Ultrastructural and immunohistochemical study of early repair of alveolar sockets after the extraction of molars from alendronate‐treated rats

The aim of the present research was to analyze ultrastructural and immunohistochemical aspects of the alveolar repair after the extraction of molars of alendronate (ALN)‐treated rats. Wistar rats received 2.5mg/kg body wt/day of ALN during 14 days previously and 7, 14 and 21 days after the extraction of the second mandibular molar. Specimens were fixed in 2% glutaraldehyde + 2.5% formaldehyde under microwave irradiation, decalcified in 4.13% EDTA and paraffin embedded for TRAP histochemistry and immunohistochemistry for OPN, BSP and endoglin, or embedded in Spurr epoxy resin for TEM analysis. Additional specimens had their soft tissues removed and were processed for scanning electron microscopy. The ALN group presented latent TRAP‐positive osteoclasts and nonresorbed alveolar crests with bacterial infection. Mild bone necrosis signs were observed at all time points studied. Ultrastructurally, empty osteocyte lacunae were observed and bone trabeculae surface presented hyalinized aspect. A significant delay in alveolar repair occurred, as well as decreased angiogenesis. ALN treatment provoked mild signs of bone necrosis, despite the high dose employed. The present findings add new information about the ultrastructural aspect of the early repair of rats under ALN treatment and highlight for giving attention when oral surgeries are performed in patients using this drug. Microsc. Res. Tech. 76:633–640, 2013. © 2013 Wiley Periodicals, Inc.     

New method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

The study of the ultrastructure of spematozoa by means of transmission electron microscopy (TEM) often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa Aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). In order to avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under a bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still-open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.     

A new method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

The study of the ultrastructure of spematozoa by means of transmission electron microscopy often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are: changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). To avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.     

An electron microscopic analysis on the ultra structural abnormalities in sperm of the common carp Cyprinus carpio L. inhabiting a polluted lake, Umiam (Meghalaya, India)

The present communication reports the ultra structural abnormalities in sperm of a fish species Cyprinus carpio inhabiting a polluted lake, Umiam in North‐East India. Transmission electron microscopy (TEM) revealed absence of differentiation between head and midpiece (neck) of some sperm while scanning electron microscopy (SEM) showed some sperm tails with highly reduced length and some sperm with folded tail. Abnormal shape of some sperm head was also revealed by Scanning electron microscopy. Detachment of membrane from some parts of the sperm head and an outward expansion of the same was observed from Transmission electron micrographs of transverse section of sperm head. The well developed mitochondria surrounding the cytoplasmic channel in the sperm tail, as observed in control were found to be drastically disorganized in fish inhabiting the polluted lake. The study suggests that the fish C. carpio inhabiting the polluted lake Umiam is under severe stress as far as its male reproductive system is concerned. The study further suggests that Electron microscopic approach is extremely important in the assessment of adverse effects of environmental pollution on fish tissue. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.     

Angiogenesis of buffalo choroid plexuses: structural and immunocytochemical study

Mammalian choroid plexuses (CPs) are vascularized structures involved in numerous exchange processes that supply nutrients and hormones to the brain, and that remove deleterious compounds and metabolites from the brain. Studies in the adult Mediterranean buffalo have investigated the morphology of CPs using histochemical and immunohistochemical techniques. To date, however, there have been no studies conducted on ruminants regarding this removal process which serves to repair functional vascular damage in the CPs. Each of these vascular repair processes is a very complex and none of these has not yet been completely understood. Then, the aim of the present study is to investigate the morphological processes during angiogenesis in the CPs of healthy adult buffaloes, utilizing transmission electron microscopy (TEM), scanning electron microscopy (SEM), and immunogold-labeling SEM analysis (biomarkers: angiopoietin-2 [Ang-2], vascular endothelial growth factor receptor-3 [VEGFR-3], and CD133). At TEM, the inner surface of the blood capillaries sometimes showed pillar-like cells, which in contact with endothelial cells formed prominences, which in turn formed neo-blood capillaries. With immunogold-labeling SEM analysis, the CP blood capillaries showed Ang-2 and VEGF-3, respectively, in positive particles and spheroid formations. In addition, the external surface of the blood capillaries showed spheroid formations that originated from the neo-vascular capillaries whose terminals formed a capillary network, positive to CD133. On the basis of these results, the following hypothesis can be made, namely, that these CPs are vascular structures which play a fundamental role in maintaining brain homeostasis and self-repairing of functional vascular damage, independently of the presence of rete mirabile in this species.     

Shear bond strength and ultrastructural interface analysis of different adhesive systems to bleached dentin

Background: It remains unclear as to whether or not dental bleaching affects the bond strength of dentin/resin restoration. Purpose: To evaluated the bond strength of adhesive systems to dentin submitted to bleaching with 38% hydrogen peroxide (HP) activated by LED‐laser and to assess the adhesive/dentin interfaces by means of SEM. Study design: Sixty fragments of dentin (25 mm²) were included and divided into two groups: bleached and unbleached. HP was applied for 20 s and photoactivated for 45 s. Groups were subdivided according to the adhesive systems (n = 10): (1) two‐steps conventional system (Adper Single Bond), (2) two‐steps self‐etching system (Clearfil standard error (SE) Bond), and (3) one‐step self‐etching system (Prompt L‐Pop). The specimens received the Z250 resin and, after 24 h, were submitted to the bond strength test. Additional 30 dentin fragments (n = 5) received the same surface treatments and were prepared for SEM. Data were analyzed by ANOVA and Tukey's test (α = 0.05). Results: There was significant strength reduction in bleached group when compared to unbleached group (P < 0.05). Higher bond strength was observed for Prompt. Single Bond and Clearfil presented the smallest values when used in bleached dentin. SEM analysis of the unbleached specimens revealed long tags and uniform hybrid layer for all adhesives. In bleached dentin, Single Bond provided open tubules and with few tags, Clearfil determined the absence of tags and hybrid layer, and Prompt promoted a regular hybrid layer with some tags. Conclusions: Prompt promoted higher shear bond strength, regardless of the bleaching treatment and allowed the formation of a regular and fine hybrid layer with less deep tags, when compared to Single Bond and Clearfil. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Ultrastructure of cyst shell and underlying membranes of three strains of the brine shrimp Artemia (Branchiopoda: Anostraca) from South India

The cyst of Artemia has shell and membranous coverings over the embryo. The membranous coverings have special adaptive features to allow the physical changes accompanying repeated hydration and dehydration cycles that might occur and adversely influence postembryonic development. Whole and slices of cryptobiotic cysts were processed for electron microscopy to study the internal details and to compare the morphological architecture of three Artemia strains of South India. Surface topography of scanning electron microscopic (SEM) studies revealed distinct button shaped structures on the cyst of Puthalam strain. Transmission electron microscopic (TEM) studies of the cysts displayed the conventional pattern of anostracan crustaceans with outer cortex and alveolar layer, cuticular membranes, and the cytoplasmic inclusions namely nucleus, yolk droplets, lipoid bodies, and mitochondria. The prominent wavy outer cortex layer of Puthalam cysts corroborates the results of SEM studies.     

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.     

Microvasculature of the buffalo (Bubalus bubalis) choroid plexuses: structural, histochemical, and immunocytochemical study

The choroid plexuses (CPs) in mammals produce the cerebrospinal fluid (CSF). In the literature, the morphology of CPs and the process that regulates the production of CSF are virtually nonexistent for domestic ruminants. Thus this study has two aims: 1. to investigate the morpho-structure of the buffalo CP microvasculature utilizing light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques, and 2. to investigate the relationship between the blood vessels and both the elongated cells and the cells with multiple protrusions located in the CPs. SEM and TEM analyses of the CPs from buffalo brain showed morphological and structural features similar those reported in other mammalian species. Moreover the blood microvasculature is the major component responsible for the formation of the CSF, secreted by the encephalic CPs. In addition the chemical composition of this fluid depends on several morpho-functional characteristics of the vascularization of the CPs. These characteristics are as follows: two shapes of the vascular organization: lamina-like and ovoid-like elongated cells of the CPs, which connect the ventricular cavities to the blood capillaries; and the CP capillaries have diverse forms. In the present study the employment of NADPHd and NOS I was taken as indirect evidence for the presence of NO for investigation their specific role in CPs. Then NOS I immunoreactivity is found in the walls of CP blood vessels demonstrating indirectly the presence of NO with a vaso-dilatatory and autoregulation function of vascular tone by cholinergic nerve stimulation of blood vessel smooth muscle.     

Ultrastructure and distribution of antennal sensilla of the chilli thrips Scirtothrips dorsalis hood (Thysanoptera: Thripidae)

The chilli thrips, Scirtothrips dorsalis Hood, is a serious pest of numerous important vegetable and ornamental crops. Various signals, especially phytochemical cues, determine the behavior of the phytophagous thrips at host selection. The sensory abilities of S. dorsalis are poorly understood although the antennae of adult are known to possess important sensory structures in orther insects. In this study, the morphology, distribution, and ultrastructure of the antennal sensilla of the S. dorsalis were examined by using scanning and transmission electron microscopy. Microscopy observations revealed that adult male and female S. dorsalis possess filiform antennae. Each antenna comprises a scape, a pedicel, and a flagellum composed of six segments without clear sexual dimorphism in the number and distribution of antennal sensilla. The scape and pedicel exhibit Böhm's bristles, sensilla chaetica, and sensilla campaniform. The external structures of these organs reveal their mechanosensory function. In the flagellum, the most represented sensilla are the multiporous sensilla basiconica, which can be divided into three types of single‐walled olfactory sensilla; three types of sensilla chaetica with mechanosensory and gustatory functions; sensilla coeloconica, which possess hollow cuticular spoke channels and represent double‐walled olfactory sensilla; sensilla capitula and sensilla cavity with thermo‐hygrosensory functions; and aporous sensilla trichodea with smooth cuticula and mechanosensory function. The putative function of described sensilla is discussed in ralation to host plant selection behavior of S. dorsalis.     

Evaluation of the effects of biodegradable nanoparticles on a vaccine delivery system using AFM, SEM, and TEM

Hepatitis B is a deadly disease, and is carried by 30% of the world's population. Antibodies are produced through a series of three manual vaccinations during infancy and childhood. However, the current needle vaccination not only induces pain in patients, but also can be inconvenient to administer. This is particularly true for the case of newborn babies. Intranasal vaccination is emerging as an alternative parenteral drug delivery method that facilitates drug delivery without causing pain. Chitosan, which is obtained through the deacetylation of chitin from crustacea, is a cationic polymer that is biodegradable, avirulent, and highly absorptive. In this study, ionic gelation between chitosan and TPP was conducted to synthesize chitosan nanoparticles with sizes of 200-400nm and a surface potential of 55-60mV, and which can be used as Hepatitis B vaccine carriers. Then, Heptatitis B antigen protein was impregnated to manufacture chitosan-recombinant gene vaccine protein (RGVP) nanoparticles. AFM, SEM, TEM, and STEM were used to analyze the manufactured nanoparticles, whose function as drug carriers and whose usefulness for intranasal vaccination were confirmed through in vivo tests with SD rats.     

Scanning and transmission electron microscopy study of the microstructural changes occurring in aluminium matrix composites reinforced with SiC particles during casting and welding: interface reactions

Processing of aluminium matrix composites (AMCs), especially those constituted by a reactive system such as Al–SiC, presents great difficulties which limit their potential applications. The interface reactivity between SiC and molten Al generates an aluminium carbide which degrades the composite properties. Scanning and transmission electron microscopes equipped with energy-dispersive X-ray spectroscopes are essential tools for determining the structure and chemistry of the Al–SiC interfaces in AMCs and changes occurring during casting and arc welding. In the present work, an aluminium–copper alloy (AA2014) reinforced with three different percentages of SiC particles was subjected to controlled remelting tests, at temperatures in the range 750–900 °C for 10 and 30 min. Arc welding tests using a tungsten intert gas with power inputs in the range 850–2000 W were also carried out. The results of these studies showed that during remelting there is preferential SiC particle consumption with formation of Al₄C₃ by interface reaction between the solid SiC particle and the molten aluminium matrix. The formation of Al₄C₃ by the same mechanism has also been detected in molten pools of arc welded composites. However, in this case there was formation of an almost continuous layer of Al₄C₃, which protects the particle against further consumption, and formation of aciculate aluminium carbide on the top weld. Both are formed by fusion and dissolution of the SiC in molten aluminium followed by reaction and precipitation of the Al₄C₃ during cooling.     

Dose–response and histopathological study,with special attention to the hypophysis,of the differential effects of domoic acid on rats and mice

The effects of the neurotoxin domoic acid (DA) in the central nervous system of rodents (essentially rats and mice) after intraperitoneal administration have been profusely studied in the past. These observations have shown that the toxin induces similar symptoms and pathology in both species, but the lethality varies greatly. This article addresses the common and specific histopathological effects in rats and mice and the difference in sensitivity of these species to DA. Various sublethal and lethal doses were employed in mice (from 3 mg/kg to 8 mg/kg) to observe their neurotoxicity by using different histological techniques, and these results were compared with the pathological effects after the administration of LD50 in rats (2.5 mg/kg). Additionally we also detected the presence of this toxin in various tissues by means of immunohistochemistry. Our results showed that rats are more vulnerable than mice to the neurotoxic effects of DA after intraperitoneal inoculation: lethality was extremely high in rats and the toxin produced hippocampal damage in rats surviving the intoxication, while lesions were not observed in DA‐inoculated mice. As for similarities between rats and mice, both displayed similar clinical signs and in both the toxin was detected in the hypophysis by immunohistochemistry, a brain region not reported to date as target of the toxin. Microsc. Res. Tech. 78:396–403, 2015. © 2015 Wiley Periodicals, Inc.     

Gross morphological and surface ultrastructural investigation on the gills of the European barracuda Sphyraena sphyraena

The present work examined 10 gill systems of European barracuda grossly and by scanning electron microscopy (SEM). Grossly, there were four pairs of the gill arches. The convex border of the gill arch carried the gills filaments, but there were abundance of spines near to its concave border and the gill rakers were absent. Laterally near to the convex border of the gill arch, SEM observations revealed that the first gill arch carried small elliptical, oval, cuboidal, and triangular groups of two shapes of spines; spearhead-like spines and canine-like spines, but the other three gill arches carried larger groups of the same shaped spines with the appearance of waterfalls. Medially near to the convex border of the first gill arch, the canine-like spines were observed only in the form of vertical rectangular groups that adhered in some areas. Laterally near to the concave border, the two shapes of spines were present in the form of longitudinal groups separated by spaces at the first gill arch, but these spaces were absent in the other three gill arches. Medially near to the concave border of the first gill arch, the two shapes of spines were presented in oval groups, while the other three gill arches were covered entirely by cuboidal groups of the two shapes of spines. The absence of the gill rakers in conjunction with an abundance of spines helps the European barracuda to control the food particles from escaping to the gill filaments to prevent its suffocation.     

An ultrastructural study of cellular response to variation in porosity in phase-pure hydroxyapatite

Hydroxyapatite has been shown to be biocompatible and bioactive. Incorporation of porosity has been shown to enhance osteointegration; however, difficulty in controlling the extent and type of porosity has limited investigation into determining the role of both macro‐ and microporosity. The current investigation reports on the synthesis of four types of phase‐pure hydroxyapatite with varying levels of porosity (HA1–HA4), and with defined levels of macro‐ and microporosities. Transmission electron microscopy was used to evaluate qualitatively the effect of these two parameters on cell–material interactions following a 30‐day incubation period. Biological mineralization was observed within vesicles and the needle‐like minerals were confirmed as hydroxyapatite using X‐ray microanalysis. This demonstrated the suitability of primary human osteoblast‐like cells as a tool to assess the extent of mineralization. Furthermore, internalization of hydroxyapatite particles was observed. Our findings show that the variation in macro‐ and microporosity does not affect the extent of cell–material interaction, with collagen synthesis evident in all samples.     

Method of platinum-carbon coating of ultrathin sections for transmission and scanning electron microscopy: an application for study of biological composites

Many biological materials are composites containing two or more components with different mechanical properties. This study is concerned with the application of a method of platinum-carbon coating (Pt/C) of ultrathin sections for TEM and SEM studies of the design of natural composite materials. The changes in profile of the ultrathin resin-embedded sections during different stages of the preparation reflect the material properties of the various components: stiffer regions deform less than softer ones. Such changes in the section profile can be visualized by the Pt/C method and used as evidence of specific material properties in particular regions of composite materials. The method increases the relief contrast, improves the 3D-view of structures, and in combination with standard TEM and SEM procedures can provide clear demonstrations of material design. The distribution of chitin crystallites in the insect cuticle and the ultrastructure of the pore canal system specialized for the transport of epidermal secretions to the cuticle surface were studied here as examples.     

An alkali digestion method to expose connective tissue fibers: A scanning electron microscopy study of rat lung

Alkali digestion has been used to remove cellular elements of tissues thus exposing the underlying connective tissue framework. We studied the action of this severe alkali treatment on the delicate tissues of rat lung. The lungs of male Sprague-Dawley rats were perfused with saline to remove blood and then inflated by fixation through the airways at 20 cm pressure. Sections of lung 2 × 5 × 5 mm were immersed in 2.5 M NaOH at 25°C for 6 h, 16 h, 24 h, 48 h, and 72 h. The alkali was changed daily. Tissues were washed to neutral with water (24 h), treated with tannic acid (1%, 3h), post-fixed with osmic acid (1%, 3 h) and processed for SEM. At 6 h, epithelial cells started to peel off the alveolar surface. At 16 h the digestion process was well advanced. At 48 h the cells were completely removed revealing the lattice network of connective tissue fibers within the alveolar surface. The method allows the complete removal of cellular elements of the lung while retaining the very fine 3D structure of the connective tissue matrix.     

Pulpal regeneration after cavity preparation,with special reference to close spatio-relationships between odontoblasts and immunocompetent cells

The regeneration process of the odontoblast cell layer incident to tooth injury, especially its relationship with immunocompetent cells in pulp healing, has not been fully understood. The purpose of the present study was to clarify this relationship between odontoblasts and immunocompetent cells in the process of pulp regeneration following cavity preparation in rat molars by immunocytochemistry for heat shock protein (Hsp) 25 as well as class II major histocompatibility complex (MHC) molecules. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin, whereas class II MHC-positive cells were predominantly located beneath the odontoblast cell layer. Cavity preparation caused the destruction of the odontoblast layer to form an edematous lesion and the shift of class II MHC-positive cells with the injured odontoblasts toward the pulp core at the affected site. Some damaged odontoblasts without apparent cytoplasmic processes, round in profile, retained the immunoreactivity for Hsp25, suggesting the survival of a part of the odontoblasts against artificial external stimuli. Twelve hours after cavity preparation, numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules. By postoperative 72 hours, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border, but the class II MHC-positive cells moved from the pulp-dentin border to the subodontoblastic layer. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts. The functional roles of Hsp 25-positive odontoblasts and immunocompetent cells such as class II MHC-positive cells in the process of pulp regeneration after cavity preparation are discussed in conjunction with our previous experimental data.