Densitometric patterns of NADPH diaphorase staining in the spinal cord of dog

Mar ala, M., Densitometric patterns of NADPH diaphorase staining in the spinal cord of dog. Biologia, Bratislava, 56: 685-693, 2001; ISSN 0006-3088 (Biologia). ISSN 1335-6399 (Biologia. Section Cellular and Molecular Biology). Segmental and laminar distribution of NADPHd activity was studied in the normal spinal cord of the dog and basic densitometric patterns of somatic, fiber-like and punctuate, non-somatic NADPHd staining were described in the gray and white matter. Prominent NADPHd activity was noted in the superficial and deep dorsal horn, pericentral region, intermediolateral cell column, Lissauer's tract and in the vertical and horizontal limbs of the medial longitudinal bundle of the ventral column in the cervical and upper thoracic segments. Key words: densitometry, NADPH diaphorase, spinal cord, dog. Introduction The use of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry alone or combined with the nitric oxide synthase immunoreactivity (NOS-IR) allowed for a morphologically distinct and topographically precise localization of small neuronal pools synthesizing, releasing and transporting NOS, an enzyme responsible for nitric oxide (NO) synthesis. The discrete loci, nuclei or solitary NOS-IR neurons have been identified not only in the cortex, brain stem and spinal cord, but also in the peripheral nervous system (Vincent & Johanson, 1983; Immunocytochemistry of the neuronal nitric oxide synthase (nNOS) showed that the occurrence of this enzyme is almost completely homotopic with the localization of neurons stained for NADPHd In the present study an attempt was made to specify the differences of somatic, fiber-like, and punctuate NADPHd staining in the gray and white matter of the spinal cord in the normal dog, including different segments and layers using the densitometric analysis. Densitometric patterns of NADPHd positivity in the undamaged spinal cord may be helpful in experimental studies aimed at a causal interpretation of changes affecting the NOS-containing neuronal pools in various experimental and pathologic conditions. Material and methods Tissue sampling, sectioning, examination of sections and the performance of the densitometric analysis Adult dogs (n = 6) of both sexes weighing 12-18 kg were used in this study. The animals were deeply anesthetized with pentobarbital (50 mg/kg, i.v.) and perfused transcardially with saline followed by freshly prepared 4% paraformaldehyde +0.1% glutaraldehyde buffered with 1M sodium phosphate, pH = 7.4. Following perfusion fixation, the spinal cords were carefully dissected out and stored in toto in the same fixative for 3-4 hours. After postfixation, the spinal cord was divided into cervical, thoracic, lumbar, sacral and coccygeal segments, and each segment was then secondarily divided into three small blocks comprising the upper, middle, and lower segmental levels, respectively. Specimens were then cryoprotected in an ascending concentration of sucrose (15-30%) with the same phosphate buffer and stored overnight at 4 • C. Frozen transverse sections (50 µm thick) were cut from all segments studied and processed for NADPH-d activity by using a modified histochemical procedure The densitometric analysis was performed using transverse sections stained for NADPHd histochemistry. Precise loci identified in the gray and white matter on transverse sections were used for the assessment of the densitometric patterns in both compartments of the spinal cor

Glycoprofiling as a novel tool in serological assays of systemic sclerosis: A comparative study with three bioanalytical methods

Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient’s quality of life. The heterogeneity of the disease also means that identification and subsequent validation of biomarkers of the disease is quite challenging. A fully validated single biomarker for diagnosis, prognosis, disease activity and assessment of response to therapy is not yet available. The main aim of this study was to apply an alternative assay protocol to the immunoassay-based analysis of this disease by employment of sialic acid recognizing lectin Sambucus nigra agglutinin (SNA) to glycoprofile serum samples. To our best knowledge this is the first study describing direct lectin-based glycoprofiling of serum SSc samples. Three different analytical methods for glycoprofiling of serum samples relying on application of lectins are compared here from a bioanalytical point of view including traditional ELISA-like lectin-based method (ELLA), novel fluorescent lectin microarrays and ultrasensitive impedimetric lectin biosensors. Results obtained by all three bioanalytical methods consistently showed differences in the level of sialic acid present on glycoproteins, when serum from healthy people was compared to the one from patients having SSc. Thus, analysis of sialic acid content in human serum could be of a diagnostic value for future detection of SSc, but further work is needed to enhance selectivity of assays for example by glycoprofiling of a fraction of human serum enriched in antibodies for individual diagnostics.Slovak Scientific Grant Agency VEGA 2/0162/14 and from the Slovak Research and Development Agency APVV 0282-11 is acknowledged. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement No. 311532 and this work has received funding from the European Union’s Seventh Framework Programme for research, Technological Development and Demonstration under grant agreement No. 317420. This publication is the result of the project implementation: Applied Research in the field of Industrial Biocatalysis, ITMS code: 26240220079 supported by the Research & Development Operational Programme funded by the ERDF. Research leading to these results was supported by BASF Slovakia