Use of Continuous Electrical Impedance Measurement for Accurate Nerve Block in Rabbits

Objective To perform an effective and safe nerve block, the needle must be placed near the target nerve while avoiding nerve damage. Our objective was to conduct an animal study to determine whether changes in electrical impedance (EI) could be used to guide the needle and achieve a safe and accurate nerve block. Methods We measured the EI of rabbit tissues during ultrasound-guided sciatic nerve block using a bipolar needle via the in-plane needle approach. The EI values and needle track on the ultrasound monitor were video-recorded. When there was a change in the EI, the needle advancement was stopped, and a stained anesthetic was injected. Subsequently, the animals were euthanized, and the anesthetic-stained tissue was examined via dissection, while the other tissue was preserved at –80°C for microscopic analysis. Results The EI remained stable as the needle advanced through the muscle (extraneural); however, it markedly decreased when the needle tip contacted the nerve or slightly punctured the epineurium (paraneural). The mean extra- and paraneural EIs were 4.92 ± 1.31 kΩ (range, 2.39–9.67 kΩ) and 2.86 ± 0.96 kΩ (range, 1.66–5.13 kΩ), respectively. Examination of the dissections and cryostat sections showed anesthetic delivery around the nerve. Conclusions EI values differed between extra- and paraneural sites, and monitoring these values allowed prediction of the needle tip location with respect to the target nerve. Real-time EI measurement could improve the nerve block.

Determination of the Involvement of Autologous Mesenchymal Bone Marrow Stem Cells in Reparative Chondrogenesis

Summary. Articular cartilage injuries are observed in 60% of arthroscopies. Degenerative changes of the articular cartilage (osteoarthritis) is the most common disease of all the joints, which affects at least 10-13% of the population over the age of 60 years. Significant progress has been made in the treatment of pain and inflammation, but the evidence base for improving the articular cartilage repair remains weak. Despite the seemingly positive results of a number of studies on the use of mesenchymal stem cells (MSCs) for the articular cartilage restoration, the question of the nature of their participation in this process remains controversial. Objective. The objective of the study was to determine the involvement of autologous mesenchymal bone marrow stem cells in reparative chondrogenesis. Materials and Methods. Experimental and morphological determination of the participation of undifferentiated autologous bone marrow MSCs in reparative chondrogenesis was carried out on fifteen rabbits. MSCs of bone marrow marked with a red fluorescent dye PKH-26 (Sigma, USA) was injected into the cavity of the injured joint on the 3rd day after the articular cartilage defect formation. The presence of the transplanted labeled autologous MSCs was examined in these animals on cryostat sections on the seventh, fourteenth and twenty-first days after implantation with fluorescence microscopy. The characteristic red glow was observed in case of the presence of labeled cells in the regenerated cartilage tissue. Results. The presence of transplanted labeled autologous MSCs on cryostat sections was determined in all studied animals. On the 7th day, the cells were arranged diffusely, without noticeable accumulations. On the 14th day, the labeled cells fluoresced in the red zone of the spectrum and created compact conglomerates mainly at the defect zone. On the 21th day, the labeled cells were found only in the regenerate zone. The data obtained indicate the direct participation of autologous MSCs in the processes of chondroreparation after the exogenous implantation into the joint cavity. Conclusions. The method of autologous bone marrow MSCs labeling using PKH-26 fluorescent dyes has shown that autologous bone marrow MSCs are directly involved in the processes of chondroreparation when they are injected intraarticularly.

Test-retest reproducibility of atomic force microscopy measurements of human trabecular meshwork stiffness

Purpose: The purpose of the present study was to quantify test-retest reproducibility of measurements of stiffness of the human trabecular meshwork (HTM) by atomic force microscopy (AFM). Methods: Eleven 40 μm radial limbal cryostat sections from a fresh human donor rim were mounted on charged slides and rehydrated at room temperature. Stiffness at four TM locations (anterior to posterior along Schlemm’s canal) was measured by AFM. At each location, a 6 x 6 grid was sampled. Indentation points were evenly distributed over a 20 μm x 20 μm area, with a rate of one load/unload cycle per second. Measurements were then repeated for calculation of test-retest variability. Results: The test-retest coefficients of variation for the four measurement locations (anterior to posterior) were 24.39, 25.28, 12.74, and 14.26%, respectively, with a notable drop in the two posterior locations compared to the anterior. The test-retest coefficient for the sections was 19.17%. For the entire eye, the test-retest coefficient of variation for the measurement of the TM stiffness was 17.13%. Young’s moduli consistently decreased from anterior to posterior location. Conclusions: Wide regional variation suggests that single value does little to fully describe the complex array of TM stiffness levels within the eye, and future studies of TM stiffness assessed by AFM should include multiple tissue samples from each eye, with documentation of the anterior-posterior location of each measurement.

Otopetrin-2 Immunolocalization in the Human Macula Utricle

Background: In the present study, we investigated the localization of otopetrin-2—a member of the otopetrin family that encodes proton-selective ion channels—in the human macula utricle using immunohistochemistry. Methods: Macula utricle were acquired at surgery from patients who required transmastoid labyrinthectomy for intractable vertigo due to Meniere’s disease (MD; n = 3) and/or vestibular drops attacks (VDA; n = 2) and from temporal bones (n = 2) acquired at autopsy from individuals with no balance disorders. Immunofluorescence staining with otopetrin-2 (rabbit affinity purified polyclonal antibody) and GFAP (mouse monoclonal antibody) to identify vestibular supporting cells was made in formalin fixed cryostat sections or whole microdissected utricle (for flat mount preparations). Secondary antibodies against rabbit and mouse were used for the identification of both proteins. Digital fluorescent images were obtained using a high-resolution laser confocal microscope. Results: Using cryostat sections and flat mount preparations otopetrin-2 immunofluorescence was seen as punctated signal throughout the supporting cells cytoplasm. GFAP immunofluorescence was present in the supporting cell cytoplasm. The distribution of otopetrin-2 was similar in the macula utricle obtained from MD, VDA, or autopsy normative patients. Conclusions: Otopetrin-2 was localized in supporting cells in a similar fashion that otopetrin-1 previously reported in the mouse macula utricle. The differential expression of otopetrin-2 in the supporting cells of the human macula utricle suggest an important role in the vestibular sensory periphery homeostasis and otolith maintenance.

Influence of Bacteriophage Infections of Microbiota on the Expression of alpha-synuclein in the Rat Intestinal Wall

The aim of the study was to identify the effect of bacteriophage microbiota infection on the expression of alpha-synuclein (A-syn) in rat small intestinal wall cells.Material and methods. The work was performed on Wistar rats, which once a day rectally injected a cocktail of bacteriophages against pathogenic bacteria or sterile saline (control). Various parts of the small intestine were frozen on dry ice, and cryostat sections were prepared to immunohistochemically investigation the localization of A-syn in the cells of the intestinal wall after 10 days of experiment.Results. A significant quantitative increase of immunopositive to A-syn lymphocytes and an increase in the expression of this protein in the neurons of the intermuscular and submucous nerve plexuses were detected.Conclusion. Bacteriophage infection of the microbiota has a pronounced effect on the expression of alpha synuclein in lymphocytes and neurons localized in the wall of the small intestine.

Seasonal variations in enzyme histochemistry of accessory sex glands in buffalo bull

The present study was conducted on vesicular, prostate and bulbourethral glands of buffalo bullcollected from the abattoir immediately after sacrifice during winter and summer season. The cryostat sections of 10-12 µm thickness were obtained at -23°C and were incubated in different substrates for demonstration of phosphatases, oxidoreductases and estrases. During winter season, the moderate to strong activity of AKPase in acinar cells and ductular epithelium was observed in vesicular, weak in prostate, but the activity was not demonstrated in bulbourethral gland, however the blood vessels showed moderate AKPase reaction in all the glands. A moderate activity of SDH, LDH and G-6-PD was observed in glandular and ductular epithelium of vesicular gland and weak to moderate activity in prostate and bulbourethral glands. The glandular and the ductular epithelium of all the glands studied were strongly positive for NADH and NADPH, whereas a weak activity for NSE was observed. There was decrease in the activity of all the enzymes during summer season which may be related to decrease physiological activity in this season.

Multiple sclerosis: Serum anti-CNS autoantibodies

Background: It is uncertain whether there are autoantibodies detectable by indirect immunofluorescence in the serum of patients with multiple sclerosis (MS). Objective: To determine whether there are anti-central nervous system (CNS) autoantibodies detectable by indirect immunofluorescence in the serum of MS patients. Methods: Sera and in some cases cerebrospinal fluid from 106 patients with multiple sclerosis, 156 patients with other neurological diseases, and 70 healthy control subjects were examined by indirect immunofluorescence using cryostat sections of rat cerebrum fixed by perfusion with paraformaldehyde. Results: Autoantibodies were detected that recognized more than 30 neuronal, glial, and mesodermal structures in 28 of 106 MS cases. Most were also detected in patients with other related and unrelated neurological diseases and several were also found in healthy controls. Novel anti-CNS autoantibodies recognizing particular sets of interneurons were detected in both normal controls and in subjects with CNS diseases. Interpretation: Serum anti-CNS autoantibodies of diverse specificities are common in MS patients. The same anti-CNS autoantibodies are not uncommon in patients with other neurological diseases. The findings provide no support for the proposition that myelin breakdown in MS is caused by exposure of intact myelin sheaths or oligodendrocytes to a pathogenic serum anti-myelin or anti-oligodendrocyte autoantibody.

Immunohistochemical characterisation of neurons in the mandibular ganglion and nerve fibres supplying the porcine mandibular gland

The present study was designed to investigate the chemical coding of neurons in the mandibular ganglion (MGn) and nerve fibres supplying the porcine mandibular gland (MGl) with the use of immunofluorescence and RT-PCR. The cryostat sections from MGn and MGl were processed for double-labelling immunohistochemistry using antisera against vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), dopamine β-hydroxylase (DβH), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), galanin (GAL), substance P (SP) and calcitonin gene-related peptide (CGRP). The MGl was found to be richly supplied by VAChT-positive nerve fibres that surrounded intra- and interlobular salivary ducts. A large number of VAChT-immunoreactive (VAChT-IR) nerve terminals were also observed around acini. Many periductal and periacinar nerve fibres stained positive for DβH. Immunoreactivity to GAL, NPY or VIP was observed in an intermediate number of nerve terminals which were associated with both salivary ducts and acini. Double-immunostaining revealed that in MGn nearly all neurons stained positive for VAChT/ChAT (98.45 ± 0.59%, mean ± SEM) and nNOS (99.71 ± 0.18%). An intermediate number of the nerve cell bodies displayed immunoreactivity to NPY or VIP (18.67 ± 0.52% and 8.11 ± 0.36%, respectively). Single GAL-IR and CGRP-positive neurons were also observed. RT-PCR revealed the presence of transcripts of ChAT, VAChT, nNOS, NPY, VIP and GAL. For SP and DβH very weak signals were observed. RT-PCR with primers targeting CGRP did not generate any PCR product.

Changes in Somatostatin-Like Immunoreactivity in the Sympathetic Neurons Projecting to the Prepyloric Area of the Porcine Stomach Induced by Selected Pathological Conditions

The aim of the present study was to define changes in the expression of somatostatin (SOM) in the sympathetic perikarya innervating the porcine stomach prepyloric area during acetylsalicylic-acid-induced gastritis (ASA) and experimentally induced hyperacidity (HCL) and following partial stomach resection (RES). On day 1, the stomachs were injected with neuronal retrograde tracer Fast Blue (FB). Animals in the ASA group were given acetylsalicylic acid orally for 21 days. On the 22nd day after FB injection, partial stomach resection was performed in RES animals. On day 23, HCL animals were intragastrically given 5 ml/kg of body weight of a 0.25 M aqueous solution of hydrochloric acid. On day 28, all pigs were euthanized. Then, 14-μm thick cryostat sections of the coeliac-superior mesenteric ganglion (CSMG) complexes were processed for routine double-labelling immunofluorescence. All pathological conditions studied resulted in upregulation of SOM-like (SOM-LI) immunoreactivity (from14.97±1.57% in control group to33.72±4.39% in the ASA group, to39.02±3.65% in the RES group, and to29.63±0.85% in the HCL group). The present studies showed that altered expression of SOM occurs in sympathetic neurons supplying the prepyloric area of the porcine stomach during adaptation to various pathological insults.