DigitalUM :: Browsing by Subject "Electron microscopy"

Browsing by Subject "Electron microscopy"

Now showing 1 - 20 of 34

Open Access
A case study of ligation induced calcification in middle cerebral artery in rat
(Murcia : F. Hernández, 2000) Tseng, M.T.; Chan, S.A.; Guo, S.Z.
A 90 rnin ligation of the middle cerebral artery (MCA) followed by 72-hour reperfusion appeared to cause calcium deposition in vascular myocytes of the tunica media and thc pcrivascular tissue 01 the Sprague Dawley rat. The presence of small ovoid to large irregularly shaped intracellular opaque dcposits were demonstrated by light and electron microscopy. Using X-ray elemental analysis the chemical nature of the deposits was found to be calcium phosphate. The functional significance of this first demonstration of acute calcification following transient ligation of the rodent MCA invites further studies.
Open Access
Adipose-derived stem cells and adipose-derived stem cell-conditioned medium modulate in situ imbalance between collagen I- and collagen V-mediated IL-17 immune response recovering bleomycin pulmonary fibrosis
(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Gonçalves Felix, Renato; Carvalho Bovolato, Ana Livia; Cotrim, Ondina Silvia; Leão, Patricia dos Santos; Batah, Sabrina Setembre; Golim, Márjorie de Assis; Velosa, Ana Paula P.; Teodoro, Walcy; Martins, Vanessa; Ferreira Cruz, Fernanda; Deffune, Elenice; Fabro, Alexandre Todorovic; Capelozzi, Vera Luiza
The immunogenic collagen V (Col V) and the proinflammatory cytokine interleukin (IL)-17 have been implicated in the pathogenesis of multiple autoimmune diseases. Col V is also up-regulated during adipogenesis and can stimulate adipocyte differentiation in vitro. Conditioned medium (CM) generated from adipose- derived mesenchymal stem cells (MSCs) reduces bleomycin (BLM)-induced lung injury in rats, suggesting a crucial role in situ of immunomodulatory factors secreted by MSCs in these beneficial effects. In the present work, we investigated this hypothesis, analyzing levels of plasma inflammatory mediators and inflammatory and fibrotic mediators in the lung tissue of BLM-injured rats after treatment with MSCs and CM. Pulmonary fibrosis was intratracheally induced by BLM. After 10 days, BLM animals were further randomized into subgroups receiving saline, MSCs, or CM intravenously. On days 14 and 21, the animals were euthanized, and the lungs were examined through protein expression of nitric oxide synthase (NOS), IL-17, transforming growth factor-β (TGF-β), vascular endothelial growth factor, endothelin-1, and the immunogenic Col V through histological quantitative evaluation and plasma levels of fibrinogen, Von Willebrand factor, and platelet-derived growth factor (PDGF). Rats that had been injected with MSCs and CM showed a significant increase in weight and significant improvements at 14 and 21 days after intravenous injection at both time points of analysis of plasma fibrinogen, PDGF, and Von Willebrand factor and NOS-2 expression, supporting an early anti-inflammatory action, thus reducing TGF-β and collagen I fibers. In contrast, intravenous injection of CM was able to significantly increase the deposition of Col V fibers and IL-17 on both day 14 and day 21 as compared with the amount observed in rats from the BLM group and MSC groups. In conclusion, this study reinforces previous observations on the therapeutic properties of MSCs and CM and is the first report to demonstrate the association of its actions with immunomodulatory biomarkers on lung tissue. We concluded that adipose-derived stem cells and adipose- derived stem cells-CM modulate an in situ imbalance between collagen I- and Col V-mediated IL-17 immune response, emerging as a promising therapeutic option for recovering from BLM pulmonary fibrosis
Open Access
Alendronate effect in esophagus, stomach and liver: An animal based pathological study
(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Papamitsou, Theodora; Sotiriou, Sotiris; Papakoulas, Apostolos; Toskas, Alexandros; Kamperis, Dimitrios; Karachrysafi, Sofia; Dietrich, Eva-Maria; Lialiaris, Stergios; Sioga, Antonia
Bisphosphonates are commonly used in clinical practice. Their effectiveness is indisputable, however their adverse effects, especially in the GI tract, are still controversial. In our report, we demonstrate pathological findings of the effect of systematic alendronate administration in esophagus, stomach and the liver of an in vivo animal model of 15 Wistar rats. Light microscopy with immunohistochemistry and electron microscopy were used. Microscopic findings of inflammation of the stomach and mild hepatic dysfunction were observed. Conclusively, alendronate can potentially affect gastric mucosa and liver function on this animal experimental model
Open Access
Butylated hydroxytoluene induces type-V collagen and overexpression of remodeling genes/proteins in experimental lung fibrosis
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2018) Martins, Vanessa; Teodoro, Walcy Rosolia; Pereira Velosa, Ana Paula; Andrade, Priscila; Farhat, Cecília; Fabro, Alexandre Todorovic; Capelozzi, Vera Luiza
Anomalous histoarchitecture with increased levels of type-V collagen (Col V) in lungs of human idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM) airway-centered interstitial fibrosis suggest that this collagen can be a possible trigger involved in the pathogenesis of these diseases. Butylated hydroxytoluene (BHT) injury model revealed a distal involvement of lung parenchyma with significant endothelial injury and fibrotic response, contrasting with the BLM airway-centered insult. We undertook this study to analyze whether BHT alters distal airway/alveolar epithelial cells (AECs) and extracellular matrix (ECM) signaling involved in the initiation and progression of pulmonary fibrosis in a different pathway concerning overexpression of Col V. Female mice C57BL/6 (n=6) were instilled intraperitoneally with 400mg/kg of BHT dissolved in 1 mL of corn oil and euthanized at day 14 or 21 after BHT administration. Morphometry, immunohistochemistry and transmission electron microscopy were performed to characterize microscopic and submicroscopic changes of AECs and endothelial cells through transforming growth factor beta (TGF-β) basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) expression. Immunofluorescence and immunogold electron microscopy were performed to characterize Col V. Quantitative polymerase chain reaction (qPCR) was used to confirm differential levels of RNA messenger. BHT lungs showed marked fibrotic areas and hyperplastic AECs. The alveolar damage caused destruction of elastic fibers and a critical increase of Col V in ECM of distal lung parenchyma. Fibrogenesis-promoting markers TGF-β, bFGF and VEGF were also overexpressed in situ, coinciding with up-regulation in remodeling enzymes, growth factors, cytokines, transduction and transcription genes. BHT alters distal lung parenchyma signaling involved in pulmonary fibrosis highlighted similarities to human IPF in a pathway involving Col V arising as a promissory model to identify effective therapeutic targets.
Open Access
The cellular expression of GABAA receptor a1 subunit during spermatogenesis in the mouse testis
(Murcia : F. Hernández, 2010) Kanbara, Kiyoto; Okamoto, Keiko; Nomura, Sakashi; Kaneko, Takeshi; Watanabe, Masahito; Otsuki, Yoshinori
GABAA receptors are pentamers in structure and are mainly composed of α, ß and γ subunits. These receptors are known to function as chloride channels. We observed α5, ß1 and γ3 subunit immunoreactivity in the mouse testes, specifically in the cytoplasm surrounding the nucleus in the spermatocytes and spermatids. In the current study, α1 subunit immunoreactivity was located in the nucleus of spermatogonia, spermatocytes and round spermatids. Immunoelectron microscopy revealed that the α1 subunit was localized within the nucleus of pachytene and diplotene spermatocytes in the area of condensed chromatin rather than extended chromatin. Protein sequence analysis revealed that the α1 subunit included DM DNA binding domains that were related to transcription factors involved in testicular differentiation in adult mice. These findings suggest that the α1 subunit may undertake a gene transcription function during the maturation of germ cells. α1 immunoreactivity was also detected within the mitochondria of spermatocytes and in the acrosome of round and elongated spermatids. Although the precise physiological role of the GABAA receptor α1 subunit in mitochondria remains unknown, we hypothesize that its function in the acrosome may be related to the acrosome reaction during fertilization or during spermatogenesis.
Open Access
Computer-aided morphometric analysis of the developing concentric structure of the human fetal intestinal tube
(Murcia : F. Hernández, 2002) Bagyánszki, M.; Kovács, É.G.; Resch, B.A.; Román, V.; Resch, B.E.; Fekete, Eva
The Image-Pro Plus 3.0 morphometric program was used to study the region-specific organization of the human fetal intestine across the radial axis of the gut at weeks 12 and 18 of gestation. The thicknesses of the epithelium, the submucosa, the muscular layers and the myenteric ganglia were measured in resin-embedded semithin sections. Statistical analysis of the collected data was performed by using the two-way ANOVA, the SNK test and the Pearson correlation. The structural changes relating to the gut morphogenesis within this developmental period were followed both light and electron microscopically. The various tissues forming the radial axis of the intestinal tube exhibited different trends concerning their individual development. The thickness of the epithelium did not change in the fetal period investigated, although the epithelial surface displayed characteristic ultrastructural changes. The thickness of the submucosal layer increased significantly, but with different dynamics along the longitudinal axis, whereas the increases in size of the muscular layers and the myenteric ganglia did not differ significantly along the longitudinal axis of the embryonic intestine. The Pearson correlation revealed a significant correlation between the development of the circular muscle layer and that of the myenteric plexus along the whole length of the intestinal tube. The epithelium, the submucosa and the longitudinal muscle layers developed independently between weeks 12 and 18 of gestation.
Open Access
Developmental regulation of GABAB receptors and downstream molecules in the mouse brain
(2025) Ana Fajardo Serrano; Rocío Alfaro Ruiz; María Llanos Martínez Poyato; Ana Esther Moreno-Martínez; Sebastián García Madrona; Alberto Roldán Sastre; Pablo Alonso-Gómez; Miriam Fernández; Ricardo Puertas-Avendaño; Ryuichi Shigemoto; Kirill A. Martemyanov; Rafael Luján; Carolina Aguado; Biología Celular e Histología
Metabotropic GABA (GABAB) receptors have modulatory functions on neuronal excitability and neurotransmitter release. To fulfil these functions, GABAB receptors form macromolecular signaling complexes with G proteins, effectors, and other associated proteins. Here we investigated the postnatal development of GABAB receptors (GABAB1 and GABAB2 subunits) in mouse brain, focusing on potential similarities in the spatial and temporal expression pattern of their associated proteins CaV2.1, Gαo, Gβ5, and RGS7, using histoblots, immunofluorescence, and immunoelectron microscopic techniques. At all ages analyzed, histoblot showed that the six proteins were widely expressed in the brain, with mostly an overlapping pattern throughout postnatal development. In the hippocampus, immunoelectron microscopy and quantitative analysis of immunoparticles for GABAB1, GABAB2, Gαo, Gβ5, and RGS7 revealed their progressive enrichment around excitatory synapses on dendritic spines of CA1 pyramidal cells toward P15. At presynaptic sites, GABAB receptors colocalize with CaV2.1, Gαo, Gβ5, and RGS7 in the active zone and extrasynaptic membranes of axon terminals, establishing synapses on dendritic spines of CA1 pyramidal cells. In the cerebellum, double immunofluorescence at P7 and P10 revealed the colocalization of GABAB1 and CaV2.1 in the whole dendritic tree of developing Purkinje cells. Immunoelectron microscopy at P15 showed that GABAB1, GABAB2, CaV2.1, Gαo, Gβ5, and RGS7 are distributed along the dendritic surface of Purkinje cells, enriched close to excitatory synapses in spines. Altogether, these data suggest that macromolecular complexes composed of GABAB1 /GABAB2/CaV2.1/ Gαo/Gβ5/RGS7 are pre-assembled during key stages of postnatal development in hippocampal and cerebellar neurons.
Open Access
Early structural alterations of intrinsic cardiac ganglionated plexus in spontaneously hypertensive rats
(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Ranceviene, Dalia; Rysevaite Kyguoliene, Kristina; Inokaitis, Hermanas; Saburkina, Inga; Plekhanova, Khrystyna; Sabeckiene, Deimante; Sabeckis, Ignas; Azukaite, Joana; Pauza, Dainius H; Pauziene, Neringa
Persistent arterial hypertension leads to structural and functional remodeling of the heart resulting in myocardial ischemia, fibrosis, hypertrophy, and eventually heart failure. Previous studies have shown that individual neurons composing the intracardiac ganglia are hypertrophied in the failing human, dog, and rat hearts, indicating that this process involves changes in cardiac innervation. However, despite a wealth of data on changes in intrinsic cardiac ganglionated plexus (GP) in late-stage disease models, little is known about the effects of hypertension on cardiac innervation during the early onset of heart failure development. Thus, we examined the impact of early hypertension on the structural organization of the intrinsic cardiac ganglionated plexus in juvenile (8-9 weeks) and adult (12-18 weeks) spontaneously hypertensive (SH) and age-matched Wistar-Kyoto (WKY) rats. GP was studied using a combination of immunofluorescence confocal microscopy and transmission electron microscopy in whole-mount preparations and tissue sections. Here, we report intrinsic cardiac GP of SH rats to display multiple structural alterations: (i) a decrease in the intracardiac neuronal number, (ii) a marked reduction in axonal diameters and their proportion within intracardiac nerves, (iii) an increased density of myocardial nerve fibers, and (iv) neuropathic abnormalities in cardiac glial cells. These findings represent early neurological changes of the intrinsic ganglionated plexus of the heart introduced by early-onset arterial hypertension in young adult SH rats.
Open Access
Electron microscopic observation of intracellular expression of mRNA and its protein product: Technical review on ultrastructural in situ hybridization and its combination with immunohistochemistry
(Murcia : F. Hernández, 2000) Matsuno, A.; Nagashima, T.; Ohsugi, Y.; Utsunomiya, H.; Takekoshi, S.; Munakata, S.; Nagao, K.; Osamura, R.Y.; Watanabe, K.
In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. Three different approaches have been applied by the investigators in this EM-ISH study: preembedding method; non-embedding method using ultrathin frozen sections; and postembedding method. In order to obtain satisfactory morphological preservation and retain the messages, we routinely utilized 6 pmthick frozen sections fixed in 4% paraformaldehyde for the preembedding method and tissues embedded in LR White resin for the postembedding method. The hybridization signal intensity by the postembedding method was lower, and non-specific signals were relatively frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of applicability and preservation of mRNA, although quantitative analysis of the expression of mRNA is rather difficult in the preembedding method. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cells using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex is also described. This ultrastructural double-staining method for mRNA and encoded protein can be expected to provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein.
Open Access
Ependymal damage in a Plasmodium yoelii yoelii lethal murine malaria model
(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2020) Rivera Fernández, Norma; Colin-Barenque, Laura; Romero Silva, Samanta E.; Salas Garrido, Gerardo; Jiménez Rosey, Samantha G.; Zepeda Rodríguez, Armando; Romero Romero, Laura P.; Menchaca Gómez, Ángeles
Malaria continues to be a major global health problem, and over 40% of the world’s population is at risk. Severe or complicated malaria is defined by clinical or laboratory evidence of vital organ dysfunction, including dysfunction of the central nervous system (CNS). The pathogenesis of complicated malaria has not been completely elucidated; however, the development of the multiorgan affection seems to play an important role in the disruption of the blood brain barrier (BBB) that protects the CNS against chemical insults. Historically, the BBB has received more attention in the pathogenesis of malaria than have the cerebrospinal fluid-brain barrier (CSFBB) and ependymal cells. This perspective may be misguided because, in the context of disease or toxicity, the CSFBB is more vulnerable to many foreign invaders than are the capillaries. Given the lack on studies of the damage to the CSFBB and ependymal epithelium in experimental murine malaria, the present study evaluated morphological changes in the ependymal cells of CD-1 male mice infected with lethal Plasmodium yoelii yoelii (Pyy) via histopathology and scanning electron microscopy (SEM). Samples were taken two, four and six days post-infection (PI). No lesions were observed upon the initial infection. By the fourth day PI, fourth ventricle ependymal samples exhibited disruptions and roughened epithelia. More severe injuries were observed at six days PI and included thickened cilia and deep separations between the ependymal intercellular spaces. In some of the analyzed areas, the absence of microvilli and cell layer detachment were observed, and some areas exhibited blebbing surfaces. The ependymal cell lesions observed in the CD1 male mice infected with lethal Pyy seemed to facilitate the paracellular permeability of the CSFBB and consequently promote the access of inflammatory mediators and toxic molecules through the barrier, which resulted in damage to the brain tissue. Understanding the mechanism of ependymal disruption during lethal murine malaria could help to elucidate the local and systemic factors that are involved in the pathogenesis of the disease and may provide essential clues for the prevention and treatment of complicated human malaria.
Open Access
Fine structure of the pecten oculi in the great horned owl (Bubo virginianus)
(Murcia : F. Hernández, 1993) Braekevelt, Charlie R.
The pecten oculi of the great horned owl (Bubo virginianus) has been examined by light and electron microscopy. The pecten in this species is of the pleated type and is small in comparison to the size of the eyeball. It consists of 7-8 accordion folds which are joined apically by a pigmented bridge of tissue. Within each fold are numerous capillaries, larger supply and drainage vessels and plentiful pleomorphic melanocytes. The capillaries are extremely specialized vessels, most of which display plentiful microfolds on both their luminal and abluminal surfaces although some capillaries shoW but a few microfolds. The endothblial cell bodies are extremely thin with most organelles located near the nucleus. Al1 capillaries are surrounded by a thick fibrillar basal lamina which is felt to be structurally important. Pericytes are a comrnon feature within these thickened basal laminae. The numerous melanocytes form an incomplete sheath around the capillaries and are also presumed to be fulfilling a structural role. While the morphology of the pecten in the great horned owl is certainly indicative of a heavy involvement in transport, when compared to the pecten in species that are more visually oriented it is smaller, displays fewer folds and a reduced number of microfolds within the capillaries.
Open Access
Fine structure of the retinal photoreceptors of the great horned owl (Bubo virginianus)
(Murcia : F. Hernández, 1993) Braekevelt, Charlie R.
The retinal photoreceptors of the great horned owl (Bubo virginianus) consist of rods, single cones and unequal double cones present in a ratio of about 30:1:2. In the light-adapted state the rods are stout cells which are not felt to undergo retinomotor movements. The rod outer segment consists of a stack of scalloped membranous discs enclosed by the cell membrane. The rod inner segment shows an ellipsoid of mitochondria and a wealth of rough endoplasmic reticulum (RER) and polysomes, Golgi zones and autophagic vacuoles but no hyperboloid of glycogen. Single cones show a slightly tapered outer segment, a heterogeneous oil droplet and an ellipsoid of mitochondria at the apex of the inner segment. Double cones consist of a larger chief member which also displays an oil droplet and a slightly smaller accessory member which does not. Both members of the double cone as well as the single cone show a prominent ellipsoid, plentiful polysomes and RER and Golgi zones in the inner segment. Neither single nor double cones possess a condensed paraboloid of glycogen but instead show plentiful scattered glycogen particles. Along the contiguous membranes between accessory and chief cones a few presumed junctional complexes are seen near the externa1 limiting membrane. Judging by their morphology in light-adaptation the cones of this species do not undergo photomechanical movements. Rods and cones (both types) have both invaginated (ribbon) and numerous superficial (conventional) synaptic sites. Rods are more numerous in this noctumally active bird than is usually noted in avian species.
Open Access
Fine structure of the retinal photoreceptors of the tiger salamander (Ambystoma tigrinum)
(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 1993) Braekevelt, C. R.
The retinal photoreceptors of the tiger salamander (Ambystoma tigrinum) have been studied by light and electron microscopy in both light- and dark-adaptation. Rods and cones are present in this duplex retina in a ratio of about 20:1. As in other urodele species these photoreceptors are very large cells. The rod outer segment is composed of bi-membranous discs of uniform diameter displaying several very deep incisures. The rod inner segment displays an ellipsoid of mitochondria and a myoid region which changes in diameter during the lighting cycle indicating that rods undergo photomechanical movements. Rod nuclei are located at all levels of the outer nuclear layer and rod spherules are large and display several invaginated and superficial synaptic sites. Cone photoreceptors while large cells are smaller than the rods. They show a smaller tapering outer segment, a large distal ellipsoid of mitochondria and a prominent paraboloid of glycogen but no oil droplet within the inner segment. Judging by the width of the myoid region which remains similar throughout the lighting cycle, cones in this species show no retino-motor responses. Cone nuclei are less electron dense than rods and are also located at all levels of the outer nuclear layer. The cone synaptic pedicle is larger than that of the rods and also shows several invaginated and superficial synaptic sites.
Open Access
Fine structure of the retinal pigment epithelium of the great horned owl (Bubo virginianus)
(Murcia : F. Hernández, 1993) Braekevelt, Charlie R.; Thorlakson, I.J.
The fine structure of the retinal epithelium (RPE), choriocapillaries and Bruch's membrane (complexus basalis) has been studied by light and electron microscopy in the great horned owl (Bubo virginianus). The RPE consists of a single layer of cuboidal cells joined laterally in the mid to basa1 region by a series of tight junctions forming part of the bloodocular barrier. Basally (sclerally) the epithelial cells show numerous deep infoldings while apically (vitreally) a wealth of microvillar processes interdigitate with the photoreceptor cells. Internally the RPE cells display a large vesicular nucleus, plentiful smooth endoplasmic reticulum (SER) and polysomes with only small scattered profiles of rough endoplasmic reticulum (RER). Numerous pleomorphic mitochondria are basally located. In the light-adapted state the melanosomes are located almost exclusively within the apical processes indicating retinomotor movements. Myeloid bodies are numerous and often show ribosomes on their outer surface. Bruch's membrane is typical of avian species in that it is pentalarninate and the lamina densa is displaced towards the choriocapillaris. The choriocapillaris itself is but minimally fenestrated facing Bruch's membrane. Most fenestrations present show a single layered diaphragm while others display a double-layered diaphragm.
Open Access
Gastrointestinal phenotype of GAD67lacZ transgenic mice with early postnatal lethality
(Murcia : F. Hernández, 2005) Krecsmarik, M.; Katarova, Z.; Bagyánszki, M.; Szabó, G.; Fekete, Eva
It has been proposed that g-aminobutyric acid (GABA) in the gut may function as a neurotransmitter, hormone and/or paracrine agent. Our aim was to examine transgenic mice of the GAD67-lacZ line with impaired postnatal growth and early postnatal lethality for gastrointestinal abnormalities. The gastrointestinal tract was dissected and processed for histology, immunohistochemistry, electron microscopy, western blotting and measurement of GAD activity. Homozygous mice of both sexes displayed an intestinal phenotype characterized by a fragile and haemorrhagic intestinal wall, a reduced number of villi, epithelial lesions and the occasional appearance of pseudostratified epithelium. The number of GABA-immunoreactive enteroendocrine cells and mucin-secreting goblet cells increased significantly relative to wild-type epithelium. The appearance of GABA-immunopositive neuronal perikarya and the lack of GABA-immunoreactive varicose fibres were observed in the enteric plexuses of transgenic mice. Tissue homogenates of transgenic mice showed higher levels of expression of GAD67 and GAD65 as compared with wild-type mice. Our results suggest that the possible reason underlying the growth impairment and postnatal lethality observed in GAD67 transgenic mice is a functional impairment of GABAergic enteric neurons and disintegration of intestinal epithelium.
Open Access
Heterogeneous ulmtrastrucmtuorf em elanosome formation in the goldfish induced by osmotic stress
(Murcia : F. Hernández, 1996) Schraerrneyer, U.; Dohms, M.; Rack, M.
In this study, melanophore cytodifferentiation in the fins of xanthic goldfish that had been exposed to osmotic stress for 18 days was investigated. It was found that multi-vesicular bodies (MVB) are not the only type of premelanosome. Granules having a homogeneous matrix also function as premelanosomes. The presence of acid phosphatase reaction product inside the melanin granules indicated that these organelles in this animal were also related to lysosomes. DOPA-oxidase of tyrosinase, the key enzyme in melanogenesis, was surprisingly not only detected in melanocytes but also in the Golgi stacks of dermal cells. Due to the mechanisms of premelanosome formation it is evident that cytoplasmic material also serves as substrate for melanogenesis. EDX microanalysis was performed to measure the ionic composition of the melanin granules. After aldehyde fixation the newly-formed melanin granules did not contain Na, but had accumulated Ca.
Open Access
Imaging the lung: the old ways and the new
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2017) Poobalasingam, Thanushiyan; Salman, David; Li, Henry; Alçada, Joana; Dean, Charlotte H.
Our understanding of lung biology can be greatly enhanced by studying embryonic and postnatal lung development, and the perturbations which occur during disease. Imaging techniques provide a unique insight into these processes. A wide variety of imaging techniques have been used to study the lungs at various stages of development and disease, ranging from histological stains to more novel techniques such as single plane illumination microscopy (SPIM), intravital microscopy (IVM), and micro-computed tomography (micro-CT). Each of these tools can be used to elicit different information about the lungs and each has its own unique advantages and disadvantages for pulmonary research. In this review we assess some of the most commonly-used and novel imaging techniques available for lung research today.
Open Access
Morphological and biochemical patterns in skeletal muscle apoptosis
(Murcia : F. Hernández, 2010) D’Emilio, A.; Biagiotti, L.; Burattini, S.; Battistelli, M.; Canonico, B.; Evangelisti, C.; Ferri, P.; Papa, S.; Martelli, A.M.; Falcieri, E.
Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.
Open Access
Peg-and-socket junctions between smooth muscle cells and endothelial cells in femoral veins are stimulated to angiogenesis by prostaglandin E2 and glycerols
(Murcia: F. Hernández, 2011) Díaz-Flores Jr., L.; Gutiérrez, R.; Sáez, F.J.; Valladares, F.; Villar, J.; Díaz-Flores, L.; Madrid Cuevas, Juan Francisco
The administration of prostaglandin (PG) E2, triacetylglycerol and glycerol induce the formation of numerous vascular buds arising from the femoral vein, as previously demonstrated by our group. In the present study, a great number of peg-and-socket junctions (PSJs) between smooth muscle cells (SMCs) (providing the pegs) and ECs (forming the sockets) were demonstrated. At the first stage, days 1 to 3, PSJs connect subendothelial penetrating processes from activated SMCs with activated ECs of the intima. Subsequently, during angiogenesis (days 4 to 6), SMCs, showing transitional aspects with pericytes, also form PSJs with intimal ECs, but also new PSJs between SMCs and sprouting ECs in the media layer were now observed. Immunohistochemically, α-smooth muscle actin (α- SMA) and H-caldesmon are positive in the cytoplasm of the SMCs, showing a higher expression in pegs. Desmin, however, although it is also positive in the cytoplasm of the SMCs, is negative in the pegs. The expression of CD34 in ECs reveals abundant positive folding that appears to correspond to the sockets. The peculiar expression of caldesmon, whose isoforms may contribute to the regulation of cell motility, and to vasculogenesis and angiogenesis, may have a role in the different mechanisms by which PSJs act in the vein wall.
Open Access
Photoreceptor fine structure in the bobtail goanna (Tiliqua rugosa)
(Murcia : F. Hernández, 1989) Braekevelt, Charlie R.
The fine structure of the retinal photoreceptors has been studied by light and electron microscopy in the bobtail goanna (Tiliqua rugosa) an Australian diurna1 lizard. The photoreceptors in this species are readily divisible into rods or cones based on morphological criteria. Single cones are the dominate cell type with a cone:rod ratio of about 80: 1. No multiple photoreceptors were present nor was a photoreceptor mosiac observed. Cones are large cells with a short, tapering outer segment. The inner segment contains a large apical oil droplet, an ellipsoid of mitochondria and a prominent paraboloid of glycogen. Rods are slender cells with a longer ncn-tapeiing outer segment. Rod inner segnlents display an ellipsoid but no oil droplet or paraboloid. The nuclei of cones are large and vesicular while rod nuclei are smaller and displayed more heterochromatin. The synaptic pedicle of cones is larger than the spherule of rods. Both rods and cones displayed both invaginated (ribbor?) and conventional synapses with the large cones having more synaptic sites. As only light-adapted specirnens were examined it is uilcertain if either rods or cones are capable of retinomotor responses.