Tissue Fixation Methods Research Articles

Investigating the microstructure of plant leaves in 3D with lab-based X-ray computed tomography

BackgroundLeaf cellular architecture plays an important role in setting limits for carbon assimilation and, thus, photosynthetic performance. However, the low density, fine structure, and sensitivity to desiccation of plant tissue has presented challenges to its quantification. Classical methods of tissue fixation and embedding prior to 2D microscopy of sections is both laborious and susceptible to artefacts that can skew the values obtained. Here we report an image analysis pipeline that provides quantitative descriptors of plant leaf intercellular airspace using lab-based X-ray computed tomography (microCT). We demonstrate successful visualisation and quantification of differences in leaf intercellular airspace in 3D for a range of species (including both dicots and monocots) and provide a comparison with a standard 2D analysis of leaf sections.ResultsWe used the microCT image pipeline to obtain estimates of leaf porosity and mesophyll exposed surface area (Smes) for three dicot species (Arabidopsis, tomato and pea) and three monocot grasses (barley, oat and rice). The imaging pipeline consisted of (1) a masking operation to remove the background airspace surrounding the leaf, (2) segmentation by an automated threshold in ImageJ and then (3) quantification of the extracted pores using the ImageJ ‘Analyze Particles’ tool. Arabidopsis had the highest porosity and lowest Smes for the dicot species whereas barley had the highest porosity and the highest Smes for the grass species. Comparison of porosity and Smes estimates from 3D microCT analysis and 2D analysis of sections indicates that both methods provide a comparable estimate of porosity but the 2D method may underestimate Smes by almost 50%. A deeper study of porosity revealed similarities and differences in the asymmetric distribution of airspace between the species analysed.ConclusionsOur results demonstrate the utility of high resolution imaging of leaf intercellular airspace networks by lab-based microCT and provide quantitative data on descriptors of leaf cellular architecture. They indicate there is a range of porosity and Smes values in different species and that there is not a simple relationship between these parameters, suggesting the importance of cell size, shape and packing in the determination of cellular parameters proposed to influence leaf photosynthetic performance.

Phagocytic intracellular digestion in amphioxus (Branchiostoma).

The digestive methods employed by amphioxus (Branchiostoma)—both intracellular phagocytic digestion and extracellular digestion—have been discussed since 1937. Recent studies also show that epithelial cells lining the Branchiostoma digestive tract can express many immune genes. Here, in Branchiostoma belcheri, using a special tissue fixation method, we show that some epithelial cells, especially those lining the large diverticulum protruding from the gut tube, phagocytize food particles directly, and Branchiostoma can rely on this kind of phagocytic intracellular digestion to obtain energy throughout all stages of its life. Gene expression profiles suggest that diverticulum epithelial cells have functional features of both digestive cells and phagocytes. In starved Branchiostoma, these cells accumulate endogenous digestive and hydrolytic enzymes, whereas, when sated, they express many kinds of immune genes in response to stimulation by phagocytized food particles. We also found that the distal hindgut epithelium can phagocytize food particles, but not as many. These results illustrate phagocytic intercellular digestion in Branchiostoma, explain why Branchiostoma digestive tract epithelial cells express typical immune genes and suggest that the main physiological function of the Branchiostoma diverticulum is different from that of the vertebrate liver.

Cryopreservation of Fish Spermatogonial Cells: The Future of Natural History Collections

As global biodiversity declines, the value of biological collections increases. Cryopreserved diploid spermatogonial cells meet two goals: to yield high-quality molecular sequence data; and to regenerate new individuals, hence potentially countering species extinction. Cryopreserved spermatogonial cells that allow for such mitigative measures are not currently in natural history museum collections because there are no standard protocols to collect them. Vertebrate specimens, especially fishes, are traditionally formalin-fixed and alcohol-preserved which makes them ideal for morphological studies and as museum vouchers, but inadequate for molecular sequence data. Molecular studies of fishes routinely use tissues preserved in ethanol; yet tissues preserved in this way may yield degraded sequences over time. As an alternative to tissue fixation methods, we assessed and compared previously published cryopreservation methods by gating and counting fish testicular cells with flow cytometry to identify presumptive spermatogonia A-type cells. Here we describe a protocol to cryopreserve tissues that yields a high percentage of viable spermatogonial cells from the testes of Asterropteryx semipunctata, a marine goby. Material cryopreserved using this protocol represents the first frozen and post-thaw viable spermatogonial cells of fishes archived in a natural history museum to provide better quality material for re-derivation of species and DNA preservation and analysis.

Brain perfusion fixation in male pigs using a safer closed system

Tissue fixation methods are well established for rodents, but not for large animals. We present a simple technique for in situ brain perfusion fixation in a male porcine model, using cervical vessels for inflow and outflow and achieving a closed system. Thirty-four pigs, aged 4.7 ± 0.6 months and weighing 60.7 ± 10.9 kg, were anaesthetised and mechanically ventilated. The ipsilateral common carotid artery and external jugular vein were dissected and constituted the inflow and outflow access, respectively. The brains were perfused and fixed in situ with heparinised saline followed by buffered formaldehyde. Then, specimens (brain, cerebellum and brainstem) were extracted and processed for histology. Fixative fluid leakage was avoided, achieving a closed system. This technique minimises the exposure to toxic chemicals such as formaldehyde and associated hazards (inherent toxicity, eye irritation), thereby increasing operators' safety. Perfusion was performed with a peristaltic pump for 20-30 minutes at an optimum rate of 0.20 l/min and required only 5 litres of the fixative. The specimens were sufficiently hardened to be extracted. High-quality tissues were available for histology analysis. This technique offers a user-friendly closed system for brain perfusion fixation which can be adapted for other tissues of the head, face and neck.

Exploration of optimal tissue fixation method for tissue-specific expression of plants

Exploration of optimal tissue fixation method for tissue-specific expression of plants

Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.

Objectives: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. Methods: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. Results: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. Conclusions: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.

Microarray comparative analysis of melanoma microRNA depending on the tissue fixation method

Cutaneous melanoma is the most aggressive form of skin cancer characterized by rapid progression, invasiveness and low survival rates. This explains the importance of optimization of early detection of this tumor. At the present time molecular and genetic methods of cancer investigation are broadly used. One of such methods is microarray that allows to analyze the expression of thousands genes in the single cell. However, this method makes high demands on the quality of samples and research procedure. Today the most common approach for tumor tissue preservation is formalin fixation followed paraffin embedding. This procedure is broadly used in clinical medicine but may affect the structure and availability of nucleic acids of the study sample. The present investigation was done to evaluate the effect of melanoma tissue fixation on the result of microRNA expression analysis by microarray. In this regard, we made comparative study of microRNA expression in melanoma samples fixed in formaldehyde and RNase inhibitor reagent based on ammonium sulfate solution. It has been shown that fixation by formalin as compared to ammonium salts based RNase inhibiting fixator alters the level of 454 microRNAs. Some miRNAs among them are playing an important role in the melanoma progression. Thus, molecular studies of skin tissues by microarray requires more thorough standardization of sample fixation methods or using of special RNA stabilizing solutions. The application of the formalin fixation method to preserve tissue for miRNA expression analysis requires further study.

Funnel‐freezing versus heat‐stabilization for the visualization of metabolites by mass spectrometry imaging in a mouse stroke model

Tissue preparation is the key to a successful matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) experiment. Rapid post-mortem changes contribute a significant challenge to the use of MSI approaches for the analysis of peptides and metabolites. In this technical note we aimed to compare the tissue fixation method ex-vivo heat-stabilization with in-situ funnel-freezing in a middle cerebral artery occlusion (MCAo) mouse model of stroke, which causes profound alterations in metabolite concentrations. The influence of the duration of the thaw-mounting of the tissue sections on metabolite stability was also determined. We demonstrate improved stability and biomolecule visualization when funnel-freezing was used to sacrifice the mouse compared with heat-stabilization. Results were further improved when funnel-freezing was combined with fast thaw-mounting of the brain sections.

Evidencia genética revela oportunidades de mejora para preparación de tejidos en análisis forenses

Introducción: los tejidos embebidos en parafina constituyen una excelente alternativa para obtener adn, especialmente cuando no es posible contar con muestras frescas o el almacenamiento y conservación del tejido no es viable, de manera que esta muestra es el único elemento disponible para realizar un cotejo. El éxito en cualquier análisis genético es contar con métodos de fijación de tejidos y de extracción de adn adecuados que permitan obtener moléculas de buena calidad y cantidad, libres de contaminantes biológicos, químicos y microbiológicos que pueden actuar como inhibidores en los procesos de amplificación por pcr. Métodos y materiales: en el laboratorio de genética del Instituto Nacional de Medicina Legal y Ciencias Forenses, Regional Suroccidente, Colombia, se realizó una prueba preliminar para la validación de la extracción de adn en tejidos embebidos en parafina, utilizando el método orgánico validado y empleado de rutina por ellaboratorio de genética de Bogotá, Colombia. Resultados y discusión: se corroboró la eficacia del método a partir del análisis de reproducibilidad, respetabilidad y concordancia en cuanto a concentración de adn, y obtención de perfiles genéticos a partir de los extractos de adn de cuatro réplicas de tres diferentes tipos de tejidos obtenidos a partir de un feto, con respecto a una muestra de sangre en soporte fta. Conclusiones: con los resultados preliminares obtenidos, se encontraron algunas oportunidades de mejora para los laboratorios que intervienen en el proceso.

P-007 The validation study of tissue specimen for limitation of endoscopic submucosal dissection indication criteria in gastric cancer

Introduction: Endoscopic submucosal dissection (ESD) is now accepted as curative treatment modality for early gastric cancer without lymph node metastasis. The indication criteria of ESD have been evaluated according to the size of lesion, depth of invasion and histology type. But there was recently controversy over limitation of ESD indication. It is not easy to validate size and depth of cancer lesion before ESD; and they are inaccurate according to tissue fixation method. The aim of this study is to validate tissue specimen by tissue fixation device according to variable resected tissue size. Methods: We made tissue fixation device for validation of ESD specimen. Two circular tissues were resected in the antrum and body of sixty porcine stomachs with 2.0cm/3.0cm/4.0cm diameter. One piece was inserted in a fixation device and the other was manually pinned on corkboard. Pressure gauge was used to measure resected specimen with equal pressure in every fixation device. After fixation for 24hrs, we examined the diameter and thickness of submucosa. Results: We validated the specimen size and depth with considering elasticity and eccentricity. The mean diameter was 23.76mm/ 32.55mm/ 44.67 mm in fixation device group and 21.50mm/ 31.61mm/ 44.20 in manual pinning group, respectively (2cm /3cm /4cm tissue). The mean thickness of submucosa using fixation device was 403.57 ± 84.40 µm/ 396.63 ± 102.51 µm/ 415.19 ± 70.79 µm and that by pinning manually was 403.57 ± 179.47 µm/ 555.04 ± 203.23 µm/ 684.93 ± 284.55 µm. (2cm /3cm /4cm tissues) Considering standard deviation, thickness in manual pinning group was much more dispersed than that in fixation device group (p value: 0.048/0.029/0.004, 2cm /3cm /4cm tissue, Fligner-Killeen test of homogeneity of variances). Conclusion: In various size resected tissues, the thickness of submucosa was relatively consistent in fixation device group, while it was much more variable in conventional fixation method group. Further study is needed for validation of early gastric cancer size related to indication of ESD.

Imaging and Mapping of Tissue Constituents at the Single-Cell Level Using MALDI MSI and Quantitative Laser Scanning Cytometry.

For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells-giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods.

Abstract 20433: Viral Myocarditis: Viral Genome Detection Yield on Endomyocardial Biopsy is Influenced by Methodological Factors

Introduction: Myocarditis is diagnosed on endomyocardial biopsy (EMB) by histological, immune-histochemical and molecular techniques to detect viral genomes. The role of EMB for the diagnosis and its contribution to patient management has been addressed by a Consensus document of American and European Cardiovascular Pathology Societies. Hypothesis: Different methods of tissue fixation and the number of myocardial samples could impact on viral genome yield on EMB. Methods: Consecutive EMBs referred at our Institution for histology/ immunohistochemistry evaluation in the time interval 1996-2012 were enrolled. Molecular investigation by RT-PCR and PCR technique was performed in all EMBs with inflammatory cardiomyopathy diagnosis at histology and immunohistochemistry. Results: A total of 467 EMBs have been diagnosed as myocarditis: 79 in pediatric (1 month to 18 years; 47M/ 32F) and 388 adult (19 to 75 years; 256M/132 F) patients (pts.). Viral etiology was identified in 28 (36%) pediatric and 101 (26%) adult pts, and the most prevalent type of viruses were enterovirus (8/28, 36%; 26/101, 24%). In a more recent subgroup of 137 EMBs (virus positive 30/137, 22%) seasonality, type of fixation and number of EMB samples were assessed. The seasonal distribution of myocarditis was higher in winter than in other seasons (33% vs. autumn 28%, spring 20% and summer 19%) without any significant difference in terms of virus positive EMBs. The number of EMB samples per pt. was ≤3 (either formalin or RNAlater) in 94/137 (69%) and >3 in 43/137 (31%), with a lower prevalence of virus positive in the former (17/94, 18% vs. 13/43, 30%). Eighty-one EMB samples were frozen while 56 were paraffin embedded, with a higher prevalence of viral genome in the former (26/81, 32% vs. 4/56, 7%; p=0.001). Conclusions: The diagnosis of myocarditis on EMB samples requires standardized protocols including molecular techniques. Viral genomes are identifiable in more than one third of pediatric and one fourth of adult cases. Methodological factors like the type of tissue fixation and the number of samples could impact on viral genome detection on EMB.

High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa.

We examined the rat and mouse epididymis using helium ion microscopy (HIM), a novel imaging technology that uses a scanning beam of He(+) ions to produce nanometer resolution images of uncoated biological samples. Various tissue fixation, sectioning and dehydration methods were evaluated for their ability to preserve tissue architecture. The cauda epididymidis was luminally perfused in vivo to remove most spermatozoa and the apical surface of the epithelial lining was exposed. Fixed epididymis samples were then subjected to critical point drying (CPD) and HIM. Apical stereocilia in principal cells and smaller apical membrane extensions in clear cells were clearly distinguishable in both rat and mouse epididymis using this technology. After perfusion with an activating solution containing CPT-cAMP, a permeant analog of cAMP, clear cells exhibited an increase in the number and size of membrane ruffles or microplicae. In contrast, principal cells did not exhibit detectable structural modifications. High-resolution HIM imaging clearly showed the ultrastructure of residual sperm cells, including the presence of concentric rings on the midpiece, and of cytoplasmic droplets in some spermatozoa. Close epithelium-sperm interactions were also detected. We found a number of sperm cells whose heads were anchored within the epididymal epithelium. In certain cases, the surface of the sperm cytoplasmic droplet was covered with vesicle-like structures whose size is consistent with that of epididymosomes. In conclusion, we describe here the first application of HIM technology to the study of the structure and morphology of the rodent epididymis. HIM technology represents a major imaging breakthrough that can be successfully applied to study the epididymis and spermatozoa, with the goal of advancing our understanding of their structure and function.

MECHANISMS OF AUTOPHAGY-MEDIATED AB SECRETION

We have recently shown that autophagy mediates the secretion of Aβ by studying autophagy-deficient APP transgenic (Tg) mice (Nilsson et al., Cell Reports). These findings have extended the previously established degradative role of autophagy in Aβ metabolism and add Aβ to the growing number of proteins secreted by autophagy. The genetic deletion of autophagy, obtained by conditional knockout (cKO) of autophagy-related protein 7 (Atg7), additionally led to accumulation of intracellular Aβ. Since the APP tg mice are based on APP overexpression, which may produce artifacts, our laboratory has developed a new generation of AD mouse models based on APP knock-in (KI) technology (Saito et al., unpublished). These mice exhibit endogenous levels of APP, but increased Aβ levels due to the introduced Swedish and Beyreuther mutations and develop Aβ plaque from 6 months of age. These mice will be used to confirm previous data obtained with APP Tg mice. In an attempt to elucidate the pathway of autophagy-mediated Aβ secretion, we used fluorescence and immunoelectron microscopy (IEM) to trace the intracellular Aβ accumulation that occurs in the autophagy deficient APP mice. By applying different tissue fixation methods we could establish protocols that allowed identification of Aβ in Golgi and in the multivesicular bodies (MVBs). To further confirm the role of autophagy in Aβ secretion, we generated autophagy-deficient APP-KI mice. Interestingly, we found that Aβ accumulated in Golgi upon deletion of autophagy. In contrast, the MVBs, which contain significant amounts of Aβ in the APP mice, exhibited drastically decreased Aβ levels in the autophagy-deficient APP mice. In agreement with the results obtained with the APP tg mice, the extracellular Aβ plaque load of autophagy-deficient APP-KI mice were drastically decreased. IEM data indicate that autophagy mediates transport of Aβ from Golgi and that secretion possibly occurs through MVBs. In addition, we could confirm that autophagy mediates the secretion of Aβ using a novel APP-KI mouse model without APP overexpression, which we use to further investigate the mechanisms of autophagy-mediated Aβ secretion.

Novel On‐Demand Bioadhesion to Soft Tissue in Wet Environments

Current methods of tissue fixation rely on mechanical-related technologies developed from the clothing and carpentry industries. Herein, a novel bioadhesive method that allows tuneable adhesion and is also applicable to biodegradable polyester substrates is described. Diazirine is the key functional group that allows strong soft tissue crosslinking and on-demand adhesion based on a free radical mechanism. Plasma post-irradiation grafting makes it possible to graft diazirine onto PLGA substrates. When the diazirine-PLGA films, placed on wetted ex vivo swine aortas, are activated with low intensity UV light, lap shear strength of up to 450 ± 50 mN cm(-2) is observed, which is one order of magnitude higher than hydrogel bioadhesives placed on similar soft tissues. The diazirine-modified PLGA thin films could be added on top of previously developed technologies for minimally invasive surgeries. The present work is focused on the chemistry, grafting, and lap shear strength of the alkyl diazirine-modified PLGA bioadhesive films.

Laser‐Capture Microdissection

Laser‐Capture Microdissection

Experimental model for the study of soft tissue fixation methods on skin-pericranium flaps in rats

To develop an experimental model to study and radiologically monitor displacement of skin flaps in the pericranium of rats subjected to traction and surgical fixation using suture anchored in a skull bone tunnel or with N-butyl-2-cyanoacrylate (Histoacryl™) surgical adhesive. Radiological markers were placed in the subcutis of Wistar rats undergoing subperiosteal detachment of the pericranium with pulling and fixation of the flap. We performed radiography on postoperative days 3, 7, 14, 21, and 45. A p-value of <0.05 was considered significant. Qualitative analysis of the data indicated that the flaps in the surgical adhesive group remained in place with no change from the immediate postoperative position. However, the flaps in the suture anchored in the skull bone tunnel group and in the control group showed similar healing, with a loss of attachment of 9.7% and 22%, respectively, compared with the immediate postoperative position. There was no quantitative difference between the groups. This experimental model created acceptable experimental conditions for testing different soft tissue fixation methods. The use of tissue fixatives contributed to better placement of pericranium-cutaneous flaps, and surgical adhesive was superior to suture anchor in the skull bone tunnel for fixation of pericranium-cutaneous flaps.

Optical parameters of the tunable Bragg reflectors in squid

Cephalopods (e.g. octopus, squid and cuttlefish) dynamically tune the colour and brightness of their skin for camouflage and communication using specialized skin cells called iridocytes. We use high-resolution microspectrophotometry to investigate individual tunable Bragg structures (consisting of alternating reflectin protein-containing, high-refractive index lamellae and low-refractive index inter-lamellar spaces) in live and chemically fixed iridocytes of the California market squid, Doryteuthis opalescens. This subcellular, single-stack microspectrophotometry allows for spectral normalization, permitting use of a transfer-matrix model of Bragg reflectance to calculate all the parameters of the Bragg stack-the refractive indices, dimensions and numbers of the lamellae and inter-lamellar spaces. Results of the fitting analyses show that eight or nine pairs of low- and high-index layers typically contribute to the observed reflectivity in live cells, whereas six or seven pairs of low- and high-index layers typically contribute to the reflectivity in chemically fixed cells. The reflectin-containing, high-index lamellae of live cells have a refractive index proportional to the peak reflectivity, with an average of 1.405 ± 0.012 and a maximum around 1.44, while the reflectin-containing lamellae in fixed tissue have a refractive index of 1.413 ± 0.015 suggesting a slight increase of refractive index in the process of fixation. As expected, incremental changes in refractive index contribute to the greatest incremental changes in reflectivity for those Bragg stacks with the most layers. The excursions in dimensions required to tune the measured reflected wavelength from 675 (red) to 425 nm (blue) are a decrease from ca 150 to 80 nm for the high-index lamellae and from ca 120 to 50 nm for the low-index inter-lamellar spaces. Fixation-induced dimensional changes also are quantified, leading us to suggest that further microspectrophotometric analyses of this iridocyte system can be used as a model system to quantify the effects of various methods of tissue fixation. The microspectrophotometry technique described can be expected to provide deeper insights into the molecular and physical mechanisms governing other biophotonically active cells and structures.

Porcine intestinal mast cells. Evaluation of different fixatives for histochemical staining techniques considering tissue shrinkage

Staining of mast cells (MCs), including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF) has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkagedifferences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from data using different fixation techniques can only be compared if the respective studyimmanent shrinkage factor has been determined and quantification results are adjusted accordingly.

Arthroscopic Capsulolabral Reconstruction for Posterior Instability of the Shoulder

Background: There are few reports in the literature detailing the arthroscopic treatment of unidirectional posterior shoulder instability. Hypothesis: Arthroscopic capsulolabral reconstruction is effective in restoring stability and function and alleviating pain in athletes with symptomatic unidirectional posterior instability. Study Design: Cohort study; Level of evidence, 2. Methods: One hundred eighty-three athletes (200 shoulders) with unidirectional recurrent posterior shoulder instability were treated with arthroscopic posterior capsulolabral reconstruction and underwent an evaluation at a mean of 36 months postoperatively. A subset of 117 shoulders of contact athletes was compared with the entire group of 200 shoulders. Patients were evaluated prospectively with the American Shoulder and Elbow Surgeons (ASES) scoring system. Stability, strength, and range of motion were evaluated preoperatively and postoperatively with standardized subjective scales. Methods of intraoperative soft tissue fixation as well as anchorless (n = 44) and anchored (n = 156) plications were recorded for each patient. Results: At a mean of 36 months postoperatively, the mean ASES score improved from 45.9 to 85.1 (P < .001). There were also significant improvements in stability, pain, and function based on previously used scales (P < .001). The contact athletes did not demonstrate any significant differences when compared with the entire cohort for any outcome measure. With regard to the method of internal fixation, patients who underwent capsulolabral plications with suture-anchors showed significantly greater improvement in ASES scores (P < .001) and a higher rate of return to play (P < .05) when compared with patients with anchorless capsulolabral plications. Conclusion: Arthroscopic capsulolabral reconstruction is an effective, reliable treatment for symptomatic, unidirectional recurrent posterior glenohumeral instability in an athletic population. Overall, 90% of patients were able to return to sport, with 64% of patients able to return to the same level postoperatively. With the incorporation of bone suture-anchors in capsulolabral reconstruction, patients had greater improvements in ASES scores and a higher rate of return to play.