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