Electron Microscopic Level Research Articles

Electroacupuncture Protects Against Cerebral Ischemia-Reperfusion Injury: Reducing Inflammatory Response and Cell Pyroptosis by Inhibiting P2X7/NLRP3/GSDMD Pathway.

To investigate the mechanism underlying the effects of Tongdu Tiaoshen electroacupuncture in the treatment of cerebral ischemia-reperfusion (I/R) injury. Sixty adult male Sprague-Dawley (SD) rats were randomly allocated to five groups (n=12): Sham, I/R, electroacupuncture (EA), BBG (P2X7R inhibitor), and MCC950 (NLRP3 inhibitor). The EA group received acupuncture at Shenting (GV24), Baihui (GV20), and Dazhui (GV14) points with a stimulation frequency of 2/5 Hz, intensity of 2 mA, and a duration of 40 min. Neurologic deficit scoring was then performed, and cerebral infarction volume was determined using triphenyl tetrazolium chloride (TTC) staining. Morphological changes in neurons in cortical areas were observed using an electron microscope, interleukin-18 (IL-18) and interleukin-1β (IL-1β) levels were assessed via the enzyme-linked immunosorbent assay (ELISA). Moreover, the expression of P2X7R/NLRP3/GSDMD pathway-related proteins and mRNAs was quantified by immunofluorescence staining, Western blot assay, and real-time fluorescence quantitative (RT-PCR) analysis. Electroacupuncture improved neurologic deficit scores, cerebral infarct size, and cell pyroptosis, and reduced IL-1β and IL-18 levels. Furthermore, electroacupuncture and inhibitor treatment significantly downregulated the expression of P2X7R, ASC, Caspase-1, NLRP3, and GSDMD involved in the P2X7/NLRP3/GSDMD signaling pathway. Tongdu Tiaoshen electroacupuncture may exert cerebral protection by inhibiting neuroinflammation through the P2X7/NLRP3/GSDMD pathway and reducing cellular pyroptosis.

Is it really descemetocele? Morphology of extremely thin membrane that remained after severe corneal melting: a case report.

The aim of this study was to report transmission electron microscopic findings of a case with whole corneal descemetocele following infective corneal ulcer for the first time in literature. A 72-year-old male patient presented with infective corneal ulcer. After resolution of the infection, corneoscleral transplantation was performed. The excised very thin corneal membrane was processed for transmission electron microscopic examination. Transmission electron microscopic examination of the specimen revealed many layered structures that consisted of two different types of cells. The first type consisted of lighter staining polygonal cells, while the second consisted of elongated cells with relatively dense staining. All cells were connected with a large number of gap or adherens junctions with intercalation of the cell membranes of adjacent cells. A haphazard distribution of cytoplasmic microfilaments were also observed in all of the cell types. There was no evidence of the presence of endothelial cells throughout the specimen. There was also no evidence of Descemet membrane presence except for a small part adjacent to iris tissue that contained some melanosomes. Although we clinically diagnosed descemetocele, Descemet membrane was not present at the electron microscopic level, and thus, the expression "descemetocele" is inappropriate.

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.

Trefoil Factor Protein 3 (TFF3) as a Guardian of the Urinary Bladder Epithelium.

SummaryTrefoil factor family (TFF) peptides have been examined primarily in the gastrointestinal tract, where they play an important role in the epithelial regeneration. The therapeutic effects of TFFs, particularly the TFF3 protein, have been well studied in humans and in animal models of gastrointestinal injury, whereas little is known about their occurrence and function in the urinary bladder. In this study, we investigated the presence, location, and function of Tff3 in the urinary bladders of wild-type mice (Tff3WT) and compared them with Tff3 knockout mice (Tff3KO) using molecular and microscopic methods at the light and electron microscopic level. Our results show that Tff3 is expressed in the superficial cells of the urothelium, where it colocalizes with the uroplakin UP1b as one of the fundamental structural components of the apical plasma membrane, which is an important component of the blood-urine permeability barrier. Analysis of the urothelium with experimentally induced injury revealed that injury is more severe in Tff3KO mice and urothelial regeneration is attenuated compared with Tff3WT mice, suggesting that Tff3 plays a fine-tuned role in homeostasis and protection of the urothelium. This study provides the first data on the precise location and function of Tff3 in the bladder epithelium. (J Histochem Cytochem XX. XXX-XXX, XXXX).

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.

COMPARATIVE HISTOLOGICAL CHARACTERISTICS OF CHANGES IN ANTERIOR ABDOMINAL WALL TISSUES AFTER IMPLANTATION OF «CAPROMESH» MESH IN COMBINATION WITH PRP AND «LIGHT» POLYPROPYLENE MESH IN AN EXPERIMENT

A systematic review of a recently published meta-analysis shows that laparoscopic and open allogeneoplasty is a safe procedure with relatively short-term and long-term results. A new minimally invasive technique has been developed that allows the mesh to be placed in the retroperitoneal space through a small transhernial incision, avoiding signifi cant trauma to the abdominal wall and contact with the abdominal cavity. However, this type of operation does not involve fi xation of the mesh in the retromuscular space, which can lead to a number of complications. The analysis of the obtained results determines the relevance of this study.Objective. To compare the features of the ultrastructural reaction of the tissues of the muscle- aponeurotic layer of tissues to the implantation of «Capromesh» in combination with PRP and polypropylene meshes in an experiment.Material and methods. The study was conducted on 16 same-sex pigs of the Vietnamese breed weighing at least 10 kg, which were divided into 2 groups. Histological and ultrastructural changes in the tissues of the muscle- aponeurotic layer of the anterior abdominal wall after implantation in the retromuscular space of «Capromesh» in combination with PRP (plasma enriched with growth factors) and polypropylene meshes were studied in the experiment.The results. A signifi cant diff erence in tissue reactions at diff erent times of the experiment on the implanted material was proven. The conducted microscopic and electron microscopic studies after the implantation of the «Capromesh» mesh in combination with PRP established that starting from the 7th day of the experiment, the infl ammatory changes in the tissues of the muscle- aponeurotic layer are not as signifi cant as during the implantation of the polypropylene mesh. Starting from the 14th day of the experiment, the signs characterizing the reduction of the infl ammatory reaction and the more intense formation of fi brous structures around the mesh material are much better expressed. On the 21st day of the experiment, at the microscopic and electron microscopic levels, a signifi cantly smaller number of areas of leukocyte infi ltration, intensive vascularization from the formation of collagen fi bers with the participation of fi broblasts around the «Capromesh» mesh material in combination with PRP was established.Conclusions. The conducted micro- and ultrastructural studies determined the priority of using the «Capromesh» mesh in combination with PRP under the conditions of material selection when performing retromuscular allogeneoplasty.

Localization and neurochemical identity of alpha1-adrenergic receptor-containing elements in the mouse locus coeruleus

The locus coeruleus (LC) is the major source for norepinephrine (NE) in the brain and projects to areas involved in learning and memory, reward, arousal, attention, and autonomic functions related to stress. There are three types of adrenergic receptors that respond to NE: alpha1-, alpha2-, and beta-adrenergic receptors. Previous behavioral studies have shown the alpha1-adrenergic receptor (α1AR) to be present in the LC, however, with conflicting results. For example, it was shown that α1ARs in the LC are involved in some of the motivational effects of stimulation of the medial forebrain bundle, which was reduced by α1AR antagonist terazosin. Another study showed that during novelty-induced behavioral activation, the α1AR antagonist prazosin reduced c-fos expression in brain regions known to contain motoric α1ARs, except for the LC, where c-fos expression was enhanced. Despite new research delineating more specific connectivity of the neurons in the LC, and some roles of the adrenergic receptors, the α1ARs have not been localized at the subcellular level. Therefore, in order to gain a greater understanding of the aforementioned studies, we used immunohistochemistry at the electron microscopic (EM) level to determine which neuronal or glial elements in the LC express the α1AR. We hypothesized, based on previous work in the ventral periaqueductal gray area, that the α1AR would be found mainly presynaptically in axon terminals, and possibly in glial elements. Single labeling immunohistochemistry at the EM revealed that about 40% of labeled elements that contained the α1AR were glial elements, while approximately 50% of the labeled neuronal elements were axon terminals or small unmyelinated axons in the LC. Double labeling immunohistochemistry found the α1AR expressed in GFAP-labeled astrocytes, in both GABAergic and glutamatergic axon terminals, and in a portion of the α1AR dendrites, colocalized with tyrosine hydroxylase (TH, a marker for noradrenergic neurons). This study sheds light on the neuroanatomical framework underlying the effects of NE and pharmaceuticals acting directly or indirectly on α1ARs in the LC.

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.

Electronic microscopic features of parafollicular cells of rat's thyroid gland after 60 days introduction of tartrazine and Mexidol®

Objective: To establish the effect of the administration of tartrazine for 60 days, as well as Mexidol® under these conditions, on the structural features of the parafollicular cells of the rat's thyroid gland at the electron microscopic level. Material and methods. Thirty white male rats weighing 200-210 g were divided into five groups six rats each. The Group I is the control; the Groups II and III — rats received tartrazine at a concentration of 750 and 1500 mg/kg for 60 days; the Groups IV and V — under similar conditions Mexidol® was administered at the rate of 50 mg/kg. Qualitative changes in parafollicular cells were studied using electron microscopy, and quantitative changes — by morphometry Results. The fine-grained or fibrous contents were detected in the cisterns of the rough endoplasmic reticulum, and areas of the destroyed matrix in some mitochondria after exposure to tartrazine. The ratio of area eu-chromatin/heterochromatin decreased in the Groups II and III by 5.7% and 56.9%, respectively, and the diameter of secretory granules — by 12.3% and 19%, respectively, compared with the Group I. The ratio of area euchromatin/het-erochromatin increased by 79.6% in Group V, and diameter of secretory granules — by 8.2% and 6.5% in the Groups IV and V respectively, compared with the data of the Groups II and III. Conclusions. The introduction of tartrazine in different doses for 60 days causes dose-dependent qualitative and quantitative changes in the ultrastructure of parafollicular cells, and the introduction of Mexidol® against this background causes a decrease in their severity.

Rabconnectin-3α/DMXL2 Is Locally Enriched at the Synaptic Ribbon of Rod Photoreceptor Synapses

Ribbon synapses reliably transmit synaptic signals over a broad signalling range. Rod photoreceptor ribbon synapses are capable of transmitting signals generated by the absorption of single photons. The high precision of ribbon synapses emphasizes the need for particularly efficient signalling mechanisms. Synaptic ribbons are presynaptic specializations of ribbon synapses and are anchored to the active zone. Synaptic ribbons bind many synaptic vesicles that are delivered to the active zone for continuous and faithful signalling. In the present study we demonstrate with independent antibodies at the light- and electron microscopic level that rabconnectin-3α (RC3α)—alternative name Dmx-like 2 (DMXL2)—is localized to the synaptic ribbons of rod photoreceptor synapses in the mouse retina. In the brain, RC3α-containing complexes are known to interact with important components of synaptic vesicles, including Rab3-activating/inactivating enzymes, priming proteins and the vesicular H+-ATPase that acidifies the synaptic vesicle lumen to promote full neurotransmitter loading. The association of RC3α/DMXL2 with rod synaptic ribbons of the mouse retina could enable these structures to deliver only fully signalling-competent synaptic vesicles to the active zone thus contributing to reliable synaptic communication.

Anatomical and Histological Evidence of Aluminum Bone Toxicity: An Experimental Study

Objectives: Aluminum has may be cytotoxic to animals and humans. It is mainly stored in bone and unfortunately, its absorption is increased with age. This study is done to define possible aluminum pathological changes in bone of aging albino rats. Methods: Twenty male albino rats aged 24 months was divided into two groups, control experimental. Experimental group received aluminum chloride for 10 weeks orally. The femur of both control and experimental group is investigated by light and electron microscope. Plain X-ray to the femur as an example to bone is also done. Results: Plain X-ray of femurs of the experimental group showed medullary bone trabeculae destruction, cortical bone resorption and sclerosis. Sections of the shaft of the femur stained with H&E confirmed X-ray gross picture and the bony cortex appeared very thin in comparison with control and showed multiple erosion cavities that may leads to bone fractures. Bone cells appeared few and highly degenerated while the periosteum was very thin and detached from the bone cortex. The bone trabeculae were highly destroyed, and the wide bone marrow spaces were filled by fat cells in some bones. At the level of electron microscope, both osteocytes and osteoblasts revealed severe degenerative changes and showed few irregular collagenous matrices. Conclusion: The degenerative changes observed in the bone of this study are most probably due to aluminum ingestion.

Excitatory and inhibitory imbalances in the trisynaptic pathway in the hippocampus in schizophrenia: a postmortem ultrastructural study.

A preponderance of evidence suggests that the hippocampus is a key region of dysfunction in schizophrenia. Neuroimaging and other studies indicate a relationship between hippocampal dysfunction and the degree of psychosis. Clinical data indicate hyperactivity in the hippocampus that precedes the onset of psychosis, and is correlated with symptom severity. In this study, we sought to identify circuitry at the electron microscopic level that could contribute to region-specific imbalances in excitation and inhibition in the hippocampus in schizophrenia. We used postmortem tissue from the anterior hippocampus from patients with schizophrenia and matched controls. Using stereological techniques, we counted and measured synapses, postsynaptic densities (PSDs), and evaluated size, number and optical density of mitochondria and parvalbumin-containing interneurons in key nodes of the trisynaptic pathway. Compared to controls, the schizophrenia group had decreased numbers of inhibitory synapses in CA3 and increased numbers of excitatory synapses in CA1; together, this indicates deficits in inhibition and an increase in excitation. The thickness of the PSD was larger in excitatory synapses in CA1, suggesting greater synaptic strength. In the schizophrenia group, there were fewer mitochondria in the dentate gyrus and a decrease in the optical density, a measure of functional integrity, in CA1. The number and optical density of parvalbumin interneurons were lower in CA3. The results suggest region-specific increases in excitatory circuitry, decreases in inhibitory neurotransmission and fewer or damaged mitochondria. These results are consistent with the hyperactivity observed in the hippocampus in schizophrenia in previous studies.

A neural tract tracing study on synaptic connections for cortical glutamatergic terminals and cervical spinal calretinin neurons in rats.

The cerebral cortex innervates motor neurons in the anterior horn of the spinal cord by regulating of interneurons. At present, nerve tracing, immunohistochemistry, and immunoelectron microscopy are used to explore and confirm the characteristics of synaptic connections between the corticospinal tract (CST) and cervical spinal calretinin (Cr) interneurons. Our morphological results revealed that (1) biotinylated dextran amine labeled (BDA+) fibers from the cerebral cortex primarily presented a contralateral spinal distribution, with a denser distribution in the ventral horn (VH) than in the dorsal horn (DH). An electron microscope (EM) showed that BDA+ terminals formed asymmetric synapses with spinal neurons, and their mean labeling rate was not different between the DH and VH. (2) Cr-immunoreactive (Cr+) neurons were unevenly distributed throughout the spinal gray matter, and were denser and larger in the VH than in the DH. At the single labeling electron microscope (EM) level, the labeling rate of Cr+ dendrites was higher in the VH than in the DH, in which Cr+ dendrites mainly received asymmetric synaptic inputs, and between the VH and DH. (3) Immunofluorescence triple labeling showed obvious apposition points among BDA+ terminals, synaptophysin and Cr+ dendrites, with a higher density in the VH than in the DH. (4) Double labeling in EM, BDA+ terminals and Cr+ dendrites presented the same pattern, BDA+ terminals formed asymmetric synapses either with Cr+ dendrites or Cr negative (Cr-) dendrites, and Cr+ dendrites received either BDA+ terminals or BDA- synaptic inputs. The average percentage of BDA+ terminals targeting Cr+ dendrites was higher in the VH than in the DH, but the percentage of BDA+ terminals targeting Cr- dendrites was prominently higher than that targeting Cr+ dendrites. There was no difference in BDA+ terminal size. The percentage rate for Cr+ dendrites receiving BDA+ terminal inputs was lower than that receiving BDA- terminal inputs, and the BDA+ terminal size was larger than the BDA- terminal size received by Cr+ dendrites. The present morphological results suggested that spinal Cr+ interneurons are involved in the regulatory process of the cortico-spinal pathway.

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.

MULTIMEDIA SYSTEMS IN TEACHING AND LEARNING AT THE DEPARTMENT OF HISTOLOGY, CYTOLOGY AND EMBRYOLOGY

Modern computer technologies are aimed at improving the quality of educational process, efficiency of providing educational information, structuring and systematization of theoretical and practical knowledge, enhancing students’ skills. Histology, cytology, and embryology are fundamental disciplines based on the deep understanding of structural organization of cells, tissues, and organs. A comprehensive innovative approach to teaching the discipline consists in using multimedia resources to support lectures and practical classes via the visualization of tissue and cellular structures on electron microscopic, light-optical and molecular levels; via the demonstration of microscopic preparations using video systems; by performing formative and summative assessment of knowledge through computer testing.
 The latest learning technologies are multimodal including graphic and audiovisual means that contributes to the better involvement of all organs of perception, thus making difference to the routine educational process in general. The digital database of histological preparations of the different magnification under the microscope, which can be used during lectures, at every practical session and during self-training, is very popular among students.
 Multimedia systems are aimed at improving teaching methods, improving the quality of delivering knowledge, fostering the personality, broadening students’ outlook and developing their intelligence. Multimedia systems include the support for lectures, practical classes and self-directed extracurricular work when preparing for classes, module test, and mastering practical skills.

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.

Immunogold for Protein Location in Chromaffin Cells.

The localization and density of any plasma membrane or intracellular protein in chromaffin cells are prerequisites for those studies designed to elucidate their contribution to cellular function within the adrenal gland and can be achieved only by immunoelectron microscopy. The most popular immunoelectron microscopic techniques involved gold particles conjugated to secondary antibodies, leading to electron-dense markers and the so-called immunogold EM method. Two main immunogold electron microscopic techniques exist: the pre-embedding immunogold, whereby the immunolabeling steps take place before samples are embedded, and the post-embedding immunogold, where the immunolabeling steps take place on embedded and sectioned samples. Pre-embedding immunogold is a very sensitive technique useful for simultaneous observation of labeled tissue at the light and electron microscopic levels. Post-embedding immunogold enables the simultaneous localization of different molecules in the cell using secondary antibodies conjugated with gold particles of different size. In this chapter, we introduce pre-embedding and post-embedding immunogold procedures used for the identification of quantitative changes in a wide range of signaling molecules in different tissues and also discuss the limitations inherent to these approaches.

Repositioning itraconazole for amelioration of bleomycin-induced pulmonary fibrosis: Targeting HMGB1/TLR4 Axis, NLRP3 inflammasome/NF-κB signaling, and autophagy

Background and aimsBleomycin (BLM) is one of the antitumor medications that had proven efficacy in the treatment of a wide range of malignant conditions. Pulmonary fibrosis which is frequently encountered during the course of bleomycin therapy may significantly reduce the potential efficacy of bleomycin in cancer therapy. This study tested the hypothesis that itraconazole may have mitigating effects on BLM-induced pulmonary fibrosis and tried to delineate the potential mechanisms of these effects. Materials and methodsIn a rat model of pulmonary fibrosis elicited by BLM, the effect of different doses of itraconazole was explored at the biochemical, histopathological, and electron microscopic levels. Key findingsItraconazole, in a dose-dependent manner, exhibited significant effects on the pro-oxidant/antioxidant balance, the inflammatory consequences, high-mobility group box 1/toll-like receptor-4 Axis, autophagy and nuclear factor kappa B/Nod-like receptor protein 3 inflammasome signaling and alleviated the histopathological, immunohistochemical, and electron microscopic perturbations induced by BLM in the pulmonary tissues. SignificanceIn view of the afore-mentioned data, itraconazole may be a promising drug that efficiently mitigates the deleterious effects of BLM on the pulmonary tissues.