Historical review: The golden age of the Golgi method in human neuropathology.

Golgi methods were used to study human neuropathology in the 1970s, 1980s, and 1990s of the last century. Although a relatively small number of laboratories applied these methods, their impact was crucial by increasing knowledge about: (1) the morphology, orientation, and localization of neurons in human cerebral and cerebellar malformations and ganglionic tumors, and (2) the presence of abnormal structures including large and thin spines (spine dysgenesis) in several disorders linked to mental retardation, focal enlargements of the axon hillock and dendrites (meganeurites) in neuronal storage diseases, growth cone-like appendages in Alzheimer disease, as well as abnormal structures in other dementias. Although there were initial concerns about their reliability, reduced dendritic branches and dendritic spines were identified as common alterations in mental retardation, dementia, and other pathological conditions. Similar observations in appropriate experimental models have supported many abnormalities that were first identified using Golgi methods in human material. Moreover, electron microscopy, immunohistochemistry, fluorescent tracers, and combined methods have proven the accuracy of pioneering observations uniquely visualized as 3D images of fully stained individual neurons. Although Golgi methods had their golden age many years ago, these methods may still be useful complementary tools in human neuropathology.

A CNN-based approach for joint segmentation and quantification of nuclei and NORs in AgNOR-stained images.

BACKGROUND AND OBJECTIVE: Oral cancer is the sixth most common kind of human cancer. Brush cytology for counting Argyrophilic Nucleolar Organizer Regions (AgNORs) can help early mouth cancer detection, lowering patient mortality. However, the manual counting of AgNORs still in use today is time-consuming, labor-intensive, and error-prone. The goal of our work is to address these shortcomings by proposing a convolutional neural network (CNN) based method to automatically segment individual nuclei and AgNORs in microscope slide images and count the number of AgNORs within each nucleus. METHODS: We systematically defined, trained and tested 102 CNNs in the search for a high-performing solution. This included the evaluation of 51 network architectures combining 17 encoders with 3 decoders and 2 loss functions. These CNNs were trained and evaluated on a new AgNOR-stained image dataset of epithelial cells from oral mucosa containing 1,171 images from 48 patients, with ground truth annotated by specialists. The annotations were greatly facilitated by a semi-automatic procedure developed in our project. Overlapping nuclei, which tend to hide AgNORs, thus affecting their true count, were discarded using an automatic solution also developed in our project. Besides the evaluation on the test dataset, the robustness of the best performing model was evaluated against the results produced by a group of human experts on a second dataset. RESULTS: The best performing CNN model on the test dataset consisted of a DenseNet-169 + LinkNet with Focal Loss (DenseNet-169 as encoder and LinkNet as decoder). It obtained a Dice score of 0.90 and intersection over union (IoU) of 0.84. The counting of nuclei and AgNORs achieved precision and recall of 0.94 and 0.90 for nuclei, and 0.82 and 0.74 for AgNORs, respectively. Our solution achieved a performance similar to human experts on a set of 291 images from 6 new patients, obtaining Intraclass Correlation Coefficient (ICC) of 0.91 for nuclei and 0.81 for AgNORs with 95% confidence intervals of [0.89, 0.93] and [0.77, 0.84], respectively, and p-values < 0.001, confirming its statistical significance. Our AgNOR-stained image dataset is the most diverse publicly available AgNOR-stained image dataset in terms of number of patients and the first for oral cells. CONCLUSIONS: CNN-based joint segmentation and quantification of nuclei and NORs in AgNOR-stained images achieves expert-like performance levels, while being orders of magnitude faster than the later. Our solution demonstrated this by showing strong agreement with the results produced by a group of specialists, highlighting its potential to accelerate diagnostic workflows. Our trained model, code, and dataset are available and can stimulate new research in early oral cancer detection.

Could Argyrophilic Nucleolar Organizer Regions count mirror DNA Ploidy in Malignant Salivary Gland Tumors?

OBJECTIVE: Nucleolar organizer regions (NORs) are DNA coils that transcribe to ribosomal RNA. The NOR-associated protein, termed argyrophilic NOR (AgNOR), was visible within the nucleus by staining with silver nitrate examination via the light microscope. AgNOR counting is a proliferation marker and may help in the diagnosis and prognosis of various neoplastic lesions. Aneuploidy (abnormal DNA content) can predict the progression, survival and prognosis of the tumors. The aim of this study was to evaluate the role of AgNORs, DNA ploidy status, and total S-phase fraction (TSPF) as prognostic parameters in malignant salivary gland tumors (MSGTs). METHODS: The current study is a retrospective study on a cohort of MSGTs (N=47), to assess AgNORs using Silver Nitrate stain, DNA index (DI), and TSPF using flow cytometry (FCM). Data including tumor size and site, lymphovascular invasion (LVI), lymph node metastasis (LNM) were collected. RESULTS: The AgNORs count was statistically significant with MSGT type. DI was found to have a significant association with tumor site, tumor size and MSGT type. In addition, TSPF was found to be significantly associated with LVI. A moderate positive correlation was noted between AgNORs count and TSPF. LNM, tumor site, high AgNORs and low DI were all associated with short disease-free survival (DFS) and poor overall survival (OS). CONCLUSION: The present study revealed that high AgNORs count, DNA aneuploidy and TSPF had a poor influence on MSGTs prognosis.

Adapted technique for demonstrating argyrophilic nucleolar organizer regions in the cytologic samples of canine mast cell tumors.

INTRODUCTION: Argyrophilic nucleolar organizer regions (AgNORs) are nonhistone argyrophilic nucleolar proteins associated with ribosomal genes found in the nucleolar organizer region that reflect cell proliferation and have an affinity for silver. AgNOR staining may be useful to evaluate prognosis in several neoplasms because higher AgNOR counts are related to higher grade tumors, metastases, and shorter survival times. OBJECTIVE: We aimed to report on a quick and practical technique to identify AgNORs adapted for use in routine cytology. MATERIALS AND METHODS: The cytopathologic diagnosis of mast cell tumor (MCT) in samples collected by fine-needle aspiration (FNA) was determined. Next, slides were impregnated with a solution containing silver nitrate; the main modification of our technique included incubation of these slides at a controlled temperature of 25 °C. Some slides were previously stained with Diff-Quik and others were only fixed with methanol. The slides were analyzed under a microscope, and the number of blackened intranuclear points (AgNORs) was counted. RESULTS: Slides prestained with Diff-Quik were easily counted compared with slides only fixed in methanol. Technical issues encountered with the methanol-fixed slides included insufficient cellularity, background precipitation, and an absence of silver impregnation. CONCLUSIONS: The technique reported in this study showed satisfactory results for AgNOR counting in cytologic smears from MCT, such as good impregnation and the elimination of background interferents. Further evaluation of this method comparing AgNOR counts with histologic examinations, tumor grades, other prognostic markers, and survival times are needed to fully evaluate the benefit of this technique.

Comparative Study of Modified Silver Nitrate Staining for the Detection of Helicobacter pylori.

BACKGROUND/AIMS: Helicobacter pylori (Hp) infection is associated with a variety of diseases, such as benign lesions, precancerous lesions, and malignant lesions, especially diseases in the digestive system. Most people with Hp infection have mild early symptoms that are not easily noticed. Therefore, the diagnosis and treatment of Hp infection is particularly important. At present, there are many methods to detection Hp infection, but there is a lack of effective detection method with high sensitivity and specificity. On the basis of the existing detection methods, the modified silver nitrate staining method in this study improved the sensitivity and specificity of Hp detection. MATERIALS AND METHODS: We selected gastric antrum and gastric angle mucosal biopsy tissues from 60 inpatients that were archived in the Pathology Department of Zhongnan Hospital of Wuhan University from July to December 2020. An Hp immunohistochemical assay, histochemical assay kit (methylene blue), and modified silver nitrate staining were used to measure the Hp infection positivity rate. RESULTS: Comparison of Hp sensitivity and specificity among the 3 methods showed that the modified silver nitrate staining method was the most excellent. The sensitivity of modified silver nitrate staining method was 98.3%, which is statistically significantly higher compared with the other 2 methods. CONCLUSION: The modified silver nitrate staining method for Hp detection is convenient and effective, and could be widely used for clinical Hp detection.

Cytological Screening Model of Normal Oral Mucosa Exposed to Carcinogens: A Pilot Study.

INTRODUCTION: Oral cytopathology is able to detect incipient cellular alterations, but it is not routinely applied to this purpose. We aimed to establish a model to screen individuals with no oral lesion exposed to smoking/alcohol, by means of the nuclear area, cell proliferation rate, and analysis of genetic damage. METHODS: In this cross-sectional pilot study, 90 patients were allocated into 3 groups: oral cancer group (patients with oral squamous cell carcinoma), tobacco/alcohol group (patients without oral lesions and exposed to these risk factors), and control group (individuals with no lesion and not exposed to tobacco and alcohol). The cytological smears performed in these individuals were stained with Papanicolaou, a silver-staining and a Feulgen reaction. The nuclei of cells were measured, and AgNORs/nucleus and micronuclei (MN) were quantified. The cutoff values were stipulated evaluating the healthy mucosa (control group) and the cancerization field mucosa (oral cancer group). RESULTS: Cutoff values for the screening of individuals exposed to carcinogens were ≥8% of nuclei larger than 100 µm2, ≥3.38 AgNOR/nucleus, and ≥3 MN per 1,000 cells. CONCLUSIONS: Nuclear area measurement and AgNORs/nucleus and MN quantification identified the incipient phase of oral carcinogenesis. A screening model for individuals without oral lesion exposed to smoking/alcohol was proposed.

Whole-organism 3D quantitative characterization of zebrafish melanin by silver deposition micro-CT.

We previously described X-ray histotomography, a high-resolution, non-destructive form of X-ray microtomography (micro-CT) imaging customized for three-dimensional (3D), digital histology, allowing quantitative, volumetric tissue and organismal phenotyping (Ding et al., 2019). Here, we have combined micro-CT with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. The resulting images enabled whole-body, computational analyses of regional melanin content and morphology. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and further allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, including subtle phenotypes not previously noticed. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, 3D computational phenomic analysis of a specific cell type at cellular resolution, with potential applications in other model organisms and melanocytic neoplasms. Advances such as this in whole-organism, high-resolution phenotyping provide superior context for studying the phenotypic effects of genetic, disease, and environmental variables.

Oral leukoplakia-epidemiological survey and histochemical analysis of 107 cases in Brazil.

OBJECTIVE: To perform an epidemiological survey comparing the cell proliferative activity of 107 cases of oral leukoplakia with their clinical and histopathological characteristics. MATERIALS AND METHODS: A cross-sectional, observational, and histological-histochemical study. The cases came from the Histopathological Diagnostic Service of UPF/RS and the School of Dentistry of Araçatuba FOA/UNESP/SP (1986-2016). The histopathological sections were stained using the silver staining (AgNOR) technique and the nuclei of 100 epithelial cells selected randomly were recorded to count the number of nucleolar organizer regions (NORs). The mean NORs per lesion were correlated to clinical and histological characteristics using ANOVA, at 5% significance. RESULTS: Most of the cases included men (62.62%), white (64.49%), and with an average age of 53.94 years. The most probable etiological factors were smoking (44.7%) and alcohol consumption (9.85%). The evolution time of most lesions was fast (33.65%), manifesting mainly in the form of plaques (70.37%) and without symptoms (58.88%). They were located mainly in the cheek mucosa (26.62%) and presented white color (66.35%), well-defined edges (59.81%), firm consistency (47.5%), and keratinized surface (49.53%). Etiological factor (p = 0.003), evolution time (p = 0.006), symptoms (p = 0.029), location (p = 0.020), consistency (p = 0.047), histopathological characteristics (p = 0.004), and superficial keratinization (p = 0.001) were statistically significant regarding the mean NORs of the leukoplakias studied. CLINICAL RELEVANCE: Oral leukoplakias caused by alcohol consumption and/or tobacco use, considering an evolution time of fewer than 12 months, asymptomatic, located in the lower lip or tongue, and with a firm consistency and increased superficial keratinization should be treated more aggressively by the clinician to avoid cancerization.

Proteome Analysis of Human Natural Killer Cell Derived Extracellular Vesicles for Identification of Anticancer Effectors.

Cancer immunotherapy is a clinically validated therapeutic modality for cancer and has been rapidly advancing in recent years. Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a viable method of controlling the immune system against cancer. Recent evidence indicates that even immune-cell-released vesicles such as NK-cell-derived exosomes also exert anticancer effect. Nevertheless, the underlying mechanisms remain elusive. In the present study, the anticancer potential of isolated extracellular vesicles (EVs) from expanded and activated NK-cell-enriched lymphocytes (NKLs) prepared by house-developed protocol was evaluated both in vitro and in vivo. Moreover, isolated EVs were characterized by using two-dimensional electrophoresis (2-DE)-based proteome and network analysis, and functional study using identified factors was performed. Our data indicated that the EVs from expanded and active NKLs had anticancer properties, and a number of molecules, such as Fas ligand, TRAIL, NKG2D, ß-actin, and fibrinogen, were identified as effector candidates based on the proteome analysis and functional study. The results of the present study suggest the possibility of NK-cell-derived EVs as a viable immunotherapeutic strategy for cancer.

Analysis of Recombinant Adeno-Associated Virus (rAAV) Purity Using Silver-Stained SDS-PAGE.

AAV virions are built from three major capsid proteins, VP1, VP2, and VP3, at a ratio of 1:1:18. On a silver-stained SDS-polyacrylamide gel, VP1, VP2, and VP3 should be the only visible bands in a highly purified recombinant adeno-associated virus (rAAV) preparation, migrating at approximately 87, 73, and 62 kDa, respectively. This protocol describes how SDS-PAGE and silver staining can be used to determine the purity of an rAAV preparation. In addition, using a highly purified rAAV preparation whose particle titer is known, this assay can be used to derive a semiquantitative estimate of the particle concentration of a test vector.

Argyrophilic nucleolar organizer regions staining for cytology smears in dogs and cats.

The argyrophilic nucleolar organizer regions (AgNORs) are cellular proliferation markers, crucial for predicting the clinical course and aggressiveness of tumors. The purpose of this study was to establish an easy and practical AgNOR staining method in the cytology of dogs and cats. Air-dried cytological slides were prepared from dogs (n=14) and cats (n=12). Acetone, formalin, ethanol and methanol were tested as fixatives for AgNOR staining. Subsequently, various methods of Romanowsky-based counterstains were tested before and after AgNOR staining. Clear and strong AgNOR spots were observed with all fixatives, and post-May-Grünwald staining was the best counterstaining method. The established method showed clear AgNOR spots even in the long-term storage samples and Romanowsky-stained ones.

Postnatal development of metallophilic macrophages in the rat thymus.

Metallophilic macrophages (MMs) are a distinct cell type of the rodent thymus. Our previous research has focused on the morphological characteristics of MMs, as well as on the molecular mechanisms involved in the development and tissue positioning of these cells. However, the postnatal development of MMs has not been sufficiently studied. In the present study, we investigated the positioning of MMs in the rat thymus between postnatal day 0 (P0) and P30. On P0, MMs were evenly distributed all over the thymic tissue-that is, the cortex, cortico-medullary zone and medulla. From P0 to P15, the number of MMs in the thymic cortex significantly decreased, and after P15, this number did not change. Thus, the present study shows that on P15, MMs almost completely disappear from the thymic cortex and show their adult position in the cortico-medullary zone and in the medulla.

Combined Application of Immunohistochemistry and Warthin-Starry Silver Stain on the Pathologic Diagnosis of Cat Scratch Disease.

PURPOSE: Cat scratch disease (CSD) is an infectious disorder caused primarily by the bacterium Bartonella henselae (B. henselae). Immunohistochemistry (IHC) and Warthin-Starry silver stain (WS) are considered to be indispensable to diagnose CSD in combination with morphologic characteristics. In this study, we retrieved and reviewed 46 cases of paraffin-embedded lymphadenitis with histologic and/or clinical suspicion of CSD between 2014 and 2018, and detected B. henselae by IHC and WS, respectively, and evaluated the application significance of IHC and WS for the detection of B. henselae and validated their values in the pathologic diagnosis of CSD. MATERIALS AND METHODS: B. henselae was detected by IHC and WS; validation of 2 methods for detecting B. henselae was evaluated by sensitivity, specificity, false-positive rate, false-negative rate, precision, negative predictive value, and agreement rate. RESULTS: Microscopically, suppurative granulomas and/or multiple stellate microabscesses were observed in the accessory cortex of lymph nodes, especially near the subcapsule. Our results showed that 80.4% (37/46) of cases were positive for B. henselae by IHC, manifesting mainly punctuate, granular, or linear to outline the shape of bacteria. However, the positive rate of B. henselae by the WS method was 52.2% (24/46). There was a significant difference between IHC and WS (P=0.023). Moreover, a positive percentage of B. henselae was 97.8% (45/46), which was detected by the combined application of IHC and WS. The combination of IHC and WS exhibited high sensitivity (97.8%) and good agreement rate (86.5%). CONCLUSION: The combined application of the IHC and WS method may have important clinical advantages, which is with the highest sensitivity and agreement rate for pathologic diagnosis of CSD.

AgNOR Pleomorphic Count as a Tumor Marker in Cervical Carcinogenesis and Feasibility of Its Introduction in Cervical Cancer Screening Programs to Discriminate High-Risk Cases of Squamous Intraepithelial Lesions of the Cervix.

BACKGROUND: AgNOR pleomorphism has been widely used for its diagnostic importance in differentiating premalignant and malignant lesions of different human neoplasms. However, an evaluation of its potential for discriminating cases of high-risk squamous intraepithelial lesions of the cervix (SIL) has been rarely attempted. AIM: The tumor marker potential of AgNOR pleomorphism counts was assessed by correlating high and low mean counts in low-grade SIL (LSIL) cases with persistence or regression of the lesion and HPV positivity. MATERIALS AND METHODS: The 115 LSIL cases selected for the study were registered from the ongoing cervical cancer screening of the rural population of Lucknow West. Silver nitrate staining for AgNOR counts and HPV DNA testing were done in all 115 cases. RESULTS: The AgNOR counts in the 115 LSIL cases revealed low counts in 92 and high counts in 23 cases. Follow-up, available in 107 cases, revealed persistence of the lesion in 21 of the 23 cases with high counts and in 4 of the 84 cases with low counts. HPV positivity showed a strong correlation with high counts. Persistence of LSIL was also more frequent with high AgNOR counts and in HPV-positive cases. CONCLUSIONS: The study showed a correlation of high mean AgNOR counts with HPV positivity and persistence of LSIL.

Quantitative analysis of AgNOR counts and pap stain in exfoliative cytology specimens of oral mucosa in bidi smokers and nonsmokers.

Background: Bidi smoking is a serious health hazard which is common throughout South Asia and parts of the Middle East. It has been strongly implicated to various benign and malignant lesions of oral cavity and oropharynx. These tobacco-filled leaves deliver more than three times the amount of nicotine, carbon monoxide, and tar as cigarettes which exert injurious effects on cells reflected in terms of accelerated proliferative activity in normal oral mucosal cells. Aim: This study aimed to compare the exfoliated cells from the oral mucosa of bidi smokers and nonsmokers, with emphasis on proliferative activity. Materials and Methods: Exfoliative smears were obtained from the oral mucosa of forty participants (twenty nonsmokers and twenty smokers) with age group ranging from 30-80 years, in and around Barwala (Haryana). The cytologic smears were evaluated using Papanicolaou (PAP) stain and AgNOR in order to evaluate the presence of cytological alterations suggestive of inflammation, dysplasia, keratinization, and proliferative activity of epithelial cells. Only PAP Class I and Class II smears were observed. Results: Comparison of the mean number of AgNORs showed a significant difference between nonsmokers and smokers. Inflammatory alterations were found in 70% of smokers and 55% of nonsmokers. A significant difference in proliferative activity was observed between smokers and nonsmokers classified as PAP Class II. Conclusion: A significant difference of AgNORs/nucleus was observed between bidi smokers and nonsmokers.

RésuméContexte: Bidi fumeurs est un grave danger pour la santé qui est commune dans toute l'Asie du Sud et certaines parties du Moyen-Orient. Il a été fortement impliqué dans diverses lésions bénignes et malignes de la cavité buccale et l'oropharynx. Ces feuilles de tabac offrent plus de trois fois la quantité de nicotine, monoxyde de carbone et de goudron que les cigarettes qui exercent des effets préjudiciables sur les cellules reflétés sous la forme d' une accélération de l'activité proliférative des cellules de la muqueuse buccale normale. Objectif: Cette étude visait à comparer les cellules exfoliées de la muqueuse orale des bidis fumeurs et non fumeurs, avec l'accent sur l'activité proliférative. Matériels et méthodes: frottis Exfoliative ont été obtenus à partir de la muqueuse orale de 40 participants (20 non-fumeurs et fumeurs) avec 20 Groupe d'âge allant de 30-80 ans, dans et autour de Barwala (Haryana). Le frottis cytologique ont été évalués à l'aide de la coloration de Papanicolaou (PAP) et d'AgNOR afin d'évaluer la présence d' altérations cytologiques évocateurs d'infl ammation, dysplasie, la kératinisation, et l'activité proliférative des cellules épithéliales. PAP uniquement les catégories I et II de Papanicolaou n'a été observé. Résultats: comparaison du nombre moyen d'AgNORs ont montré une différence entre les non-fumeurs et les fumeurs. Des modifications ont été trouvés infl ammatory dans 70% des fumeurs et 55% des non-fumeurs. Une différence dans l'activité proliférative a été observée entre les fumeurs et les non-fumeurs PAP, le niveau d'emploi de la classe II. Conclusion: une différence de AgNORs/noyau a été observée entre fumeurs et non-fumeurs bidi. Mots-clés: AgNOR, frottis exfoliative, les fumeurs.

Role of FNA and Special Stains in Rapid Cytopathological Diagnosis of Pulmonary Nocardiosis.

BACKGROUND: Nocardia, a gram-positive aerobic bacillus of the Actinomycetales family, is a significant opportunistic pathogen in immunocompromised individuals. Clinical and radiological features of pulmonary nocardiosis are nonspecific and can be misdiagnosed as tuberculosis, pneumocystis, staphylococcal or fungal infections, or as malignancy. Aspiration cytology with special stains is a quick and effective approach for accurate diagnosis. MATERIALS AND METHODS: We present 7 cases of pulmonary nocardiosis, admitted to the pathology department in a tertiary-care hospital in Punjab. Clinical findings, immune status, laboratory tests, chest radiographs, and computed tomography scans were reviewed. Cytologically, special stains like 1% Ziehl-Neelsen (ZN), 20% ZN, periodic acid-Schiff (PAS), Grocott methenamine silver (GMS), and reticulin stains were studied along with May-Grünwald Giemsa, Papanicolaou, and hematoxylin and eosin. RESULTS: All the patients were immunocompromised. The radiological changes were nonspecific. Cytomorphology showed acute and chronic inflammatory infiltrates with necrosis. None of the cases showed well-defined granulomas. GMS, modified 1% ZN and, Gordon and Sweet reticulin stains highlighted the delicate filamentous bacteria in all cases. PAS and 20% ZN stain for tuberculous bacilli were uniformly negative. CONCLUSION: FNAC can provide a quick and accurate diagnosis of nocardiosis and thereby facilitate timely medical management.

Detection of Borrelia in Ixodes scapularis ticks by silver stain, immunohistochemical and direct immunofluorescent methods.

BACKGROUND: Lyme disease (LD) is one of the most common tick-borne diseases caused by several Borrelia species of spirochetes. Ixodes scapularis is responsible for the transmission of LD in the northeastern United States. The rate of infection varies with the duration of tick attachment to the host; however, this information may be unknown. In skin biopsies, it is often difficult to identify spirochetes. Testing of ticks is not routinely performed. Treatment is established by clinical presentation. OBJECTIVE: To test paraffin-embedded I. scapularis ticks for Borrelia by different methods. MATERIALS/METHODS: We examined 20 paraffin-embedded ticks by silver stain, immunohistochemical (IHC) and direct immunofluorescent (DIF) methods and compared the percentage of positivity with DIF results from the Connecticut Agricultural Experiment Station. RESULTS: The results were similar by DIF, which proved to be the most sensitive method, followed by the silver stain and IHC. CONCLUSION: We found that the identification of spirochetes in paraffin-embedded ticks was less difficult than in tissue with a comparable turnaround time to that of a routine biopsy. Timely identification of Borrelia in ticks may influence the clinical management of the patients.

Stress-induced tRNA cleavage and tiRNA generation in rat neuronal PC12 cells.

Transfer RNA (tRNA) plays a role in stress response programs involved in various pathological conditions including neurological diseases. Under cell stress conditions, intracellular tRNA is cleaved by a specific ribonuclease, angiogenin, generating tRNA-derived fragments or tRNA-derived stress-induced RNA (tiRNA). Generated tiRNA contributes to the cell stress response and has potential cell protective effects. However, tiRNA generation under stress conditions in neuronal cells has not been fully elucidated. To examine angiogenin-mediated tiRNA generation in neuronal cells, we used the rat neuronal cell line, PC12, in combination with analysis of SYBR staining and immuno-northern blotting using anti-1-methyladenosine antibody, which specifically and sensitively detects tiRNA. Oxidative stress induced by arsenite and hydrogen peroxide caused tRNA cleavage and tiRNA generation in PC12 cells. We also demonstrated that oxygen-glucose deprivation, which is an in vitro model of ischemic-reperfusion injury, induced tRNA cleavage and tiRNA generation. In these stress conditions, the amount of generated tiRNA was associated with the degree of morphological cell damage. Time course analysis indicated that generation of tiRNA was prior to severe cell damage and cell death. Angiogenin over-expression did not influence the amount of tiRNA in normal culture conditions; however, it significantly increased tiRNA generation induced by cell stress conditions. Our findings show that angiogenin-mediated tiRNA generation can be induced in neuronal cells by different cell stressors, including ischemia-reperfusion. Additionally, detection of tiRNA could be used as a potential cell damage marker in neuronal cells. Cover Image for this issue: doi: 10.1111/jnc.14191.

CD38 is Required for Dendritic Organization in Visual Cortex and Hippocampus.

Morphological screening of mouse brains with known behavioral deficits can give great insight into the relationship between brain regions and their behavior. Oxytocin- and CD38-deficient mice have previously been shown to have behavioral phenotypes, such as restrictions in social memory, social interactions, and maternal behavior. CD38 is reported as an autism spectrum disorder (ASD) candidate gene and its behavioral phenotypes may be linked to ASD. To address whether these behavioral phenotypes relate to brain pathology and neuronal morphology, here we investigate the morphological changes in the CD38-deficient mice brains, with focus on the pathology and neuronal morphology of the cortex and hippocampus, using Nissl staining, immunohistochemistry, and Golgi staining. No difference was found in terms of cortical layer thickness. However, we found abnormalities in the number of neurons and neuronal morphology in the visual cortex and dentate gyrus (DG). In particular, there were arborisation differences between CD38-/- and CD38+/+ mice in the apical dendrites of the visual cortex and hippocampal CA1 pyramidal neurons. The data suggest that CD38 is implicated in appropriate development of brain regions important for social behavior.

Nuclear and membrane estrogen receptor antagonists induce similar mTORC2 activation-reversible changes in synaptic protein expression and actin polymerization in the mouse hippocampus.

AIMS: Estrogens play pivotal roles in hippocampal synaptic plasticity through nuclear receptors (nERs; including ERα and ERß) and the membrane receptor (mER; also called GPR30), but the underlying mechanism and the contributions of nERs and mER remain unclear. Mammalian target of rapamycin complex 2 (mTORC2) is involved in actin cytoskeleton polymerization and long-term memory, but whether mTORC2 is involved in the regulation of hippocampal synaptic plasticity by ERs is unclear. METHODS: We treated animals with nER antagonists (MPP/PHTPP) or the mER antagonist (G15) alone or in combination with A-443654, an activator of mTORC2. Then, we examined the changes in hippocampal SRC-1 expression, mTORC2 signaling (rictor and phospho-AKTSer473), actin polymerization (phospho-cofilin and profilin-1), synaptic protein expression (GluR1, PSD95, spinophilin, and synaptophysin), CA1 spine density, and synapse density. RESULTS: All of the examined parameters except synaptophysin expression were significantly decreased by MPP/PHTPP and G15 treatment. MPP/PHTPP and G15 induced a similar decrease in most parameters except p-cofilin, GluR1, and spinophilin expression. The ER antagonist-induced decreases in these parameters were significantly reversed by mTORC2 activation, except for the change in SRC-1, rictor, and synaptophysin expression. CONCLUSIONS: nERs and mER contribute similarly to the changes in proteins and structures associated with synaptic plasticity, and mTORC2 may be a novel target of hippocampal-dependent dementia such as Alzheimer's disease as proposed by previous studies.