Ultrastructural Evaluation Research Articles

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil–injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

The Effects of Experimental Intratympanic Steroid Administration on Organ of Corti Type 1 Spiral Ganglion.

Introduction It is unclear how effective is the intratympanic (IT) steroid treatment on organ of Corti type 1 spiral ganglion, its optimal dosage and frequency of administration. The effect of dexamethasone on cochlear functions in individuals with a normal hearing ability is also unknown. Objective The aim of this study was to evaluate, at the electrophysiological and ultrastructural levels, the effect of IT dexamethasone administration in guinea pigs with normal hearing on organ of Corti type 1 spiral ganglion. Methods A total of 20 guinea pigs ( n = 40 ears) whose hearing was detected to be normal by electrophysiological tests were included in the study and randomly divided into 6 groups. Four groups were considered study groups, while 2 groups were considered control groups. Dexamethasone was administered intratympanically in doses of 2 mg/mL and 4 mg/mL in the guinea pigs in the study groups. The animals in the control groups received physiological saline in equal doses as the study groups. All interventions were performed under general anesthesia, and the electrophysiological tests were repeated following the IT injections. Results No statistically significant differences were found among the groups when the IT injections were evaluated in terms of the electrophysiological measurements ( p > 0.05). The ultrastructural evaluation showed a cellular mitochondrial increase in the spiral ganglions of the cochlea in the groups in which dexamethasone was administered in a dose of 4 mg/mL. Conclusion According to the findings of this study, it can be suggested that the IT injection of dexamethasone is safe, and when applied in a dose of 4 mg/mL, it increases metabolic activity at the cellular level.

Histopathological and Ultrastructural evaluation of the Effect of Electronic Cigarette Smoke on the Submandibular Gland of Albino Rats

Introduction: E-cigarettes (E-cigs) have received a considerable attention within the last years and are among the newest products for reduction of tobacco injury. Recently, it has been postulated that they are not considered a safe substitute for smoking and may have a limited degree of effectiveness in arresting smoking. Aim: The aim of this study was to detect the histopathological and ultrastructural changes of tissue architecture of the submandibular glands in rats after passive E-cig exposure.Methods: 12 adult male albino rats were divided into three groups (four rats each). Group I (Control group): Rats were exposed to fresh air. Group II (E-cigarette 4 weeks): Each rat was exposed to passive E-cig smoke for duration of one hour daily. The exposure time was divided into 6 equal intervals of 10 mins /hr, 7 days a week for four successive weeks. Group III (E-cigarette 12 weeks): Rats of this group were exposed to the same dose and manner of treatment of E-cig smoke as in group II for 12 weeks duration. Animals were sacrificed following each treatment period and specimens were prepared for histological examination and transmission electron microscopic study.Results: H & E results revealed signs of atrophy and degeneration within the parenchymal elements together with dilatation and congestion of blood vessels with interstitial hemorrhage in 4 weeks E-cig exposed rats. More deleterious changes with marked distortion of the gland’s architecture were detected in 12 weeks E-cig exposed group. These data were supported by the electron microscopic results which reflected degenerative changes in cytoplasmic organelles. Conclusion: E-cig is not safe and its use to replace cigarette smoking cannot be recommended.

In-vitro construction of endometrial-like epithelium using CD146+ mesenchymal cells derived from human endometrium

Endometrial CD146+ cells were purified, using magnetic activated cell sorting, and then embedded and cultured in a collagen–matrigel scaffold on top of myometrial smooth muscle cells for 10 days. At the end of culture period, the differentiation and formation of the epithelial-like cells were confirmed by morphological and ultrastructural evaluations, and analysis by reverse transcription polymerase chain reaction of the specific expression of genes: osteopontin (SPP1), matrix metalloproteinase 2, zonula occludens 1, laminin alpha 2 and collagen type IV; and by western blotting of CD9 protein. The results showed that the human endometrial mesenchymal CD146+ cells were able to produce endometrial glandular tube-like structures in vitro. Ultrastructural observation revealed some projections on the apical surfaces, appearance of basal lamina-like structures on the basal surface, and tight junctions and desmosomes on the lateral surfaces of the epithelial-like cells. The expression of studied genes at RNA level and CD9 at protein level confirmed the formation of endometrial epithelial-like cells. This culture system may have potential applications in cell therapy and in studies on human embryo implantation.

New insights on mitochondrial heterogeneity observed in prepared mitochondrial samples following a method for freeze-fracture and scanning electron microscopy

Mitochondria are dynamic intracellular organelles with diverse roles in tissue- and cell type-specific processes, extending beyond bioenergetics. In keeping with this array of functions, mitochondria are described as heterogeneous both between and within tissue types based on multiple parameters, including a broad spectrum of morphological features, and new research points toward a need for the evaluation of mitochondria as isolated organelles. Although transmission electron microscopy (TEM) is commonly used for the evaluation of mitochondria in tissues and renders mitochondrial structures in ultra-thin sections in two-dimensions, additional information regarding complex features within these organelles can be ascertained using scanning electron microscopy (SEM), which allows for analysis of phenotypic differences in three-dimensions. One challenge in producing mitochondrial images for evaluation of ultrastructure using SEM has been the ability to reliably visualize important intramitochondrial features, the inner membrane and cristae structures, on a large-scale (e.g. multiple mitochondria) within a sample preparation, as mitochondria are enclosed within a double membrane. This can be overcome using a ‘freeze-fracture’ technique in which mitochondrial preparations are snap-frozen followed by application of intense pressure to break open the organelles, revealing the intact components within. Previously published reports using freeze-fracture strategies for mitochondrial SEM have demonstrated feasibility in whole tissue specimens, but a detailed methodology for SEM analysis on isolated mitochondrial fractions has not been reported. By combining previously reported tissue freeze-fracture strategies, along with utilizing the depth of field created by SEM, herein we present a complete method reliant on the freeze-fracture of mitochondrial fractions prepared by differential centrifugation to produce a comprehensive and direct evaluation of three-dimensional mitochondrial ultrastructure by SEM. Image analysis of internal mitochondrial features demonstrates heterogeneity in mitochondrial ultrastructure from a single sample preparation.

Technical feasibility of isolated Bowman layer graft preparation by femtosecond laser: a pilot study.

To evaluate the technical feasibility of isolated Bowman layer (BL) graft preparation by femtosecond laser (FSL) and to compare the ultrastructural morphology to manually dissected grafts. Five whole globes were placed in custom-made eye holders and debrided of epithelium. After programming a dissection depth of 20 μm, the FSL was docked into position and 5 isolated BL grafts were created. From 5 additional globes, corneoscleral buttons were procured, mounted in artificial anterior chambers, and stripped of BL via the previously described manual technique. Three specimens from both series were randomly selected and assigned to transmission electron microscopy for ultrastructural evaluation and thickness measurements. All dissections were uneventful and 10 total grafts were produced: 5 by FSL and 5 by manual dissection. Mean graft thickness was 37 (±8.6) μm (n = 3) for the FSL group and 11.7 (±1.6) μm (n = 3) for the manually dissected group. Transmission electron microscopy revealed a thick but relatively smooth posterior cut edge in the FSL group, versus a virtually isolated BL with irregular rests of dispersed stroma in the manually dissected group. Femtosecond laser may have potential for harvesting intact BL and with a smooth posterior surface, but accompanied by variable amounts of anterior stroma owing to technical limitations.

Accuracy of Nasal Nitric Oxide Measurement as a Diagnostic Test for Primary Ciliary Dyskinesia. A Systematic Review and Meta-analysis.

Primary ciliary dyskinesia (PCD) is a rare disorder causing chronic otosinopulmonary disease, generally diagnosed through evaluation of respiratory ciliary ultrastructure and/or genetic testing. Nasal nitric oxide (nNO) measurement is used as a PCD screening test because patients with PCD have low nNO levels, but its value as a diagnostic test remains unknown. To perform a systematic review to assess the utility of nNO measurement (index test) as a diagnostic tool compared with the reference standard of electron microscopy (EM) evaluation of ciliary defects and/or detection of biallelic mutations in PCD genes. Ten databases were searched for reference sources from database inception through July 29, 2016. Study inclusion was limited to publications with rigorous nNO index testing, reference standard diagnostic testing with EM and/or genetics, and calculable diagnostic accuracy information for cooperative patients (generally >5 yr old) with high suspicion of PCD. Meta-analysis provided a summary estimate for sensitivity and specificity and a hierarchical summary receiver operating characteristic curve. The Quality Assessment of Diagnostic Accuracy Studies-2 tool was used to assess study quality, and Grading of Recommendations Assessment, Development, and Evaluation was used to assess the certainty of evidence. In 12 study populations (1,344 patients comprising 514 with PCD and 830 without PCD), using a reference standard of EM alone or EM and/or genetic testing, summary sensitivity was 97.6% (92.7-99.2) and specificity was 96.0% (87.9-98.7), with a positive likelihood ratio of 24.3 (7.6-76.9), a negative likelihood ratio of 0.03 (0.01-0.08), and a diagnostic odds ratio of 956.8 (141.2-6481.5) for nNO measurements. After studies using EM alone as the reference standard were excluded, the seven studies using an extended reference standard of EM and/or genetic testing showed a summary sensitivity of nNO measurements of 96.3% (88.7-98.9) and specificity of 96.4% (85.1-99.2), with a positive likelihood ratio of 26.5 (5.9-119.1), a negative likelihood ratio of 0.04 (0.01-0.12), and a diagnostic odds ratio of 699.3 (67.4-7256.0). Certainty of the evidence was graded as moderate. nNO is a sensitive and specific test for PCD in cooperative patients (generally >5 yr old) with high clinical suspicion for this disease. With a moderate level of evidence, this meta-analysis confirms that nNO testing using velum closure maneuvers has diagnostic accuracy similar to EM and/or genetic testing for PCD when cystic fibrosis is ruled out. Thus, low nNO values accompanied by an appropriate clinical phenotype could be used as a diagnostic PCD test, though EM and/or genetics will continue to provide confirmatory information.

Ultrastructural examination of skin biopsies may assist in diagnosing mitochondrial cytopathy when muscle biopsies yield negative results.

Ultrastructural evaluation of skin biopsies has been utilized for diagnosis of mitochondrial disease. This study investigates how frequently skin biopsies reveal mitochondrial abnormalities, correlates skin and muscle biopsy findings, and describes clinical diagnoses rendered following the evaluation. A retrospective review of surgical pathology reports from 1990 to 2015 identified skin biopsies examined by electron microscopy for suspected metabolic disease. A total of 630 biopsies were included from 615 patients. Of these patients, 178 also underwent a muscle biopsy. Of the 630 skin biopsies, 75 (12%) showed ultrastructural abnormalities and 34 (5%) specifically showed mitochondrial abnormalities including increased size (n=27), reduced or abnormal cristae (n=23), dense matrices (n=20), and increased number (n=8). Additional findings included lysosomal abnormalities (n=13), lipid accumulation (n=2) or glycogen accumulation (n=1). Of the 34 patients with mitochondrial abnormalities on skin biopsy, 20 also had muscle biopsies performed and nine showed abnormalities suggestive of a mitochondrial disorder including absent cytochrome oxidase staining (n=2), increased subsarcolemmal NADH, SDH, or cytochrome oxidase staining (n=1), or ultrastructural findings including large mitochondrial size (n=5), abnormal mitochondrial structure (n=5), and increased mitochondrial number (n=4). The most common presenting symptoms were intellectual disability (n=13), seizures (n=12), encephalopathy (n=9), and gastrointestinal disturbances (n=9). At last known follow-up, 12 patients had a definitive diagnosis of a mitochondrial disorder. One patient each had Complex I deficiency, Complex III deficiency, Charcot-Marie-Tooth disease, pyruvate dehydrogenase deficiency, and Phelan-McDermid syndrome. Our results suggest that skin biopsy sometimes yields diagnostic clues suggestive of a mitochondrial cytopathy in cases with a negative muscle biopsy.

More histologic and ultrastructural degenerative signs in the subscapularis tendon and the joint capsule in male patients with shoulder impingement

PurposeThe purpose of the present study was to analyze biopsy samples from the subscapularis tendon and from the joint capsule from male patients with shoulder impingement syndrome (SAIS) and compare them with samples from male patients with post-traumatic recurrent shoulder instability. The hypothesis of the study was that patients with SAIS would have more histologic and ultrastructural degenerative changes in their subscapularis tendon and joint capsule than patients with post-traumatic recurrent shoulder instability.MethodsMale patients scheduled for surgery, with either subacromial decompression or Bankart reconstruction, were included. Four biopsies from each patient were obtained from the capsule and four from the subscapularis tendon during arthroscopic surgery. The histologic characteristics and the presence of glycosaminoglycans were assessed using the light microscope, and the ultrastructure was assessed using a transmission electron microscope.ResultsEight patients, median age 53 (45–74) years (p < 0.0001), were included in the impingement group, and 12 patients, median age 27 (22–48) years, were included in the instability group. The histologic assessment revealed significantly higher cellularity and total degeneration score in the capsule (p = 0.016 and p = 0.014 respectively) in patients with subacromial impingement compared with the instability patients. The corresponding finding was not made for the subscapularis tendon. The ultrastructural evaluation revealed that the instability patients had more fibrils with a large diameter (indicating less degeneration) in both the subscapularis tendon and the capsule compared with the impingement patients (p < 0.0001).ConclusionMale patients with subacromial impingement have more histologic and ultrastructural degenerative changes in their shoulder compared with patients with post-traumatic recurrent shoulder instability.Clinical relevanceIt appears that in patients with subacromial impingement, the whole shoulder joint is affected and not only the subacromial space. It is the opinion of the authors that intra-articular therapeutic injections could be tried more often in these patients.Level of evidenceIII.

Pathology of Naturally Occurring Bacillary Hemoglobinuria in Cattle

Clostridium haemolyticum causes bacillary hemoglobinuria (BH), an infectious and usually fatal disease that occurs mostly in cattle, which is clinically characterized by jaundice, hemoglobinuria, and anemia. The trematode Fasciola hepatica has been commonly reported as the main predisposing factor that triggers this condition. The authors evaluated 20 naturally occurring cases of bovine BH to characterize the pathology and pathogenesis of the disease. Grossly, the most consistent finding was a large, frequently single focus of necrosis surrounded by a red to purple halo, observed most frequently on the parietal surface of the right and left hepatic lobes. Other findings were jaundice, dark-brown discoloration of kidneys, and red urine in the urinary bladder. Microscopically, characteristic lesions were locally extensive, necrotizing hepatitis with thrombosis and numerous intralesional Gram-positive rod-shaped bacteria, and acute renal tubular necrosis. By immunohistochemistry, many hepatocytes outside the necrotic focus in the liver were positive for activated caspase 3, suggesting that those cells were undergoing apoptosis. Ultrastructural evaluation revealed hepatocyte necrosis, hemolysis, and clumps of vegetative and sporulating bacilli within the liver. Polymerase chain reaction for the C. haemolyticum beta toxin gene was positive in randomly selected liver samples. No gross or microscopic lesions indicative of fascioliasis were detected in the liver of any animal, suggesting that other yet undetermined predisposing factors were associated with these cases of BH.

Aspectos morfológicos e ultraestruturais de células sanguíneas de Crotalus durissus terrificus

RESUMO: A avaliação hematológica, de importância comprovada como um meio auxiliar de diagnóstico ao clínico de pequenos animais domésticos, vem se tornando comum em animais selvagens não apenas para a clínica, mas para a avaliação do manejo e como estudo auxiliar para a fisiologia das várias espécies. Tendo em vista o aumento da demanda para a produção de várias drogas de importância farmacêutica, a criação de serpentes peçonhentas vem se tornando comum a ponto destes animais já serem reconhecidos como sendo de produção. O conhecimento do manejo e da clínica destes animais ainda é escasso e a mortalidade é elevada nos criatórios, tornando urgente a ampliação destes. Embora alguns estudos hematológicos já tenham sido realizados em cascavéis (Crotalus durissus) os dados analisados ainda são insipientes, notadamente em relação à caracterização das células do sangue e poucos estudos em microscopia eletrônica foram realizados em serpentes. Com o objetivo de caracterizar as células sanguíneas morfologicamente, sob microscopia óptica e ultraestrutural, foram coletadas amostras de sangue de 52 de indivíduos da subespécie Crotalus durissus terrificus para a realização de esfregaços sanguíneos e avaliação ultraestrutural. Concluiu-se que a coloração hematológica de Giemsa permite a avaliação morfológica e a diferenciação das células sanguíneas em serpentes assim como a visualização de hemoparasitos. A avaliação ultraestrutural permite evidenciar as organelas celulares e a diferenciação entre as células, inclusive entre os tipos leucocitários, porém ainda são necessários outros estudos para que seja elucidada a hipótese da existência dos eosinófilos na espécie estudada assim como é necessária melhor caracterização dos grânulos dos azurófilos para que se confirme uma possível diferença entre os monócitos típicos e os azurófilos.

Polydeoxyribonucleotide, an Adenosine-A2A Receptor Agonist, Preserves Blood Testis Barrier from Cadmium-Induced Injury.

Cadmium (Cd) impairs blood-testis barrier (BTB). Polydeoxyribonucleotide (PDRN), an adenosine A2A agonist, has positive effects on male reproductive system. We investigated the effects of PDRN on the morphological and functional changes induced by Cd in mice testes. Adult Swiss mice were divided into four groups: controls administered with 0.9% NaCl (1 ml/kg, i.p., daily) or with PDRN (8 mg/kg, i.p. daily), animals challenged with Cd chloride (CdCl2; 2 mg/kg, i.p, daily) and animals challenged with CdCl2 (2 mg/kg, i.p., daily) and treated with PDRN (8 mg/kg, i.p., daily). Experiments lasted 14 days. Testes were processed for biochemical, structural, and ultrastructural evaluation and hormones were assayed in serum. CdCl2 increased pERK 1/2 expression and Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) levels; it decreased testosterone (TE) and inhibin-B levels and induced structural damages in extratubular compartment and in seminiferous epithelium, with ultrastructural features of BTB disruption. Many TUNEL-positive germ cells were present. CdCl2 increased tubular TGF-β3 immunoreactivity and reduced claudin-11, occludin, and N-cadherin immunoreactivity. PDRN administration reduced pERK 1/2 expression, FSH, and LH levels; it increased TE and inhibin-B levels, ameliorated germinal epithelium changes and protected BTB ultrastructure. Few TUNEL-positive germ cells were present and the extratubular compartment was preserved. Furthermore, PDRN decreased TGF-β3 immunoreactivity and enhanced claudin-11, occludin, and N-cadherin immunoreactivity. We demonstrate a protective effect of PDRN on Cd-induced damages of BTB and suggest that PDRN may play an important role against Cd, particularly against its harmful effects on gametogenesis.

Chronic exposure to ethanol causes steatosis and inflammation in zebrafish liver

AIMTo evaluate the effects of chronic exposure to ethanol in the liver and the expression of inflammatory genes in zebrafish.METHODSZebrafish (n = 104), wild type, adult, male and female, were divided into two groups: Control and ethanol (0.05 v/v). The ethanol was directly added into water; tanks water were changed every two days and the ethanol replaced. The animals were fed twice a day with fish food until satiety. After two and four weeks of trial, livers were dissected, histological analysis (hematoxilin-eosin and Oil Red staining) and gene expression assessment of adiponectin, adiponectin receptor 2 (adipor2), sirtuin-1 (sirt-1), tumor necrosis factor-alpha (tnf-a), interleukin-1b (il-1b) and interleukin-10 (il-10) were performed. Ultrastructural evaluations were conducted at fourth week.RESULTSExposing zebrafish to 0.5% ethanol developed intense liver steatosis after four weeks, as demonstrated by oil red staining. In ethanol-treated animals, the main ultrastructural changes were related to cytoplasmic lipid particles and droplets, increased number of rough endoplasmic reticulum cisterns and glycogen particles. Between two and four weeks, hepatic mRNA expression of il-1b, sirt-1 and adipor2 were upregulated, indicating that ethanol triggered signaling molecules which are key elements in both hepatic inflammatory and protective responses. Adiponectin was not detected in the liver of animals exposed and not exposed to ethanol, and il-10 did not show significant difference.CONCLUSIONData suggest that inflammatory signaling and ultrastructural alterations play a significant role during hepatic steatosis in zebrafish chronically exposed to ethanol.

Toxoplasma gondii plaque assays revisited: Improvements for ultrastructural and quantitative evaluation of lytic parasite growth

Lytic growth of intracellular Toxoplasma gondii tachyzoite stages over a period of days results in plaques within mononolayers of host cells. Plaque assays are in frequent use to isolate single clones and to investigate invasion, replication and egress over a longer time frame. To allow correlating plaque morphology and/or size with ultrastructural examination of individual parasites we introduce a simple protocol for correlative light and electron microscopy (CLEM) of entire plaques. We also illustrate the advantages of visualizing only the boundaries of plaques by staining for infected cells (‘positive staining’) rather than the traditional staining of the intact cell monolayer, thus outlining the area of lysed cells (‘negative staining’). Tachyzoites expressing β-galactosidase of Escherichia coli are an easy to visualize histochemical marker for this purpose. Quantitative measurements of plaque area with our compiled user-friendly ImageJ macros are compared to commercial software for ease and shown to be more accurate for some applications. Finally, a chemically defined medium is shown to be superior to the fetal bovine serum-containing medium for plaque assays, resulting in larger plaques. The reported additions and changes of the plaque assay procedure offer improved ways to analyze subtle differences in invasion, pathogen growth and egress. Our chemically defined medium will improve standardization of e.g. drug screening assays.

A combination of lactic acid bacteria regulates Escherichia coli infection and inflammation of the bovine endometrium

Uterine function in cattle is compromised by bacterial contamination and inflammation after calving. The objective of this study was to select a combination of lactic acid bacteria (LAB) to decrease endometrium inflammation and Escherichia coli infection. Primary endometrial epithelial cells were cultured in vitro to select the most favorable LAB combination modulating basal tissue inflammation and E. coli infection. Supernatants were obtained to determine expression of pro-inflammatory cytokines, and E. coli infection was evaluated after harvesting the tissue and plate counting. The selected LAB combination was tested in uterus explants to assess its capacity to modulate basal and acute inflammation (associated with E. coli infection). The combination of Lactobacillus rhamnosus, Pediococcus acidilactici, and Lactobacillus reuteri at a ratio of 25:25:2, respectively, reduced E. coli infection in vitro with (89.77%) or without basal tissue inflammation (95.10%) compared with single LAB strains. Lactic acid bacteria treatment reduced CXCL8 and IL1B expression 4.7- and 2.2-fold, respectively, under acute inflammation. Ex vivo, the tested LAB combination reduced acute inflammation under E. coli infection, decreasing IL-8, IL-1β, and IL-6 up to 2.2-, 2.5-, and 2.2-fold, respectively. In the total inflammation model, the LAB combination decreased IL-8 1.6-fold and IL-6 1.2-fold. Ultrastructural evaluation of the tissue suggested no direct interaction between the LAB and E. coli, although pathological effects of E. coli in endometrial cells were greatly diminished or even reversed by the LAB combination. This study shows the promising potential of LAB probiotics for therapeutic use against endometrial inflammation and infection.

Effect of long-term selection for egg production on eggshell quality of Japanese quail (Coturnix japonica)

An experiment was conducted to evaluate egg quality and ultrastuctural measurements of eggshell using a Scanning Electron Microscope (SEM) in 2 lines (selected and control) of Japanese quail. A selection program was applied over 22 consecutive generations for higher egg production and lower broken egg percentage. The results revealed that the females of the selected line produced significantly (P < 0.01) higher egg mass compared to that of the control line. Also, the selection procedure significantly improved feed conversion ratio. The eggshells of the selected line had a higher breaking strength compared to those of the control line, although there was no difference between them in shell thickness. Significantly higher wet (P < 0.01) and dry (P < 0.05) eggshell percentages were found in the selected line. In general, the eggshells of the selected line had a lower total score (good) of ultrastructural evaluation compared to the control line. According to scanning electron microscope data, the incidence of certain structural variants was more common in eggshells of the control line suggesting poor shell strength. The incidence of alignment was more prevalent in control eggshells compared to selected ones, suggesting lower resistance to breakage. Late fusion and large interstitial spaces of the palisade layer indicating decreased resistance to fracture were observed in control eggshells. It could be concluded that the improvement eggshell quality may be caused by the long-term selection for lower cracked and broken egg rates from generation to generation.

Immunomodulatory effect of gelatin-coated silver nanoparticles in mice: Ultrastructural evaluation

ABSTRACTSilver nanoparticles (SNP) are used in many pharmaceutical, cosmetic, and industrial products already available in the market. Although they are considered relatively safe, many toxic and pathological alterations in different organs including immune organs were reported after SNP administration. In this study, 10-week-old male mice (n = 20) were divided into two groups. Ten mice received greenly synthesized gelatin-coated silver nanoparticles in a dose of 10 mg/kg body weight for five consecutive days while the other 10 received 0.5 ml of distilled water daily for 5 days and kept as control. At the sixth day, all mice were sacrificed; blood and tissue samples were collected and prepared for pathological analysis. Liver and kidney lesions were in the form of degenerative and inflammatory changes. Interestingly, the immune organs were drastically affected by SNP treatment. Severe hyperplasia of the Peyer’s patches was noticed in the intestines of intoxicated animals both in gross and microscopic examination. Spleen was enlarged and showed large number of megakaryocytes. The particles were encountered in membrane-bound phagosomes inside macrophages in different organs like lungs and spleen. Blood picture complied to morphological findings with an increase in monocytes and eosinophils accompanied by drop in the platelets count in the intoxicated animals.

The Application of Paraphenylenediamine Staining for Assessment of Phospholipidosis.

Drug-induced phospholipidosis is characterized by intracellular accumulation of phospholipids with lamellar bodies in cells exposed to xenobiotics. Demonstration of the lamellar bodies by transmission electron microscopy (TEM) is the hallmark for a definitive diagnosis of phospholipidosis. However, the preparation of tissue samples for TEM and their ultrastructural evaluation are technically challenging and time consuming. Paraphenylenediamine (PPD) is essentially a fat stain, and the staining mechanism is based upon the osmication of unsaturated lipids. Thus, the application of PPD staining to osmicated tissue samples is considered an optimal way to identify lipids. We evaluated the potential of PPD staining to localize phospholipid accumulations on osmium-fixed semi-thin tissue sections of the lung, kidney, and liver, which were affected with phospholipidosis, under a light microscope. PPD staining revealed the presence of PPD positive dark fine granular material in the cytoplasm for all affected tissues examined, which correlated ultrastructurally with lamellar bodies as well as a light microscopic finding of cytoplasmic vacuolation. The great advantage of PPD is that it can be incorporated into the protocol for standard TEM tissue preparation and significantly improve the efficiency of TEM work. In conclusion, PPD provides a simple, sensitive, and reliable method for visualizing phospholipid accumulation on light microscopy and represents an easy tool to study drug-induced phospholipidosis.

Disseminated Histiocytic Sarcoma in an African Hedgehog (Atelerix albiventris)

Disseminated histiocytic sarcoma (HS) was diagnosed on post-mortem examination of a 1.5-year-old African hedgehog (Atelerix albiventris) that was presented in poor physical condition and with diarrhoea. Leucocytosis and a hypoechoic abdominal mass were noted on haematological and ultrasonographical examinations. Gross pathological, histopathological, immunohistochemical and ultrastructural evaluation of the mass supported a diagnosis of disseminated HS. To our knowledge, this report represents the first documentation of disseminated HS in this species.

Influence of blood-brain barrier permeability on O-(2-18F-fluoroethyl)-L-tyrosine uptake in rat gliomas.

O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) is an established tracer for the diagnosis of brain tumors with PET. This study investigates the influence of blood-brain barrier (BBB) permeability on 18F-FET uptake in two rat glioma models and one human xenograft model. F98 glioma, 9L gliosarcoma or human U87 glioblastoma cells were implanted into the striatum of 56 Fischer or RNU rats. Thereafter, animals were divided into a control group and a group receiving injections of the glucocorticoid dexamethasone (Dex). After 12-13 days of tumor growth animals received injection of Evans blue dye (EBD) to visualize BBB disturbance and underwent 18F-FET PET followed by autoradiography. Time activity curves, standardized uptake values (SUV) and Tumor-to-brain ratios (TBR) of 18F-FET uptake [18-61min post injection (p.i.)] were evaluated using a volume-of-Interest (VOI) analysis. BBB disturbance was quantitatively evaluated by EBD fluorescence. The membrane gaps of blood vessel endothelial tight junctions were measured using electron microscopy to visualize ultrastructural BBB alterations in one untreated and one Dex treated F98 glioma. Data were analyzed by two-way ANOVAs. In Dex treated animals EBD extravasation was significantly reduced in 9L (P < 0.001) and U87 (P = 0.008) models and showed a trend in F98 models (P = 0.053). In contrast, no significant differences of 18F-FET uptake were observed between Dex treated animals and control group except a decrease of the TBR in the 9L tumor model in PET (P < 0.01). Ultrastructural evaluation of tumor blood vessel endothelia revealed significant reduction of the cleft diameter between endothelial cells after Dex treatment in F98 model (P = 0.010). Despite a considerable reduction of BBB permeability in rat gliomas after Dex treatment, no relevant changes of 18F-FET uptake were noted in this experimental study. Thus, 18F-FET uptake in gliomas appears to be widely independent of the permeability of the BBB.