Good Morphological Preservation Research Articles

Long-Term Porcine Retina Explants as an Alternative to InVivo Experimentation.

The porcine retina represents an optimal model system to study treatment approaches for inherited retinal dystrophies owing to close anatomical similarities to the human retina, including a cone enriched visual streak. The aim of this work was to establish a protocol to keep explants in culture for up to 28days with good morphological preservation. Two to four retina explants per eye were obtained from the central part of the retina and transferred onto a membrane insert with the photoreceptors facing down. Different medium compositions using Neurobasal-A medium containing 100 or 450mg/dL glucose and combinations of fetal calf serum, B-27 with or without insulin and N-2 were tested. We developed a tissue quality score with robust markers for different retinal cell types (protein kinase C alpha, peanut agglutinin and 4',6-diamidino-2-phenylindol). Retinae were kept until 28days with only little degradation. The best results were attained using Neurobasal-A medium containing 100mg/dL glucose supplemented with B-27 containing insulin and N-2. For an easy preparation process, it is necessary to minimize transport time and keep the eyes on ice until dissected. Heat-mediated decontamination by the butcher has to be avoided. Using a standardized protocol, porcine retina explants represent an easy to handle intermediate model between in vitro and in vivo experimentation. This model system is robustly reproducible and contributes to the implementation of the 3R principle to minimize animal experimentation. This model can be used to test future therapeutic approaches for inherited retinal dystrophies.

Cryo-fluorescence micro-optical sectioning tomography for volumetric imaging of various whole organs with subcellular resolution

SummaryOptical visualization of complex microstructures in the entire organ is essential for biomedical research. However, the existing methods fail to accurately acquire the detailed microstructures of whole organs with good morphological and biochemical preservation. This study proposes a cryo-fluorescence micro-optical sectioning tomography (cryo-fMOST) to image whole organs in three dimensions (3D) with submicron resolution. The system comprises a line-illumination microscope module, cryo-microtome, three-stage refrigeration module, and heat insulation device. To demonstrate the imaging capacity and wide applicability of the system, we imaged and reconstructed various organs of mice in 3D, including the healthy tongue, kidney, and brain, as well as the infarcted heart. More importantly, imaged brain slices were performed sugar phosphates determination and fluorescence in situ hybridization imaging to verify the compatibility of multi-omics measurements. The results demonstrated that cryo-fMOST is capable of acquiring high-resolution morphological details of various whole organs and may be potentially useful for spatial multi-omics.

Time-related changes in cell morphology and biomarker immunoreactivity for cells stored in a buffer-based cell medium.

Buffer-based cell media (BBCM) are a valuable tool in the post-collection processing of cytology samples, though with poorly defined effects on cell properties. In this study, time-related changes in cell morphology and biomarker immunoreactivity were evaluated for cells stored at room temperature in a BBCM prepared with bovine serum albumin (BSA) and ethylene diamine tetraacetic acid (EDTA). Cytospins were prepared at five consecutive 24-hour intervals (0, 24, 48, 72, 96) from three human cell lines (MCF7, SK-MEL-28, FaDu) suspended and stored in BBCM. Preservation of cell morphology was evaluated on Papanicolaou-stained cytospins from the percentages of apoptotic cells. Preservation of immunoreactivity was evaluated for cytokeratins, oestrogen receptors, Ki67, and melanoma markers from the percentages of cells positive for the corresponding immunocytochemical reactions. Cell morphology was well preserved for the majority of cells of the three lines stored for 24 and 48hours (93%, 97%, 98% and 62%, 81%, 88%, respectively), while the majority of cells were apoptotic after 72 and 96hours (70%, 47%, 39% and 77%, 70%, 59%, respectively). The immunoreactivity of cytokeratins remained unchanged during the entire 96hours, while that of melanoma markers (S100, HMB45, Melan-A) decreased by 27%, 2%, and 3%, respectively. The immunoreactivity of oestrogen receptors and Ki67 decreased by 29% and 17% after the first 24hours, and was completely lost after 96hours. A BBCM with the addition of BSA and EDTA facilitates good preservation of cell morphology and immunoreactivity of biomarkers for up to 48hours at room temperature.

Iterative envelope mean fractal dimension filter for the separation of crackles from normal breath sounds

This paper presents a new method of separating pulmonary crackles from normal breath sounds: the iterative envelope mean fractal dimension (IEM-FD) filter. Crackles are an important physiological parameter for evaluating lung condition of an individual and their automatic separation from normal breath sounds can provide an objective way of diagnosing or monitoring different cardiopulmonary diseases. The filter combines the new iterative envelope mean (IEM) method with the established fractal dimension (FD) technique. The IEM method estimates the non-stationary and stationary parts of the lung sound signal and then the FD technique is applied to the estimated non-stationary output of the IEM method for further refining the separation process. The IEM-FD filter is tested using a publicly available dataset and, compared with an established crackle separation technique. The IEM-FD achieves high accuracy for crackle detection in the presence of noise with SNR >= -1 dB for fine crackles and SNR > +1 dB for coarse crackles, and has low computational cost, with minimal under- or over- estimation and good preservation of crackle morphology. The method is shown to have an overall performance suitable for automated analysis to determine accurately the number and characteristics of pulmonary crackles in a recorded lung sound.

Cryo-Gel embedding compound for renal biopsy biobanking

Optimal preservation and biobanking of renal tissue is vital for good diagnostics and subsequent research. Optimal cutting temperature (OCT) compound is a commonly used embedding medium for freezing tissue samples. However, due to interfering polymers in OCT, analysis as mass spectrometry (MS) is difficult. We investigated if the replacement of OCT with Cryo-Gel as embedding compound for renal biopsies would enable proteomics and not disturb other common techniques used in tissue diagnostics and research. For the present study, fresh renal samples were snap-frozen using Cryo-Gel, OCT and without embedding compound and evaluated using different techniques. In addition, tissue samples from normal spleen, skin, liver and colon were analyzed. Cryo-Gel embedded tissues showed good morphological preservation and no interference in immunohistochemical or immunofluorescent investigations. The quality of extracted RNA and DNA was good. The number of proteins identified using MS was similar between Cryo-Gel embedded samples, samples without embedding compound and OCT embedded samples. However, polymers in the OCT disturbed the signal in the MS, while this was not observed in the Cryo-Gel embedded samples. We conclude that embedding of renal biopsies in Cryo-Gel is an excellent and preferable alternative for OCT compound for both diagnostic and research purposes, especially in those cases where proteomic analysis might be necessary.

Comparing Different Cross-Section Cutting Methods for SEM Analysis of Membrane-Electrodes Assemblies

The observation of membrane-electrodes assemblies (MEAs) in cross-section is a basic tool for their morphological characterization, and the study of changes occurring in the different layers by operation in a fuel cell. But the preparation of a well-defined and representative cross-section plane in the MEAs is difficult due to a layered structure with very different mechanical properties among the layers, including a Nafion membrane, two catalyst layers, and two gas diffusion layers. For this reason, the preparation of samples for cross-sectional observation requires appropriate cutting techniques able to leave a cross-sectional plane showing the morphology of each layer with minor changes, ie. avoiding uneven surfaces, mixing of layers, porosity and morphology alterations, and/or delamination. It is possible that no single method may accomplish with all requirements, so different cross sectioning procedures must be used to have a complete characterization. With this aim, different preparation methods for cross-section analysis of MEAs have been tested in order to make a comparative analysis, showing advantages and disadvantages for each of the methods. The preparation procedures analyzed are cutting with sharp-edge, laser-cutting, embedded-mechanical polishing, and focused ion beam (FIB). Results are shown in the figure. Cutting with a sharp edge (Fig. a) is a quick and easy method that may provide good cross-sectional observation and morphology preservation if an appropriate tool is used and careful handling. However, the probability for a successful preparation is not high due to frequent intermixing of layers, delamination, and uneven surface. Laser-cutting with a CO2 laser (Fig. b)) occurs by burning and vaporization of the material, leaving a plane with a clear damage at a micrometric scale. Especially Nafion membranes and carbon clothes showed severe alteration of the phases in the cutting plane, so this method cannot be used for a micrometric study of the morphology of the layers. Mechanical polishing of an embedded MEA a resin (Fig.c) provides, on the other hand, better preserved layers and more reproducible results; the morphology of powder layers, like the catalyst layer and microporous layer, may be altered because of the rearrangement of particles during the polishing process. Better preservation of multilayered samples can be obtained with the FIB technique (Fig.d), although the observation profile cannot be extended beyond a few millimeters. The FIB cross-cutting technique, that may be combined with a cross section polishing with argon gas, appears to better preserve the morphology of the layers and in a reproducible way. Acknowledgements. This work was supported by the Ministerio de Economía y Competitividad of Spain, and Fondo Europeo de Desarrollo Regional (FEDER), Project E-LIG-E, ENE2015-70417-P (MINECO/FEDER). Figure 1

Oxygenated UW Solution Decreases ATP Decay and Improves Survival After Transplantation of DCD Liver Grafts

Donation after circulatory death (DCD) liver grafts are known to be predisposed to primary nonfunction and ischemic cholangiopathy. Many DCD grafts are discarded because of older donor age or long warm ischemia times. Thus, it is critical to improve the quality of DCD liver grafts. Here, we have tested whether an enriched oxygen carrier added to the preservation solution can prolong graft survival and reduce biliary damage. We assessed the adenosine triphosphate (ATP) content decay of mouse liver grafts after cold ischemia, warm ischemia, and combined warm+cold ischemia. In addition, we used a rat model of liver transplantation to compare survival of DCD grafts preserved in high-oxygen solution (preoxygenated perfluorocarbon [PFC] + University of Wisconsin [UW] solution) versus lower oxygen solution (preoxygenated UW solution). Adenosine triphosphate levels under UW preservation fall to less than 10% after 30 minutes of warm ischemia. Preoxygenated UW solution with PFC reached a significantly higher PaO2. After 45 minutes of warm ischemia in oxygenated UW + PFC solution, grafts showed 63% higher levels of ATP (P = 0.011). In addition, this was associated with better preservation of morphology when compared to grafts stored in standard UW solution. Animals that received DCD grafts preserved in higher oxygenation solution showed improved survival: 4 out of 6 animals survived long-term whereas all control group animals died within 24 hours. The additional oxygen provided by PFC during static cold preservation of DCD livers can better sustain ATP levels, and thereby reduce the severity of ischemic tissue damage. PFC-based preservation solution extends the tolerance to warm ischemia, and may reduce the rate of ischemic cholangiopathy.

Performance of 4 Immunohistochemical Phosphohistone H3 Antibodies for Marking Mitotic Figures in Breast Cancer.

Phosphohistone H3 (PHH3) has been suggested to facilitate and improve mitotic activity assessment in breast cancer and other tumor entities, but the reliability of respective immunohistochemical antibodies has not yet been compared for routine purposes. Our aim was to test the performance of 4 different PHH3 antibodies on a series of highly proliferating breast cancers with good preservation of morphology. Four commercially available PHH3 antibodies were tested on 9 grade 3 invasive breast cancers processed in the same batch. We analyzed the number of antibody stained and nonstained mitotic figures as well as the total of cells observed in 10 high power fields per tumor to calculate sensitivity, specificity, and accuracy of the respective antibodies for staining mitotic figures, taking morphologically defined mitotic figures as gold standard. Sensitivity, specificity, and accuracy of the respective PHH3 antibodies for staining mitotic figures were 54.51%, 99.98%, and 98.79% for Cell Marque, 87.48%, 67.62%, and 67.47% for Epitomics, 98.62%, 99.73%, and 99.49% for Merck 06-570, and 99.74%, 99.52%, and 99.51% for Merck 09-797, respectively. Sensitivity was lowest for telophase. In statistical analysis, the Cell Marque antibody demonstrated significantly lower sensitivity and Epitomics substantially lower sensitivity and specificity than Merck 06-570 and Merck 09-797 antibodies (P<0.0001, respectively). Performance and reliability varied significantly between the 4 tested antibodies. For faster identification of mitotic hot spots and as potential marker in digital image analysis, the Merck antibodies seem to be most suitable.

PAXgene-fixed paraffin-embedded sample is applicable to laser capture microdissection with well-balanced RNA quality and tissue morphology.

Assessing how gene expression analysis by RNA sequencing (RNA-Seq) correlates to a unique morphology is increasingly necessary, and laser capture microdissection (LCM) is a critical research tool for discovering the genes responsible in a region of interest (ROI). Because RNA-Seq requires high-quality RNA, a sample preparation procedure that can preserve morphology and give the required quality of RNA is essential. A PAXgene®-fixed paraffin-embedded (XFPE) block can satisfy the need for high-quality RNA, but there are few reports on adapting the method for LCM, such as how small an ROI is analyzable by RNA-Seq. In this study, we confirmed the morphology and preservation of RNA in XFPE and then assessed the relationship between the size of pieces cut by LCM and their RNA quality. In XFPE, the morphology was similar to that in alcohol-based fixed samples, the quality of the RNA extracted from a whole sample was excellent, that is equivalent to that of a fresh frozen sample, and the quality was maintained over one year later. Three sizes of pieces—large (25,000 µm2), medium (5,000 µm2), and small (1,000 µm2)—were cut by LCM so that the total areas of the sections cut per size were the same. RNA quality was found to be best preserved when tissue was cut into pieces of over 5,000 µm2. In summary, XFPE exhibits good morphology and excellent preservation of RNA quality. Furthermore, it can be a good tool when used with LCM and RNA-Seq, giving well-balanced RNA quality and tissue morphology in the ROI.

How to obtain good morphology and antigen detection in the same tissue section?

Most human and animal biopsy samples are routinely embedded in paraffin since this enables the pathologist or researcher to obtain excellent morphology and simplifies storage. Nevertheless, in many cases, the antigen of interest cannot be detected in paraffin section. The alternative available for good immunohistochemistry is preparation of cryosections, which usually provide decent antigen preservation and are frequently used for immunofluorescence. However, cryosections often do not provide efficient morphological details of tissues and cells for pathologic evaluation. In order to obtain good antigen preservation and improve tissue and cell morphology after freezing, we tested three different fixations and freezing methodologies and compared them to routine formaldehyde fixation and paraffin embedding. As a model system, we selected the epithelium of the rat urinary bladder and trachea. On all samples, haematoxylin and eosin staining was performed as well as immunofluorescence with antibodies against tight junction protein ZO-1 and against intermediate filament cytokeratin 7. The best compromise between morphology and immunofluorescence was obtained with "sucrose impregnation prior to freezing" method. Moreover, this procedure is also quicker in comparison to standard paraffin section preparation. To check the clinical relevance of our study, this method was used for human biopsy samples of neoplastic urothelial and bronchial mucosa lesions. Besides good immunofluorescence results, the morphology of these samples was well preserved. We therefore propose that cryosection preparation with sucrose impregnation prior to freezing should be further exploited in other clinical and veterinary applications, since it enables good morphology and antigen preservation.

Ultrathin Colloidal Cesium Lead Halide Perovskite Nanowires.

Highly uniform single crystal ultrathin CsPbBr3 nanowires (NWs) with diameter of 2.2 ± 0.2 nm and length up to several microns were successfully synthesized and purified using a catalyst-free colloidal synthesis method followed by a stepwise purification strategy. The NWs have bright photoluminescence (PL) with a photoluminescence quantum yield (PLQY) of about 30% after surface treatment. Large blue-shifted UV-vis absorption and PL spectra have been observed due to strong two-dimensional quantum confinement effects. A small angle X-ray scattering (SAXS) pattern shows the periodic packing of the ultrathin NWs along the radial direction, demonstrates the narrow radial distribution of the wires, and emphasizes the deep intercalation of the surfactants. Despite the extreme aspect ratios of the ultrathin NWs, their composition and the resulting optical properties can be readily tuned by an anion-exchange reaction with good morphology preservation. These bright ultrathin NWs may be used as a model system to study strong quantum confinement effects in a one-dimensional halide perovskite system.

Organotypic slice cultures of human gastric and esophagogastric junction cancer.

Gastric and esophagogastric junction cancers are heterogeneous and aggressive tumors with an unpredictable response to cytotoxic treatment. New methods allowing for the analysis of drug resistance are needed. Here, we describe a novel technique by which human tumor specimens can be cultured ex vivo, preserving parts of the natural cancer microenvironment. Using a tissue chopper, fresh surgical tissue samples were cut in 400 μm slices and cultivated in 6‐well plates for up to 6 days. The slices were processed for routine histopathology and immunohistochemistry. Cytokeratin stains (CK8, AE1/3) were applied for determining tumor cellularity, Ki‐67 for proliferation, and cleaved caspase‐3 staining for apoptosis. The slices were analyzed under naive conditions and following 2–4 days in vitro exposure to 5‐FU and cisplatin. The slice culture technology allowed for a good preservation of tissue morphology and tumor cell integrity during the culture period. After chemotherapy exposure, a loss of tumor cellularity and an increase in apoptosis were observed. Drug sensitivity of the tumors could be assessed. Organotypic slice cultures of gastric and esophagogastric junction cancers were successfully established. Cytotoxic drug effects could be monitored. They may be used to examine mechanisms of drug resistance in human tissue and may provide a unique and powerful ex vivo platform for the prediction of treatment response.

Enhancement of Human Adipose-Derived Stem Cell Expansion and Stability for Clinical use

Co-culture techniques associating both dermal fibroblasts and epidermal keratinocytes have shown to have better clinical outcome than keratinocyte culture alone for the treatment of severe burns. Since fat grafting has been shown to improve scar remodelling, new techniques such as cell-therapy-assisted surgical reconstruction with isolated and expanded autologous adipose-derived stem cells (ASCs) would be of benefit to increase graft acceptation. Therefore, integrating ASCs into standardized procedures for cultured skin grafting could be of benefit for the patient if cell quality and quantity could be maintained. The purpose of this study was to evaluate ASC processing from adult tissue with simple isolation (without enzymatic steps), expansion (low density of 325-3,000 cells/cm 2 ) and storage conditions to assure methods to enhance the cellular resistance when transferred back to the patient. Co-culture with cell-banked skin progenitor cells (FE002-SK2) showed an increase of 40-50% ASCs yield at high passages alongside with a better preservation of morphology, proper adipogenic and osteogenic differentiation and efficient biocompatibility with 3D collagen scaffolds. ASCs can be considered as a valuable additional cell source to be delivered in biological bandages to the patient in a need of tissue reconstruction such as burn patients.

Organotypic slice cultures of human gastric cancer (GC) and esophagogastric junction adenocarcinoma (AEG): A new technology to study treatment response, resistance, and tumor heterogeneity.

76 Background: GC and AEG have an unpredictable response to cytotoxic treatment and a poor prognosis. There is an urgent need for new research methods allowing for the determination of chemotherapy sensitivities, the analysis of resistance mechanisms and tumor heterogeneity. Here, we describe a novel technique extending our recent findings in other tumors (Gerlach et al. 2014; Merz et al. 2013), by which cancer specimens can be cultured in vitro and maintained in their natural micro-environment. Methods: Using a tissue chopper, fresh surgical and endoscopic tissue samples from GC and AEG were cut in 400 µm thick slices and cultivated in 6-well plates for up to 6 days. The slices were then fixed, embedded in paraffin and cut for routine histopathology and immunohistochemistry. Cytokeratin stains (CK8 and AE1/3) were used for determining tumor cellularity, ki-67 for proliferation, and cleaved caspase 3 staining for apoptosis. The slices were examined under naïve condition and following in-vitro exposure to 5-FU, cisplatin or docetaxel over a period of 2-4 days. Results: GC and AEG slice cultures from resection specimens (n=14) and endoscopic biopsies (n=17) revealed a good preservation of tissue morphology and tumor cell integrity during the culture period in most cases. The stroma and the tumor cellularity remained stable over at least 4 days, proving the viability of cancer in slice cultures. The amount of sampled tissue from endoscopic biopsies was identified as a critical determinant for the feasibility of slice cultures. During treatment of cultures with chemotherapy, a significant loss of tumor cellularity and an increase of apoptotic cells were observed, although a systematic and reproducible read-out still needs to be established. Conclusions: Slice cultures of GC and AEG were successfully established. They can be expected to provide a unique and powerful in vitro platform for the determination of sensitivities of a given tumor towards chemotherapy, to examine mechanisms of drug-resistance and to analyze tumor heterogeneity in patient samples.

An effective and practical immunohistochemical protocol for bone specimens characterized by hyaluronidase and pepsin predigestion combined with alkaline phosphatase-mediated chromogenic detection.

The aim of this study was to provide an effective procedure for immunohistochemistry (IHC) investigations of bone specimens. Samples from rat femoral and human vertebral bone were processed with a detailed and effective IHC protocol summarized here. First, a novel antigen retrieval (AR) method of hyaluronidase combined pepsin predigestion (H+P) was established and the optimal concentration and pH value for AR of bone specimens were determined. Second, the newly developed method was compared with existing AR methods (boiling in sodium citrate, hyaluronidase predigestion (H) and pepsin predigestion (P), with PBS only as the negative control) using two chromogenic detection systems (horseradish peroxidase (HRP) and alkaline phosphatase (AP)) to evaluate their efficacy in obtaining the best IHC results for bone samples. Considering the drawbacks of significant shrinking and detachment from slide for heat retrieval methods and the only moderate immunolabeling for H and P, H+P was the optimal AR method for IHC of bone specimens with the advantages of both good morphological preservation and strong immunoreactivity. Moreover, AP-mediated chromogenic detection was superior to HRP-labeled chromogenic detection due to significantly less non-specific staining. In conclusion, we presented an effective and practical IHC protocol for bone specimens characterized by H+P predigestion combined with AP-mediated chromogenic detection. Finally, a detailed troubleshooting guide was provided for common mistakes that occur during IHC processing of the bone tissue samples.

Detection of replication sites in plant cell nuclei by using semithin sections

A novel approach for detection of replication sites in plant cells nuclei is described. The included nucleotide (EdU) was revealed by using “click” chemistry in semithin sections of the material embedded in acrylic resin. The use of the proposed protocol allows (1) to achieve good preservation of cell morphology, (2) to work with any tissues, and (3) to obtain high resolution during microscopy (especially, axial).

The practicalities of using tissue slices as preclinical organotypic breast cancer models

Models considering breast cancer complexity cannot be easily or accurately replicated in routine cell line or animal models. We aimed to evaluate the practicality of organotypic tissue slice culture in...

Sexing of Dog Sperm by Fluorescence &lt;i&gt;In Situ&lt;/i&gt; Hybridization

Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.

The Non-Crosslinking Fixative RCL2®-CS100 is Compatible with Both Pathology Diagnosis and Molecular Analyses

Formalin is the key agent for tissue fixation and pathological diagnosis. However, it poorly preserves nucleic acids and this can impair molecular studies. An alternative to formalin would be a fixative which can allow both morphologic and molecular analyses. To assess the suitability of such a fixative, breast (n = 11) and colon (n = 12) tumor samples were fixed in the non cross-linking RCL2®-CS100 fixative and compared to paired formalin-fixed and to frozen samples, the current standards for histology and molecular analyses, respectively. Sections from RCL2®-CS100-fixed samples showed good preservation of cellular and architectural morphology, suitable for routine diagnosis. Although some antibodies required change in the immunohistochemical procedures, quality of the immunohistochemical staining was comparable to that obtained after formalin fixation. HER2 chromogenic in situ hybridization was also successfully performed. High quality DNA could be isolated from RCL2®-CS100-fixed cancer tissues as evidenced by successful amplification of large DNA fragment, CGH array, KRAS and microsatellites genotyping. The quality of RNA from RCL2®-CS100-fixed samples was slightly decreased in comparison to that of RNA isolated from frozen samples, as evidenced by a decreased RNA integrity number but remained exploitable for molecular assays. Our results support the use of the RCL2®-CS100 fixative for histological diagnosis and recovery of high-quality nucleic acids for molecular applications. However, specific procedures for tissue handing and processing, essential to provide high-quality specimens, could limit its use to small target lesions which cannot be frozen without impairing their pathological evaluation.

Histopathological analysis of a 15‐year user of an auditory brainstem implant

Auditory brainstem implants (ABIs) can provide highly beneficial hearing sensations to individuals deafened by bilateral vestibular schwannomas (neurofibromatosis type 2). Relatively little is known about the status of stimulated neurons after long-term ABI use. Direct examination of the cochlear nuclear complex (CN) of one 5-year ABI user indicated no deleterious effect. Recently, we examined the brainstem of a patient who used his ABI daily for 15 years with excellent performance. There was good preservation of CN cell size, morphology, and packing density, a very favorable sign considering that a number of infants are now receiving ABIs.