Semi-Automated Live Tracking of Microglial Activation in CX3CR1GFP Mice During Experimental Autoimmune Encephalomyelitis by Confocal Scanning Laser Ophthalmoscopy

Confocal scanning laser ophthalmoscopy (cSLO) is a non-invasive technique for real-time imaging of the retina. We developed a step-by-step protocol for the semi-automatic evaluation of myeloid cells in cSLO images from CX3CR1GFP mice, expressing green fluorescent protein (GFP) under control of the endogenous CX3C chemokine receptor 1 locus. We identified cSLO parameters allowing us to distinguish animals with experimental autoimmune encephalomyelitis (EAE) from sham-treated/naïve animals. Especially cell count (CC) and the total microglial area (SuA) turned out to be reliable parameters. Comparing the cSLO results with clinical parameters, we found significant correlations between the clinical EAE score and the SuA and of the inner retinal layer thickness, measured by optical coherence tomography, with the CC as well as the SuA. As a final step, we performed immunohistochemistry to confirm that the GFP-expressing cells visualized by the cSLO are Iba1 positive and validated the step-by-step protocol against manual counting. We present a semi-automatic step-by-step protocol with a balance between fast data evaluation and adequate accuracy, which is optimized by the option to manually adapt the contrast threshold. This protocol may be useful for numerous research questions on the role of microglial polarization in models of inflammatory and degenerating CNS diseases involving the retina.

Biofilm Formation of Multidrug-Resistant MRSA Strains Isolated from Different Types of Human Infections

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens causing chronic infections, mainly due to its capacity to form biofilms. However, the mechanisms underlying the biofilm formation of MRSA strains from different types of human infections are not fully understood. MRSA strains isolated from distinct human infections were characterized aiming to determine their biofilm-forming capacity, the biofilm resistance to conventional antibiotics and the prevalence of biofilm-related genes, including, icaA, icaB, icaC, icaD, fnbA, fnbB, clfA, clfB, cna, eno, ebpS, fib and bbp. Eighty-three clinical MRSA strains recovered from bacteremia episodes, osteomyelitis and diabetic foot ulcers were used. The biofilm-forming capacity was evaluated by the microtiter biofilm assay and the biofilm structure was analyzed via confocal scanning laser microscopy. The antimicrobial susceptibility of 24-h-old biofilms was assessed against three antibiotics and the biomass reduction was measured. The metabolic activity of biofilms was evaluated by the XTT assay. The presence of biofilm-related genes was investigated by whole-genome sequencing and by PCR. Despite different intensities, all strains showed the capacity to form biofilms. Most strains had also a large number of biofilm-related genes. However, strains isolated from osteomyelitis showed a lower capacity to form biofilms and also a lower prevalence of biofilm-associated genes. There was a significant reduction in the biofilm biomass of some strains tested against antibiotics. Our results provide important information on the biofilm-forming capacity of clinical MRSA strains, which may be essential to understand the influence of different types of infections on biofilm production and chronic infections.

Multimodal Imaging of West African Crystalline Maculopathy in an Igbo Man

Purpose: This report describes a case of West African crystalline maculopathy. Methods: A case report is presented. Results: A 71-year-old Nigerian man was referred for evaluation of bilateral crystalline retinal deposits seen on routine examination. The patient had no acute visual symptoms and no significant ocular history except for cataract extraction and intraocular lens implantation in both eyes. Dilated fundocscopic examination was notable for bilateral greenish-yellow, foveocentric intraretinal crystalline deposits, which were visible on color fundus photography, multicolor confocal scanning laser ophthalmoscopy, and spectral-domain optical coherance tomography. The crystalline deposits were not associated with abnormal short-wavelength autofluorescence or fluorescein angiography findings. Conclusions: A diagnosis of West African crystalline maculopathy was made after other causes of crystalline maculopathy were excluded.

MUSCULATURE AND NEUROTRANSMITTERS IN THE DIGESTIVE SYSTEM OF TREMATODES

In this paper we analyzed the results of our own and published data concerning the presence of muscle elements in various parts of the digestive system in adult and larval forms of trematodes. The data on the localization of the circular and longitudinal muscle fibers in the pharynx, esophagus, and intestine of various representatives of trematodes are presented. The results of immunocytochemical studies indicate the presence the serotonergic and peptidergic (FMRFamidergic) nerve elements in the parts of the digestive system of trematodes. The available literature date is supplemented by the studies conducted on Prodistomum alaskense, a representative of the family Lepocreadiidae, an intestinal parasite of deep-sea fish (Zaprora silenus and Aptocyclus ventricosus). The localization of the serotoninergic and FMRFamidergic nervous structures was identified using immunocytochemical methods and the confocal scanning laser microscopy. For musculature staining the TRITC (tetramethylrhodamine isothiocyanate) – conjugated phalloidin was used. The preparations were examined using a fluorescence microscope and a confocal scanning laser microscope. The analysis of the data obtained and the information available in the literature suggests that the muscular system of the digestive tract is well developed in trematodes of various taxonomic groups. The musculature of the digestive system of trematodes is innervated by serotonergic and peptidergic (FMRFamidergic) nerve elements, which are involved in the regulation of the contractile activity of various parts of the digestive system of trematodes.

Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of μ-opioid peptide receptors

The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.