Light Microscopic Level Research Articles

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.

Alpha-T-catenin is expressed in peripheral nerves as a constituent of Schwann cell adherens junctions.

The adherens junction component, alpha-T-catenin (αTcat) is an established contributor to cardiomyocyte junction structure and function, but recent genomic studies link CTNNA3 polymorphisms to diseases with no clear cardiac underpinning, including asthma, autism and multiple sclerosis, suggesting causal contributions from a different cell-type. We show Ctnna3 mRNA is highly expressed in peripheral nerves (e.g. vagus and sciatic), where αTcat protein enriches at paranodes and myelin incisure adherens junctions of Schwann cells. We validate αTcat immunodetection specificity using a new Ctnna3-knock-out fluorescence reporter mouse line yet find no obvious Schwann cell loss-of-function morphology at the light microscopic level. CTNNA3/Ctnna3 mRNA is also abundantly detected in oligodendrocytes of the central nervous system via public databases, supporting a general role for αTcat in these unique cell-cell junctions. These data suggest that the wide range of diseases linked to CTNNA3 may be through its role in maintaining neuroglial functions of central and peripheral nervous systems. This article has a corresponding First Person interview with the co-first authors of the paper.

Altered PLD3 exonuclease activity impacts on lysosomal and mitochondrial homeostasis

AbstractBackgroundPhospholipase D3 (PLD3) is a single‐pass type II membrane protein that is majorly localized to late endosomes/lysosomes (LE/Lys) where it functions as a 5'‐3' exonuclease that degrades ssDNA. Whereas PLD3 has been identified as risk factor for late‐onset Alzheimer’s disease (LOAD), knockout studies have resulted in conflicting data regarding its impact on the amyloid precursor protein metabolism. Moreover, the substrates for PLD3 in LE/Lys remain unexploredMethodCRISPR/Cas9 gene editing was used to generate PLD3 knockout (KO) SH‐SY5Y cells that were subsequently stably rescued with wild‐type PLD3 and coding‐variants (M6R & V232M). All cell lines were evaluated for morphological and functional alterations of the LE/Lys compartment, including lipid profiling and analysis of the nucleotide content of LE/Lys magnetically isolated from the different cell lines. We optimized a PLD3 activity assay and analyzed the LE/Lys nucleotide content as a means to identify PLD3 substrates.ResultWe report that a PLD3 KO and LOAD‐associated risk variants lead to a significant lysosomal pathology, comprising an increase in the size of LE/Lys, more LAMP1 immunoreactivity, a reduced catabolic activity, an increased propensity for leakage and a marked increase in cholesterol content and in storage lipids. At the ultrastructural level, this is accompanied with the appearance of multilamellar bodies, sometimes including mitochondrial remnants. This is suggestive for a defective mitophagy, which we confirm on the light microscopy level using mKeima assays. Furthermore, we provide support for a role of PLD3 in the homeostatic degradation of mtDNA. We argue that when PLD3’s role is compromised, this leads to a build‐up of the lysosomal nucleotide content, impacting on the normal functioning of LE/Lys. We are currently analysing alterations in downstream cellular signaling pathways, including the TLR9 cascade.ConclusionCollectively, our findings subscribe a key role for PLD3 exonuclease activity in lysosomal homeostasis. Its dysfunction, as occurs in LOAD, leads to the co‐occurrence of lysosomal and mitochondrial defects.

A novel murine model of autoimmune dysautonomia by α3 nicotinic acetylcholine receptor immunization.

We aimed to establish a novel murine model of autoimmune autonomic ganglionopathy (AAG), which represents autoimmune dysautonomia, associated with MHC class II to understand its pathomechanism and the pathogenicity of nicotinic acetylcholine receptor (nAChR) antibodies. The amino acid sequence of the mouse nAChRα3 protein was analyzed using an epitope prediction tool to predict the possible MHC class II binding mouse nAChRα3 peptides. We focused on two nAChRα3 peptides in the extracellular region, and experimental AAG (EAAG) was induced by immunization of C57BL/6 mice with these two different peptides. EAAG mice were examined both physiologically and histologically. Mice with EAAG generated nAChRα3 antibodies and exhibited autonomic dysfunction, including reduced heart rate, excessive fluctuations in systolic blood pressure, and intestinal transit slowing. Additionally, we observed skin lesions, such as alopecia and skin ulcers, in immunized mice. Neuronal cell density in the sympathetic cervical ganglia in immunized mice was significantly lower than that in control mice at the light microscopic level. We interpreted that active immunization of mice with nAChRα3 peptides causes autonomic dysfunction similar to human AAG induced by an antibody-mediated mechanism. We suggested a mechanism by which different HLA class II molecules might preferentially affect the nAChR-specific immune response, thus controlling diversification of the autoantibody response. Our novel murine model mimics AAG in humans and provides a useful tool to investigate its pathomechanism.

MESNA HAS ANTIOXIDANT POTENTIAL ON INTESTINE, LIVER, KIDNEY AND LUNG INJURY INDUCED BY INTESTINAL ISCHEMIA/REPERFUSION

An ischemia/reperfusion (IR) injury of small intestine is a serious and common condition that is a result of the blockage of the superior mesenteric artery (SMA) due to some significant clinical problems. In the present study, we evaluated the effect of Mesna on systemic injury induced by IR in small intestine and liver, kidney and lung of rats. Thirty-two Wistar albino female rats were randomly divided into four groups as control, ischemia, IR (Sham) and IR+Mesna. Ischemia period was executed by clamping SMA for 2 h and after when reperfusion was permitted by removing the clamp from SMA for 2 h. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) activities were measured spectrophotometrically in the tissues. Microscopic examination was performed with Hematoxylin-Eosin (H&E) staining for light microscopy and double staining (Uranyl acetate and Reynolds’lead citrate) for electron microscopy. Oxidative damage was determined in ischemia (p < 0.05) and IR groups (p < 0.05) by Chiu score in small intestine and also antioxidant enzyme activities in all groups. Significant recovery in SOD, GPx and CAT activities in IR+Mesna group was determined but the effect of oxidative damage was not reduced significantly histopatologically. Mesna treatment caused recovery in SOD, GPx and CAT activities but not achieved an improvement in the histopathologic findings in level of light microscopy in IR damages but contains the signs of improving at electron microscopy level in Mesna dose and application time of IR.

Responses to Ice Formation and Reasons of Frost Injury in Potato Leaves

Potato leaves are ice-tolerant but are frost-damaged at −3 °C. Freezing occurs in two steps, a first non-destructive freezing event and a second independent lethal event. Localization of ice, and whether cells freeze-dehydrate after the first freezing event remains unknown. The cause of frost damage during the second freezing event lacks experimental evidence. Cytological responses of mesophyll cells were examined during ice formation using cryo-microscopic techniques after high-pressure freeze-fixation and freeze-substitution. CO2 gas exchange on frozen leaves revealed functional responses, but also frost damage. After the first freezing event, gas exchange was uninterrupted. Consequently, intercellular spaces are free of ice, and ice may be restricted to xylem vessels. The cellular shape of the mesophyll cells was unchanged, cells did not freeze-dehydrate but were supercooled. When thawed after the first freezing event, leaves were initially photoinhibited but regained photosynthesis. During the second freezing event, cells froze intracellularly, and some palisade parenchyma cells remained intact for a prolonged time. Intracellular ice caused complete destruction of cells, and chloroplasts became invisible at the light microscopic level. When thawed after the second freezing, leaves were unable to regain photosynthesis. Consequently, freezing avoidance is the only viable strategy for potatoes to survive frost.

Is the central mesencephalic reticular formation a purely horizontal gaze center?

Historically, the central mesencephalic reticular formation has been regarded as a purely horizontal gaze center based on the fact that electrical stimulation of this region produces horizontal saccades, it provides monosynaptic input to medial rectus motoneurons, and cells recorded in this region often display a peak in firing when horizontal saccades are made. We tested the proposition that the central mesencephalic reticular formation is purely a horizontal gaze center by examining whether this region also supplies terminals to superior rectus and levator palpebrae superioris motoneurons, both of which fire when making vertical eye movements. The experiments were carried out using dual tracer techniques at the light and electron microscopic level in macaque monkeys. Injections of biotinylated dextran amine or Phaseolus vulgaris leukoagglutinin into the central mesencephalic reticular formation produced anterogradely labeled terminals that were in synaptic contact with superior rectus and levator palpebrae superioris motoneurons that had been retrogradely labeled. These results indicate that this region is not purely connected with horizontal gaze motoneurons. In addition, we found that the number of contacts on vertical gaze motoneurons increased with more rostral injections involving the mesencephalic reticular formation adjacent to the interstitial nucleus of Cajal. This suggests that there is a caudal to rostral gradient for horizontal to vertical saccades, respectively, represented within the midbrain reticular formation. Finally, we utilized post-embedding immunohistochemistry to show that a portion of the labeled terminals were GABAergic, indicating they likely originate from downgaze premotor neurons.

Molecular landscape of canine soft tissue sarcoma

Canine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features including hemangiosarcoma, lymphangiosarcoma, histiocytic sarcoma, synovial sarcoma, leiomyosarcoma, and rhabdomyosarcoma. Histologically, canine STS sub‐types are difficult to discern at the light microscopic level due to their overlapping microscopic features. Thus, genomic, and transcriptomic profiling of canine STS may prove more valuable in differentiating the diverse sub‐types of mesenchymal neoplasms within this grouping. To this purpose we sought to characterize the mutation and expression profiles of canine STS. In this study we used whole exome capture (Agilent Sure Select Canine V2) to sequence 29 tumors, grade I‐III, from non‐visceral locations, and matched normal samples, along with RNAseq of tumor samples. For the WES data, 40‐170 million 150 bp paired‐end reads were obtained from Illumina sequencing which were mapped against CanFam3.1 genome via BWA and short variants were called and annotated Mutect2 and VEP tools, respectively. The median depth of sequencing for normals and tumors were 97X (range: 35X–128X) and 110X (range: 22X–133X), respectively. Additionally, RNAseq reads were sequenced from 29 tumors and mapped against CanFam3.1 genome via STAR and gene count data was generated by HTSeq‐count tool. The total number of somatic variants identified across 29 samples ranged from 275 to 4,196, of which, 6% to 31% were annotated as cDNA variants. The protein coding mutations per callable megabase ranged from 0.3 to 37.3. Known cancer genes (COSMIC database) with recurrent mutations were KMT2D(21% of the samples), TP53 (21%), CNTNAP2(14%) genes. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B (PDGFB) and collagen genes. These gene fusions were confirmed through Sanger sequencing of amplified cDNA. To delineate the transcriptomic sub‐types, we used hierarchical and K‐means clustering methods. This resulted in 4 groups with similar expression profiles comprising of 11, 4, 8 and 6 samples in each. Further, genes set variation analysis (GSVA) was used to profile the distinct pathways across all 4 clusters. Preliminary data indicates that the 11‐sample and 6‐sample clusters had up‐regulated non‐homologous end joining pathway and immune response pathways, respectively. Further study of genes and pathways that are enriched in sample clusters will help in categorizing the canine STS tumors for diagnosis and treatment.

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy.

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

Crinum latifolium extract inhibits lipopolysaccharide-induced inflammation in human macrophages

Abstract Introduction: Crinum latifolium is a natural plant showing the anti-inflammatory effects. We aimed to evaluate the effects of Crinum latifolium extract on proinflammatory cytokine production and maturation of early and late endosomes in the lipopolysaccharide (LPS)-induced human macrophages at light microscopic and biochemical levels. Material and Methods: The effects of Crinum latifolium extract in human macrophages stimulated with LPS was evaluated to optic densities of the lysosome-associated membrane protein-1 (LAMP-1) and early endo-some antigen 1 (EEA1) by immunohistochemistry staining and to level of the proinflammatory cytokine interleukin (IL)-6 by enzyme-linked immunosorbent assay (ELISA). Results: Crinum latifolium group exhibited a significant decreased in the levels of IL-6 in the supernatant as compared to the LPS group for 2 and 72 hours (p<0.0001). Crinum latifolium group exhibited a significant increased in the optic density of the EEA1 and LAMP-1 in coverslips as compared to the control and lipopolysaccharide groups after 2 and 72 hours (p<0.0001). Conclusions: Crinum latifolium may a therapeutic plant in the inflammatory diseases, such as sepsis, through anti-inflammatory effects, such as decrease in production of the proinflammatory cytokine and increase in maturations of the early and late endosomes in macrophages.

Explainable Biomarkers for Automated Glomerular and Patient-Level Disease Classification.

Pathologists use multiple microscopy modalities to assess renal biopsy specimens. Besides usual diagnostic features, some changes are too subtle to be properly defined. Computational approaches have the potential to systematically quantitate subvisual clues, provide pathogenetic insight, and link to clinical outcomes. To this end, a proof-of-principle study is presented demonstrating that explainable biomarkers through machine learning can distinguish between glomerular disorders at the light-microscopy level. The proposed system used image analysis techniques and extracted 233 explainable biomarkers related to color, morphology, and microstructural texture. Traditional machine learning was then used to classify minimal change disease (MCD), membranous nephropathy (MN), and thin basement membrane nephropathy (TBMN) diseases on a glomerular and patient-level basis. The final model combined the Gini feature importance set and linear discriminant analysis classifier. Six morphologic (nuclei-to-glomerular tuft area, nuclei-to-glomerular area, glomerular tuft thickness greater than ten, glomerular tuft thickness greater than three, total glomerular tuft thickness, and glomerular circularity) and four microstructural texture features (luminal contrast using wavelets, nuclei energy using wavelets, nuclei variance using color vector LBP, and glomerular correlation using GLCM) were, together, the best performing biomarkers. Accuracies of 77% and 87% were obtained for glomerular and patient-level classification, respectively. Computational methods, using explainable glomerular biomarkers, have diagnostic value and are compatible with our existing knowledge of disease pathogenesis. Furthermore, this algorithm can be applied to clinical datasets for novel prognostic and mechanistic biomarker discovery.

Gonadal Development in European Eel Populations of North Adriatic Lagoons at Different Silvering Stages

The European eel (Anguilla anguilla, Linnaeus 1758) is a catadromous fish with significant cultural, scientific, and commercial value. The protection of this species is particularly difficult because the biology of the eel life cycle remains unknown in many aspects. The European eel (A. anguilla) population has declined alarmingly over the past 30 years; this condition has led to questions about the long-term welfare of this species. This work aims to perform a histological analysis on gonad samples from European eels coming from four different lagoons of the North Adriatic at different stages of silvering, and to evaluate the maturation of the gonads. For this study, a total of 859 wild eels were captured from four different lagoons using the “lavoriero”. Subsequently, the biometric parameters were collected. Seventy-nine female eels were randomly selected, dissected, and the gonads were removed for histological analysis. Sections of 4 µm were cut and stained with hematoxylin and eosin (H&E). Histological observations of germ cells at the light microscopy level allowed for the characterization of six steps of oocyte maturation. Valle di Comacchio had the highest levels of oocyte maturation, while Valle Ca’ Pasta had the lowest. Eels with silver index III had an oocyte maturation nearly equal to that of eels at stages IV and V of silvering. Considering the results, we can affirm that eels from North Adriatic lagoons have high oocyte maturation levels and high GSI value indices at silvering stage III. The following experimental work shows that the levels of oocyte maturation are higher even at lower silver index levels. It can be hypothesized that in these eels, the transition from yellow to silver eel occurs faster in relation to the high trophic availability in North Adriatic lagoons.

Membrane traffic and Ca2+ signals in ciliates

A Paramecium cell has as many types of membrane interactions as mammalian cells, as established with monoclonal antibodies by R. Allen and A. Fok. Since then, we have identified key players, such as SNARE proteins, Ca2+ -regulating proteins, including Ca2+ -channels, Ca2+ -pumps, Ca2+ -binding proteins of different affinity, etc., at the molecular level, probed their function and localized them at the light and electron microscopy level. SNARE proteins, in conjunction with a synaptotagmin-like Ca2+ -sensor protein, mediate membrane fusion. This interaction is additionally regulated by monomeric GTPases whose spectrum in Tetrahymena and Paramecium has been established by A. Turkewitz. As known from mammalian cells, GTPases are activated on membranes in conjunction with lumenal acidification by an H+ -ATPase. For these complex molecules, we found in Paramecium an unsurpassed number of 17 a-subunit paralogs which connect the polymeric head and basis part, V1 and V0. (This multitude may reflect different local functional requirements.) Together with plasmalemmal Ca2+ -influx channels, locally enriched intracellular InsP3 -type (InsP3 R, mainly in osmoregulatory system) and ryanodine receptor-like Ca2+ -release channels (ryanodine receptor-like proteins, RyR-LP), this complexity mediates Ca2+ signals for most flexible local membrane-to-membrane interactions. As we found, the latter channel types miss a substantial portion of the N-terminal part. Caffeine and 4-chloro-meta-cresol (the agent used to probe mutations of RyRs in man during surgery in malignant insomnia patients) initiate trichocyst exocytosis by activating Ca2+ -release channels type CRC-IV in the peripheral part of alveolar sacs. This is superimposed by Ca2+ -influx, that is, a mechanism called "store-operated Ca2+ -entry" (SOCE). For the majority of key players, we have mapped paralogs throughout the Paramecium cell, with features in common or at variance in the different organelles participating in vesicle trafficking. Local values of free Ca2+ -concentration, [Ca2+ ]i , and their change, for example, upon exocytosis stimulation, have been registered by flurochromes and chelator effects. In parallel, we have registered release of Ca2+ from alveolar sacs by quenched-flow analysis combined with cryofixation and X-ray microanalysis.

Characterization of the GfaABC1D promoter to selectively target astrocytes in the rhesus macaque brain

BackgroundThe study of astrocytic functions in non-human primates (NHPs) has been hampered by the lack of genetic tools to selectively target astrocytes. Viral vectors with selective and efficient transduction of astrocytes could be a potent tool to express marker proteins, modulators, or sensors in NHP astrocytes, but the availability of thoroughly characterized astrocytic selective promoter sequences to use in these species remains extremely limited. New MethodWe describe the specificity and efficiency of an astrocyte-specific promoter, GfaABC1D in the brain of the rhesus macaque, with emphasis in basal ganglia regions. AAV5-pZac2.1-GfaABC1D-tdTomato was locally injected into the globus pallidus external segment (GPe) and putamen. The extent, efficiency, and specificity of transduction was analyzed with immunohistochemistry at the light and electron microscope levels. ResultsThe GfaABC1D promoter directed the expression of tdTomato in an astrocyte-specific manner in directly or indirectly targeted regions (including both segments of the globus pallidus, putamen, subthalamic nucleus and cortex). Comparison with Existing MethodsDue to its small size, the GfaABC1D promoter is advantageous over other previously used glial fibrillary acidic protein-based promoter sequences, facilitating its use to drive expression of various transgenes in adeno-associated viruses (AAV) or other viral vectors. ConclusionGfaABC1D is an efficient promoter that selectively targets astrocytes in the monkey basal ganglia and expands the viral vector toolbox to study astrocytic functions in non-human primates.

Biochemical and histopathological investigation of the protective effects of melatonin and vitamin E against the damage caused by acetamiprid in Balb-c mouse testicles at light and electron microscopic level.

The protective effects of melatonin (Mel) and vitamin E (Vit E) against the negative effects of acetamiprid (Acmp) on testicles, reproductive hormones, and oxidative stress parameters were investigated in the present study. A total of 50 Balb-c male mice were used in 7 groups; 6 mice in the control groups (distilled water, corn oil, ethanol), and 8 in other groups (Acmp, Acmp + Mel, Acmp + Vit E, Acmp + Vit E + Mel). After the experiment, which lasted 21days, hematoxylin eosin (H&E), periodic acid Schiff (PAS), and caspase-3 immunohistochemical (IHC) staining was performed on the testicular tissues. Also, the tissues were examined ultrastructurally with the transmission electron microscopy (TEM). In the Acmp group, there were decreased seminiferous tubule diameter and epithelial thickness, epithelial degeneration, decreased spermatozoa in the lumen, decreased PAS-positive staining in the seminiferous epithelial basement membrane, edema in the interstitial area, and hydropic degeneration in Leydig cells. Caspase-3 immunoreactivity was higher than in the other groups. TEM examination showed degeneration in tubule cells, lysosomal accumulation in cells of the spermatogenic line, vacuolizations with myelin figures, and necrosis. Hydropic degeneration, electron-dense lipid vacuoles, and chromatolysis were evident in the Leydig cell cytoplasm. In Sertoli cells, electron-dense lysosomal deposits were noted. In biochemical terms, there were decreased tissue glutathione (GSH) and total antioxidant status (TAS), and increased malondialdehyde (MDA) and total oxidant status (TOS). Plasma luteinizing hormone (LH), follicular stimulating hormone (FSH), and testosterone levels were decreased. In the groups with melatonin, vitamin E, and both were applied together, tissue damage, and apoptotic cell death were reduced at both light microscopic and ultrastructural levels. In biochemical terms, there were decreased oxidative parameters and increased hormonal parameters. It was found that vitamin E was more effective in decreasing oxidative parameters and increasing antioxidative parameters when compared to melatonin, and hormonal parameters increased at a higher level in the Acmp + Vit E group than in all groups. As a result, it was found that exposure to Acmp caused damage to testicular tissue, induced oxidative stress in testicles, and decreased plasma LH, FSH, and testosterone levels, and although vitamin E is more effective than melatonin in preventing this damage, both are effective.

Comparison of Microwave Treatment and Pressure Cooker Methods for Antigen Retrieval Techniques in Immunohistochemistry

Background: Immunohistochemistry (IHC) is a method which is capable of detecting antigens or antibodies on the cells. The antigen antibody complex formed as a result of this reaction can be visualized at a light microscopic level. The main procedure involves application of a primary antibody which is targeted against a selected tissue antigen. Immunohistochemistry implements various polyclonal and monoclonal antibodies which help to identify the tissue location of the disease. Antigen retrieval (AR) is a key step in IHC which can be done by two methods: 1] Pressure cooker treatment 2] Microwave treatment method. This study aims to compare antigen retrieval efficacy and quality of immunohistochemical sections obtained by these two methods.
 Methodology: This prospective analytical study will be conducted in the histopathology and immunohistochemistry divisions of department of Pathology, J.N.M.C. Wardha. The study will evaluate immunohistochemical sections during which antigen retrieval would be done either by microwave treatment method or autoclave method in 30 cases. All the cases will be divided in two groups. For Group 1 - antigen retrieval will be performed by microwave treatment method. For Group 2 - antigen retrieval will be done by autoclave method. Qualitative evaluation and comparison of the sections obtained by these 2 methods will be done. Statistical evaluation will be done by SPSS software version 22.0, Prism 6.0 analysis software. Chi square test will be used for comparison.
 Results: A significant difference is expected in the between the efficacy of antigen retrieval by pressure cooker method and microwave treatment.
 Conclusion: Conclusions will be drawn on careful analysis of results.

Neurotensin Attenuates Nociception by Facilitating Inhibitory Synaptic Transmission in the Mouse Spinal Cord.

Neurotensin (NT) is an endogenous tridecapeptide in the central nervous system. NT-containing neurons and NT receptors are widely distributed in the spinal dorsal horn (SDH), indicating their possible modulatory roles in nociception processing. However, the exact distribution and function of NT, as well as NT receptors (NTRs) expression in the SDH, have not been well documented. Among the four NTR subtypes, NTR2 is predominantly involved in central analgesia according to previous reports. However, the expression and function of NTR2 in the SDH has not yet been directly elucidated. Specifically, it remains unclear how NT-NTR2 interactions contribute to NT-mediated analgesia. In the present study, by using immunofluorescent histochemical staining and immunohistochemical staining with in situ hybridization histochemical staining, we found that dense NT- immunoreactivity (NT-ir) and moderate NTR2-ir neuronal cell bodies and fibers were localized throughout the superficial laminae (laminae I-II) of the SDH at the light microscopic level. In addition, γ-aminobutyric acid (GABA) and NTR2 mRNA were colocalized in some neuronal cell bodies, predominantly in lamina II. Using confocal and electron microscopy, we also observed that NT-ir terminals made both close contacts and asymmetrical synapses with the local GABA-ir neurons. Second, electrophysiological recordings showed that NT facilitated inhibitory synaptic transmission but not glutamatergic excitatory synaptic transmission. Inactivation of NTR2 abolished the NT actions on both GABAergic and glycinergic synaptic release. Moreover, a behavioral study revealed that intrathecal injection of NT attenuated thermal pain, mechanical pain, and formalin induced acute inflammatory pain primarily by activating NTR2. Taken together, the present results provide direct evidence that NT-containing terminals and fibers, as well as NTR2-expressing neurons are widely distributed in the spinal dorsal horn, GABA-containing neurons express NTR2 mainly in lamina II, GABA coexists with NTR2 mainly in lamina II, and NT may directly increase the activity of local inhibitory neurons through NTR2 and induce analgesic effects.

A comparison of the retinal structure in the zebra-snout seahorse (Hippocampus barbouri Jordan & Richardson, 1908) between juveniles and adults in captivity

The activity of the sensory organ in the eye structure of the teleost fish is essential as it plays an important role in regulating fish-feeding behaviours. Unfortunately, the above information of zebra-snout seahorse Hippocampus barbouri, an aquaculture species in Thailand, has not been described. In this study, the eye structure, together with the retinal structure of juvenile [5th and 20th day after birth (DAB)] and adult (35th DAB), H. barbouri reared in captivity was investigated. All DABs were carried out and histologically observed. Light microscopic level explored the external-lateral surface of eye structure of H. barbouri, which consisted of the external, middle, and inner layers, as similarly reported in other teleost species. A well-differentiated retinal and photoreceptor cell layer were observed at 35th DAB compared to that at other DABs. This feature might be adequate to support the base of the increased feeding activity of adult seahorse in captivity for further research.

Viral-Like Particles Are Associated With Endosymbiont Pathology in Florida Corals Affected by Stony Coral Tissue Loss Disease

Stony coral tissue loss disease (SCTLD) was first documented in 2014 near the Port of Miami, Florida, and has since spread north and south along Florida’s Coral Reef, killing large numbers of more than 20 species of coral and leading to the functional extinction of at least one species, Dendrogyra cylindrus. SCTLD is assumed to be caused by bacteria based on presence of different molecular assemblages of bacteria in lesioned compared to apparently healthy tissues, its apparent spread among colonies, and cessation of spread of lesions in individual colonies treated with antibiotics. However, light microscopic examination of tissues of corals affected with SCTLD has not shown bacteria associated with tissue death. Rather, microscopy shows dead and dying coral cells and symbiotic dinoflagellates (endosymbionts) indicating a breakdown of host cell and endosymbiont symbiosis. It is unclear whether host cells die first leading to death of endosymbionts or vice versa. Based on microscopy, hypotheses as to possible causes of SCTLD include infectious agents not visible at the light microscopy level or toxicosis, perhaps originating from endosymbionts. To clarify this, we examined corals affected with SCTLD and apparently healthy corals using transmission electron microscopy. Endosymbionts in SCTLD-affected and apparently healthy corals consistently had varying degrees of pathology associated with elongated particles compatible in morphology with filamentous positive single-stranded RNA viruses of plants termed anisometric viral-like particles (AVLP). There was apparent progression from early to late replication of AVLP in the cytoplasm of endosymbionts adjacent to or at times within chloroplasts, with morphologic changes in chloroplasts consistent with those seen in plant cells infected by viruses. Coral host cell pathology appeared limited to massive proliferation and lysis of mucus cells. Based on these findings, we hypothesize that SCTLD is a viral disease of endosymbionts leading to coral host death. Efforts to confirm the presence of a virus associated with SCTLD through other means would be appropriate. These include showing the presence of a virus through molecular assays such as deep sequencing, attempts to grow this virus in the laboratory through culture of endosymbionts, localization of virus in tissue sections using immunohistochemistry or in situ hybridization, and experimental infection of known-virus-negative corals to replicate disease at the gross and microscopic level.

Short-term fenofibrate treatment improves ultrastructure of hepatocytes of old rats.

Fenofibrate (FN) is a hypolipemic drug used for the treatment of mixed dyslipidemia. Since in our previous study FN administration to young and old rats adversely affected the serum activity of liver marker enzymes, we decided to examine the effects of FN on liver ultrastructure of young and old animals. Young and old rats were fed standard rodent chow supplemented with 0.1% FN for 30 days. Liver samples obtained from animals under full anesthesia were processed by routine methods to obtain ultrathin and histological sections for the examination by light microscopy (LM) and transmission electron microscopy (TEM). Furthermore, liver lysates were analyzed by Western blotting for the expression of the autophagy-related proteins LC3A/B and beclin 1. The ultrastructure of hepatocytes in both age groups was well-preserved, with the presence of abundant mitochondria, numerous peroxisomes and lysosomes, glycogen stored in the form of rosettes, and occasionally autolysosomes. However, hepatocytes of old control rats contained less mitochondria and peroxisomes, and more lipid droplets than cells of young animals. The effects of FN on liver ultrastructure were age-depended. FN increased the relative number of mitochondria and peroxisomes in the hepatocytes of old, and did not affect their number in young rats. Moreover, FN decreased and increased the relative number of lipid droplets in the hepatocytes of old and young rats, respectively. At the LM level, Oil Red O staining revealed smaller and larger lipid droplets within hepatocytes and non-parenchymal liver cells. In the livers of young and old rats lipid droplets were distributed mainly in the periportal zones of hepatic lobules. Morphometric analysis confirmed that livers of control old rats contained more lipid-stainable areas than those of young ones; however, no effect of FN was observed either in young or old rats. Despite larger size of autolysosomes and autophagic vacuoles in hepatocytes of old rats, the expression of autophagy-related proteins did not differ in the livers of control and fenofibrate-treated young and old animals. The results of our study suggest that fenofibrate, apart from its hypolipemic action, may have beneficial effect on the energy metabolism in the liver of old individuals by increasing the number of mitochondria and peroxisomes in hepatocytes.