, the amount of HC ranged from 1 per cent to 85 per cent of WBC count. All patients, except two, had phenotype B. In these last two, TRAP as well as phenotype B could not be documented due to low HC numbers in their exams. Cytoplasmatic projections and the absecence of lamellar ribosomic complex were the most frequent ultrastructural findings, even in those patients with the lowest HC numbers. Based on these features, TEM is an efficient method for searching for HC at HCL diagnosis and during the course of the disease.