Search

Purpose To describe characteristics of the vitreomacular interface (VMI) in traumatic macular holes (TMH) compared to idiopathic macular holes (IMH) using immunofluorescence and electron microscopy, and to correlate with clinical data. Methods For immunocytochemical and ultrastructural analyses, premacular tissue with internal limiting membrane (ILM) and epiretinal membrane (ERM) was harvested during vitrectomy from 5 eyes with TMH and 5 eyes with IMH. All specimens were processed as flat mounts for phase-contrast microscopy, interference and fluorescence microscopy, and transmission electron microscopy (TEM). Primary antibodies were used against microglial and macroglial cells. Clinical data was retrospectively evaluated. Results Surgically excised premacular tissue of eyes with TMH showed a less pronounced positive immunoreactivity for anti-glutamine synthetase, anti-vimentin and anti-IBA1 compared to eyes with IMH. Cell nuclei staining of the flat-mounted specimens as well as TEM presented a lower cell count in eyes with TMH compared to IMH. All detected cells were found on the vitreal side of the ILM. No collagen fibrils were seen in specimens of TMH. According to patients’ age, intraoperative data as well as spectral-domain optical coherence tomography (SD-OCT) analysis revealed an attached posterior vitreous in the majority of TMH cases (60%), whereas all eyes with IMH presented posterior vitreous detachment. Conclusion The vitreomacular interface in TMH and IMH shows significant differences. In TMH, glial cells are a rare finding on the vitreal side of the ILM.

The application of both fluorescence and electron microscopy results in a powerful combination of imaging modalities called "correlative light and electron microscopy" (CLEM). Whereas conventional transmission electron microscopy (TEM) tomography is only able to image sections up to a thickness of ~300nm, scanning transmission electron microscopy (STEM) tomography at 200kV allows the analysis of sections up to a thickness of 900nm in three dimensions. In the current study we have successfully integrated STEM tomography into CLEM as demonstrated for human retinal pigment epithelial 1 (RPE1) cells expressing various fluorescent fusion proteins which were high-pressure frozen and then embedded in Lowicryl HM20. Fluorescently labeled gold nanoparticles were applied onto resin sections and imaged by fluorescence and electron microscopy. STEM tomograms were recorded at regions of interest, and overlays were generated using the eC-CLEM software package. Through the nuclear staining of living cells, the use of fluorescently labeled gold fiducials for the generation of overlays, and the integration of STEM tomography we have markedly extended the application of the Kukulski protocol (Kukulski et al., 2011, 2012). Various fluorescently tagged proteins localizing to different cellular organelles could be assigned to their ultrastructural compartments. By combining STEM tomography with on-section CLEM, fluorescently tagged proteins can be localized in three-dimensional ultrastructural environments with a volume of at least 2.7×2.7×0.5μm.

To describe characteristics of the vitreomacular interface (VMI) in traumatic macular holes (TMH) compared to idiopathic macular holes (IMH) using immunofluorescence and electron microscopy, and to correlate with clinical data.For immunocytochemical and ultrastructural analyses, premacular tissue with internal limiting membrane (ILM) and epiretinal membrane (ERM) was harvested during vitrectomy from 5 eyes with TMH and 5 eyes with IMH. All specimens were processed as flat mounts for phase-contrast microscopy, interference and fluorescence microscopy, and transmission electron microscopy (TEM). Primary antibodies were used against microglial and macroglial cells. Clinical data was retrospectively evaluated.Surgically excised premacular tissue of eyes with TMH showed a less pronounced positive immunoreactivity for anti-glutamine synthetase, anti-vimentin and anti-IBA1 compared to eyes with IMH. Cell nuclei staining of the flat-mounted specimens as well as TEM presented a lower cell count in eyes with TMH compared to IMH. All detected cells were found on the vitreal side of the ILM. No collagen fibrils were seen in specimens of TMH. According to patients' age, intraoperative data as well as spectral-domain optical coherence tomography (SD-OCT) analysis revealed an attached posterior vitreous in the majority of TMH cases (60%), whereas all eyes with IMH presented posterior vitreous detachment.The vitreomacular interface in TMH and IMH shows significant differences. In TMH, glial cells are a rare finding on the vitreal side of the ILM.

The application of both fluorescence and electron microscopy results in a powerful combination of imaging modalities called "correlative light and electron microscopy" (CLEM). Whereas conventional transmission electron microscopy (TEM) tomography is only able to image sections up to a thickness of ~300nm, scanning transmission electron microscopy (STEM) tomography at 200kV allows the analysis of sections up to a thickness of 900nm in three dimensions. In the current study we have successfully integrated STEM tomography into CLEM as demonstrated for human retinal pigment epithelial 1 (RPE1) cells expressing various fluorescent fusion proteins which were high-pressure frozen and then embedded in Lowicryl HM20. Fluorescently labeled gold nanoparticles were applied onto resin sections and imaged by fluorescence and electron microscopy. STEM tomograms were recorded at regions of interest, and overlays were generated using the eC-CLEM software package. Through the nuclear staining of living cells, the use of fluorescently labeled gold fiducials for the generation of overlays, and the integration of STEM tomography we have markedly extended the application of the Kukulski protocol (Kukulski et al., 2011, 2012). Various fluorescently tagged proteins localizing to different cellular organelles could be assigned to their ultrastructural compartments. By combining STEM tomography with on-section CLEM, fluorescently tagged proteins can be localized in three-dimensional ultrastructural environments with a volume of at least 2.7×2.7×0.5μm.

Machine learning classifications of first-episode psychosis (FEP) using neuroimaging have predominantly analyzed brain volumes. Some studies examined cortical thickness, but most of them have used parcellation approaches with data from single sites, which limits claims of generalizability. To address these limitations, we conducted a large-scale, multi-site analysis of cortical thickness comparing parcellations and vertex-wise approaches. By leveraging the multi-site nature of the study, we further investigated how different demographical and site-dependent variables affected predictions. Finally, we assessed relationships between predictions and clinical variables. 428 subjects (147 females, mean age 27.14) with FEP and 448 (230 females, mean age 27.06) healthy controls were enrolled in 8 centers by the ClassiFEP group. All subjects underwent a structural MRI and were clinically assessed. Cortical thickness parcellation (68 areas) and full cortical maps (20,484 vertices) were extracted. Linear Support Vector Machine was used for classification within a repeated nested cross-validation framework. Vertex-wise thickness maps outperformed parcellation-based methods with a balanced accuracy of 66.2% and an Area Under the Curve of 72%. By stratifying our sample for MRI scanner, we increased generalizability across sites. Temporal brain areas resulted as the most influential in the classification. The predictive decision scores significantly correlated with age at onset, duration of treatment, and positive symptoms. In conclusion, although far from the threshold of clinical relevance, temporal cortical thickness proved to classify between FEP subjects and healthy individuals. The assessment of site-dependent variables permitted an increase in the across-site generalizability, thus attempting to address an important machine learning limitation.

Mammalian podocytes, the key determinants of the kidney's filtration barrier, differentiate from columnar epithelial cells and several key determinants of apical-basal polarity in the conventional epithelia have been shown to regulate podocyte morphogenesis and function. However, little is known about the role of Crumbs, a conserved polarity regulator in many epithelia, for slit-diaphragm formation and podocyte function. In this study, we used Drosophila nephrocytes as model system for mammalian podocytes and identified a conserved function of Crumbs proteins for cellular morphogenesis, nephrocyte diaphragm assembly/maintenance, and endocytosis. Nephrocyte-specific knock-down of Crumbs results in disturbed nephrocyte diaphragm assembly/maintenance and decreased endocytosis, which can be rescued by Drosophila Crumbs as well as human Crumbs2 and Crumbs3, which were both expressed in human podocytes. In contrast to the extracellular domain, which facilitates nephrocyte diaphragm assembly/maintenance, the intracellular FERM-interaction motif of Crumbs is essential for regulating endocytosis. Moreover, Moesin, which binds to the FERM-binding domain of Crumbs, is essential for efficient endocytosis. Thus, we describe here a new mechanism of nephrocyte development and function, which is likely to be conserved in mammalian podocytes.

Background Machine learning classifications of first-episode psychosis (FEP) using neuroimaging have predominantly analyzed brain volumes. Some studies examined cortical thickness data, but most of them have used parcellation approaches with data from single sites, which limits claims of generalizability. To address these limitations, we conducted a large-scale, multi-site analysis of cortical thickness comparing parcellations and vertex-wise approaches. By leveraging the multi-site nature of the study, we further investigated how different demographical and site-dependent variables affected predictions. Finally, we assessed relationships between the predictions and clinical variables. Methods 428 subjects (147 females, mean age 27.14) with FEP and 448 (230 females, mean age 27.06) healthy controls were enrolled in 8 centers by the ClassiFEP group. All subjects underwent a structural MRI (sMRI) session and were clinically assessed. Cortical thickness parcellation (68 areas) and full cortical maps (20484 vertices) were extracted. Supervised (linear Support Vector Machine) classification was used to differentiate FEP from HC, within a repeated nested Cross-Validation (CV) framework through the NeuroMiner software. In both inner and outer CVs, a 10-fold CV cycle was employed. We performed repeated nested CV at the outer cross-validation cycle by randomly permuting the participants within their groups (10 permutations) and repeating the CV cycle for each of these permutations. As feature preprocessing, regression of covariates (age, sex, and site), Principal Component Analysis and Scaling were applied. All preprocessing steps were implemented within the CV. Further analyses were conducted by stratifying the sample for MRI scanner, sex and by performing random resampling with increasingly reduced sample sizes. Results Vertex-wise thickness maps outperformed parcellation-based methods with a balanced accuracy (BAC) of 66.2% and an Area Under the Curve of 72%, compared to a BAC of 59% and an Area Under the Curve of 61% obtained with the ROI-based approach. The two BACs were significantly different based on the McNemar’s Test. By stratifying our sample for MRI scanner, we increased the overall BAC to more than 70% and we also increased generalizability across sites. Temporal areas resulted the most influential regions in the classification. The predictive decision scores presented significant correlations with age at onset, duration of treatment and the presence of affective vs non-affective psychosis. Discussion Cortical thickness could represent a valid measure to classify FEP subjects, showing temporal areas as potential markers in the early stages of psychosis. The assessment of site-dependent variables allowed us to increase the across-site generalizability of the model, thus attempting to address an important machine learning limitation, especially in the framework of large multi-site cohort and big data analysis.

Background The self-disturbances (SDs) concept is considered to be part of the Schneider’s first rank symptoms, i.e., thought-withdrawal, thought-insertion, thought-broadcasting, somatic-passivity experiences, mental/motor automatisms, disrupted unitary self-experience (Mishara et al., 2014). SDs were originally described by W. Mayer-Gross (1920), who observed them in psychotic patients. Methods We classified Mayer-Gross’ findings on SDs into the following categories: experience is new/compelling (aberrant salience), reduced access/importance of autobiographical past, cognitions/emotions occur independently from self’s volition, foreign agents have power over self and developed an SDs scale based on these categories and cognitive domains (perception, motor, speech, thinking etc.). Scale is applied as a measure of the frequency of the experiences. In our current study on phenomenology and neurobiology of psychotic symptoms, we administered the scale to a study group of patients with schizophrenia (N=84) and healthy volunteers (N=170). Further, the resting state fMRI was performed and the group was divided into two subgroups with (N=13) and without self-disturbances (N=10) and in healthy individuals (N=39). Results We found substantial differences in the frequency of self-disturbances in patients with schizophrenia compared to healthy controls (total score differences, Z=-5.83, p< 0.001). On a neural level, patients with self-disturbances experienced a decreased functional brain connectivity of the default mode and salience networks as compared to the patients without self-disturbances and healthy controls. The differences were mainly explained by the factor ‘’foreign agents’’ and the novelty of the experience. Discussion The scale identifies self-disturbances in schizophrenia and confirms self-related processing in patients with schizophrenia to be associated with altered activation in the cortical midline structures. Supported by the grant projects MH CR AZV 17-32957A and MEYS NPU4NUDZ: LO1611.

Due to its structural and molecular similarities to mammalian podocytes, the Drosophila nephrocyte emerged as a model system to study podocyte development and associated diseases. Similar to podocytes, nephrocytes establish a slit diaphragm between foot process-like structures in order to filter the hemolymph. One major obstacle in nephrocyte research is the distinct visualization of this subcellular structure to assess its integrity. Therefore, we developed a specialized dissection and fixation protocol, including high pressure freezing and freeze substitution techniques, to improve the preservation of the intricate ultrastructural details necessary for electron microscopic assessment. By means of scanning transmission electron microscopy (STEM) tomography, a three-dimensional dataset was generated to further understand the complex architecture of the nephrocyte channel system. Moreover, a staining protocol for immunolabeling of ultrathin sections of Epon-embedded nephrocytes is discussed, which allows the reliable detection of GFP-tagged fusion proteins combined with superior sample preservation. Due to the growing number of available GFP-trap fly lines, this approach is widely applicable for high resolution localization studies in wild type and mutant nephrocytes.

Background The auditory verbal hallucinations (AVH) prevalence among patients diagnosed with schizophrenia is more than 70% (Hugdahl et al, 2008). The brain mechanisms of AVH are not yet clarified. In the currect study, we explore the specific brain connections that are usually associated with perception and reception of the speech, being present in patients with AVHs. Methods In total 70 patients with first psychotic episode, 41 with auditory hallucinations and 29 patients who had never experienced hallucinations were included into the study based on the following criteria: non-hallucinated patients were defined using PANSS scores (Hallucinations P3 scored as 1)/hallucinated patients (PANSS P3 score 3). All patients underwent resting MRI scans. We analysed the resting state functional connectivity between temporal cortices including TPJ structures (supramarginal gyrus and angular gyrus, parahypocampal gyrus) and predefined thalamic nuclei that are parts of the auditory pathways, specifically: the medial geniculate nuclei(MGB), the mediodorsal nuclei (MDN), having a big spectrum of connections with the limbic system and the reticular thalamic nuclei (RTN) which is a main source of inhibitory signals (using detailed atlas of thalamus, Morel, 2003). Results The analysis revealed the increased connectivity between mediodorsal nucleus L and Hesch gyrus (L, R), pahahippocampal gyrus (L,R) as well as between mediodorsal nucleus (R) and parahipocampal gyrus (L). The decreased connectivity was detected between medial geniculate body (L), Medial geniculate body (R) and inferior parietal lobule (L) as well as between medial geniculate body (L) x parahyppocampal gyrus (L) (FDR corr p=0.05). Discussion We confirmed our hypothesis on the altered connectivity between thalamic nuclei and auditory cortex (Heshl gyrus, parahippocampal gyrus) and inferior parietal cortex in patients with AVHs. The findings go in line with other studies on functional dysconnectivity with right hippocampal formation and mediodorsal thalamus compared to patients without lifetime AHs (Diederen et al, 2010; Shinn et al, 2013). The patterns of decreased connections presumably indicate a sensory defect: decreased connectivity between medial geniculate nuclei (main auditory pathway) and parahippocampal gyrus (memory retrieval) and inferior parietal cortex (interpretation of sensory information, Radua et al, 2010). The patterns of increased connections presumably imply alterations of emotional processing (projections from mediodorsal nuclei to auditory cortex) and deserve further investigation.

In the present study we evaluated attributional style which refers to how individuals explain the causes for positive and negative events in their lives in patients with first episode of schizophrenia with and without paranoid ideation. 43 patients with first episode of psychosis and 37 matched normal controls completed Ambiguous Intentions Hostility Questionnaire (AIHQ) (Combs et al. 2007). Between group comparison of AIHQ scores showed a notable tendency to show aggressive response in overall patients group. We obtained significant elevation of hostility and blame biases scores in intentional and accidental situations in patients with paranoid ideation while the patients with non-paranoid ideation showed greater hostility and blame biases only in accidental situations as compared to controls. Correlations with positive and negative symptoms were obtained. Our findings suggest that patients with first episode of psychosis exhibit difficulties of the attribution biases which are interconnected with symptoms and thus indicate a trait-deficit of attributional style.