Multi-Scale Investigation of Human Renal Tissue in Three Dimensions.

Histopathology as a diagnostic mainstay for tissue evaluation is strictly a 2D technology. Combining and supplementing this technology with 3D imaging has been proposed as one future avenue towards refining comprehensive tissue analysis. To this end, we have developed a laboratory-based X-ray method allowing for the investigation of tissue samples in three dimensions with isotropic volume information. To assess the potential of our method for micro-morphology evaluation, we selected several kidney regions from three patients with cystic kidney disease, obstructive nephropathy and diabetic glomerulopathy. Tissue specimens were processed using our in-house-developed X-ray eosin stain and investigated with a commercial microCT and our in-house-built NanoCT. The microCT system provided overview scans with voxel sizes of [Formula: see text] and the NanoCT was employed for higher resolutions including voxel sizes from [Formula: see text] to 210 nm. We present a methodology allowing for a precise micro-morphologic investigation in three dimensions which is compatible with conventional histology. Advantages of our methodology are its versatility with respect to multi-scale investigations, being laboratory-based, allowing for non-destructive imaging and providing isotropic volume information. We believe, that after future developmental work this method might contribute to advanced multi-modal tissue diagnostics.

Histological Evidence of Wound Healing Improvement in Rats Treated with Oral Administration of Hydroalcoholic Extract of Vitis labrusca.

Plant extracts rich in phenolic compounds have been demonstrated to accelerate wound healing, but their use by oral route has been poorly studied. The leaves of Vitis labrusca are rich in phenolic acids and flavonoids. The goal of this study was to assess the healing properties of the oral administration of hydroalcoholic extract of V. labrusca leaves (HEVL) in a murine model. HEVL was obtained by Soxhlet and dynamic maceration, and their yield and phenolic acids and flavonoid contents were determined. For the wound healing assay, 8 mm wounds were performed on the back of 48 Wistar rats, assigned into four groups (n = 12): CTR (distilled water), HEVL100, HEVL200, and HEVL300 (HEVL at 100, 200, and 300 mg/kg, respectively). On days 7 and 14, wound closure rates were assessed, and the healing wounds were subjected to histological analysis. Soxhlet-obtained extract was selected for the wound healing assay because it provided a higher yield and phenolic acid and flavonoid contents. HEVL significantly reduced leukocytosis in the peripheral blood (p < 0.05), accelerated wound closure (p < 0.05), and improved collagenization (p < 0.05) on day 7, as well as enhanced the epidermal tissue thickness (p < 0.001) and elastic fiber deposition on day 14 (p < 0.01). Furthermore, HEVL promoted an increase in the histological grading of wound healing on both days 7 and 14 (p < 0.01). The doses of 200 and 300 mg/kg provided better results than 100 mg/Kg. Our data provide histological evidence that the oral administration of HEVL improves wound healing in rodents. Therefore, the extract can be a potential oral medicine for healing purposes.

Penetration Depth in Nanoparticles Incorporated Radiofrequency Hyperthermia into the Tissue: Comprehensive Study with Histology and Pathology Observations.

In present study, the effective penetration of radiofrequency (RF) induced gold decorated iron oxide nanoparticles (GS@IONPs) hyperthermia was investigated. The effective penetration depth of RF also the damage potency of hyperthermia was evaluated during histopathology observations which were done on the chicken breast tissue and hepatocellular carcinoma (HCC) models. The thermal damages are well- documented in our previous cellular study which was engaged with potency of RF hyperthermia in Epithelial adenocarcinoma (MCF-7) and fibroblast (L-929) cells deaths [1]. In recent work, PEGylated iron oxide nanoparticles (IONPs) were used as base platform for gold magnetic nanoparticles (GS@IONPs) formation. The 144.00015 MHz, 180W RF generator was applied for stimulating the nanoparticles. The chicken breast tissue and the hepatocellular tumor model was considered in the experimental section. In histology studies, the structural changes also the effective penetration depth of RF induced nanoparticles was observed through microscopic monitoring of the tissue slices in histology observations (Gazi medical school). The highest damage level was seen in 8.0 µm tissue slices where lower damages were seen in depth of 1.0 cm and more inside tissue. The histology observations clarified the effective penetration depth of RF waves and irreversible damages in the 2.0 cm inside the tissue.

Section Plane Effects on Morphometric Values of Microcomputed Tomography.

OBJECTIVES: Histomorphometry is the established gold standard for inspection of trabecular microstructures in biomaterial research. However, microcomputed tomography can provide images from the perspective of various section planes. The aim of the present study was to evaluate the effects of different section planes, which may cause bias in two-dimensional morphometry, on the morphometric values of microcomputed tomography. METHODS: A socket preservation technique was performed on the extracted premolar area of 4 beagle dogs. After an 8-week healing period, a total of 16 specimens were obtained and analyzed with conventional histomorphometry and microtomographic morphometry. Using the original images of the histologic specimens for comparison, the most similar tomographic image was selected by trial and error. Then, the section plane was then moved with ±79 µm parallel offsets and rotated ±10° around the center from the occlusal view. The images were compared in terms of bone, graft, and noncalcified area, and the concordance correlation coefficient (CCC) was calculated. RESULTS: There was a high CCC in the comparison between histomorphometric images and the most similar microtomographic images. However, the CCC value was low in the comparisons with both parallel movement and rotation. Our results demonstrate that the sectioning plane has a significant effect on measurements. CONCLUSION: Two-dimensional morphometric values for biomaterial research should be interpreted with caution, and the simultaneous use of complementary 3-dimensional tools is recommended.

Salivary Transferrin Levels in Patients with Oral Lichen Planus

Objective: To compare salivary transferrin levels between patients with oral lichen planus (OLP) and healthy subjects. Material and Methods: In this descriptive, analytical, crosssectional study, 11 patients with OLP and 22 healthy subjects were selected after matching in terms of age and gender. OLP was confirmed by two oral medicine specialists based on clinical and histopathological criteria. Salivary samples were collected by spitting. The patients were asked to collect their saliva in their oral cavity and then evacuate it into sterilized Falcon tubes. The procedure was repeated every 60 seconds for 5-15 minutes. A total of 5 mL of saliva was collected using this method. The samples were collected from 8 to 9 in the morning in a fasting state to avoid circadian changes. The collected salivary samples were immediately placed next to ice and transferred to the laboratory to be centrifuged at 4°C at 800 g to isolate squamous cells and cellular debris. Then the samples were frozen at -80°C until the samples were prepared. An ELISA kit was used to determine salivary transferrin levels. Data were analyzed with descriptive statistics (means and standard deviations) and t-test for independent groups using SPSS 17. Statistical significance was set at p<0.05. Results: The mean salivary transferrin concentrations in patients with OLP and healthy subjects were 0.9055±0.28229 and 1.5932±0.80041 mg/dL, respectively (p<0.05). Conclusion: The salivary transferrin levels in patients with OLP were significantly lower than those in healthy subjects.

Evaluation of an extract of the Punica granatum flower as a biological stain of rat tissues: a preliminary study.

Natural dyes and especially hematoxylin, which is herbal, are widely used in staining tissues. The aim of this study is to evaluate the staining status of different tissues taken from rats with pomegranate flower extract. For this reason, 2 Wistar albino rats, one male and one female, were used as stain biomaterial. A histological follow up procedure was applied to the lung, kidney, liver, and heart tissue samples taken from the rats and the unstained preparates of these tissues were prepared. As the source of the dye, the dry flowers of Punica granatum (PG) obtained from local markets of Kayseri were used. Each tissue sample underwent the same staining procedure with the same temperature, duration, and dye solution. Before and after the staining procedure, × 40 images of the tissue preparates were taken using a light microscope. Generally, different tones of staining were observed in the nuclei and cytoplasms of all cells and epithelium cells. Staining in parts specific to each tissue occurred. For example, there were light stains on the glomerular cells and the Bowman capsule in the kidney tissue Differences in staining can only be explained by molecular diversity differences in tissue. However, in order to improve the initial staining results obtained in this study, it is possible that working with different temperatures, pH values, mordant substances, and dye that the dye molecules in the extract will provide more vivid colors with different molecules in the tissues.

A comprehensive evaluation for the treatment of lupus nephritis.

Systemic lupus is the prototypic human autoimmune disease. It is a kaleidoscope of autoreactivities, with clear indications of both a genetic and environmental basis. Indeed, it is a disease that can manifest in virtually every tissue and organ and can also be found spontaneously in a number of animal species, including dogs, cats and horses. Moreover, there are multiple murine models of lupus, the first of which, New Zealand Black (NZB) mice, were discovered in 1959. Despite an enormous effort from scientists in multiple disciplines, the etiology of lupus remains elusive and the introduction of new therapies has been disappointing. Fortunately, significant advances have occurred to help patients through the general principles of internal medicine, including antibiotics, dialysis, and of course use of steroids and immunosuppressive agents. However, the magic bullet has yet to be discovered. One of the major causes of morbidity in lupus remains lupus nephritis and there has been significant effort and encouragement in understanding the pathogenesis, renal histologic classification, and use of therapeutic protocols to induce and sustain remission of lupus nephritis. Indeed, the first use of evidence-based clinical trials in lupus was initiated by Dr. Alfred D. Steinberg at NIH in pioneering studies involving either oral or intravenous pulses of cyclophosphamide, azathioprine or corticosteroids alone and/or some combination. Cyclophosphamide intravenously proved to be superior and the use of cyclophosphamide in combination with methylprednisolone remained the standard protocol for the treatment of lupus nephritis for decades. Although alternative therapies have been introduced, including mycophenolate mofetil, the use of therapies first pioneered at NIH may still be considered standard of care in the appropriate indications. More targeted therapies are much desired. In this review we provide a comprehensive overview of lupus nephritis and the evolution of clinical treatments.

Sampling the Rat Mesenteric Artery.

Vascular injury can be induced by different classes of drug candidates, and it can affect the mesenteric vasculature. Sampling of the mesenteric vessels in the rat is crucial for proper assessment of potential adverse or pharmacologic effects of drugs in nonclinical rodent studies. To date, several sampling and processing techniques for the histopathologic evaluation of the mesenteric artery in rodents have been described and used in studies with candidate drugs that may affect the vascular system. However, most of those techniques require a significant amount of time and effort. A less labor-intensive, time-consuming, and expensive technique that allows examination of the mesentery vasculature with abundant longitudinal and cross sections of the vessels when examined microscopically was developed and presented here.

In Vitro Propagation of Withania somnifera (L.) Dunal.

Withania somnifera (L.) Dunal known as Ashwagandha is commonly used in traditional Indian medicine system. It possesses immense therapeutic value against a large number of ailments such as mental diseases, asthma, inflammation, arthritis, rheumatism, tuberculosis, and a variety of other diseases including cancer. The therapeutic potential of W. somnifera is due to the presence of secondary metabolites mainly, tropane alkaloids and withanolides (steroidal lactones). The growing realization of commercial value of the plant has initiated a new demand for in vitro propagation of elite chemotypes of Withania. Micropropagation which is an important tool for rapid multiplication requires optimization of number of factors such as nutrient medium, status of medium (solid and liquid), type of explant, and plant growth regulators. Similarly, an efficient and reproducible in vitro regeneration system which is a prerequisite for the development of genetic transformation protocol requires precise manipulation of various intrinsic and extrinsic factors.

Seeing the forest and trees: whole-body and whole-brain imaging for circadian biology.

Recent advances in methods for making mammalian organs translucent have made possible whole-body fluorescent imaging with single-cell resolution. Because organ-clearing methods can be used to image the heterogeneous nature of cell populations, they are powerful tools to investigate the hierarchical organization of the cellular circadian clock, and how the clock synchronizes a variety of physiological activities. In particular, methods compatible with genetically encoded fluorescent reporters have the potential to detect circadian activity in different brain regions and the circadian-phase distribution across the whole body. In this review, we summarize the current methods and strategy for making organs translucent (removal of lipids, decolourization of haemoglobin and adjusting the refractive index of the specimen). We then discuss possible applications to circadian biology. For example, the coupling of circadian rhythms among different brain regions, brain activity in sleep-wake cycles and the role of migrating cells such as immune cells and cancer cells in chronopharmacology.

Re-purposing of histological tissue sections for corroborative western blot analysis of hypothalamic metabolic neuropeptide expression following delineation of transactivated structures by Fos immuno-mapping.

Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow.

Diagnostic value of high-resolution peripheral quantitative computed tomography (HR-pQCT) in the qualitative assessment of cribra orbitalia: a preliminary study.

Cribra orbitalia are a porotic or sieve-like lesions in the bony orbital roof. This characteristic has frequently been detected in palaeopathological skulls from many parts of the world and has been the object of extensive research. Our objective was to determine if high-resolution peripheral quantitative computed tomography (HR-pQCT) could produce reliable information in the study of cribra orbitalia. Seven skulls displaying cribra orbitalia were investigated by HR-pQCT. The two-dimensional slices were compared with histological sections. The HR-pQCT images and histological sections showed similar results, i.e. two groups of lesions with different characteristics. HR-pQCT can be of great value in palaeopathological research. It is a nondestructive, fast and precise technique that allows an easy evaluation of the bone architecture without destruction of the sample.

Histochemical evaluation of alkaloids in rhizome of Coptis chinensis using laser microdissection and liquid chromatography/mass spectrometry.

Traditional macroscopic and microscopic identification methods of medicinal materials are economical and practical, but usually experience-based due to few chemical supports. Here histochemical evaluation on bioactive components of Coptidis Rhizoma (CR) in anatomic sections using laser microdissection and liquid chromatography-mass spectrometry (LMD-LC-MS) was developed to correlate the inner quality and outer features of materials from different growing areas. Results of a total 33 peaks representing potential different alkaloids were detected and 8 common peaks were identified as the major alkaloids, namely magnoflorine, thalifendine, columbamine, epiberberine, jatrorrhizine, coptisine, palmatine, and berberine. Six major alkaloids were quantified in the top and middle sections of raw materials and in their tissues and cells at the same time. Histochemical analyses showed consistent results with direct determination in raw materials and explained the reason why top sections of all samples contained higher contents of alkaloids by giving out attributions of each alkaloid in different anatomic sections. Besides, results manifested the distribution and accumulation rules of alkaloids in diverse tissues and cells of CR. This study demonstrates an effective and scientific way to correlate bioactive components and morphological features of medicinal materials, which is beneficial to future research, agriculture and application.

Neuropathology standards: what constitutes an optimal histomorphologic evaluation of the nervous system in general toxicity studies.

The identification of neurotoxicity is a critical issue in drug development, and toxicologic pathologists play an important role in this effort. Neuropathology is a specialized area of toxicologic pathology in which a substantial number of nonroutine techniques and methods have been developed, and there are undoubtedly many instances in which these specialized procedures have helped characterize a neuropathologic lesion. Routine histopathologic methods employed in general toxicologic pathology studies are needed to identify the complete range of possible neuropathologic changes; once identified, many of these changes can be better defined by specialized techniques, such as immunohistochemistry, to confirm cell types involved. Sometimes, when neurotoxicity is expected, dedicated studies can be designed a priori and optimized for detection of the anticipated effects. However, when neurotoxicity arises unexpectedly or there is uncertainty around the potential for neurotoxicity, the decision of what to do can become more difficult. Recommendations to go ahead and perform the "optimal" study design that would accommodate all the potentially useful specialized techniques for characterizing a neuropathologic change are sometimes not practical and can be unnecessary and potentially detrimental to other end points in the study. In addition, there is not always agreement on when specialized techniques would be required and which ones should be used when necessary. Two techniques in particular that are commonly recommended to help facilitate the detection of neuropathologic lesions are perfusion fixation and the Fluoro-Jade stain.

Histology without xylene.

After the hazardous effects of xylene became indisputable in the 1970s, many potential substitutes became available, some with as many if not more hazards. This article discusses the inadequacy of 5 vegetable oils as substitutes, as well as the characteristics of 22 D-limonene-based substitutes, all less effective in their chemical role, some capable of inducing health problems, and costing more than twice as much as xylene. Some of the 35 alkane-based substitutes discussed are effective for tissue processing, less toxic, with a cost about the same as xylene, but are not very effective for dewaxing and other staining tasks. Isopropanol (2-propanol) alone or mixed with molten paraffin is a technically acceptable and cost-effective substitute for xylene for tissue processing, but in this study, we demonstrate that the best clearing agents from the sectioning quality and diagnostic value point of view, with automated or manual protocols, are mixtures of 5:1 and 2:1 isopropanol and mineral oil, followed by undiluted mineral oil, all at 50 degrees C, making them a safer and cheaper substitute than xylene. Using a 1.7% dishwasher soap aqueous solution at 90 degrees C to dewax before staining and oven drying the stained sections before coverslipping will eliminate xylene from the staining tasks. Tissue processors retorts and conduits can be dewaxed with a 2% solution of a strong glassware laboratory detergent. These 4 methodologies will make the histology laboratory xylene-free but, due to the natural resistance to change, many histotechs will be reluctant to adopt them if they think that their technical expertise could be jeopardized, and the only way these changes will succeed is if the pathologists, as stewards of the histology laboratory, commit to their implementation.

The use of histologically defined specific biomarkers in drug development with special reference to the glutathione S-transferases.

Failure of drug candidates late in development is very expensive. This can be reduced by using more specific biomarkers of effect and toxicity during the preclinical and development testing. However, traditional toxicity tests have not been developed to study toxicology and so may lack sufficient sensitivity and specificity hence the search for new biomarkers using the many "-omics" technologies. Important aspects of useful biomarkers are that their origin is known and localised so that one knows what is being observed. Furthermore, biomarkers with a localised origin are less likely to be subject to background variation and have a wider dynamic range. This paper will discuss how using biomarkers with a known cellular origin, toxic effects can be found earlier and at lower doses of compound.

The morphology of murine oncornaviruses following different methods of preparation for electron microscopy.

The effect of different preparative procedures for electron microscopy on the size and shape of murine oncornaviruses has been studied. With conventional negative staining procedures using neutral sodium phosphotungstate, both murine mammary tumor virus and murine leukemia virus appeared in head-and-tail forms, with a peak head diameter of 122 and 130 nm, respectively. Negative staining with uranyl accetate gave round virions with peak diameters of 148 and 130 nm. Prefixed virus was round with peak diameters of 141 and 130 nm, respectively, in phosphotungstate, and 148 and 117 nm, respectively, in uranyl acetate. With thin sections, the peak diameters were 143 and 123 nm. The preservation of the spherical shape of the virus was obtained by glutaraldehyde fixation dehydration in alcholic solutions of uranyl acetate, and critical point drying. Under these conditions the viruses had peak diameters of 99 and 82 nm, respectively. The size of murine mammary tumor virus has always been found to be larger than murine leukemia virus in all preparations except for negative staining with neutral sodium phosphotungstate. Shadowing of the virion preparations revealed considerable flattening of the particles in all cases except for critical point drying. Negatively stained preparations did not cast any shadow, and thus thethickness of the particles could not be evaluated. Virus can be reversibly converted from spherical to head-and-tail forms by altering osmotic strength. Under most of the conditions used, murine mammary tumor virus gave a bimodal size distribution with significant numbers of particles that were smaller than the major virus size.