Phosphotungstic Acid Staining Research Articles

Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy.

Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Visualization of rat tendon in three dimensions using micro-Computed Tomography

Micro-computed tomography (CT) is an X-ray-based imaging modality that produces three-dimensional (3D), high-resolution images of whole-mount tissues, but is typically limited to dense tissues, such as bone. The X-rays readily pass-through tendons, rendering them transparent. Contrast-enhancing chemical stains have been explored, but their use to improve contrast in different tendon types and across developmental stages for micro-CT imaging has not been systematically evaluated. Therefore, we investigated how phosphotungstic acid (PTA) staining and tissue hydration impacts tendon contrast for micro-CT imaging. We showed that PTA staining increased X-ray absorption of tendon to enhance tissue contrast and obtain 3D micro-CT images of immature (postnatal day 21) and sexually mature (postnatal day 50) rat tendons within the tail and hindlimb. Further, we demonstrated that tissue hydration state following PTA staining significantly impacts soft tissue contrast. Using this method, we also found that tail tendon fascicles appear to cross between fascicle bundles. Ultimately, contrast-enhanced 3D micro-CT imaging will lead to better understanding of tendon structure, and relationships between the bone and soft tissues.•Simple tissue fixation and staining technique enhances soft tissue contrast for tendon visualization using micro-CT.•3D tendon visualization in situ advances understanding of musculoskeletal tissue structure and organization.

Transmission electron microscopy of dermal collagen using ethanolic phosphotungstic acid staining

Transmission electron microscopy of dermal collagen using ethanolic phosphotungstic acid staining

X-ray micro-computed tomography of Xenopus tadpole reveals changes in brain ventricular morphology during telencephalon regeneration.

Xenopus tadpoles serve as an exceptional model organism for studying post-embryonic development in vertebrates. During post-embryonic development, large-scale changes in tissue morphology, including organ regeneration and metamorphosis, occur at the organ level. However, understanding these processes in a three-dimensional manner remains challenging. In this study, the use of X-ray micro-computed tomography (microCT) for the three-dimensional observation of the soft tissues of Xenopus tadpoles was explored. The findings revealed that major organs, such as the brain, heart, and kidneys, could be visualized with high contrast by phosphotungstic acid staining following fixation with Bouin's solution. Then, the changes in brain shape during telencephalon regeneration were analyzed as the first example of utilizing microCT to study organ regeneration in Xenopus tadpoles, and it was found that the size of the amputated telencephalon recovered to >80% of its original length within approximately 1 week. It was also observed that the ventricles tended to shrink after amputation and maintained this state for at least 3 days. This shrinkage was transient, as the ventricles expanded to exceed their original size within the following week. Temporary shrinkage and expansion of the ventricles, which were also observed in transgenic or fluorescent dye-injected tadpoles with telencephalon amputation, may be significant in tissue homeostasis in response to massive brain injury and subsequent repair and regeneration. This established method will improve experimental analyses in developmental biology and medical science using Xenopus tadpoles.

Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy

Rapid determination of drug efficacy against bacterial pathogens is needed to detect potentially resistant bacteria and allow for more rational use of antimicrobials. As an indicator of the antimicrobial effect for rapid detection, we found changes in image brightness in antimicrobial-affected bacteria by scanning electron microscopy (SEM). The cell envelopes of unaffected bacteria were stained with phosphotungstic acid (PTA), whereas the entire cells of affected bacteria were stained. Since tungsten density increases backscattered electron intensity, brighter bacterial images indicate lethal damage. We propose a simplified method for determining antimicrobial efficacy by detecting damage that occurs immediately after drug administration using tabletop SEM. This method enabled the visualization of microscopic deformations while distinguishing bacterial-cell-envelope damage on gram-negative bacteria due to image-brightness change. Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa were exposed to imipenem and colistin, which affect the cell envelope through different mechanisms. Classification of single-cell images based on brightness was quantified for approximately 500 bacteria per sample, and the bright images predominated within 5 to 60 min of antimicrobial treatment, depending on the species. Using intracellular PTA staining and characteristic deformations as indicators, it was possible to determine the efficacy of antimicrobials in causing bacterial-cell-envelope damage.

Gene expression detection in developing mouse tissue using in situ hybridization and µCT imaging

High resolution and noninvasiveness have made soft-tissue X-ray microtomography (µCT) a widely applicable three-dimensional (3D) imaging method in studies of morphology and development. However, scarcity of molecular probes to visualize gene activity with µCT has remained a challenge. Here, we apply horseradish peroxidase-assisted reduction of silver and catalytic gold enhancement of the silver deposit to insitu hybridization in order to detect gene expression in developing tissues with µCT (here called GECT, gene expression CT). We show that GECT detects expression patterns of collagen type II alpha 1and sonic hedgehogin developing mouse tissues comparably with an alkaline phosphatase-based detection method. After detection, expression patterns are visualized with laboratory µCT, demonstrating that GECT is compatible with varying levels of gene expression and varying sizes of expression regions. Additionally, we show that the method is compatible with prior phosphotungstic acid staining, a conventional contrast staining approach in µCT imaging of soft tissues. Overall, GECT is a method that can be integrated with existing laboratory routines to obtain spatially accurate 3D detection of gene expression.

Phosphotungstic acid-enhanced microcomputed tomography for quantitative visualization of mouse mammary gland morphology.

Even though current techniques provide two-dimensional (2D) imaging of the mouse mammary gland, they fail to achieve high-resolution three-dimensional (3D) reconstruction and quantification. The objective of this study is to establish and evaluate quantitative visualization of the mouse mammary epithelium through microcomputed tomography (microCT) using phosphotungstic acid (PTA) as a contrast agent. Ex vivo microCT scan images of the mouse mammary glands were obtained following staining by PTA, whereas for quantification we adapted volumetric parameters that are used for assessing trabecular bone morphometry and can be structurally applicable in the mammary ductal system. The proposed method was validated in distinct developmental stages and upon short-term treatment with synthetic progesterone, using the carmine alum staining for comparison. We demonstrate a simple PTA staining procedure that allows high contrast 3D imaging of mammary glands and quantitation of mammary duct structures using microCT. We validated the proposed method in distinct developmental stages, such as at puberty, adult mice, pregnancy as well as upon progesterone treatment. Compared with carmine alum staining, the microCT analysis provided higher resolution 2D and 3D images of the mammary gland morphology, with lower background that enabled the detection of subtle changes. This work is the first study that employs PTA-enhanced microCT for 3D imaging and volumetric analysis of mouse mammary glands. Our results establish PTA-enhanced microCT as a useful tool for comparative studies of the mouse mammary gland morphology that can apply in mutant mice and for the preclinical evaluation of pharmaceuticals in breast cancer models.

Phosphotungstic acid (PTA) preferentially binds to collagen- rich regions of porcine carotid arteries and human atherosclerotic plaques observed using contrast enhanced micro-computed tomography (CE-µCT).

Background and aims: Atherosclerotic plaque rupture in the carotid artery can cause small emboli to travel to cerebral arteries, causing blockages and preventing blood flow leading to stroke. Contrast enhanced micro computed tomography (CEμCT) using a novel stain, phosphotungstic acid (PTA) can provide insights into the microstructure of the vessel wall and atherosclerotic plaque, and hence their likelihood to rupture. Furthermore, it has been suggested that collagen content and orientation can be related to mechanical integrity. This study aims to build on existing literature and establish a robust and reproducible staining and imaging technique to non-destructively quantify the collagen content within arteries and plaques as an alternative to routine histology. Methods: Porcine carotid arteries and human atherosclerotic plaques were stained with a concentration of 1% PTA staining solution and imaged using MicroCT to establish the in situ architecture of the tissue and measure collagen content. A histological assessment of the collagen content was also performed from picrosirius red (PSR) staining. Results: PTA stained arterial samples highlight the reproducibility of the PTA staining and MicroCT imaging technique used with a quantitative analysis showing a positive correlation between the collagen content measured from CEμCT and histology. Furthermore, collagen-rich areas can be clearly visualised in both the vessel wall and atherosclerotic plaque. 3D reconstruction was also performed showing that different layers of the vessel wall and various atherosclerotic plaque components can be differentiated using Hounsfield Unit (HU) values. Conclusion: The work presented here is unique as it offers a quantitative method of segmenting the vessel wall into its individual components and non-destructively quantifying the collagen content within these tissues, whilst also delivering a visual representation of the fibrous structure using a single contrast agent.

Pre-Sowing Treatments, Seed Components and Water Imbibition Aids Seed Germination of Gloriosa superba

Gloriosa superba L. is a horticulturally and medicinally important plant. Its seeds have poor, erratic, and deferred germination. The detailed seed structure components and water imbibition mechanism facilitating the process of seed germination in G. superba remain unexplored. Therefore, it is essential to develop methods to ensure consistent and enhanced seed germination in G. superba. Various pre-sowing treatments along with the Brunauer-Emmett-Teller (BET) surface area analysis and 3D X-ray micro-tomography (micro-T) were employed to elucidate seed structure components, porosity network, and the water imbibition mechanism during germination in G. superba. The study revealed that consistent and significantly improved seed germination (>85%) was observed using the pre-sowing treatment mechanical scarification followed by 24 h water soaking in G. superba. BET and micro-T showed that the tegmen of the seed coat exhibited porosity (21%) with a well-connected porosity network (17.50%) that helped in water movement through hilum, which was confirmed by phosphotungstic acid staining. However, the sarcotesta and endosperm were water-impermeable due to their negligible porosity. Multidisciplinary techniques such as BET and micro-T along with conventional methodologies can be employed to address the seed coat structure, porosity, and water imbibition mechanism aiding seed germination. Mechanical scarification enabled the water to penetrate internal seed layers through the permeable tegmen via the reticulate pore network, which significantly improved seed germination. The developed seed germination method can produce a large number of plants in less time and conserve the natural populations of this high-value medicinally important species.

Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining

Multiple stains have been historically utilized in electron microscopy to provide proper contrast and superior image quality enabling the discovery of ultrastructures. However, the use of these stains in microbiological viability assessment has been limited. Phosphotungstic acid (PTA) staining is a common negative stain used in scanning electron microscopy (SEM). Here, we investigate the feasibility of a new SEM-PTA assay, aiming to determine both viable and dead microbes. The optimal sample preparation was established by staining bacteria with different PTA concentrations and incubation times. Once the assay conditions were set, we applied the protocol to various samples, evaluating bacterial viability under different conditions, and comparing SEM-PTA results to culture. The five minutes 10% PTA staining exhibited a strong distinction between viable micro-organisms perceived as hypo-dense, and dead micro-organisms displaying intense internal staining which was confirmed by high Tungsten (W) peak on the EDX spectra. SEM-PTA viability count after freezing, freeze-drying, or oxygen exposure, were concordant with culture. To our knowledge, this study is the first contribution towards PTA staining of live and dead bacteria. The SEM-PTA strategy demonstrated the feasibility of a rapid, cost-effective and efficient viability assay, presenting an open-view of the sample, and providing a potentially valuable tool for applications in microbiome investigations and antimicrobial susceptibility testing.

Evaluation of Histopathological Effects of Smoked Marijuana on Albino Rats and Its Oxidative Stress Indices

Smoking marijuana (cannabis Savita), a psychoactive substance may result in side effect on living cells of the system and oxidative stress may meditate some of these effects. This study aims to evaluate the acute and chronic histopathological effect on wistar albino rat as well as oxidative stress indices and some of the haematological parameters evaluation. This study was conducted at the animal house of Ebonyi State University, Presco campus Abakaliki. The experimental animals were share into control group, acute and chronic administered group which was housed in the temperature and humidity controlled facility of 22°C+1, standard laboratory mice chow diet with tap water freely available at ad libitum to the animals. The weight of the male wistar albino rat were taken twice daily which range from 150g to 200g after which was exposed to smoke of burnt wrapped 2mg of marijuana morning and evening for 21 days for experimental animals in group B and 42 days for group C animals respectively. The acute histopathological effect and that of chronic of the lungs of smoked marijuana wistar albino rat displayed histomorphological changes with Haematoxylin and Eosin, methanamine silver staining and phosphotungstic acid staining method when compared with the control lung sections that shows normal histomorphological pattern with the present of alveolar pneumocytes and alveolar space. Evaluation of the lungs of smoked marijuana rat shows increased MDA and decreased level of CAT, SOD, GSH and GPx but examining some of the haematological parameters show elevated Twbc and platelet while packed cell volume decreased the level as compared with control animals. I hereby concluded that marijuana smoked is a potent source of cellular oxidative stress that contributes significantly to cellular injury, dysfunction, and pulmonary edema as well as histomorphological changes from the lungs of marijuana smokers and disorder in some of the haematological parameters.

Contrast-enhanced nano-CT reveals soft dental tissues and cellular layers.

To introduce a methodology designed to simultaneously visualize dental ultrastructures, including cellular and soft tissue components, by utilizing phosphotungstic acid (PTA) as a contrast-enhancement agent. Sound third molars were collected from healthy human adults and fixed in 4% buffered paraformaldehyde. To evaluate the impact of PTA in concentrations of 0.3%, 0.7% and 1% on dental soft and hard tissues for CT imaging, cementum and dentine-pulp sections were cut, dehydrated and stained with immersion periods of 12, 24h, 2days or 5days. The samples were scanned in a high-resolution nano-CT device using pixel sizes down to 0.5µm to examine both the cementum and pulpal regions. Dental cementum and periodontium as well as odontoblasts and predentine were made visible through PTA staining in high-resolution three-dimensional nano-CT scans. Different segments of the tooth required different staining protocols. The thickness of the cementum could be computed over the length of the tooth once it was made visible by the PTA-enhanced contrast, and the attached soft tissue components of the interior of the tooth could be shown on the dentine-pulp interface in greater detail. Three-dimensional illustrations allowed a histology-like visualization of the sections in all orientations with a single scan and easy sample preparation. The segmentation of the sigmoidal dentinal tubules and the surrounding dentine allowed a three-dimensional investigation and quantitative of the dentine composition, such as the tubular lumen or the ratio of the tubular lumen area to the dentinal surface. The staining protocol made it possible to visualize hard tissues along with cellular layers and soft tissues in teeth using a laboratory-based nano-CT technique. The protocol depended on both tissue type and size. This methodology offers enhanced possibilities for the concomitant visualization of soft and hard dental tissues.

Insights from Contrast‐Enhanced MicroCT into the Formation of the Marsupial Hindlimb

As the sister clade to placental mammals, marsupials offer insight into a completely separate experiment in mammalian evolution. As with many placental groups (e.g., ungulates, rodents, and lagomorphs), several marsupial groups like kangaroos and bandicoots evolved elongate limbs with reduced digit numbers for locomoting in open, arid habitats. However, unlike most placental lineages which specialized their limbs from a cursorial ancestor, marsupial lineages evolved their specialized hindlimbs from a grasp-climbing ancestor that was morphologically similar to extant didelphid marsupials. To examine the developmental changes involved in marsupial limb diversification we used contrast-enhanced microcomputed tomography (CE microCT) to characterise postnatal limb development in the grey short-tailed opossum and the tammar wallaby. We compared phosphotungstic acid (PTA) and Ruthenium Red (RR) as contrast agents for differentiating bone and cartilage in microCT scans. PTA staining was effective at contrasting soft tissues but it was difficult to differentiate bone and cartilage tissues without time-consuming manual segmentation. Preliminary results show that RR stains soft-tissues much more slowly than PTA and skin removal is required for stain penetration of postnatal specimens. Comparison of limb segment proportions between the opossum and tammar wallaby provides evidence that macropods undergo a pronounced late postnatal elongation of the distal hindlimb elements, especially the calcaneus, metatarsals and phalanges. Digital segmentation of the hindfoot of an early postnatal PTA-stained specimen of the tammar wallaby provides evidence that a cartilaginous condensation anterior to the reduced second and third toe is morphologically more similar to a prehallax than a first toe. Potential implications for the evolvability of tetrapod digit number and the ancestral constraints of arboreality on limb adaptation are considered.

Liquid-phase ASEM imaging of cellular and structural details in cartilage\xa0and bone formed during endochondral ossification: Keap1-deficient osteomalacia

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1−/− femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

Propagation phase-contrast micro-computed tomography allows laboratory-based three-dimensional imaging of articular cartilage down to the cellular level

High-resolution non-invasive three-dimensional (3D) imaging of chondrocytes in articular cartilage remains elusive. The aim of this study was to explore whether laboratory micro-computed tomography (micro-CT) permits imaging cells within articular cartilage. Bovine osteochondral plugs were prepared four ways: in phosphate-buffered saline (PBS) or 70% ethanol (EtOH), both with or without phosphotungstic acid (PTA) staining. Specimens were imaged with micro-CT following two protocols: 1) absorption contrast (AC) imaging 2) propagation phase-contrast (PPC) imaging. All samples were scanned in liquid. The contrast to noise ratio (C/N) of cellular features quantified scan quality and were statistically analysed. Cellular features resolved by micro-CT were validated by standard histology. The highest quality images were obtained using propagation phase-contrast imaging and PTA-staining in 70% EtOH. Cellular features were also visualised when stained in PBS and unstained in EtOH. Under all conditions PPC resulted in greater contrast than AC (p<0.0001 to p=0.038). Simultaneous imaging of cartilage and subchondral bone did not impede image quality. Corresponding features were located in both histology and micro-CT and followed the same distribution with similar density and roundness values. Three-dimensional visualisation and quantification of the chondrocyte population within articular cartilage can be achieved across a field of view of several millimetres using laboratory-based micro-CT. The ability to map chondrocytes in 3D opens possibilities for research in fields from skeletal development through to medical device design and treatment of cartilage degeneration.

The utility of micro-computed tomography for the non-destructive study of eye microstructure in snails

Molluscan eyes exhibit an enormous range of morphological variation, ranging from tiny pigment-cup eyes in limpets, compound eyes in ark clams and pinhole eyes in Nautilus, through to concave mirror eyes in scallops and the large camera-type eyes of the more derived cephalopods. Here we assess the potential of non-destructive micro-computed tomography (µ-CT) for investigating the anatomy of molluscan eyes in three species of the family Solariellidae, a group of small, deep-sea gastropods. We compare our results directly with those from traditional histological methods applied to the same specimens, and show not only that eye microstructure can be visualised in sufficient detail for meaningful comparison even in very small animals, but also that μ-CT can provide additional insight into gross neuroanatomy without damaging rare and precious specimens. Data from μ-CT scans also show that neurological innervation of eyes is reduced in dark-adapted snails when compared with the innervation of cephalic tentacles, which are involved in mechanoreception and possibly chemoreception. Molecular tests also show that the use of µ-CT and phosphotungstic acid stain do not prevent successful downstream DNA extraction, PCR amplification or sequencing. The use of µ-CT methods is therefore highly recommended for the investigation of difficult-to-collect or unique specimens.

Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM

The malformation and disordered remodeling of bones induce various diseases, including osteoporosis. We have developed atmospheric SEM (ASEM) to directly observe aldehyde-fixed bone tissue immersed in radical scavenger buffer without thin sectioning. The short procedure realized the observation of bone mineralization surrounded by many cells and matrices in natural aqueous buffer, decreasing the risk of changes. In osteoblast primary cultures, mineralization was visible without staining. Correlative energy dispersive X-ray spectrometry indicated the formation of calcium phosphate mineral. Fixed bone was sectioned, and the section surface was inspected by ASEM. Mineralized trabeculae of talus spongy bone were directly visible. Associated large and small cells were revealed by phosphotungstic acid staining, suggesting remodeling by bone-absorbing osteoclasts and bone-rebuilding osteoblasts. In tibia, cortical bone layer including dense grains, was bordered by many cells with protrusions. Tissue immuno-EM performed in solution for the first time and anti-cathepsin-K antibody, successfully identified osteoclasts in femur spongy bone. A microfluidics chamber fabricated on the silicon nitride film window of an ASEM dish allowed mineralization to be monitored in vitro; calcium phosphate crystals as small as 50 nm were imaged. ASEM is expected to be widely applied to study bio-mineralization and bone-remodeling, and to help diagnose bone-related diseases.

Three‐dimensional visualization of cranial soft tissues in embryonic and early postnatal mice using phosphotungstic acid enhanced microCT

Acquiring three‐dimensional imaging of animal specimens utilized in genetic, developmental, and evolutionary studies has typically been based on microCT (bone and mineralized tissues) and magnetic resonance microscopy (soft and un‐mineralized tissues). However, recent advances in microCT imaging have allowed for 1–5 μm resolution for small biological specimens, while typical microMRI imaging can generally only produce data with a resolution of approximately 30–40 μm. In order to achieve high‐resolution 3D visualization of soft tissues, the use of various contrast agents have been proposed, including iodine, osmium, and phosphotungstic acid (PTA). We investigated protocols for using PTA to stain soft tissues of the head in embryonic and early postnatal mice to enhance visualization of soft tissues by microCT imaging. Protocols for mice between embryonic day 13.5 and postnatal day 7 have been developed that allow for the visualization, segmentation, quantification, and analysis of soft tissue structures. Stained specimens were mounted in a 50:50 mix of polyester and paraffin waxes within a small tube and scanned on the GE v|tome|x L300 scanner in the Center for Quantitative Imaging at Pennsylvania State University with voxel dimensions between 1 and 10 μm, depending on specimen size. Multiple soft tissue structures, including the eye, inner ear, cartilage, muscle, glandular tissue, and brain, are easily differentiated in the resultant scans. Images of PTA‐stained specimens can be superimposed with microCT images of the same specimen before PTA staining to enable the study of relationships between hard and soft tissues in the same individual. This approach to both 2D and 3D visualization of soft tissues in murine models has immediate implications for understanding developmental processes and integration between hard and soft tissues of the cranium.Support or Funding InformationNational Science Foundation Grant BCS‐1731909, and NIDCR R01DE027677 and R01 DE022988This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of glial cells molecules in the optic nerve of adult dromedary camel (Camelus dromedarius): A histological and immunohistochemical analysis.

The optic nerve (ON) is an important organ in the visual system of animals, which transfers electrical impulses towards the brain from the retina. High enrichment of glial cells in ON is known to support neuron and regulate retinal homoeostasis. However, research on immunohistochemical of glial cells proteins in the camel is scanty in available literature. Hence, the current work is an attempt to investigate the histomorphology of camel ON with regard to the expression patterns of glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and Iba1 for the three glial subtypes, namely astrocytes, oligodendrocytes and microglia, respectively. Optic nerves from fourteen dromedary camels were dissected and preserved in 10% formalin. Then, the paraffin-embedding sections were subjected for histochemical and immunohistochemical analysis. Our results demonstrated that ON axons aggregate into fascicles that surrounded by light and densely stained glial cells. Then, we examined the myelin sheath using Heidenhain's and Mallory's phosphotungstic acid staining. Immunoassay results revealed that GFAP is enriched in the ON and distributed evenly, whereas MBP and Iba1 were present at scanty levels. Further analysis of mRNA level of GFAP, MBP and Iba1 in the ON confirmed an elevation of GFAP expression compared to MBP and Iba1. We further found partial co-localization of different types of glial cells that reflect their coordinated function in the ON. Although our data provide the first evidence for differential expression pattern of glial proteins, further molecular studies still required to reveal the specific function of these molecules in the camel ON.

PD31-10 HYDROXYAPATITE IN PEYRONIE’S PLAQUES

You have accessJournal of UrologySexual Function/Dysfunction: Peyronie’s Disease I1 Apr 2017PD31-10 HYDROXYAPATITE IN PEYRONIE’S PLAQUES Thomas Schmid, Robin Pourzal, Joseph Temple, and Laurence Levine Thomas SchmidThomas Schmid More articles by this author , Robin PourzalRobin Pourzal More articles by this author , Joseph TempleJoseph Temple More articles by this author , and Laurence LevineLaurence Levine More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1388AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES About a third of Peyronie′s Disease (PD) patients have evidence of tunica albuginea mineralization at presentation on B-mode ultrasound. The nature of both small and large mineral deposits in PD plaques was characterized by Energy Dispersive X-ray Spectroscopy (EDS) and micro CT. METHODS Tissue specimens were collected from PD patients after surgery with patients′ consent and institutional approval and stored frozen. X-rays were used to identify post-surgical specimens with mineralization. The soft tissues in PD plaques with mineral foci were digested with 0.2% pronase for 2h followed by 0.025% bacterial collagenase P overnight at 37 oC. Mineral foci resistant to enzyme digestion were gold coated and analysed by EDS using a JEOL JSM electron microscope. Other PD plaques were fixed with ethanol and phosphotungstic acid (PTA) and imaged by micro CT. RESULTS Mineral foci in PD plaques were resistant to enzyme digestions by the general protease pronase and subsequent digestion by bacterial collagenase. When the mineral content of the enzyme resistant foci were analysed by EDS they showed a Ca/P ratio of 1.80 +/- 0.09. The Ca/P ratios were very similar for both large and small foci. For comparison rabbit tibial bone treated in a similar manner had Ca/P ratio of 1.76 +/- 0.09. The location of the mineral within the collagenous plaques was imaged using micro CT with PTA as a contrast agent to stain the collagen fibers. Micro CT images of the mineralized plaques showed mineral deposition at the interface between collagenous layers with different degrees of PTA stain contrast. CONCLUSIONS Mineral foci in PD plaques were enzyme resistant. The high Ca/P ratio (1.80) of mineral deposits in PD plaques were indicative of mature hydroxyapatite (Ca/P 1.67) found in bone. This high ratio was similar in both small and large mineral foci in the PD enzyme resistant tissues. The location of the mineral deposits and adjacent unmineralized collagenous matrix could readily be visualized by micro CT when PTA was used to stain the collagen fibers. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e586-e587 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Thomas Schmid More articles by this author Robin Pourzal More articles by this author Joseph Temple More articles by this author Laurence Levine More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...