Bone marrow cells of swine: collection and separation

Bone marrow is a source of stem cells for greater and easier access, which is widely studied as a provider of hematopoietic and mesenchymal cells for various purposes, mainly therapeutic by the advances in research involving cell therapy. The swine is an animal species commonly used in the pursuit of development of experimental models. Thus, this study aimed to standardize protocol for collection and separation of bone marrow in swines, since this species is widely used as experimental models for various diseases. Twelve animals were used, which underwent bone marrow puncture with access from the iliac crest and cell separation by density gradient followed by a viability test with an average of 98% of viable cells. Given our results, we can ensure the swine as an excellent model for obtaining and isolation of mononuclear cells from bone marrow, stimulating several studies addressing the field of cell therapy.     

Immunolocalization indicates that both original and regenerated lizard tail tissues contain populations of long retaining cells,putative stem/progenitor cells

The regeneration of the tail in lizards is likely sustained by stem/progenitor cells located in the stump after amputation of the tail. This microscopic and ultrastructural study shows the localization of 5‐bromo‐deoxy‐uridine (5BrdU)‐long retaining labeled cells in different tissues of the tail stump. These putative stem/progenitor cells are sparsely detected in the epidermis of scales, adipose tissue, intermuscle connective septa, myosatellite cells, and perichondrion of the vertebrae. Most of 5BrdU‐labeled cells are present in the bone marrow of vertebrae as hemocytoblasts and reticulate cells, whereas more numerous myelocytes and polychromatophilic erythroblasts show a variable level of nuclear labeling. 5BrdU and tritiated‐thymidine labeled and unlabeled hemopoietic cells are seen in circulating vessels and in the blastema where their maturation is completed. This observation indicates that the entire differentiation span of both white and red blood cells, at least during tail regeneration, lasts longer than 4 weeks. Labeled polychromatophilic erythroblasts and heterophilic and basophilic myelocytes are present in the synusoidal vessels of the regenerating tail. This study indicates that extravasating blood cells involved in immunity make large part of the forming blastema cell population, but are replaced by mesenchymal cells of different origin. The presence of long retaining labeled cells in tissues of the tail stump is likely connected to the production of blastema mesenchymal cells. Although no direct cell‐lineage study has been done, histological, immunocytochemical, and autoradiographic studies have indicated that it is from these tissues that proliferating cells appear mainly localized after tail amputation and blastema formation. Microsc. Res. Tech. 78:1032–1045, 2015. © 2015 Wiley Periodicals, Inc.     

Fate of donor bone marrow cells in medial collateral ligament after simulated autologous transplantation

A potential strategy to enhance ligament healing by transplantation of mesenchymal stem cells (MSCs), which are demonstrated to differentiate into fibroblast-like cells in vitro, is presented. The objective of this study was to follow transplanted nucleated cells from bone marrow, which contain MSCs, in the healing medial collateral ligament (MCL) over time, and to examine their phenotype and survivability. It was hypothesized that MSCs in nucleated cells from bone marrow would differentiate into fibroblast-like cells in the healing ligament following adaptation to the environment. The transplantation model employed in this study eliminates the immune response to a donor by the recipient using a transgenic rat (donor), which does not produce foreign protein from transgenes, and its wild-type rat (recipient) in order to simulate autologous transplantation. The MCL of the wild-type rat was ruptured, where 1 x 10(6) nucleated cells of bone marrow from the transgenic rat were injected. The transgenes in transplanted nucleated cells were detected throughout the healing MCL for 28 days by in situ hybridization. At 3 days, many donor cells were evident in the injury site and fascial pocket, and some were found in the midsubstance. Morphologically, transplanted cells with elongated nuclei were found at the ruptured edge of the midsubstance and surface of the unruptured site after 3 days. At 28 days, these cells continued to survive in the healing MCL. Their shapes were similar to those of surrounding recipient MCL fibroblasts. Thus, transplanted cells might differentiate into fibroblasts. Therefore, it was demonstrated that there is a potential for nucleated cells from bone marrow to serve as a vehicle for therapeutic molecules as well as to be a source in enhancing healing of ligaments.     

Extracellular breakdown of collagen by mice decidual cells. A cytochemical and ultrastructural study.

The interaction of antimesometrial decidual cells and collagen fibrils was studied by light microscopy and ultrastructural cytochemistry in fed and acutely fasted mice on days 9-11 of pregnancy. Fibrillar elements in the extracellular space consisted of collagen fibrils and filamentous aggregates (disintegrating collagen fibrils). Intracellular vacuoles exhibited typical collagen immersed in electron-translucent material (clear vacuoles) and faint cross-banded collagen immersed in electron-opaque material (dark vacuoles). Fibrillar elements showed extracellular acid phosphatase activity which was stronger in the region of mature decidua than in predecidual cells region in all animals; it was conspicuous in mature decidua of fasted animals. Intracellular acid phosphatase activity was observed in dark vacuoles and lysosomes, and was absent in clear vacuoles in all cells studied. Since acid phosphatase activity reflects the presence of lysosomal hydrolases in general, the results indicate that breakdown of extracellular collagen occurs by release of lysosomal enzymes by decidual cells and also by internalization of collagen for intracellular degradation in fed and fasted mice. Collagen breakdown may be part of the process of tissue remodeling in mature and predecidual regions, however, in mature decidua, collagen breakdown is enhanced and may therefore contribute to nutrition of the fetus, specially in acutely fasted mice.     

Cytotoxicity of fipronil on mice liver cells

In the present study, mice livers were examined following exposure to different doses of fipronil (15, 25, and 50 mg/kg). Histological and histochemical techniques were used to determine the cytotoxic potential of this compound and to assess the damage it caused to livers. Mice were divided into four groups: control group and groups I, II, and III were exposed to 15, 25, and 50 mg/kg fipronil, respectively. Our findings revealed cytological, morphohistological, and histochemical alterations in liver cells of animals from groups I, II, and III compared to group control animals. These changes included Kupffer-cell proliferation, hepatocyte hypertrophy, accumulation and distribution of proteins, polysaccharides, lipids, and vacuoles in the cytoplasm of hepatocytes, and congestion of blood vessels. These phenotypes mainly characterize the following: (a) autophagic processes, (b) steatosis, and (c) cell death by necrosis, which demonstrate the damage caused by fipronil on nontarget organisms in artificial conditions.     

极低频电磁场对免疫低下小鼠免疫功能影响的实验研究

目的：研究极低频电磁场对环磷酰胺所致的免疫抑制小鼠免疫功能的影响。方法：极低频电磁场连续全身作用4周龄免疫低下小鼠60天后,测定小鼠血清抗体（IgG、IgM、IgA）、补体（C3、C4）的含量、腹腔巨噬细胞吞噬功能、胸腺指数和脾脏指数。结果显示：极低频电磁场对免疫低下小鼠的免疫功能有增强作用。     

Effects of prenatal stress on male offspring sexual maturity.

Prenatal stimulations have been shown to have long-term effects on at reproductive activity. We evaluated the influence of the prenatal stress on the hypothalamic-pituitary-gonad (HPG) axis in male offsprings from mothers with high number of offsprings per litter (HNL) and low number of offsprings per litter (LNL) after hypothesizing that the number of offsprings per litter may modify the effect of the prenatal stress on the HPG of adult offsprings. Pregnant Wistar rats were used for this study. Immobilization (IMO) stress was used, 30 min, 3 times per week, from the 5th to 21st day of pregnancy. The weight of adrenal and gonads, and the corticosterone (COR), testosterone (TES) and luteinizing hormone (LH) plasmatic levels were analyzed in the male offspring at 30, 45 and 70 days of age. The offspring males coming from LNL showed a decrease in testicle weight and TES levels, without changes in the plasmatic LH levels. However, the offspring of HNL showed a decrease of LH levels. It is possible to conclude that in LNL prenatal stress would produce alterations to gonadal level, while in HNL the effect of stress would be evident at pituitary level.     

Histological evaluation on bone regeneration of dental implant placement sites grafted with a self-setting alpha-tricalcium phosphate cement

This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R. Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology.     

Effects of amorphous layers on ADF-STEM imaging

A study of high-resolution ADF imaging in uncorrected and aberration-corrected STEMs was carried out by multislice simulation. The presence of amorphous layers at the surface of a crystalline specimen is shown to significantly alter the visibility of the atomic columns. After propagating through an amorphous layer a portion of the beam passes without any alteration while scattered electrons introduce a Gaussian background. The dependence of the image contrast on the crystal structure, orientation and the types of the atoms present in the crystal was studied. In the case of uncorrected probes an amorphous layer thicker than 200 A is necessary to achieve considerable reduction of the visibility of the atomic columns, but with aberration-corrected probes only 60 A is necessary. With changes in defocus, crystalline specimens with amorphous layers on the top can also be imaged and high-resolution ADF images can be obtained. An amorphous layer at the beam entry surface affects the ADF image more than that of an amorphous layer at the exit surface. Approximately linear reduction of the contrast (with a slop of 1) is expected with increased thickness of amorphous layer.     

Silver deposition on freeze-dried cells allows subcellular localization of cholesterol with imaging TOF-SIMS

Imaging time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) was used for characterization and subcellular localization of organic ions in leucocytes adhering to glass surfaces. The cells were fixed by freeze drying in 0.15 m ammonium formate buffer at pH 7.2–7.4. The freeze‐dried cells were sputter‐coated with silver, and the silver surface was analysed with imaging TOF‐SIMS. TOF‐SIMS spectra were recorded by scanning the primary ion beam over the analysis area and acquiring positive mass spectra of the ions leaving the surface. The relative brightness of each pixel within the analysis area reflects the signal intensity of a selected ion in that pixel. Data were collected separately at high mass resolution m/Δm > 7000 and at high lateral resolution (= 0.5 µm). The images were analysed by principal component analysis (PCA). The glass‐adhering cells showed a well defined attachment area with a diameter of up to 20 µm, and an equally well defined cell body, containing the nucleus, with a diameter of 8–10 µm. On the raw data images, the obtained cholesterol distributions were consistent with a higher cholesterol content of the cell membrane in the attachment area than in the cell body. Using PCA analysis, silver‐cationized molecular cholesterol was found localized mainly in the attachment area of the cells. Cholesterol was also seen at higher concentration in circular spots of ≤ 1 µm in diameter, probably representing caveolae.     

极低频脉冲电磁场对小鼠免疫功能的影响

目的：研究极低频脉冲电磁场对小鼠免疫功能的影响。方法：磁场强度为4高斯,频率为5Hz的极低频脉冲电磁场连续全身作用4周龄小鼠60天后,测定小鼠血清抗体（IgG、IgM、IgA）的含量、腹腔巨噬细胞吞噬功能、胸腺指数和脾脏指数。结果：磁场组的IgG、IgM含量、脾脏指数显著高于对照组（p〈0．01）;磁场组腹腔内巨噬细胞吞噬率显著高于对照组（p〈0．05）。结论：磁场强度为4高斯,频率为5Hz的极低频脉冲电磁场对小鼠的免疫功能有增强作用。     

Cell-surface ultrastructural changes during the in vitro neuron-like differentiation of rat bone marrow-derived mesenchymal stem cells

The neuron-like differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) has been extensively studied. However, the alternations of the cell-surface ultrastructures and the membrane tension/reservoir of the cells during this differentiation process are poorly understood. Therefore, atomic force microscopy (AFM) was utilized in this study to observe the cell-surface ultrastructural changes among rat bone marrow-derived mesenchymal stem cells (rBMMSCs), partially differentiated cells, and fully differentiated neuron-like cells. By analyzing the stiffness of plasma membranes, lamellipodial extensions, average heights of small membrane protrusions and relatively larger uplifted structures, and peak-peak spacing among protrusions and/or uplifted structures, we found that the membrane reservoir may potentially decrease upon the differentiation from rBMMSCs to partially differentiated cells and to fully differentiated neuron-like cells. The results may help to better understanding the membrane tension of various types of cells and related biological processes, such as membrane traffic, cell adhesion, motility, differentiation, among others. The data also implies that AFM may be a useful tool for evaluating membrane reservoir by imaging cell-surface ultrastructures.     

Effects of substrates on biofilm formation observed by atomic force microscopy

Formation of biofilm is known to be strongly dependent on substrates including topography, materials, and chemical treatment. In this study, a variety of substrates are tested for understanding biofilm formation. Sheets of aluminum, steel, rubber, and polypropylene have been used to examine their effects on formation of Pseudomonas aeruginosa biofilm. In particular, the morphological variation, transition, and adhesiveness of biofilm were investigated through local measurement by atomic force microscopy (AFM). Mechanism of removing biofilm from adhering to substrate is also analyzed, thus the understanding of the mechanism can be potentially useful to prevent the biofilm formation. The results reveal that formation of biofilm can remain on rough surface regardless of substrates in hot water, which may easily induce extra-polymeric substances detachment from bacterial surface. By probing using AFM, local force–distance characterization of extra-cellular materials extracted from the bacteria can exhibit the progress of the biofilm formation and functional complexities.     

Effects of microwave irradiation and chemical fixation on the localization of perisinusoidal cells in rat liver by gold impregnation

Modified gold impregnation is one of the methods that are used in light microscopical demonstration of hepatic perisinusoidal cells. This method has some disadvantages, such as restriction of fixation time to 16 h, which allows limited time for processing the tissues, especially when dealing with a large amount of material, and a long impregnation time (16–24 h). We investigated the effect of prolonged fixation on the staining of sections, to shorten the time needed for gold impregnation by using microwave irradiation. Liver specimens were fixed in Baker's calcium–formalin for different periods of time. After fixation, frozen sections were impregnated in gold chloride solution either at room temperature or in a microwave oven. The staining quality of the sections which had been impregnated in the microwave oven for a much shorter time were equal to or even superior to the ones impregnated at room temperature. Prolonging the fixation time up to 7 days did not affect the staining results by microwave irradiation, whereas satisfactory results were not obtained from sections stained at room temperature and fixed for more than 3 days. We conclude that microwave irradiation can be used to shorten the impregnation time in gold chloride solution and the duration of fixation can be prolonged up to 3 days in the original method and up to 7 days when microwave irradiation is used during impregnation.     

Effects of the ascorbic acid supplementation on NADH-diaphorase myenteric neurons in the duodenum of diabetic rats.

We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate the neuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.     

Effects of tilt on high-resolution ADF-STEM imaging

A study of the effects of small-angle specimen tilt on high-resolution annular dark field images was carried out for scanning transmission electron microscopes with uncorrected and aberration-corrected probes using multislice simulations. The results indicate that even in the cases of specimen tilts of the order of 1 degree a factor of 2 reduction in the contrast of the high-resolution image should be expected. The effect holds for different orientations of the crystal. Calculations also indicate that as the tilted specimen gets thicker the contrast reduction increases. Images simulated with a low-angle annular dark field detector show that tilt effects are more pronounced in this case and suggest that these low-angle detectors can be used to correct specimen tilt during scanning transmission electron microscopes operation.