Form, synapses and orientation topography of a new cell type in layer 6 of the cat's primary visual cortex.

Here we report the morpho-functional features of a novel type of deep-layer neuron. The neuron was selected from a large pool of intracellularly labelled cells based on the large cell body, numerous spine-free dendrites with an overall interneuron morphology. However, the axon gave off long-range axons up to 2.8 mm from the parent soma in layers 5/6 before entering the white matter. The boutons were uniformly distributed along the axon without forming distinct clusters. Dendritic length, surface area and volume values were at least 3 times larger than any known cortical neuron types with the exception of giant pyramidal cells of layer 5. Electron microscopy of the boutons revealed that they targeted dendritic spines (78%) and less frequently dendritic shafts (22%). Nearly half of the postsynaptic dendrites were immunopositive to GABA. Superimposing the axonal field on the orientation map obtained with optical imaging showed a preponderance of boutons to cross-orientations (38%) and an equal representation of iso- and oblique orientations (31%). The results suggest an integrating role for the layer 6 stellate neuron which projects to a functionally broad range of neurons in the deep cortical layers and to other cortical and/or subcortical regions.

Application of the mirror technique for block-face scanning electron microscopy.

The mirror technique adapted for electron microscopy allows correlating neuronal structures across the cutting plane of adjoining light microscopic sections which, however, have a limited thickness, typically less than 100 µm (Talapka et al. in Front Neuroanat, 2021, https://doi.org/10.3389/fnana.2021.652422 ). Here, we extend the mirror technique for tissue blocks in the millimeter range and demonstrate compatibility with serial block-face electron microscopy (SBEM). An essential step of the methodological improvement regards the recognition that unbound resin must be removed from the tissue surface to gain visibility of surface structures. To this, the tissue block was placed on absorbent paper during the curing process. In this way, neuronal cell bodies could be unequivocally identified using epi-illumination and confocal microscopy. Thus, the layout of cell bodies which were cut by the sectioning plane can be correlated with the layout of their complementary part in the adjoining section processed for immunohistochemistry. The modified mirror technique obviates the spatial limit in investigating synaptology of neurochemically identified structures such as neuronal processes, dendrites and axons.

Application of the Mirror Technique for Three-Dimensional Electron Microscopy of Neurochemically Identified GABA-ergic Dendrites.

In the nervous system synaptic input arrives chiefly on dendrites and their type and distribution have been assumed pivotal in signal integration. We have developed an immunohistochemistry (IH)-correlated electron microscopy (EM) method - the "mirror" technique - by which synaptic input to entire dendrites of neurochemically identified interneurons (INs) can be mapped due preserving high-fidelity tissue ultrastructure. Hence, this approach allows quantitative assessment of morphometric parameters of synaptic inputs along the whole length of dendrites originating from the parent soma. The method exploits the fact that adjoining sections have truncated or cut cell bodies which appear on the common surfaces in a mirror fashion. In one of the sections the histochemical marker of the GABAergic subtype, calbindin was revealed in cell bodies whereas in the other section the remaining part of the very same cell bodies were subjected to serial section EM to trace and reconstruct the synaptology of entire dendrites. Here, we provide exemplary data on the synaptic coverage of two dendrites belonging to the same calbindin-D28 K immunopositive IN and determine the spatial distribution of asymmetric and symmetric synapses, surface area and volume of the presynaptic boutons, morphometric parameters of synaptic vesicles, and area extent of the active zones.

Primary uterine NK-cell lymphoma, nasal-type: a unique malignancy of a prominent cell type of the endometrium.

Natural killer (NK) cells host in the human endometrium with dedicated role in reproductive physiology. Interestingly, malignant transformation of these specialized cells has not been presented thus far. Here we report a primary endometrial NK-cell lymphoma of a 48 year-old patient presenting with irregular bleeding. The endometrial curetting showed a dense lymphomatous infiltrate demonstrating highly infiltrative aggressive features with characteristic angiocentric, partially angiodestructive growth pattern and accompanying focal necroses. The lymphoma cells displayed a CD3ε/CD56/TIA-1/granzyme-B-positive and CD5/CD4/CD8/TCRγδ-negative immunophenotype, proved to be positive for Epstein-Barr virus by EBER in situ hybridization, and revealed no clonal T-cell receptor gene rearrangement. The diagnosis of uterine extranodal NK-cell lymphoma, nasal-type was made. Clinically, the disease was limited to the uterus at diagnosis, but progressed rapidly, and the patient died within 5 months due disseminated lymphoma, irrespective of intensive chemotherapy. Genuine NK-cell lymphomas occurring in the uterus as primary site seem to be rare making the therapeutic decisions extremely complicated.

Reduced paraoxonase1 activity is a risk for atherosclerosis in patients with systemic lupus erythematosus.

Excessive lipid peroxidation is a major factor of accelerated atherosclerosis, observed in patients with systemic lupus erythematosus (SLE). We aimed at the present study to determine the paraoxonasel (PON1) and arylesterase activities, and lipid-profile in 37 SLE patients and 30 age-/sex-matched controls. Association was analyzed between PON1 activity and SLEDAI, CRP, anti-oxLDL, and antiphospholipid antibody (aPL) levels, steroid dose, and atherothrombotic events. The age of patients was 40.8 +/- 13.9 year, follow-up time 6.7 +/- 6.2 year, SLEDAI 2 (0-15). PON1 and arylesterase activities were measured spectrophotometrically using paraoxon and phenyl acetate as substrates, respectively. Phenotypic distribution of PON1 was determined by dual substrate method. We measured antioxLDL and aPL levels by ELISA, the CRP by automated immunoassay. PON1 activity (121.9 +/- 65.9 U/mL) was reduced significantly (P < 0.001) in SLE as compared to control (188.1 +/- 78.9 U/mL), but arylesterase activity was not different. A negative correlation was found between PON1 activity and age. PON1 activity did not correlate with other measured parameters. Reduced PON1 activity associated with clinical atherothrombotic complications (P < 0.01). High activity BB phenotype was not present in SLE. Lipid parameters (TC, LDL-C, HDL-C, ApoAI, and ApoB) were within normal range in both groups. Results indicated reduced PON1 activity in lupus patients despite long disease duration and low inflammatory activity, and it was evidenced as a risk for atherosclerotic complications. As the arylesterase activity was normal, further examinations are required to find other mechanisms, such as anti-PON1 antibodies, genetic polymorphisms, and difference in distribution of HDL-subfractions or enzyme abnormalities in HDL remodeling.

Reduced flow-mediated vasodilation as a marker for cardiovascular complications in lupus patients.

Systemic lupus erythematosus is associated with accelerated atherosclerosis and increased cardiovascular morbidity and mortality. Objectives were to determine endothelial dysfunction with a non-invasive method in lupus patients and to analyse correlation with risk factors and atherosclerotic complications. Sixty-one SLE patients and 26 healthy age- and sex-matched control subjects were entered into the study. The diameters of brachial artery at rest, during reactive hyperaemia, and after glyceril trinitrate administration, as well as the intima-media thickness of the common carotid artery were measured using high-resolution B-mode ultrasonography. Demographic characteristics, lipid profile, paraoxonase activity, concentration of anti-phospholipid antibodies and anti-oxLDL were assessed together with atherosclerotic complications. The endothelium dependent vasodilation (FMD) was significantly impaired in SLE patients as compared to controls. The absolute difference of vessel diameter (Deltad) was 0.25+/-0.15 mm vs. 0.38+/-0.16 mm (p=0.001), and Deltad as in percent of the rest diameter was 7.31+/-5.2% vs. 9.86+/-3.87% (p=0.013) in lupus patients and controls, respectively. Nitrate mediated dilation (NMD) did not differ. FMD negatively correlated with age, systolic and diastolic blood pressure in SLE, but did not show significant correlation with the other examined parameters. However, FMD significantly differed between SLE patients with (5.54+/-4.36%) and without (8.81+/-5.28%) cardiovascular complications (p=0.01). The determination of flow-mediated vasodilation is a useful method to detect endothelial dysfunction in lupus patients, as reduced capacity of brachial artery may distinguish between SLE patients and healthy subjects, as well as lupus patients with and without atherosclerotic vascular complications.