Light Microscopic Level Research Articles

The complex neurochemistry of the cockroach antennal heart

The innervation of the antennal heart of the cockroach Periplaneta americana was studied with immunocytochemical techniques on both the light and electron microscopic levels. The antennal heart is innervated by two efferent systems, both using one biogenic amine in combination with neuropeptides. In one, we found co-localization of serotonin with proctolin and allatostatin. These fibers most likely originate from paired neurons located in the suboesophageal ganglion. In the second system, we found octopamine co-localized with the short neuropeptide F. The source of this second system is dorsal unpaired median (DUM) neurons, also located in the suboesophageal ganglion. The possible effects of these neuromediators on different targets are discussed.

Quantitation of the physicochemical properties of myelin using Nile Red fluorescence spectroscopy.

Myelin is a vital structure that is key to rapid saltatory conduction in the central and peripheral nervous systems. Much work has been done over the decades examining the biochemical composition and morphology of myelin at the light and electron microscopic levels. Here we report a method to study myelin based on the fluorescent probe Nile Red. This lipophilic dye readily partitions into live and chemicallyfixed myelin producing bright, well-resolved images of the sheath. Using spectral confocal microscopy, a complete emission spectrum of Nile Red fluorescence can be acquired for each pixel in an image. The solvatochromic properties of Nile Red cause its emission spectrum to change depending on the polarity of its local environment. Therefore, measuring spectral shifts can report subtle changes in the physicochemical properties of myelin. We show differences in myelin polarity in central versus peripheral nervous system and in different regions of central nervous system white matter of the mouse brain, together with developmental and sex variations. This technique is also well suited for measuring subtle changes in myelin properties in live exvivo white matter specimens. We also demonstrate how light deprivation induces a myelin polarity change in adult mouse optic nerve underscoring a continuing myelin plasticity in response to axonal activity well into adulthood. The Nile Red spectroscopic method allows measurement of subtle physicochemical changes in myelin that can importantly influence its electrical properties and by extension, conduction velocities in axons.

Unveiling the placental secrets: Exploring histopathological changes and TROP2 expression in intrahepatic cholestasis of pregnancy

IntroductionGestational cholestasis, also known as intrahepatic cholestasis of pregnancy (ICP) or obstetric cholestasis, is a liver disease that can manifest in late pregnancy. Trophoblast cell surface antigen (TROP2) is a type I transmembrane glycoprotein identified in placental trophoblast cells that plays a critical role in trophoblast invasion of the decidua upon implantation into the placenta. Our study aims to investigate the role of TROP2 in pregnancy cholestasis. Materials and methodsStudy groups: Group 1 (control group) (n = 10): consists of healthy normal pregnant women without any disease, Group 2 (cholestasis group) (n = 10): consists of pregnant women diagnosed with cholestasis. After routine histological follow-up, hematoxylin and eosin staining and TROP2 immunostaining were performed and scored. ResultsIn the cholestasis group, in contrast to the control group, thrombus structures were observed in the intervillous space. In the cholestasis group compared to the control group, villus mesenchymal connective tissue cells, capillary endothelium and trophoblasts around the villus showed significantly stronger anti-TROP2 staining (p < 0.05). DiscussionCholestasis, a condition that may manifest during pregnancy, may be associated not only with observable pathological changes in placental tissues at the light microscopic level, but also with an increase in TROP2 expression. Given the critical role of TROP2 in trophoblast invasion during placental implantation, we hypothesize that TROP2 may serve as a key marker of the cholestatic processes occurring during pregnancy.

Considerations on the evolutionary biology and functions of eosinophils: what the "haeckel"?

The origins and evolution of the eosinophilic leukocyte have received only scattered attention since Paul Ehrlich first named this granulocyte. Studies suggest that myeloperoxidase, expressed by granulocytes, and eosinophil peroxidase diverged some 60 to 70 million years ago, but invertebrate to vertebrate evolution of the eosinophil lineage is unknown. Vertebrate eosinophils have been characterized extensively in representative species at light microscopic, ultrastructural, genetic, and biochemical levels. Understanding of eosinophil function continues to expand and includes to date regulation of "Local Immunity And/Or Remodeling/Repair" (the so-called LIAR hypothesis), modulation of innate and adaptive immune responses, maintenance of tissue and metabolic homeostasis, and, under pathologic conditions, inducers of tissue damage, repair, remodeling, and fibrosis. This contrasts with their classically considered primary roles in host defense against parasites and other pathogens, as well as involvement in T-helper 2 inflammatory and immune responses. The eosinophils' early appearance during evolution and continued retention within the innate immune system across taxa illustrate their importance during evolutionary biology. However, successful pregnancies in eosinophil-depleted humans/primates treated with biologics, host immune responses to parasites in eosinophil-deficient mice, and the absence of significant developmental or functional abnormalities in eosinophil-deficient mouse strains under laboratory conditions raise questions of the continuing selective advantages of the eosinophil lineage in mammals and humans. The objectives of this review are to provide an overview on evolutionary origins of eosinophils across the animal kingdom, discuss some of their main functions in the context of potential evolutionary relevance, and highlight the need for further research on eosinophil functions and functional evolution.

Cytology in cnidaria using Exaiptasia as a model.

A need exists for additional methods to examine cnidaria at the cellular level to aid our understanding of health, anatomy, and physiology of this important group of organisms. This need is particularly acute given that disease is emerging as a major factor in declines of ecologically important functional groups such as corals. Here we describe a simple method to process cnidarian cells for microscopic examination using the model organism Exaiptasia. We show that this organism has at least 18 cell types or structures that can be readily distinguished based on defined morphological features. Some of these cells can be related back to anatomic features of the animal both at the light microscope and ultrastructural level. The cnidome of Exaiptasia may be more complex than what is currently understood. Moreover, cnidarian cells, including some types of cnidocytes, phagocytize cells other than endosymbionts. Finally, our findings shed light on morphologic complexity of cell-associated microbial aggregates and their intimate intracellular associations. The tools described here could be useful for other cnidaria.

Curcumin and gallic acid have a synergistic protective effect against ovarian surface epithelium and follicle reserve damage caused by autologous intraperitoneal ovary transplantation in rats

The objective of this study to examine the effects of curcumin and gallic acid use against oxidative stress damage in the autologous intraperitoneal ovarian transplantation model created in rats on ovarian follicle reserve, ovarian surface epithelium, and oxidant-antioxidant systems. 42 adult female Sprague Dawley rats (n=7) were allocated into 6 groups. Group 1 served as the control. In Group 2, rats underwent ovarian transplantation (TR) to their peritoneal walls. Group 3 received corn oil (CO) (0.5 ml/day) one day before and 14 days after transplantation. Group 4 was administered curcumin (CUR) (100 mg/kg/day), Group 5 received gallic acid (GA) (20 mg/kg/day), and Group 6 was treated with a combination of curcumin and gallic acid via oral gavage after transplantation. Rats were sacrificed on the 14th postoperative day, and blood along with ovaries were collected for analysis. The removed ovaries were analyzed at light microscopic, fluorescence microscopic, and biochemical levels. In Group 2 and Group 3, while serum and tissue Total Oxidant Levels (TOS) and Oxidative Stress Index (OSI) increased, serum Total Antioxidant Levels (TAS) decreased statistically significantly (p˂0.05) compared to the other groups (Groups 1, 4, 5, and 6). The ovarian follicle reserve was preserved and the changes in the ovarian surface epithelium and histopathological findings were reduced in the antioxidant-treated groups (Groups 4, 5, and 6). In addition, immunofluorescence examination revealed that the expression of Cytochrome C and Caspase 3 was stronger and Ki-67 was weaker in Groups 2 and 3, in comparison to the groups that were given antioxidants. It can be said that curcumin and gallic acid have a histological and biochemical protective effect against ischemia-reperfusion injury due to ovarian transplantation, and this effect is stronger when these two antioxidants are applied together compared to individual use.

A Quantitative Assessment of Apoptotic Index with Varying Histological Degree of Dysplasia and Squamous Cell Carcinoma of Oral Cavity - A Light Microscopic Study.

The aim of the present study was to quantitatively assess the apoptotic index at a light microscopic level in hyperkeratosis with varying histological degrees of dysplasia and different grades of oral squamous cell carcinoma (OSCC) and its correlation with prognostic significance. The study included the histo-pathologically diagnosed cases of hyperkeratosis with varying degrees of dysplasia and different grades of OSCC, which were retrieved from the archives of the department, and clinical details were retrieved from the case records of the patients. The sample size consisted of 80 biopsied cases, divided into seven groups of 10 experimental cases and one group of 10 cases of normal mucosa as a control group. The age range of 50-59 shows maximum patients of hyperkeratosis with varying degrees of dysplasia and OSCC. Maximum apoptotic bodies were seen in cases of severe dysplasia and well-differentiated cases of OSCC. There was an observational difference noticed in apoptotic bodies between the patients who had less than 1-year disease-free survival rate. The apoptotic cells are being seen using routine light microscopy techniques with hematoxylin and eosin staining. In different grades of squamous cell carcinoma, a decrease in the apoptotic count was noticed from well-differentiated squamous cell carcinoma toward poorly differentiated squamous cell carcinoma cases. Increased mean apoptotic bodies were observed in patients showing less than 1 year of survival.

Disynaptic Inhibitory Cerebellar Control Over Caudal Medial Accessory Olive.

The olivocerebellar system, which is critical for sensorimotor performance and learning, functions through modules with feedback loops. The main feedback to the inferior olive comes from the cerebellar nuclei (CN), which are predominantly GABAergic and contralateral. However, for the subnucleus d of the caudomedial accessory olive (cdMAO), a crucial region for oculomotor and upper body movements, the source of GABAergic input has yet to be identified. Here, we demonstrate the existence of a disynaptic inhibitory projection from the medial CN (MCN) to the cdMAO via the superior colliculus (SC) by exploiting retrograde, anterograde, and transsynaptic viral tracing at the light microscopic level as well as anterograde classical and viral tracing combined with immunocytochemistry at the electron microscopic level. Retrograde tracing in Gad2-Cre mice reveals that the cdMAO receives GABAergic input from the contralateral SC. Anterograde transsynaptic tracing uncovered that the SC neurons receiving input from the contralateral MCN provide predominantly inhibitory projections to contralateral cdMAO, ipsilateral to the MCN. Following ultrastructural analysis of the monosynaptic projection about half of the SC terminals within the contralateral cdMAO are GABAergic. The disynaptic GABAergic projection from the MCN to the ipsilateral cdMAO mirrors that of the monosynaptic excitatory projection from the MCN to the contralateral cdMAO. Thus, while completing the map of inhibitory inputs to the olivary subnuclei, we established that the MCN inhibits the cdMAO via the contralateral SC, highlighting a potential push-pull mechanism in directional gaze control that appears unique in terms of laterality and polarity among olivocerebellar modules.

Establishment of Mouse Orthotopic Urinary Bladder Tumor Model and Its Analysis by Light and Electron Microscopy.

Mouse tumor models are an important tool in cancer research, and the orthotopic cancer cell transplantation model is the most widely used among them. Methods for establishing tumor models may differ in many ways, including the selection of cancer cell lines and the type of urinary bladder pretreatment. Here, we describe our mouse orthotopic bladder tumor model using a labeled MB49 urothelial cancer cell line and chemical pretreatment with the cationic polypeptide poly-L-lysine to traumatize the bladder epithelium. Double labeling of MB49 cancer cells by their transduction with GFP and internalization of metal nanoparticles allows the study of their implantation process from the first hours to several days after intravesical injection, as well as the analysis of developed tumors after 3weeks. Thus, our model provides a comprehensive analysis of the early and late stages of tumor development in the bladder at the light and electron microscopic level.

The internal anatomy of the scale mite Pterygosoma pseudotrapelus (Trombidiformes, Pterygosomatidae)

This study is the first description of the internal anatomy of the scale mites using the example of parasitizing Pterygosoma pseudotrapelus (Bochkov et al., 2009) – a parasite of agamid lizards. Data on histology of the integument, digestive tract, excretory organs, prosomal glands, tracheae, and body-cavity tissues of females, supplemented by the ultrastructure of major organs, are discussed in comparison with information from other groups of the Acari. The female reproductive system was examined only at the light microscopic level. The shape of mite body is adapted to the existence under the scales of the reptile, where the parasite spends its entire life. The width of the adult females is nearly twice their length, resulting in the eccentric position of certain internal organs: the large reproductive system is oriented across the body, so that the ovary with developing oocytes occupies the right side of the idiosoma, while oviduct, seminal receptacle, and vagina are displaced to the left. The structure of the tracheal system and the podocephalic complex of P. pseudotrapelus is similar to that of Tetranychoidea, a group also regarded as one of the lower taxa in the cohort Raphignathina. A number of previously unknown primitive traits identified in the study confirm a relatively basal position of the scale mites within Raphignathina. These include a panoistic ovary, the postcolon instead of an excretory organ of more advanced forms, and 2 pairs of podocephalic acinar glands against the general trend of their reduction in this cohort. An unpaired tracheal gland was found independent from the podocephalic system. The coxal glands lack a filtering sacculus and their epithelium forms mutual interdigitations with the surrounding modified ventricular wall.

Ultrastructure of the female pedal gonad in Phoxichilidium femoratum (Chelicerata, Pycnogonida)

Phoxichilidium femoratum is a common species of sea spiders – a small and unique group of chelicerates with unusual adult anatomy. In particular, substantial parts of the reproductive system in pycnogonids (unlike euchelicerates) are located in the appendages. Existing studies of pycnogonid gonads are often limited to light-microscopic level, cover a small range of species, and focus on the contents of the gonad diverticula. Ultrastructural data are rare and contradictory, and the organisation of the gonad wall and the gonoducts is unknown. Here we present a detailed light and transmission electron microscopy-based examination of the pedal portion of the adult female reproductive system in Phoxichilidium femoratum Rathke, 1799. We describe its gross anatomy and the ultrastructure of the gonad diverticulum, oviduct and gonopore, as well as development of the oocytes. Each gonad diverticulum is enclosed in the extracellular matrix of the horizontal septum and bears some internal cellular lining. However, neither the gonad lining, nor the septum sheath cells, ever form a continuous epithelial layer. Oocytes, which undergo maturation in the diverticulum, remain, until very late in the process, attached to the gonad wall though specialised stalk cells. Interestingly, stalk cells do not participate in egg envelope or yolk formation: both are synthesized endogenously in the oocytes. The oviduct is supplied with musculature, which assists in egg transport to the gonopore, whereas the gonopore itself is surrounded by specialised glands.

Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research.

The gold-standard fixative for immunohistochemistry is 4% formaldehyde; however, it limits antibody access to target molecules that are buried within specialized neuronal components, such as ionotropic receptors at the postsynapse and voltage-gated ion channels at the axon initial segment, often requiring additional antigen-exposing techniques to detect their authentic signals. To solve this problem, we used glyoxal, a two-carbon atom di-aldehyde. We found that glyoxal fixation greatly improved antibody penetration and immunoreactivity, uncovering signals for buried molecules by conventional immunohistochemical procedures at light and electron microscopic levels. It also enhanced immunosignals of most other molecules, which are known to be detectable in formaldehyde-fixed sections. Furthermore, we unearthed several specific primary antibodies that were once judged to be unusable in formaldehyde-fixed tissues, allowing us to successfully localize so far controversial synaptic adhesion molecule Neuroligin 1. Thus, glyoxal is a highly effective fixative for immunostaining, and a side-by-side comparison of glyoxal and formaldehyde fixation is recommended for routine immunostaining in neuroscience research.

Morphology of the syrinx of three species of birds from Brazilian cerrado (Psittacara leucophthalmus, Rhynchotus rufescens and Cariama cristata): Gross anatomy and light microscopy study.

The aim of this study was to describe the morphology of the trachea and syrinx at macroscopic and light microscopy levels of three species of birds from different orders that inhabit the Brazilian cerrado. For that, five adult specimens (three males and two females of each species) of white-eyed parakeet (Psittacara leucophthalmus), red-winged tinamou (Rhynchotus rufescens) and red-legged seriema (Cariama cristata) were used. The trachea and syrinx of the birds were collected and destined for anatomical and histological studies. The trachea of the studied birds presented an elongated path and originated in the larynx and extended caudally to the syrinx. No sexual dimorphism was observed in the syrinx of the studied species, probably because it is associated with their song, which is very similar between males and females of these species. The findings of this study allowed us to classify the syrinx as tracheal in the white-eyed parakeet and tracheobronchial in the red-winged tinamou and red-legged seriema. In general, the morphological features of the trachea and syrinx were similar to those described for other species of birds, such as the presence of intrinsic and extrinsic syringeal muscles, and the lateral and medial tympaniform membranes, which would represent important anatomical structures in sound production through vibration during expiration and eventual inspiration. The morphological structure of the syrinx in the three avian species of the Brazilian cerrado is consistent with the ability of these avian species to perform a potential vocalization, especially the red-legged seriema that emits characteristic sounds very loud and can carry several kilometres.

Localization of Substance P-Immunoreactive Nerve Structures in the Rat Colon

The tachykinins, including their major representative substance P (SP), are an essential component of its non-adrenergic, non-cholinergic transmission of the myenteric plexus of the enteric nervous system. Hence, the understanding of the role that SP plays in the conduction of a wide variety of motility and secretory reflexes lies in the evidence-based understanding of the sites of its expression. In the present study, the presence, distribution and staining intensity of the SP-positive nerve structures in the myenteric plexus of the proximal and distal colon of the adult rat have been investigated by means of immunohistochemistry at the light microscopic level. We observed an abundance of intensely stained beaded SP-immunopositive fibres, surrounding the SP-immunonegative myenteric neuronal cell bodies in a basket-like manner. Fine bundles of SP-immunostained varicose fibres were also registered in the adjacent smooth muscle layers. The statistical analysis revealed discernible prevalence of the staining intensity in the proximal colon, compared to the distal one. In summary, our findings provide immunohistochemical evidence for the occurrence of abundant SP-immunopositive nerve structures in the colonic region of the rat. Taken together with previous morphological and pharmacological research the results of this study suggest that the SP-mediated neurotransmission is of physiological and likely of pathological significance for the examined region.

Pathology and infectious agents of unionid mussels: A primer for pathologists in disease surveillance and investigation of mortality events.

Freshwater mussels are one of the most imperiled groups of organisms in the world, and more than 30 species have gone extinct in the last century. While habitat alteration and destruction have contributed to the declines, the role of disease in mortality events is unclear. In an effort to involve veterinary pathologists in disease surveillance and the investigation of freshwater mussel mortality events, we provide information on the conservation status of unionids, sample collection and processing techniques, and unique and confounding anatomical and physiological differences. We review the published accounts of pathology and infectious agents described in freshwater mussels including neoplasms, viruses, bacteria, fungi, fungal-like agents, ciliated protists, Aspidogastrea, Digenea, Nematoda, Acari, Diptera, and Odonata. Of the identified infectious agents, a single viral disease, Hyriopsis cumingii plague disease, that occurs only in cultured mussels is known to cause high mortality. Parasites including ciliates, trematodes, nematodes, mites, and insects may decrease host fitness, but are not known to cause mortality. Many of the published reports identify infectious agents at the light or ultrastructural microscopy level with no lesion or molecular characterization. Although metagenomic analyses provide sequence information for infectious agents, studies often fail to link the agents to tissue changes at the light or ultrastructural level or confirm their role in disease. Pathologists can bridge this gap between identification of infectious agents and confirmation of disease, participate in disease surveillance to ensure successful propagation programs necessary to restore decimated populations, and investigate mussel mortality events to document pathology and identify causality.

Structural plasticity of the axon initial segment in rat hippocampal granule cells following high frequency stimulation and LTP induction.

The axon initial segment (AIS) is the site of action potential initiation and important for the integration of synaptic input. Length and localization of the AIS are dynamic, modulated by afferent activity and contribute to the homeostatic control of neuronal excitability. Synaptopodin is a plasticity-related protein expressed by the majority of telencephalic neurons. It is required for the formation of cisternal organelles within the AIS and an excellent marker to identify these enigmatic organelles at the light microscopic level. Here we applied 2 h of high frequency stimulation of the medial perforant path in rats in vivo to induce a strong long-term potentiation of dentate gyrus granule cells. Immunolabeling for βIV-spectrin and synaptopodin were performed to study structural changes of the AIS and its cisternal organelles. Three-dimensional analysis of the AIS revealed a shortening of the AIS and a corresponding reduction of the number of synaptopodin clusters. These data demonstrate a rapid structural plasticity of the AIS and its cisternal organelles to strong stimulation, indicating a homeostatic response of the entire AIS compartment.

Effect of the Anticancer Drug Doxorubicin (Adriamycin) on Antioxidant Studies and Ultrastructural Investigation in the Liver, Kidney, and Heart Tissues of Male Rats

Doxorubicin is a well-known antineoplastic agent that has proved to be successful in the treatment of various types of cancer. I used rats as the model to evaluate the effect of doxorubicin on antioxidant studies and ultrastructural investigations in the liver, kidney, and heart tissues. Male albino rats were given 1.0 mg/kg body weight of the anticancer drug doxorubicin intraperitoneally three times a week for 52 days. This was for a total of 18 doses. Control animals received 52 doses of 0.5 ml of saline over 52 days. The body weights of rats injected with doxorubicin experienced a significant decrease after the last dose compared to the control group of rats. In this study, the weights of the heart, kidneys, and liver were measured. Except for cardiac tissues, the protein content in the aforementioned tissues in treated rats was significantly different from the control. Glutathione (GSH) levels in the kidneys of experimental rats were not significantly lower (7.946 ± 0.781) compared to controls (8.06 ± 0.74) but there was a non-significant increase in GSH levels in the liver (17.095 ± 1.066) compared to controls (13.8 ± 1.3). In addition, the mean GSH levels in doxorubicin-treated hearts were significantly lower (7.9462 ± 0.781) compared to controls (8.06 ± 0.74). Lipid peroxidation (Lpx) and malondialdehyde content (MDA), a marker of lipid peroxidation, were found in much lower concentrations in the liver organ of the doxorubicin-treated group (0.0162 ± 0.00086) as compared to (0.20 ± 0.02) controls, and MDA content in the kidney was decreased (0.0239 ± 0.0003) compared to control rats (0.31 ± 0.03), as well as heat production (0.0398 ± 0.00097) compared to (47.451 ± 1.708) controls. Glutathione reductase (GR) levels were significantly elevated in the same tissue treatment group. Glutathione-S-Transferase (G-S-T) activity was assessed and significantly increased in all tissues in the doxorubicin model. Glutathione peroxidase (GPx) activity showed a significant decrease in all the above tissues after doxorubicin injection. The catalase (CAT) activity of doxorubicin was greatly increased in one treated rat. In the doxorubicin-treated group, levels of cytochrome p450 (CYTp450) were significantly decreased in liver and kidney tissue and significantly elevated in heart tissue. After doxorubicin treatment, cytochrome b5 (CYTb5) levels in liver tissues increased significantly (837.177± 61.197) compared to controls (615 ± 37.0), and the contents of cytochrome b5 in rats' kidneys increased significantly (447.685 ± 35.215) compared to controls (2605.5± 259.2). and cytochrome b5 in heart tissues was lower (165.352± 8.7) when compared to controls (88± 0.4). The results showed that there were few obvious changes in histological, ultrastructural, and biochemical changes in liver tissue in the doxorubicin model. Long-term doxorubicin treatment in kidney tissue results in no significant changes at the light microscopic level, but the electron microscopic level reveals no change from a histological point of view.

Assembly and regulation of the mammalian mRNA processing body.

Messenger RNA processing bodies (P-bodies) are cytoplasmic membrane-free organelles that contain proteins involved in mRNA silencing, storage and decay. The mechanism by which P-body components interact and the factors that regulate the stability of these structures are incompletely understood. In this study, we used a fluorescence-based, two-hybrid assay to investigate interactions between P-body components that occur inside the cell. LSm14a, PATL1, XRN1, and NBDY were found to interact with the N-terminal, WD40-domain-containing portion of EDC4. The N-terminus of full-length PATL1 was required to mediate the interaction between EDC4 and DDX6. The C-terminal, alpha helix-domain- containing portion of EDC4 was sufficient to mediate interaction with DCP1a and CCHCR1. In the absence of endogenous P-bodies, caused by depletion of LSm14a or DDX6, expression of the portion of EDC4 that lacked the N-terminus retained the ability to form cytoplasmic dots that were indistinguishable from P-bodies at the level of UV light microscopy. Despite the absence of endogenous P-bodies, this portion of EDC4 was able to recruit DCP1a, CCHCR1 and EDC3 to cytoplasmic dots. The results of this study permit the development of a new model of P-body formation and suggest that the N-terminus of EDC4 regulates the stability of these structures.

Calcium Dynamics, WUSCHEL Expression and Callose Deposition during Somatic Embryogenesis in Arabidopsis thaliana Immature Zygotic Embryos.

In this work, we studied the induction of somatic embryogenesis in Arabidopsis using IZEs as explants. We characterized the process at the light and scanning electron microscope level and studied several specific aspects such as WUS expression, callose deposition, and principally Ca2+ dynamics during the first stages of the process of embryogenesis induction, by confocal FRET analysis with an Arabidopsis line expressing a cameleon calcium sensor. We also performed a pharmacological study with a series of chemicals know to alter calcium homeostasis (CaCl2, inositol 1,4,5-trisphosphate, ionophore A23187, EGTA), the calcium-calmodulin interaction (chlorpromazine, W-7), and callose deposition (2-deoxy-D-glucose). We showed that, after determination of the cotiledonary protrusions as embryogenic regions, a finger-like appendix may emerge from the shoot apical region and somatic embryos are produced from the WUS-expressing cells of the appendix tip. Ca2+ levels increase and callose is deposited in the cells of the regions where somatic embryos will be formed, thereby constituting early markers of the embryogenic regions. We also found that Ca2+ homeostasis in this system is strictly maintained and cannot be altered to modulate embryo production, as shown for other systems. Together, these results contribute to a better knowledge and understanding of the process of induction of somatic embryos in this system.

Corneal changes in Fuchs endothelial corneal dystrophy and bullous keratopathy

Evaluation of structural and immunohistochemical features of cornea in Fuchs endothelial corneal dystrophy (FECD) and bullous keratopathy (BK). Group 1 - 44 patients (46 eyes) with FECD, group 2 - 42 patients (42 eyes) with BK. All patients underwent keratoplasty. Preoperative anterior segment optical coherence tomography (AS-OCT, RTVue-100, Optovue, USA) was performed. Endothelium-Descemet membrane (EDM) complexes, corneal buttons were obtained intraoperatively. Morphological (H&E staining) and immunohistochemical (primary antibodies to pancytokeratin, vimentin, fibronectin) studies were performed at the light microscope level (Leica DM-2500, Leica Application Suite V4.8, Leica Microsystems, Switzerland). A direct correlation is found between the results of DM analysis in vivo with OCT and ex vivo with light microscopy. DM thickness (AS-OCT) was significantly greater in FECD (23.0 [19.0; 27.0] μm), than in BK (13.0 [12.0; 14.0] μm). Morphological study of EDM and corneal buttons showed similar difference in DM thickness: 17.9 [16.1; 20.0] μm in FECD and 11.9 [11.3; 13.0] μm in BK. Irregular optical density of stroma is a feature of edema and local fibrosis. In FECD and BK pancytokeratin is expressed in epithelial and endothelial cells, vimentin - in keratocytes, macrophages and vascular endothelium, fibronectin - in DM. In FECD, vimentin is expressed in endothelial cells. FECD and BK are associated with different DM' and endothelium' abnormalities, which lead to similar changes of stroma and epithelium. AS-OCT is a useful method of FECD and BK in vivo diagnostics and the selection of treatment option.