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.

SEM/FIB Imaging for Studying Neural Interfaces.

Tissue and neural engineering for various regenerative therapies are rapidly growing fields. Of major interest is studying the complex interface between cells and various 3D structures by scanning electron microscopy with focused ion beam. Notwithstanding its unrivaled resolution, the optimal fixation, dehydration, and staining protocols of the samples while preserving the complex cell interface in its natural form, are highly challenging. The aim of this work was to compare and optimize staining and sample drying procedures in order to preserve the cells in their "life-like state" for studying the cell interface with either 3D well-like structures or gold-coated mushroom-shaped electrodes. The process involved chemical fixation using a combination of glutaraldehyde and formaldehyde, followed by gentle drying techniques in which we compared four methods: (critical point drying, hexamethyldisiloxane, repeats of osmium tetroxide-thiocarbohydrazide [OTOTO], and resin) in order to determine the method that best preserves the cell and cell interface morphology. Finally, to visualize the intracellular organelles and membrane, we compared the efficacy of four staining techniques: osmium tetroxide, osmium tetroxide and salts, osmium and uranyl acetate, and OTOTO. Experiments were performed on embryonic stem cell-derived photoreceptor precursors, neural cells, and a human retinal pigment epithelial cell line, which revealed that the optimal processing combination was resin drying and OTOTO staining, as manifested by preservation of cell morphology, the lowest percentage of cellular protrusion breakage as well as a high-quality image. The obtained results pave the way for better understanding the cell interface with various structures for enhancing various biomedical applications.

Use of scanning electron microscopy in the cochlea of guinea pigs.

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 (7.5mg/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.

A micro-CT-based method for quantitative brain lesion characterization and electrode localization.

Lesion verification and quantification is traditionally done via histological examination of sectioned brains, a time-consuming process that relies heavily on manual estimation. Such methods are particularly problematic in posterior cortical regions (e.g. visual cortex), where sectioning leads to significant damage and distortion of tissue. Even more challenging is the post hoc localization of micro-electrodes, which relies on the same techniques, suffers from similar drawbacks and requires even higher precision. Here, we propose a new, simple method for quantitative lesion characterization and electrode localization that is less labor-intensive and yields more detailed results than conventional methods. We leverage staining techniques standard in electron microscopy with the use of commodity micro-CT imaging. We stain whole rat and zebra finch brains in osmium tetroxide, embed these in resin and scan entire brains in a micro-CT machine. The scans result in 3D reconstructions of the brains with section thickness dependent on sample size (12-15 and 5-6 microns for rat and zebra finch respectively) that can be segmented manually or automatically. Because the method captures the entire intact brain volume, comparisons within and across studies are more tractable, and the extent of lesions and electrodes may be studied with higher accuracy than with current methods.

[The synoviorthesis: a reappraisal]. / Les synoviortheses: etat des lieux.

BACKGROUND: The evolution of the rheumatologic practice involved a handing-over in question of the place and methods of application of the synoviorthesis. The last innovations, in particular the appearance of the bio-therapies, allowed a better control of inflammatory rheumatism thus making it possible to better select arthritis likely to profit precociously from a synoviorthesis before the installation of major articular destruction. AIM: Through a general review of the literature, we recall in this work the various means of synoviorthesis, their current indications and their results. METHODS: An extensive electronic search of the relevant literature was carried out using MEDLINE. Key words used for the final search were: synoviorthesis, osmic acid, radiosynoviorthesis, arthritis, treatment. RESULTS: This systematic review allowed us to conclude that fields of application of the synoviorthesis is in addition widens because of the interesting results to see spectacular this technique in some other affections such as the haemophilia. In addition we have compared the efficiency and the tolerance of the different methods of synoviorthesis. CONCLUSION: The synoviorthesis constitutes a tempting therapeutic alternative of share its effectiveness and its good tolerance so much so that it constitutes an undeniable factor of articular protection. Its fields of application widened. Thus on the good knowledge of the indications and the precautions necessary to its realization its success.

Glândula submandibular de ratos com envelhecimento: observações ao microscópio eletrônico de varredura de alta resolução / Submandibular gland of rats with ageing: observations with high resolution scanning electron microscopy

As características tridimensionais dos componentes intracelulares de células acinares e de ductos foram reveladas usando o método ósmio-DMSO-ósmio. As amostras foram maceradas em solução de tetróxido de ósmio diluído após a fratura na solução de dimetil sulfoxido. As lamelas do retículo endoplasmático granular são reveladas entremeadas por várias mitocôndrias. As lamelas do retículo endoplasmático granular são localizados ao redor dos núcleos na porção basal e estas estruturas são observadas em imagens tridimensionais de microscopia eletrônica de alta resolução.

The three-dimensional characteristics of the intracellular components of acinar and ductal cells were revealed using the osmium-DMSO-osmium method. The samples were macerated in diluted osmium after fractured in DMSO solution. The stacks of the rough endoplasmic reticulum are revealed intermingling by several mitochondria. The lamellae of the rough endoplasmic reticulum are located around the nuclei at basal portion and these structures are shown in three-dimensional HRSEM images.

Joint aspiration and injection.

Joint aspiration/injection is an invaluable procedure for the diagnosis and treatment of joint disease. The knee is the commonest site to require aspiration although any non-axial joint is accessible for obtaining synovial fluid. Septic arthritis and crystal arthritis can be readily diagnosed by aspirating synovial fluid. Intra-articular injection of long-acting insoluble corticosteroids produces rapid resolution of inflammation in most injected joints and is a well established procedure in rheumatological practice. The technique involves only a knowledge of basic anatomy and should not be unduly painful for the patient. Provided sterile equipment and a sensible, aseptic approach are used it is a safe procedure. This chapter addresses the indications, technical principals, expected benefits and risks of intra-articular corticosteroid injection. The use of other intra-articular injections including osmic acid, radioisotopes and hyaluronic acid, which are less universally utilised than intra-articular corticosteroid, will also be addressed.

[Chemical synovectomy in arthritis by intra-articular injection of osmic acid].

18 patients suffering from persistent synovitis despite medical therapy were treated with an intra-articular injection of osmic acid. After 1 year of follow-up, 68% had good results and there were no complications nor detectable radiographic evidence of disease progression. Because osmic acid is almost as effective as surgical synovectomy and is cheap and easy to administer, it can be recommended as the first choice for treatment of corticosteroid-resistant arthritis in the early stages of the disease.

Comparison of the efficacy of nonsurgical synovectomy (synoviorthesis) and joint lavage in knee osteoarthritis with effusions.

OBJECTIVE: Radioactive or chemical synovectomy (synoviorthesis) is widely used as local therapy for inflammatory joint disease in France. The objective of this retrospective study was to compare the efficacy of osmic acid or radiation synovectomy with that of joint lavage for the treatment of knee osteoarthritis with effusions. PATIENTS AND METHODS: All study patients met American College of Rheumatology criteria for knee osteoarthritis, which was symptomatic despite conservative therapy including local corticosteroid injections. Fifty-four patients were treated by synoviorthesis (osmic acid, n = 16; yttrium 90, n = 76) and 45 by joint lavage (total 67 lavages). RESULTS: Thirty two per cent of the patients in the synoviorthesis group had a good or excellent outcome after six months. Results were better with yttrium 90 than with osmic acid. Improvements were most marked in patients with chondrocalcinosis. Efficacy was negatively correlated with the femorotibial lesions but not with the patellofemoral lesions. Patients with knee alignment disorders had poorer outcomes. In the joint lavage group, 30% of the knees showed improvements after three months and results were significantly better after three and six months when the lavage was followed by an injection of triamcinolone hexacetonide. No side effects were recorded. CONCLUSION: Our data suggest that chemical or radiation synovectomy or joint lavage followed by injection of a delayed-action steroid may be useful for the treatment of knee osteoarthritis with chronic or recurrent effusions.

[Intradiscal injection of hypertonic saline, phenol-glycerin and osmic acid for the treatment of lumbar disc herniation: an experimental study].

UNLABELLED: The present study was designed to investigate the possible clinical application of hypertonic saline (HS), phenol in glycerin (PHG) and osmic acid (OSA) for intradiscal therapy. MATERIALS & METHODS: HS in several concentrations, 10% PHG and 4% OSA were separately injected into the lumbar intervertebral discs of 60 Japanese white rabbits. Additionally, these substances were placed directly on the dura of the spinal cord of 48 guinea pigs. The animals were sacrificed periodically and were submitted to histological examination using light microscopy. RESULTS: HS caused localized necrosis of the nucleus pulposus cells in a concentration-related fashion. Some discs decreased their height. With time, all the discs generally regained their normal histology. Following administration of 10% PHG, the area of necrosis of the nucleus pulposus cells was more extensive than that by HS, but the regenerative or reparative reaction was not so brisk. Examination of the discs treated with 4% OSA demonstrated severe changes in the nucleus pulposus and the inner annulus fibrosus with resultant disc-space narrowing. The reparative tissue seen after injection of OSA was fibrocartilage in nature. No histological change was seen in the surrounding tissue including the neural tissue following administration of any of the substances. DISCUSSION: Chymopapain is the substance most frequently used for clinical chemonucleolysis. The major clinical complication with chymopapain has been anaphylaxis. The present substances have been used in other clinical applications without reports of anaphylaxis. In this report, HS was shown to hold the potential for reducing intradiscal pressure without induction of scar tissue or significant loss of disc function. PHG and OSA caused considerable but circumscribed histological damage to the disc tissue, but had no such effect on the neural tissues. These data suggested that HS, PHG and OSA may have clinical applications as agents in intradiscal therapy.

Effect of intraarticular osmic acid on synovial membrane volume and inflammation, determined by magnetic resonance imaging.

The changes in MR-determined synovial membrane volume, early synovial enhancement, and cartilage and bone erosions after osmic acid knee synovectomy were studied. Gadolinium-DTPA enhanced magnetic resonance imaging (MRI) of 18 knees with persistent arthritis was performed before and 1 month after treatment. The synovial membrane volume was significantly reduced (median -52%) in all 9 patients brought into clinical remission (p < 0.01), while no significant change was found in patients with clinical relapse. The early synovial enhancement was not significantly changed. MRI revealed progressive erosive changes in 2 patients. The time of relapse was correlated to a MR-erosion score, but not to early synovial enhancement or volumes of synovium or effusion (Spearman tests). MRI-determined synovial membrane volumes and early synovial enhancement may be objective quantitative markers of inflammation. MR-scores of cartilage and bone erosions are sensitive to progressive changes occurring within a month.

Intraarticular osmic acid in postinfectious arthritis persisting after treated bacterial arthritis.

We describe 4 patients successfully treated with intraarticular osmic acid for post-infectious arthritis persisting after apparent eradication of bacteria from the infected joint. In addition the bactericidal effect of osmic acid in vitro is demonstrated. We conclude that treatment with osmic acid carries no risk of iatrogenic infection; on the contrary, an antibacterial effect seems likely.

Knee arthroscopy after yttrium or osmic acid injection.

This study presents the macroscopic and histologic results of 35 knee arthroscopies performed on patients with rheumatoid arthritis, some months after an yttrium or osmic acid intraarticular injection. The procedure was most often performed after a failure of the injection or a relapse of synovitis. Arthroscopy provides an understanding of the cause of synoviorthesis failure--insufficient action of the product on the synovitis or its poor diffusion, fibri-nonecrotic deposits, or cartilaginous lesions--and may be used both diagnostically and therapeutically.

Protection of cartilage in chemical synovectomy by perfusion.

The authors examined the effect of osmium tetroxide (OsO4) on the synovial membrane and cartilage of the rat knee joints by light-, polarization-, and electron microscope. They found that the synovial membrane undergoes severe damage after a 10 min exposure to OsO4. Removing the OsO4 from the joint cavity by perfusion after the 10 min exposure, there was no concomitant damage to the cartilage. Based on experimental results, the authors recommend in human therapy the exposure of the joints to OsO4 for no longer than 10 min and complete removal by exhaustive perfusion.