Angioarchitecture of the ventral surface of the tongue from Wistar rats

The goal of this study was to describe the angioarchitecture of the ventral surface of the tongue from Wistar rats using a vascular corrosion casting technique associated with scanning electron microscopy (SEM). Six Wistar rats were used for the vascular casting method with Mercox resin. Following the resin polymerization, the tongue of each animal was dissected and corroded in a 5% sodium hydroxide solution. Once the corrosion and drying of the specimens were completed, the specimens were mounted on aluminum stubs, coated with carbon and gold and analyzed under SEM. The results showed that the ventral surface of the tongue presents simple, even and abundant vasculature constituted by a vascular plexus consisting of a superficial vascular network and by the ranine veins. The superficial vascular network, made up of the ascending and descending branches, presents as a loose network, with little morphological variation between the capillary loops.     

Contribution to the study of the vasculature of submandibular and sublingual glands and lymph nodes of rats by corrosion cast technique combined with scanning electron microscopy

The study of anatomical structures in their normal state allows the identification of pathological changes that can occur in them. Angiogenesis and the vasculature have been widely studied, mainly because of their association with the development of neoplasms. One of the methods applied for such purposes is the corrosion cast technique, which provides a copy of the vessels with normal as well as pathological structures. The replica of the vasculature provided by this technique allows the three-dimensional analysis of vessels by means of scanning electron microscopy. The aim of the present study was to demonstrate, by means of corrosion casts, the angioarchitecture of the submandibular and sublingual glands and lymph nodes. Scanning electron microscopy showed that the three structures have distinct vascular patterns. The corrosion cast technique can be employed in the study of the angioarchitecture of the submandibular and sublingual glands and lymph nodes, but requires specific precautions. The removal of the structures en bloc and the handling of the replicas with the aid of a stereoscopic magnifier reduce the risk of fractures.     

New corrosion cast media and its ability for SEM and light microscope investigation

SEM of corrosion casts (CC) provides the opportunities to study the vessels and ducts in the phyllogenetic and ontogenetic (age‐related) settings, as well as the pathogenesis, compensation, and sanogenesis in different diseases and experimental models. Along with the refinement of SEM CC, the requirements toward casting media (CM) as nontoxicity, low viscosity, quick polymerization, resistance to corrosion solutions, availability, and so on, gradually has developed. We aimed to adapt the sets widely used in dental practice toward the modern requirements to the CC. The following ratio of the components of Protacryl‐M and Aycryl‐C sets were used for the preparation CM—0.25 g MAYCRYL Powder +0.08 g Benzoyl Peroxide +5.0 ml Protacryl‐M liquid component +0.2 Redont Colour (dye concentrate). The obtained solidifying mass was injected in the blood vessels and biliary ducts of the adult Wistar white rats. The SEM of CC of different organs’ vascular networks, as well as a biliary tract, reveals that offered CM excellently replicates the forms and branching features of studied tubular structures of all sizes and gives the adequate imprinting of their luminal surfaces. Besides, CM may provide the replication of perivascular spaces and give the casts having no analogous in the appropriate literature. The CM prepared by us perfectly reproduces all possibilities of famous rubbers widely used for the casting of different vascular–ductular structures. Besides, it presents the new implications, which should be implemented in the profound research of the connective‐tissue skeleton of different organs.     

Distinct vascular zones in the canine prostate

The vascular bed of canine prostate was studied and detailed distinct vascular zones were visualized on corrosion casts by scanning electron microscopy. This study was performed because of scarce information about the zonal vascularization of the prostate gland in dogs. There are no studies for three-dimensional microvascular distribution of the capsular vessels and the capsular microvascular trabeculae. SEM (vascular corrosion casting method) was used to show 3D angioarchitecture of the prostate gland. The lobules on the dorsal and lateral surface of the gland were numerous but small. Their small size is probably due to the abundant blood supply in the region. Few but large ventral lobules were observed. Three prostatic zones were clearly defined: capsular, parenchyma, and urethral. The diameter of the venous blood vessel compared to arterial vessels of the capsule was smaller. Two types of arteries were observed in trabeculae: direct and branched. The direct arteries were straight, with only a few branches. The branched arteries contained many bifurcations, with the vessel's diameter decreasing gradually. The trabeculae capillary network formed loops, with frequent sphincter-like constrictions and pouch-like protuberances.     

The effect of the anti‐angiogenic drug sunitinib malate on the vascular architecture of oral squamous cell carcinoma

The effects of anti‐angiogenic therapies in guiding tumor angioarchitecture prompted us to examine the modifications in the vascular network of the oral squamous cell carcinoma (SCC) produced by the multitargeted tyrosine kinase inhibitor sunitinib malate. Twelve Syrian hamsters had their right buccal pouches submitted to tumor induction with dimethylbenzanthracene and carbamide peroxide for 55 days. The animals were then divided into two groups of six animals each; group I was treated with sunitinib malate and group II (control) was remained untreated. After 4 weeks, the hamsters had their vascular networks casted by Mercox^® resin and analyzed by scanning electron microscopy. The qualitative study of the vascular network of the control tumor‐bearing pouches showed images of intussusception and sprouting angiogenesis, flattened blood vessels, abrupt variations in their diameter, and a tortuous course. The samples treated with sunitinib exhibited a qualitative reduction of the signs of vascular proliferation. In addition, these casts presented an attenuation of the morphological features observed in the untreated tumor‐bearing pouches. Quantitative analysis demonstrated that the pouches treated with sunitinib did not show a decrease (P > 0.05) in the vascular diameter and intervessel distances when compared with the control group. The results of the present study suggest that sunitinib may act on the vascular network of oral SCC, normalizing the blood vessels. However, further experiments should be performed in order to determine a judicious dose of this anti‐angiogenic therapy. Microsc. Res. Tech. 77:250–256, 2014. © 2014 Wiley Periodicals, Inc.     

Systemically alendronate was incorporated into dental tissues but did not cause morphological or mechanical changes in rats teeth

This study evaluated the effect of the systemic use of sodium alendronate in rats in vivo. Forty‐five Wistar rats aged 36 to 42 days and weighing 200 to 230 g were randomly assigned to a control group (n = 20), which received distilled water, and an experimental group (n = 25), which received 2 weekly doses of 1 mg/kg of chemically pure sodium alendronate. The animals were killed after 60 days of treatment. The tibias were removed for analysis of bone mineral density by dual‐energy X‐ray absorptiometry (DXA). Then, the maxillary incisors were extracted for analysis of the mineralized dental tissues using fluorescence spectroscopy (FS), scanning electron microscopy (SEM), bright field microscopy (BFM), and cross‐sectional microhardness (CSMH) testing. DXA and CSMH data were subjected to statistical analysis by Kruskal‐Wallis test (5% significance level). The experimental group presented higher bone mineral density than the control group by DXA. FS analysis revealed presence of alendronate in the mineralized dental tissues of the specimens of the experimental group. Significant morphological differences were not found by SEM and BFM. Enamel and dentin (100 and 300 μm from the dentinoenamel junction) CSMH data did not show significant difference between the control and experimental groups. Based on the obtained results, we conclude that while alendronate increased the bone mineral density and was incorporated into the mineralized dental tissues it did not cause significant alterations in the morphology and microhardness of rat incisor enamel and dentin. Microsc. Res. Tech. 75:1265–1271, 2012. © 2012 Wiley Periodicals, Inc.     

Estimation of length and surface of anisotropic capillaries

Surface density (S_V) and length density (L_V) of myocardial capillaries have hitherto been estimated from their profile boundary length (B_A) and their numerical density (Q_A) on transverse sections by the simplifying assumptions of the Krogh model (perfectly anisotropic, straight, unbranched capillaries with constant cross-sectional area). As the capillaries actually are partially anisotropic, curved, branching cylinders with variable cross-sectional area, a geometrical bias arises from the model-reality discrepancies. We have applied and compared two methods to overcome these inconsistencies: (1) estimation of L_V and S_V by a more realistic model (the Dimroth-Watson distribution); (2) estimation of L_V and S_V from isotropic uniform random (IUR) sections. Twelve male Wistar rats were fixed by retrograde vascular perfusion. One pair of longitudinal and transverse sections, and six IUR sections per animal were selected at random from the left ventricular papillary muscles. Ultrathin sections were silver-impregnated and studied by light microscopic morphometry. Nearly identical estimates of L_V and S_V were found by both methods. The model-based estimation provides biologically meaningful anisotropy constants, but it presupposes knowledge of the anisotropy axis. The IUR method provides no measure of anisotropy, but it can be applied in tissues where the anisotropy axis is not known. Both methods are equally efficient and practically unbiased in S_V estimation, but the model-based estimation is far more efficient in L_V estimation.     

Electron microscopic observation of the sagittal structure of Drosophila mature sperm

Observation of sperm development and determination of their morphological characteristics are very important to the understanding of phylogenetic relationships and the study of sperm function during fertilization. Although ultrastructural studies of sperm development in the testes of the fruit fly Drosophila have been performed, there are few reports describing electron microscopic morphology of mature sperm, that is, those released from the testes to the seminal vesicles. Here, we present the first report of the sagittal organization of Drosophila sperm head and neck regions by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The head and tail structures of a mature sperm, for example, the acrosome, nucleus, and flagellum, were easy to distinguish by the morphological characteristics of the sperm surface by SEM. The morphological relationships between the surface and internal structures of mature sperm were confirmed by observing longitudinal sections with TEM. Our approach overcame the technical difficulties involved in sample preparation for electron microscopic observation of the Drosophila mature sperm head, and therefore, this study serves as an important foundation for future genetic dissection of sperm ultrastructure and function in male sterile mutants. Microsc. Res. Tech. 77:661–666, 2014. © 2014 Wiley Periodicals, Inc.     

Adhesive interface and bond strength of endodontic sealers to root canal dentine after immersion in phosphate‐buffered saline

This study evaluated the bond strength (BS) and the adhesive interface of four endodontic sealers to root canal dentine, before, and after immersion in phosphate buffered saline (PBS) to simulate an in vivo environment. Eighty roots were instrumented using ProTaper rotatory files, under irrigation with 17% EDTA and 1% NaOCl. Posteriorly were divided into four groups (n = 20) according to the sealer used: Endofill, AH Plus, Sealapex, and MTA Fillapex. Each group was divided into two subgroups (n = 10) and stored at 37°C immersed in water for 7 days and in PBS for 60 days. From each subgroup, 1 mm thick sections were obtained. One section of each region (coronal, middle, and apical) was submitted to the push‐out test and failures were observed. Twelve sections of each subgroup (four from each region) were evaluated under SEM. Three‐way ANOVA evaluation for BS showed significant differences between groups and regions (P < 0.0001), but not between subgroups (P > 0.05). AH Plus had significantly higher BS than the others sealers, regardless of the analyzed subgroup (Tukey's test, P < 0.5). The most common failures were adhesive to dentine and cohesive of the sealer. The SEM evaluation (Kruskal–Wallis, Mann–Whitney) showed homogeneous adhesive interface formed and sealer tags in all groups with significant statistical differences with AH Plus, regardless of PBS immersion. AH Plus was superior to the other sealers for both BS and quality of interface formation. Immersion in PBS did not interfere on BS or adhesive interface of the sealers tested. Microsc. Res. Tech. 77:1015–1022, 2014. © 2014 Wiley Periodicals, Inc.     

Comparison of different irrigation activation techniques on smear layer removal: An in vitro study

The purpose of this study was to evaluate the efficiency of different irrigation activation techniques on smear layer removal. About 80 single‐rooted human maxillary central teeth were decoronated to a standardized length.The samples were prepared by using ProTaper system to size F4 and divided into eight equal groups (n = 10) according to the final irrigation activation technique; distilled water was used as an irrigant in Group 1. The other groups were treated with 2.5% NaOCl and 17% EDTA, respectively. Conventional syringe irrigation (CSI) was used in Group 2. Irrigation solutions were activated using passive ultrasonic irrigation (PUI, Group 3), EndoVac apical negative pressure (ANP, Group 4), diode laser (Group 5), Nd:YAG laser (Group 6), Er:YAG laser (Group 7), and Er:YAG laser using with photon‐induced photoacoustic streaming (PIPS?, Group 8). Teeth were split longitudinally and subjected to scanning electron microscope (SEM). PIPS showed the best removal of smear layer when compared with PUI, ANP, Nd:YAG, and Er:YAG, but the difference was not statistically significant (P > 0.05). Smear layer scores obtained with PIPS technique were statistically significant different from those of obtained with control, CSI and diode laser groups (P < 0.05). All experimental irrigation techniques except ANP and diode laser removed smear layer more effectively at the coronal and middle levels compared to the apical level (P < 0.05). Irrigation activated/delivered techniques except diode laser have a positive effect on removing of smear layer. Microsc. Res. Tech. 78:230–239, 2015. © 2015 Wiley Periodicals, Inc.     

Ultrastructural changes in the cemento‐enamel junction caused by acidic beverages: An in vitro study

The present in vitro study was aimed at evaluating the morphological changes in the cemento‐enamel junction (CEJ) after exposure to acidic beverages using the scanning electron microscopy (SEM). The initial pH and titratable acidity (TA) was analyzed from follow groups: (I) Coca cola, (II) orange juice, (III) Cedevita, (IV) Red Bull, (V) Somersby cider, and (VI) white wine. The CEJ samples (n = 64), obtained from unerupted third molars, were allocated to one control (artificial saliva, n = 16) and six experimental groups (n = 8). The experimental samples were immersed in beverages (50 ml) for 15 min, three times daily, 10 days, and in artificial saliva between immersions. SEM analysis was performed in a blind manner, according to scoring scale. One‐way ANOVA and Tukey's post hoc tests, as well as Kruskal–Wallis and Mann–Whitney U test used for statistical analysis. The pH values of the acidic beverages ranged from 2.65 (Coca cola) to 3.73 (orange juice), and TA ranged from 1.90 ml (Coca cola) to 5.70 ml (orange juice) of NaOH to reach pH 7.0. The SEM analysis indicated statistically significant differences between the control samples and those immersed in acidic beverages. The Groups IV, I, and II, showed the highest CEJ damage grade while those of the Group VI were the lowest. All the tested acidic beverages caused morphological changes in the CEJ with a smaller or larger exposure of dentine surface, and were not always related to the pH or TA of acidic beverages.     

In vivo biocompatibility versus degree of conversion of resin‐reinforced cements in different time periods

This study focused on test the null hypothesis that there is no difference between the degree of conversion and biocompatibility of different resin reinforced glass ionomer cements (RRGICs). Forty‐eight male Wistar rats were used, distributed into four groups (n = 12), as follows: Group C (Control, polyethylene), Group FOB (Fuji Ortho Band), Group UBL (Ultra band Lok), and Group MCG (Multicure Glass), in subcutaneous tissue. The events of edema, necrosis, granulation tissue, multinuclear giant cells, young fibroblasts, and collagen formation were analyzed at 7, 15, and 30 days. The degree of conversion was evaluated by the Fourier method. Biocompatibility and degree of conversion were assessed using the Kruskal–Wallis and Dunn tests, and ANOVA and Tukey's test, respectively (P < 0.05). It was observed that, there was significant difference between Groups FOB and UBL for the presence of young fibroblasts at 15 days (P = 0.034) and between the Control and MCG Groups for the presence of multinucleated giant cells at 30 days (P = 0.009). Monomer conversion increased progressively until day 30, with significant difference between Group FOB and Groups UBL and MCG (P = 0.013) at 15 days. The null hypothesis was partially accepted, Fuji Ortho Band showed a less monomer conversion and a smaller number of young fibroblasts in the time of 15 days. Microsc. Res. Tech. 77:335–340, 2014. © 2014 Wiley Periodicals, Inc.     

Microvasculature of the cerebral cortex: A vascular corrosion cast and immunocytochemical study

In mammals, the cerebral cortex microvasculature (CCM) of the neopallium plays important roles in the physiological and pathological processes of the brain. The aim of the present work is to analyze the CCM by use of the SEM‐vascular corrosion cast technique, and to examine the immunocytochemical characteristics of the CCM in adult domestic ruminants (cattle, buffalo, and sheep) by using the SEM‐immunogold technique. The CCM originated from the very small, finger‐like terminal branches of the macrovasculature of the brain. The superficial cortical arterioles were more numerous than the deep straight arterioles which proceeded toward the white matter. The surface casts of the arterioles and capillaries of the cerebral cortex showed ring‐shaped formations in the arterioles and at the origin of the capillaries. All capillaries down‐stream from these ring‐shaped formations were flaccid. Casts of the capillaries showed wrinkles due to the presence of endothelial folds, which is characteristic of varying blood pressure. Formations having intense anti‐GIFAP immunoreactivity were frequently evident along the course of the blood capillaries in the cerebral cortex. These formations were probably astrocytes that might regulate the cerebral microcirculation based on physiological and pathological stimuli, such as neuronal activation. Microsc. Res. Tech. 77:257–263, 2014. © 2014 Wiley Periodicals, Inc.     

Tissue integrity,costs and time associated with different agents for histological bone preparation

The selection of an appropriate demineralizing solution in pathology laboratories depends on several factors such as the preservation of cellularity, urgency of diagnostic and financial costs. The aim of this study was to test different decalcification bone procedures in order to establish the best value of these in formalin‐fixed and paraffin‐embedded samples. Femurs were removed from 13 adult male Wistar rats to obtain 130 bone disks randomly divided into five groups that were demineralized in different concentrations of nitric acid (Group I); formic acid (Group II); acetic acid (Group III); EDTA, pH7.4 (Group IV) and Morsés solution (Group V). Serial, 3‐μm‐thick sections were obtained and stained with hematoxylin‐eosin to calculate the percentage of osteocyte‐occupied lacunae. The sections were also stained with Masson's trichrome in conjunction with picrosirius red under polarized light followed by a semi‐quantitative analysis to verify the adjacent muscle‐to‐bone integrity and preservation of collagen fibres. The highest percentage of osteocyte‐occupied lacunae was found with 10% acetic acid solution (95.64 ± 0.95%) and Group I (nitric acid) demanded the shorter time (0.8–5.7days). Of all solutions, 5% nitric acid incurred the lowest cost to achieve complete demineralization compared with other solutions (p < .001). Group IV (EDTA) had the highest integrity of muscle and collagen type I and III (P < 0.01). Demineralization with 10% acetic acid was the most effective at preserving bone tissue, while 5% EDTA was the best at maintaining collagen and adjacent muscle to bone. In conclusion, nitric acid at 5% showed the most efficient result as it balanced both time and cost as a demineralizing solution.     

The microvascular cast as a three-dimensional tissue skeleton: visualization of rapid morphological changes in tissues of the rat uterus

A new interpretation of microvascular corrosion casting is described using examples from casts of the rat uterus taken during the oestrous cycle and early pregnancy. The casts are considered as representing a three-dimensional model of the tissue vascular space; this characteristic model or tissue skeleton is used to demonstrate transient morphological changes that may be difficult to visualize by other techniques, that may be obscured by other structures, or destroyed by conventional aldehyde fixation procedures. This new application, when supplemented by conventional histology to determine vessel distribution within the tissue and to confirm observations first made by casting, provides a technique for visualizing three-dimensional morphological changes in tissues that is accurate, relatively simple and far more rapid than the laborious method of histological serial sectioning and three-dimensional tissue reconstruction.     

Effect of chitosan and Dysphania ambrosioides on the bone regeneration process: A randomized controlled trial in an animal model

The focus of this triple‐blind study was on evaluating the effect of chitosan combined with Dysphania ambrosioides (A) extract on the bone repair process in vivo. In total, 60 male Wistar rats (Rattus norvegicus albinus) weighing between 260 and 270 g were randomly selected for this study and distributed into four groups (n = 15). Group C (chitosan), Group CA5 (chitosan + 5% of D. ambrosioides), Group CA20 (chitosan + 20% of D. ambrosioides), and Group CO (Control‐Blood clot). In each animal, bone defects measuring 2 mm in diameter were performed in both tibias for placement of the substances. After 7, 15, and 30 days, the animals were sedated and sacrificed using the cervical dislocation technique and the tissues were analyzed under optical microscope relative to the following events: inflammatory infiltrate, necrosis, osteoclasts, osteoblasts, fibroblasts, periosteal, and endosteal bone formation. The data were evaluated to verify distribution using the Kolmogorov–Smirnov test, and variance, using the Levene test; as distribution was not normal, data were subjected to the Kruskal–Wallis and Dunn nonparametric tests (p < .05). A significant inflammatory infiltrate was observed in Group CA5 (p = .008) in the time interval of 7 days, and in Group C at 15 (p = .009) and 30 (p = .017) days. Osteoblastic activity was more significant in Group CA20 (p = .027) compared with CA5 in the time interval of 7 days. Group CA20 demonstrated a significantly higher endosteal and periosteal bone formation value in the time interval of 7 (p = .013), 15 (p = .004), and 30 days (p = .008) compared with the other groups. The null hypothesis was refuted, bone regeneration was faster in spheres with an association of chitosan and 20% extract, and complete bone repair occurred clinically at 15 days and histologically at 30 days. The spheres proved to be a promising method for the biostimulation of alveolar bone repair and bone fractures.     

Evaluation of microvasculature at the auditory midbrain–the benefits of sectioning at a tangential angle

Vascular remodeling in the brain occurs as a plastic change following neural over‐activity. The auditory midbrain (or inferior colliculus, IC) is an ideal place to study sound‐induced vascular changes because it is the brain's most vascularized structure and it is tonotopically organized. However, its micro‐vascular pattern remains poorly understood. Since the IC is a sphere‐like structure, the histological assessment of vasculature could depend on the angle of sectioning. Here, we studied the effects of cutting the IC at different angles on microvascular assessment, specifically: micro‐vascular density and the shape of microvascular lumen. Photomicrographs were taken from 5 µm toluidine blue‐stained histological sections obtained at two angles of sectioning: (a) the conventional coronal sectioning, and (b) a novel “tangential” sectioning (tangential to the dorso‐medial surface of the IC). Results showed that the tangential sections, in comparison with the coronal sections, yielded (a) a higher count of micro‐vascular density and (b) a higher proportion of round‐shaped micro‐vascular lumens. This discrepancy in results between two cut angles is likely related to the spatial pattern of blood vessels supplying the IC. We propose that the tangential sectioning should be adopted as standard for the accurate study of microvasculature in the IC. Microsc. Res. Tech. 78:105–110, 2015. © 2014 Wiley Periodicals, Inc.     

Effect of Chenopodium ambrosioides on the healing process of the in vivo bone tissue

The focus of this double‐blind randomized study was on evaluating the effect of an aqueous extract of Mastruz (Chenopodium ambrosioides L.) on the bone repair process in vivo. In total, 36 male Wistar rats were randomly selected for this study, and divided into 3 groups (n = 12): Group HS (Hemostatic Sponge), Group SM (Hemostatic Sponge with Mastruz) and Group BC (Blood Clot). In each animal, bone defects measuring 2 mm in diameter were performed in both tibias for placement of the substances. After 3 and 10 days, the animals were sacrificed, and the tissues were analyzed under an optical microscope relative to the following events: inflammatory infiltrate; necrosis; young fibroblasts; osteoclastic and osteoblastic activity; endosteal and periosteal bone formation; and bone repair. The results were assessed by using Kruskal–Wallis and Mann–Whitney tests (p < .05). Inflammatory infiltrate demonstrated difference between Groups SM and BC in the time interval of 3 days (p = .004); an event related to the presence of the fibrin sponge and liquid of the extract, which induced a foreign body initial reaction. The presence of young fibroblasts (p = .003), osteoclastic (p = .003), and osteoblastic (p = .020) activity was statistically significant between Groups HS and BC in the time interval of 10 days; performance was related to the presence of the sponge within bone. As regards injured bone tissue repair, Group SM demonstrated a higher level of regenerative capacity (p = 0.004), due to a larger quantities of endosteal and periosteal bone formation, demonstrated in Group SM. The aqueous extract of mastruz stimulated bone neoformation, presenting wound closure with bone tissue at the end of 10 days.