Improved procedure for histological identification of osteoid matrix in decalcified bone

Several improvements on the original method of Yoshiki and coworkers for histological identification of osteoid matrix in decalcified bone are described in this report. The first, fixation of bone with neutral buffered formalin, a popular and stable fixative, should produce better tissue morphology and ensure easy handling in any laboratory. The second is a simple test for aged cyanuric chloride. Aged reagents show poor or no solubility in methanol and have almost no effect on differential staining of osteoid matrix. The third is an application of an organic acid solution in place of neutral EDTA for bone decalcification. Reduced decalcification time with the acid results in rapid preparation of bone sections. Neutral formalin fixation, immersion in the cyanuric chloride solution, decalcification with an organic acid, and hematoxylin and eosin staining, all quite routine laboratory procedures, yield high quality results for identification of osteoid matrix in bone sections.     

The study of morphological and histological changes in tissue cultures ofmatricaria inodora L

This paper deals with some morphological and histological changes observed during regular intervals inMatricaria inodora L. tissue cultures derived from leaf expiants. The expiants were cultivated on Murashige-Skoog’s culture medium supplemented with 1.0 mg 1⁻¹ 2,4-dichlorophenoxyacetic acid. The callus formation started about a week after isolation. During the second week the meristematic centers were differentiated from which root and shoot apices were later formed. During long term cultivation under the same culture conditions the inhibition of development of shoot apices took place. Only roots of unorganized growth have been regenerated. The influence of culture conditions on morphogenetic potential is discussed.     

The value of autopsy and other histological examinations for the safety of tissue transplantation

Directive 2006/17/EC requires that all available medical information, including autopsy reports, is evaluated before releasing tissues for transplantation. The aim of this study was to investigate whether evaluation of results of autopsy and other histological examinations contributes to the safety of tissue transplantation. From the files of all deceased Dutch donors, from whom tissues were retrieved in a 6-month period, results of autopsy and other histological examinations (remnant heart after valve donation and biopsies obtained during retrieval) were evaluated for contraindications for transplantation. Of 758 donors at least one tissue was considered suitable for transplantation at initial assessment. 637 Donors donated corneas, 256 skin, 177 heart valves and 61 musculoskeletal tissues. On 220 donors (29.0%) autopsy was done. Of seven donors no autopsy results were requested, since a contraindication was detected earlier in the medical screening. In 19 donors with autopsy (8.9%) general or tissue-specific contraindications were detected. There were no differences in distribution of detected contraindications among donors who donated different tissues. For 136 donors (17.9%) results of histological examinations other than autopsy were available; results of examination of remnant hearts for all, brain autopsy for two (0.3%) and retrieval biopsy for four donors (0.5%). Contraindications were detected in nine of these donors with histology results other than autopsy (6.6%). For 402 donors (53%) no histological examinations were done. Evaluation of results of autopsy and other histological examinations improves the safety of tissue transplantation for all types of tissues. In donors without autopsy alternative histological examinations can contribute to enhance the safety of tissue transplantation.     

Evaporation process in histological tissue sections for neutron autoradiography

The analysis of the distribution and density of nuclear tracks forming an autoradiography in a nuclear track detector (NTD) allows the determination of ¹⁰B atoms concentration and location in tissue samples from Boron Neutron Capture Therapy (BNCT) protocols. This knowledge is of great importance for BNCT dosimetry and treatment planning. Tissue sections studied with this technique are obtained by cryosectioning frozen tissue specimens. After the slicing procedure, the tissue section is put on the NTD and the sample starts drying. The thickness varies from its original value allowing more particles to reach the detector and, as the mass of the sample decreases, the boron concentration in the sample increases. So in order to determine the concentration present in the hydrated tissue, the application of corrective coefficients is required. Evaporation mechanisms as well as various factors that could affect the process of mass variation are outlined in this work. Mass evolution for tissue samples coming from BDIX rats was registered with a semimicro analytical scale and measurements were analyzed with software developed to that end. Ambient conditions were simultaneously recorded, obtaining reproducible evaporation curves. Mathematical models found in the literature were applied for the first time to this type of samples and the best fit of the experimental data was determined. The correlation coefficients and the variability of the parameters were evaluated, pointing to Page’s model as the one that best represented the evaporation curves. These studies will contribute to a more precise assessment of boron concentration in tissue samples by the Neutron Autoradiography technique.     

Robust automated tumour segmentation on histological and immunohistochemical tissue images

Tissue microarray (TMA) is a high throughput analysis tool to identify new diagnostic and prognostic markers in human cancers. However, standard automated method in tumour detection on both routine histochemical and immunohistochemistry (IHC) images is under developed. This paper presents a robust automated tumour cell segmentation model which can be applied to both routine histochemical tissue slides and IHC slides and deal with finer pixel-based segmentation in comparison with blob or area based segmentation by existing approaches. The presented technique greatly improves the process of TMA construction and plays an important role in automated IHC quantification in biomarker analysis where excluding stroma areas is critical. With the finest pixel-based evaluation (instead of area-based or object-based), the experimental results show that the proposed method is able to achieve 80% accuracy and 78% accuracy in two different types of pathological virtual slides, i.e., routine histochemical H&E and IHC images, respectively. The presented technique greatly reduces labor-intensive workloads for pathologists and highly speeds up the process of TMA construction and provides a possibility for fully automated IHC quantification.     

Chronic histological effects of ultrasonic hyperthermia on normal feline brain tissue

The histopathological changes associated with ultrasonic heating of normal cat brain have been correlated with thermal distributions. Ultrasound energy was applied for 50 min at different intensities to generate tissue temperatures from 42 to 48 degrees C. Animals were sacrificed at various intervals from 1 to 56 days. The organization and resolution of thermal damage was characterized by three stages of histopathological changes within the nervous tissue. The acute stage (Days 1-3) was defined by (1) extensive coagulation necrosis, (2) pyknosis of neuronal elements in the gray matter, (3) edema and vacuolation in the white matter, and (4) polymorphonuclear leukocytes. The subacute stage (Days 3-21) was characterized by (1) the appearance of lipid-laden macrophages, (2) liquefaction of the necrotic regions, (3) fibroblastic proliferation, and (4) vascular proliferation with some perivascular inflammatory infiltration (lymphocytes). Lastly, the chronic stage (Days 21-56) was defined by (1) fibrosis (reticulin and collagen formation) and (2) gliosis (reactive astrocytic proliferation) occurring around the fluid-filled necrotic center. Analysis of these data has also included a study of the lesion size versus the dose (temperature for 50 min) of heating. The results demonstrate a significant linear dose-response correlation. The results of this study indicate that the histological appearance and time course of repair of thermal injury in the normal brain tissue are analogous to acute brain necrosis resulting from cerebral infarction, except the thermal damage does not result in significant hemorrhage.     

TdTomato and EGFP identification in histological sections: insight and alternatives

Abstract

Tandem dimer Tomato (tdTomato) provides a useful alternative to enhanced green fluorescent protein (eGFP) for performing simultaneous detection of fluorescent protein in histological sections together with fluorescence immunohistochemistry (IHC). eGFP has many properties that make it useful for cell labeling; however, during simultaneous fluorescence IHC, the usefulness of eGFP may be limited. This limitation results from a fixation step required to identify eGFP in histological tissue sections that can mask antibody epitopes and adversely affect staining intensity. An alternative fluorescent protein, tdTomato, may assist concurrent detection of fluorescent protein within tissue sections and fluorescence IHC, because detection of tdTomato does not require tissue fixation. Tissue sections were obtained from various organs of mice ubiquitously expressing eGFP or tdTomato that were either unfixed or fixed with 4% paraformaldehyde. These tissues later were combined with fluorescence IHC. Both eGFP and tdTomato displayed robust signals in fixed frozen sections. Only tdTomato fluorescence, however, was detected in unfixed frozen sections. Simultaneous detection of fluorescence IHC and fluorescent protein in histological sections was observed only in unfixed frozen tdTomato tissue. For this reason, tdTomato is a useful substitute for eGFP for cell labeling when simultaneous fluorescence IHC is required.     

Optical coherence tomography for rapid tissue screening and directed histological sectioning

In pathology, histological examination of the "gold standard" to diagnose various diseases. It has contributed significantly toward identifying the abnormalities in tissues and cells, but has inherent drawbacks when used for fast and accurate diagnosis. These limitations include the lack of in vivo observation in real time and sampling errors due to limited number and area coverage of tissue sections. Its diagnostic yield also varies depending on the ability of the physician and the effectiveness of any image guidance technique that may be used for tissue screening during excisional biopsy. In order to overcome these current limitations of histology-based diagnostics, there are significant needs for either complementary or alternative imaging techniques which perform non-destructive, high resolution, and rapid tissue screening. Optical coherence tomography (OCT) is an emerging imaging modality which allows real-time cross-sectional imaging with high resolutions that approach those of histology. OCT could be a very promising technique which has the potential to be used as an adjunct to histological tissue observation when it is not practical to take specimens for histological processing, when large areas of tissue need investigating, or when rapid microscopic imaging is needed. This review will describe the use of OCT as an image guidance tool for fast tissue screening and directed histological tissue sectioning in pathology.     

Effect of histological fixation on carbohydrate-rich tissue compounds and their periodate consumption

Summary The recovery of the periodate-reactive material in the rat liver and human costal cartilage after fixation and decalcination was estimated, determing the periodate consumption rate of the sections. Carnoy solution gave the best recovery, about from 60 to 75 per cent in the case of the rat liver and about 87 to 97 per cent in the case of cartilage. Formaldehyde yielded somewhat lover recovery, and addition of 80 per cent ethanol to it did not significantly increase the preservation of the carbohydrates. Direct determination of the periodate consumption rate offers a simple and reliable method for estimation of the recovery of the periodate-reactive material in histological samples.     

Minocycline impairment of both osteoid tissue removal and osteoclastic resorption in a synchronized model of remodeling in the rat

In addition to their antibacterial effects, tetracyclines may inhibit interstitial collagenase activity and bone resorption. These properties were assessed morphometrically using minocycline (25 and 50 mg/kg/day given by the IM route) in a rat model of synchronized remodeling in which osteoclastic resorption peaks 4 days after the activating event (the extractions of the upper molars) along the antagonist mandibular cortex, a zone undergoing physiologically active formation. During the first 2 days of activation, minocycline at the two doses impaired very significantly the disorganization of both the osteoid seam and the layer of osteoblasts, a prerequisite to give osteoclasts access to the mineralized bone surface. The number of readily identifiable osteoblasts decreased slightly during this period, suggesting that minocycline prevented their transformation into lining cells. Their synthetic activity, as estimated by the size of the cells and their nucleus, appeared relatively preserved too, mostly with the higher dose. At the peak of osteoclascia, the bone surfaces undergoing remodeling were significantly decreased in the minocycline-treated groups. The resorption surface was reduced (P < 0.0003) as well as the number of osteoclasts (P < 0.0007), which were also significantly smaller. Their resorbing activity was dramatically affected as well: they excavated lacunae whose area was significantly reduced by over 70%. In addition, formation was still a prominent activity in the treated animals. These data are compatible with the inhibition at the early stages of activation of an osteoblast-secreted collagenase whose action may be the elimination of the osteoid seam. The inhibition of an osteoclast collagenase and/or of a bone matrix bound-collagenase may be responsible for the reduction in lacunar size. A direct effect of minocycline on osteoclast resorptive activity may also participate in the low resorption profile, as tetracyclines are known to interfere with the intracellular [Ca²⁺]. © 1996 Wiley-Liss, Inc.