Changes in the Metabolic Profile of Melatonin Synthesis-Related Indoles during Post-Embryonic Development of the Turkey Pineal Organ.

Research on age-dependent changes in pineal activity has been limited almost exclusively to melatonin (MLT). This study determined, for the first time, the alterations occurring in the metabolic profile of MLT synthesis-related indoles during the post-embryonic development period in birds. Turkeys reared under a 12 h light/dark cycle were euthanized at 2 h intervals for 24 h at the ages of 2, 7, 14, and 28 days and 10, 20, 30, and 45 weeks. The results showed prominent changes in the metabolic profile of indoles during development and could be distinguished in four stages. The first stage, from hatching to the age of 2 weeks, was characterized by a decrease in the 5-hydroxytryptophan concentration and an increase in the concentrations of serotonin (5-HT), MLT, 5-methoxyindoleacetic acid, and 5-methoxytryptamine (5-MTAM). During the second stage, around the age of 1 month, the concentrations of N-acetylserotonin (NAS) and MLT reached a maximum. The synthesis and degradation of 5-HT were also the highest. The third stage, around the age of 10 weeks, was characterized by decreased levels of 5-HT (approximately 50%) and 5-hydroxyindoleacetic acid and a high level of 5-MTAM. The last stage, covering the age of 20 to 45 weeks, was characterized by a large decrease in the synthesis, content, and degradation of 5-HT. Despite these changes, there were no prominent differences in the nocturnal levels of NAS and MLT between the third and fourth stages. The concentrations of all tryptophan derivatives showed daily fluctuations until the age of 45 weeks.

Embryonic Development of Avian Pineal Secretory Activity-A Lesson from the Goose Pineal Organs in Superfusion Culture.

The embryonic ontogeny of pineal secretory activity in birds has been investigated almost exclusively in chickens. This study aimed to characterize this process in domestic geese. The pineal organs of embryos aged 18-28 days were incubated in superfusion culture under different light conditions for 4-5 days and treated with norepinephrine (NE). Melatonin (MLT) was measured by radioimmunoassay and other indoles by HPLC with fluorescence detection. Additionally, pineal organs were collected from embryos at 14-28 days of age and used to measure catecholamines by HPLC with electrochemical detection. MLT secretion increased with embryo age, most intensively between the 22nd and 24th days of life. The daily changes in MLT secretion under the 12 L:12D cycle occurred on the first day of culture, starting from an embryonic age of 24 days. MLT secretion was controlled by the light-dark cycle in all age groups studied. However, exposure to light during the scotophase did not alter the secretion of MLT. The endogenous oscillator expressed its activity in regulating MLT secretion in the pineal organs of embryos aged 24 days and older but could not generate a rhythm after one cycle. The rhythm of 5-hydroxytryptophan release during the first day of culture was found in the pineal organs of all embryos, while the rhythmic release of N-acetylserotonin and 5-methoxyindole acetic acid started at the age of 24 days. The proportion of released indoles changed with embryo age. NE caused a decrease in MLT secretion and provoked an increase in serotonin release. Incubation of the pineal organs induced the development of MLT secretory machinery and its diurnal rhythmicity. The pineal content of catecholamines increased prominently at the end of embryonic development.

Metabolism of Melatonin Synthesis-Related Indoles in the Turkey Pineal Organ and Its Modification by Monochromatic Light.

The metabolism of pineal indoles is closely related to alterations in the light and dark phases of a daily cycle. Recent research showed important interspecies differences in the pineal biochemistry, and a strong impact of monochromatic light on many physiological processes in birds. Therefore, the aims of study were to characterize the metabolism of melatonin-synthesis indoles in the pineal organ of the domestic turkey, and to determine the changes occurring in this metabolism under the influence of different wavelengths and intensities of light. For this purpose, 3-week-old turkeys were kept under 16 lx white light, or under blue, green, and red light with intensities of 16, 32, and 64 lx during the photophase, and after 7 d were sacrificed at 4 h intervals. The activities of melatonin-synthesizing enzymes and the contents of indoles were measured in the same pineal organ. The results revealed that the activities of tryptophan hydroxylase and arylalkylamine N-acetyltransferase, and the levels of all tryptophan derivatives had significant daily changes in birds kept under each light condition used. The profile of pineal indole metabolism in 4-week-old turkeys was characterized by high-amplitude rhythms in the activity of arylalkylamine N-acetyltransferase and the contents of N-acetylserotonin and melatonin, equal relative amounts of serotonin and 5-hydroxyindoleacetic acid, and higher content of melatonin than N-acetylserotonin. The monochromatic light significantly modified the pineal indole metabolism, and its effects were dependent on the color and intensity of light. Pronounced changes occurred in the level of serotonin synthesis and the daily rhythm course of melatonin synthesis.

Roles of Direct Photoreception and the Internal Circadian Oscillator in the Regulation of Melatonin Secretion in the Pineal Organ of the Domestic Turkey: A Novel In Vitro Clock and Calendar Model.

The regulation of melatonin secretion in the avian pineal organ is highly complex and shows prominent interspecies differences. The aim of this study was to determine the roles of direct photoreception and the internal oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. The pineal organs were collected from 12-, 13- and 14-week-old female turkeys reared under a 12 L:12 D cycle with the photophase from 07.00 to 19.00, and were incubated in superfusion culture for 3-6 days. The cultures were subjected to different light conditions including 12 L:12 D cycles with photophases between 07.00 and 19.00, 13.00 and 01.00 or 01.00 and 13.00, a reversed cycle 12 D:12 L, cycles with long (16 L:8 D) and short (8 L:16 D) photophases, and continuous darkness or illumination. The pineal organs were also exposed to light pulses of variable duration during incubation in darkness or to periods of darkness during the photophase. The secretion of melatonin was determined by direct radioimmunoassay. The turkey pineal organs secreted melatonin in a well-entrained diurnal rhythm with a very high amplitude. Direct photoreception as an independently acting mechanism was able to ensure quick and precise adaptation of the melatonin secretion rhythm to changes in light-dark conditions. The pineal organs secreted melatonin in circadian rhythms during incubation in continuous darkness or illumination. The endogenous oscillator of turkey pinealocytes was able to acquire and store information about the light-dark cycle and then to generate the circadian rhythm of melatonin secretion in continuous darkness according to the stored data. The obtained data suggest that the turkey pineal gland is highly autonomous in the generation and regulation of the melatonin secretion rhythm. They also demonstrate that the turkey pineal organ in superfusion culture is a valuable model for chronobiological studies, providing a highly precise clock and calendar. This system has several features which make it an attractive alternative to other avian pineal glands for circadian studies.

Embryonic Ontogeny of 5-Hydroxyindoles and 5-Methoxyindoles Synthesis Pathways in the Goose Pineal Organ.

The aim of this study was to characterize the embryonic ontogeny of 5-hydroxyindoles and 5-methoxyindoles synthesis pathways in the goose pineal organ. The study was performed on embryos aged 14-28 days, which have been incubated under a 12L:12D cycle. The pineal organs were collected for measurements of indole content by HPLC every 6 h on embryonic day (ED) 14, ED 16, ED 18 and ED 22 or every 2 h on ED 24, ED 26 and ED 28. The level of tryptophan showed no significant changes during development and no day-night variations. The content of 5-hydroxytryptophan increased between ED 14 and ED 26. It was significantly higher during scotophase than during photophase starting from ED 14. The serotonin content was low during the early stages of development (ED 14-ED 18) and prominently increased from ED 20. The serotonin levels also showed day-night differences; however, they were less conspicuous than those of 5-hydroxytryptophan. The changes in the level of 5-hydroxyindole acetic acid were similar to those of serotonin. 5-Hydroxytryptophol was measurable from ED 18. Levels of N-acetylserotonin, which were detectable for the first time on ED 16, prominently increased between ED 22 and ED 28 and showed significant day-night differences from ED 20. Melatonin was detectable from ED 18. Like N-acetylserotonin, its content increased rapidly between ED 22 and ED 28, and from ED 20 showed diurnal variations. 5-Methoxyindole acetic acid and 5-methoxytryptophol occurred at measurable levels from ED 18 and ED 26, respectively. The obtained results showed that embryonic development of indole metabolism in the goose pineal organ starts with the beginning of serotonin synthesis. The processes of serotonin acetylation and 5-hydroxyindoles methylation were turned on later. Diurnal rhythmicity develops very early in the embryonic pineal organ of the goose when the eggs are incubated under a 12 h light: 12 h dark schedule. Two processes are responsible for generation of the diurnal rhythms of 5-hydroxyindoles and 5-methoxyindoles: (i) hydroxylation of tryptophan and (ii) acetylation of serotonin.

Effects of Streptozotocin-Induced Diabetes on the Pineal Gland in the Domestic Pig.

Several observations from experiments in rodents and human patients suggest that diabetes affects pineal gland function, including melatonin secretion; however, the accumulated data are not consistent. The aim of the present study was to determine the effects of streptozotocin-induced diabetes on the pineal gland in the domestic pig, a species widely used as a model in various biomedical studies. The study was performed on 10 juvenile pigs, which were divided into two groups: control and diabetic. Diabetes was evoked by administration of streptozotocin (150 mg/kg of body weight). After six weeks, the animals were euthanized between 12.00 and 14.00, and the pineal glands were removed and divided into two equal parts, which were used for biochemical analyses and for preparation of explants for the superfusion culture. The pineal contents (per 100 µg protein) of serotonin, 5-hydroxyindole acetic acid, 5-hydroxytryptophol, 5-methoxyindole acetic acid, 5-methoxytryptophol, and 5-methoxytryptamine were significantly lower in diabetic pigs than in control pigs. In contrast, the level of N-acetylserotonin was significantly higher in diabetic animals. No significant differences were found in the level of melatonin between control and experimental pigs. The amounts of 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, and 3,4-dihydroxyphenylacetic acid were significantly lower in the pineal glands of diabetic animals. The level of vanillylmandelic acid was higher in diabetic pigs. No differences were observed in the level of basal and NE-stimulated release of N-acetylserotonin or melatonin between the pineal explants prepared from control and experimental animals. In vitro treatment with insulin was ineffective. In conclusion, streptozotocin-induced diabetes affects both indole metabolism and adrenergic neurotransmission in the pig pineal gland.

Feline Infectious Peritonitis: Immunohistochemical Features of Ocular Inflammation and the Distribution of Viral Antigens in Structures of the Eye.

Feline infectious peritonitis (FIP) is a serious, widely distributed systemic disease caused by feline coronavirus (FCoV), in which ocular disease is common. However, questions remain about the patterns of ocular inflammation and the distribution of viral antigen in the eyes of cats with FIP. This study characterized the ocular lesions of FIP including the expression of glial fibrillary acidic protein and proliferating cell nuclear antigen by Müller cells in the retina in cases of FIP and to what extent macrophages are involved in ocular inflammation in FIP. Immunohistochemistry for FCoV, CD3, CD79a, glial fibrillary acidic protein, calprotectin, and proliferating cell nuclear antigen was performed on paraffin sections from 15 naturally occurring cases of FIP and from controls. Glial fibrillary acidic protein expression was increased in the retina in cases of FIP. Müller cell proliferation was present within lesions of retinal detachment. Macrophages were present in FIP-associated ocular lesions, but they were the most numerous inflammatory cells only within granulomas (2/15 cats, 13%). In cases of severe inflammation of the ciliary body with damage to blood vessel walls and ciliary epithelium (3/15, 20%), some macrophages expressed FCoV antigens, and immunolabeling for calprotectin on consecutive sections suggested that these FCoV-positive macrophages were likely to be recently derived from blood. In cases of severe and massive inflammation of most ocular structures (4/15, 26%), B cells and plasma cells predominated over T cells and macrophages. These results indicate that gliosis can be present in FIP-affected retinas and suggest that breakdown of the blood-ocular barrier can allow FCoV-bearing macrophages to access the eye.

Intraperitoneal exposure of whitefish to microcystin-LR induces rapid liver injury followed by regeneration and resilience to subsequent exposures.

To date, there has been no systematic approach comprehensively describing the sequence of pathological changes in fish during prolonged exposure to microcystin-LR (MC-LR). Towards this aim, juvenile whitefish individuals received an intraperitoneal injection with pure MC-LR, and the injection was repeated every week to maintain continuous exposure for 28days. During the exposure period, growth and condition of the fish were assessed based on biometric measurements. Additionally, selected biochemical markers were analysed in the fishes' blood, and their livers were carefully examined for morphological, ultrastructural, and molecular changes. The higher dose of MC-LR (100µg·kg-1) caused severe liver injury at the beginning of the exposure period, whereas the lower dose (10µg·kg-1) caused less, probably reversible injury, and its effects began to be observed later in the exposure period. These marked changes were accompanied by substantial MC-LR uptake by the liver. However, starting on the 7th day of exposure, cell debris began to be removed by phagocytes, then by 14th day, proliferation of liver cells had markedly increased, which led to reconstruction of the liver parenchyma at the end of the treatment. Surprisingly, despite weekly-repeated intraperitoneal injections, MC-LR did not accumulate over time of exposure which suggests its limited uptake in the later phase of exposure. In support, mRNA expression of the membrane transport protein oatp1d was decreased at the same time as the regenerative processes were observed. Our study shows that closing of active membrane transport may serve as one defence mechanism against further MC-LR intoxication.

Diurnal profiles of melatonin synthesis-related indoles, catecholamines and their metabolites in the duck pineal organ.

This study characterizes the diurnal profiles of ten melatonin synthesis-related indoles, the quantitative relations between these compounds, and daily variations in the contents of catecholamines and their metabolites in the domestic duck pineal organ. Fourteen-week-old birds, which were reared under a 12L:12D cycle, were killed at two-hour intervals. The indole contents were measured using HPLC with fluorescence detection, whereas the levels of catecholamines and their metabolites were measured using HPLC with electrochemical detection. All indole contents, except for tryptophan, showed significant diurnal variations. The 5-hydroxytryptophan level was approximately two-fold higher during the scotophase than during the photophase. The serotonin content increased during the first half of the photophase, remained elevated for approximately 10 h and then rapidly decreased in the middle of the scotophase. N-acetylserotonin showed the most prominent changes, with a more than 15-fold increase at night. The melatonin cycle demonstrated only an approximately 5-fold difference between the peak and nadir. The 5-methoxytryptamine content was markedly elevated during the scotophase. The 5-hydroxyindole acetic acid, 5-hydroxytryptophol, 5-methoxyindole acetic acid and 5-methoxytryptophol profiles were analogous to the serotonin rhythm. The norepinephrine and dopamine contents showed no significant changes. The DOPA, DOPAC and homovanillic acid levels were higher during the scotophase than during the photophase. Vanillylmandelic acid showed the opposite rhythm, with an elevated level during the daytime.

Histology and Ultrastructure of the Esophagus in European Beaver (Castor fiber) Displays Features Adapted to Seasonal Changes in Diet.

The European beaver is a herbivorous rodent whose diet changes seasonally, and in winter consists of large quantities of woody plants. It is distinguished among other mammals by a unique organization of the stomach that comprises the cardiogastric gland and by the unusual process of mucus formation in the gastric mucosa. The aim of study was to (i) characterize the structure of the beaver esophagus with particular attention to the mucosal epithelium; (ii) compare the histological structure of the esophagi collected in spring, summer, and winter; (iii) provide preliminary data on the structure of the esophagus in beaver fetuses. The study was conducted on esophagi of 18 adult beavers captured in Poland in April, August, and December, and on 3 fetal organs. The results obtained in adults show that the mucosa is lined with thick stratified squamous keratinized epithelium with a structure similar to that of the skin epidermis. Ultrastructural studies reveal the presence of multiple lamellar and non-lamellar bodies in granular cells, whose morphology and location gradually change while reaching the upper epithelial layers. The muscularis mucosa comprises a layer of longitudinally oriented bundles of smooth muscle cells. Both mucosa and submucosa do not comprise any glands. The thick muscularis externa consists mainly of internal circular and external longitudinal layers of striated muscle fibers. The keratinized layer of mucosa epithelium was 2-3-fold thicker in esophagi collected in winter than in those collected in spring and summer, while the epithelial cell layer thickness remained unchanged regardless of the season. Immunolabeling for proliferating cell nuclear antigen shows a higher index of epithelium proliferation in esophagi collected in winter than in spring and summer. No seasonal differences were noted in other layers of the esophagus. Fetal organs have epithelium covered with a keratinized layer, thinner than in adults, and the muscularis externa comprises both striated and smooth muscle cells.

Low-Intensity Blue Light Exposure Reduces Melanopsin Expression in Intrinsically Photosensitive Retinal Ganglion Cells and Damages Mitochondria in Retinal Ganglion Cells in Wistar Rats.

This study investigated the effect of low-intensity blue light on the albino Wistar rat retina, including intrinsically photosensitive retinal ganglion cells (ipRGCs). Three groups of nine albino Wistar rats were used. One group was continuously exposed to blue light (150 lx) for 2 d (STE); one was exposed to 12 h of blue light and 12 h of darkness for 10 d (LTE); one was maintained in 12 h of white light (150 lx) and 12 h of darkness for 10 d (control). Melanopsin (Opn4) was immunolabelled on retinal whole-mounts. To count and measure Opn4-positive ipRGC somas and dendrites (including Sholl profiles), Neuron J was used. Retinal cryosections were immunolabeled for glial fibrillary acid protein (GFAP) and with terminal deoxynucleotidyl transferase dUTP nick-end labelling for apoptosis detection. LTE reduced the length of Opn4-positive ipRGC dendrites (p = 0.03) and decreased Opn4-immunoreactivity in ipRGC outer stratifying dendrites. LTE and STE decreased the complexity of dendritic arborization (Sholl profile; p < 0.001, p = 0.03, respectively), increased retinal GFAP immunoreactivity (p < 0.001, p = 0.002, respectively), and caused outer segment vesiculation and outer nuclear layer apoptosis. Ultrastructural analysis showed that LTE damaged mitochondria in retinal ganglion cells and in the inner plexiform layer. Thus, LTE to low-intensity blue light harms the retinas of albino Wistar rats.

The Use of Chitin from the Molts of Mealworm (Tenebrio molitor) for the Removal of Anionic and Cationic Dyes from Aqueous Solutions.

The possibility of using chitin from the molts of an insect-ealworm (Tenebrio molitor) to remove anionic (RB5, RY84) and cationic dyes (BV10, BR46) from aqueous solutions was investigated. The scope of the research included, among others: Characteristics of chitin from mealworms (FTIR, SEM, pHPZC), the effect of pH on sorption efficiency, sorption kinetics (pseudo-first, pseudo-second order, intramolecular diffusion models) and the determination of the maximum sorption capacity (Langmuir and Freundlich models). The sorption efficiency of anionic dyes on chitin from mealworm was the highest at pH 2-3, and for cationic dyes at pH 6. The equilibrium time of sorption of anionic dyes was 240-300 min and for cationic dyes it was 180-240 min. The experimental data on dye sorption kinetics was best described by the pseudo-second order model. The maximum sorption capacity of chitin from the mealworm for the anionic dyes RB5 and RY84 was 121.15 mg/g and 138.55 mg/g, respectively, and was higher than with some carbon-based materials (literature data). In the case of cationic dyes, the sorption capacity of the tested chitin was lower and reached 3.22 mg/g and 59.56 mg/g for BV10 and BR46, respectively.

Isolation of Citrus lemon extracellular vesicles: Development and process control using capillary electrophoresis.

A new and scalable method for the isolation of extracellular vesicles (EV) from Citrus lemon juice samples was developed. The methodology included preliminary preconcentration of the sample using ultrafiltration (UF) followed by size-exclusion chromatography (SEC) purification and final preconcentration of the eluates. Transmission electron microscopy and proteomic analysis showed that isolates contained exosome-like vesicles, exocyst-positive organelle (EXPO), and microvesicles. The efficiency of certain isolation steps was evaluated with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A good correlation between CE, BCA, and NTA results was shown. The application of CE enabled the detection of soluble contaminants, macromolecular aggregates, and vesicles' heterogeneity. The fluorescent staining of encapsulated nucleic acids was proposed for the identity confirmation of EV detected in CE. The study demonstrates the CE as a comprehensive tool for monitoring of the EV isolation process.

Novel insights into the nervous system affected by prolonged hyperglycemia.

Multiple molecular pathways including the receptor for advanced glycation end-products-diaphanous related formin 1 (RAGE-Diaph1) signaling are known to play a role in diabetic peripheral neuropathy (DPN). Evidence suggests that neuropathological alterations in type 1 diabetic spinal cord may occur at the same time as or following peripheral nerve abnormalities. We demonstrated that DPN was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. More than 500 differentially expressed genes (DEGs) belonging to multiple functional pathways were identified in diabetic spinal cord and of those the most enriched was RAGE-Diaph1 related PI3K-Akt pathway. Only seven of spinal cord DEGs overlapped with DEGs from type 1 diabetic sciatic nerve and only a single gene cathepsin E (CTSE) was common for both type 1 and type 2 diabetic mice. In silico analysis suggests that molecular changes in spinal cord may act synergistically with RAGE-Diaph1 signaling axis in the peripheral nerve. KEY MESSAGES: Molecular perturbations in spinal cord may be involved in the progression of diabetic peripheral neuropathy. Diabetic peripheral neuropathy was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. In silico analysis revealed that PI3K-Akt signaling axis related to RAGE-Diaph1 was the most enriched biological pathway in diabetic spinal cord. Cathepsin E may be the target molecular hub for intervention against diabetic peripheral neuropathy.

Profiles of Rho, Opn4, c-Fos, and Birc5 mRNA expression in Wistar rat retinas exposed to white or monochromatic light.

Despite concern over potential retinal damage linked to exposure to light-emitting-diode (LED) light (particularly blue light), it remains unknown how exposure to low-intensity monochromatic LED light affects the expression of rhodopsin (Rho, a photopigment that mediates light-induced retinal degeneration), melanopsin (Opn4, a blue-light sensitive photopigment), c-Fos (associated with retinal damage/degeneration), and Birc5 (anti-apoptotic). This study investigated the mRNA expression profiles of these genes under exposure to white and monochromatic light (blue, red, green) in the retinas of albino rats under a cycle of 12 h of light and 12 h of darkness. In each group, 32 Wistar rats were exposed to one type of monochromatic-LED or white-fluorescent light for 7 day (150 lx). Retinal samples were taken for qPCR analysis and light and electron microscopy. Blue and green light exposure markedly decreased expression of Rho and Opn4 mRNA and increased expression of Birc5 and c-Fos mRNA (P < 0.05). In retinas from the blue-light group, loss and vesiculation of photoreceptor outer segments were visible, but not in retinas from the red-light and control group. Measurements of the photoreceptor inner and outer segments length revealed, that this length was significantly decreased in the blue- and green-light exposure groups (P < 0.02), but not in the red-light exposure group. Increased expression of Birc5 and decreased expression of Rho and Opn4 after exposure to blue and green light may be early responses that help to reduce light-induced retinal damage.

Immortalization of swine umbilical vein endothelial cells (SUVECs) with the simian virus 40 large-T antigen.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis.

Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies.

The development of field-emission scanning electron microscopes for high-resolution imaging at very low acceleration voltages and equipped with highly sensitive detectors of backscattered electrons (BSE) has enabled transmission electron microscopy (TEM)-like imaging of the cut surfaces of tissue blocks, which are impermeable to the electron beam, or tissue sections mounted on the solid substrates. This has resulted in the development of methods that simplify and accelerate ultrastructural studies of large areas and volumes of biological samples. This article provides an overview of these methods, including their advantages and disadvantages. The imaging of large sample areas can be performed using two methods based on the detection of transmitted electrons or BSE. Effective imaging using BSE requires special fixation and en bloc contrasting of samples. BSE imaging has resulted in the development of volume imaging techniques, including array tomography (AT) and serial block-face imaging (SBF-SEM). In AT, serial ultrathin sections are collected manually on a solid substrate such as a glass and silicon wafer or automatically on a tape using a special ultramicrotome. The imaging of serial sections is used to obtain three-dimensional (3D) information. SBF-SEM is based on removing the top layer of a resin-embedded sample using an ultramicrotome inside the SEM specimen chamber and then imaging the exposed surface with a BSE detector. The steps of cutting and imaging the resin block are repeated hundreds or thousands of times to obtain a z-stack for 3D analyses.

Norepinephrine Is a Major Regulator of Pineal Gland Secretory Activity in the Domestic Goose (Anser anser).

This study determined the effect of norepinephrine and light exposure on melatonin secretion in goose pineal explants. Additionally, it investigated changes in the content of norepinephrine, dopamine, and their metabolites [3,4-dihydroxyphenylacetic acid; vanillylmandelic acid (VMA); homovanillic acid] in goose pineal glands in vivo under 12 h of light and 12 h of darkness (LD), a reversed cycle (DL), constant light (LL), and constant darkness (DD). In vitro content of melatonin was measured by radioimmunoassay; contents of catecholamines and their metabolites were measured by high-performance liquid chromatography. Exposure of pineal explants to LD or DL established rhythmic melatonin secretion; this rhythm was much better entrained with norepinephrine exposure during photophase than without it. When the explants were kept in LL or DD, the rhythm was abolished, unless NE was administered during natural scotophase of a daily cycle. In vivo, norepinephrine and dopamine levels did not display rhythmic changes, but their respective metabolites, HMV and VMA, displayed well-entrained diurnal rhythms. These results indicate that norepinephrine and sympathetic innervation play key roles in regulation of pineal secretory activity in geese, and that pineal levels of VMA and HMV provide precise information about the activity of sympathetic nerve fibers in goose pineal glands.

Diurnal Rhythm of Plasma Melatonin Concentration in the Domestic Turkey and Its Regulation by Light and Endogenous Oscillators.

The aim of this study was to characterize the diurnal rhythm of plasma melatonin (MLT) concentration and its regulation by light and endogenous oscillators in 10-week-old domestic turkeys. Three experiments were performed to examine (i) the course of daily changes in plasma MLT concentration in turkeys kept under a 12 h light: 12 h dark (12L:12D) cycle; (ii) the influence of night-time light exposure lasting 0.5, 1, 2, or 3 h on the plasma MLT level; and (iii) the occurrence of circadian fluctuations in plasma MLT levels in birds kept under continuous dim red light and the ability of turkeys to adapt their pineal secretory activity to a reversed light-dark cycle (12D:12L). The plasma MLT concentration was measured with a direct radioimmunoassay. The plasma MLT concentration in turkeys kept under a 12L:12D cycle changed significantly in a daily rhythm. It was low during the photophase and increased stepwise after the onset of darkness to achieve the maximal level in the middle of the scotophase. Next, it decreased during the second half of the night. The difference between the lowest level of MLT and the highest level was approximately 18-fold. The exposure of turkeys to light during the scotophase caused a rapid, large decrease in plasma MLT concentration. The plasma MLT concentration decreased approximately 3- and 10-fold after 0.5 and 1 h of light exposure, respectively, and reached the day-time level after 2 h of exposure. In turkeys kept under continuous darkness, the plasma MLT level was approximately 2.5-fold higher at 02:00 h than at 14:00 h. In birds kept under 12D:12L, the plasma MLT level was significantly higher at 14:00 h than at 02:00 h. The results showed that plasma MLT concentrations in 10-week-old turkeys have a prominent diurnal rhythm, which is endogenously generated and strongly influenced by environmental light.

Effects of Deoxynivalenol and Zearalenone on the Histology and Ultrastructure of Pig Liver.

The purpose of this study was to determine the effects of single and combined administrations of deoxynivalenol (DON) and zearalenone (ZEN) on the histology and ultrastructure of pig liver. The study was performed on immature gilts, which were divided into four equal groups. Animals in the experimental groups received DON at a dose of 12 µg/kg body weight (BW) per day, ZEN at 40 µg/kg BW per day, or a mixture of DON (12 µg/kg BW per day) and ZEN (40 µg/kg BW). The control group received vehicle. The animals were killed after 1, 3, and 6 weeks of experiment. Treatment with mycotoxins resulted in several changes in liver histology and ultrastructure, including: (1) an increase in the thickness of the perilobular connective tissue and its penetration to the lobules in gilts receiving DON and DON + ZEN; (2) an increase in the total microscopic liver score (histology activity index (HAI)) in pigs receiving DON and DON + ZEN; (3) dilatation of hepatic sinusoids in pigs receiving ZEN, DON and DON + ZEN; (4) temporary changes in glycogen content in all experimental groups; (5) an increase in iron accumulation in the hepatocytes of gilts treated with ZEN and DON + ZEN; (6) changes in endoplasmic reticulum organization in the hepatocytes of pigs receiving toxins; (7) changes in morphology of Browicz-Kupffer cells after treatment with ZEN, DON, and DON + ZEN. The results show that low doses of mycotoxins used in the present study, even when applied for a short period, affected liver morphology.