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Martin Marsala - One of the best experts on this subject based on the ideXlab platform.

  • the effect of a spinal cord hemisection on changes in Nitric Oxide Synthase pools in the site of injury and in regions located far away from the injured site
    Cellular and Molecular Neurobiology, 2006
    Co-Authors: Nadežda Lukacova, Maria Kolesarova, Karolina Kucharova, Jaroslav Pavel, Dalibor Kolesar, Jozef Radoňak, Martin Marsala, Malgorzata Chalimoniuk, Józef Langfort, J Marsala
    Abstract:

    : 1. The present study was designed to examine the Nitric Oxide Synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury. 2. A radioassay detection was used to determine Ca(2+)-dependent and inducible Nitric Oxide Synthase activities. Somal, axonal and neuropil neuronal Nitric Oxide Synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal Nitric Oxide Synthase immunoreactivity was made by an image analyser. The level of neuronal Nitric Oxide Synthase protein was measured by the Western blot analysis. 3. Our data show the increase of inducible Nitric Oxide Synthase activity and a decrease of Ca(2+)-dependent Nitric Oxide Synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible Nitric Oxide Synthase activity was increased at both time points. Ca(2+)-dependent Nitric Oxide Synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the Nitric Oxide Synthase activities and in the level of neuronal Nitric Oxide Synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal Nitric Oxide Synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal Nitric Oxide Synthase immunoreactive staining in the ventral part of rostrally oriented segments. 4. Our findings provide evidence that the changes in Nitric Oxide Synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of Nitric Oxide Synthase activity and neuronal Nitric Oxide Synthase immunoreactivity, measured segment by segment shows that Nitric Oxide may play a significant role in the stepping cycle in the quadrupeds.

  • Immunohistochemical, Histochemical and Radioassay Analysis of Nitric Oxide Synthase Immunoreactivity in the Lumbar and Sacral Dorsal Root Ganglia of the Dog
    Cellular and Molecular Neurobiology, 2006
    Co-Authors: Nadežda Lukacova, Dalibor Kolesar, Martin Marsala, Jozef Maršala
    Abstract:

    In this study, immunohistochemistry for neuronal Nitric Oxide Synthase (bNOS-IR), nicotinamide adenine dinucleotide phosphate diaphorase histochemistry (NADPHd) and Nitric Oxide Synthase radioassay were used to study the occurrence, number and distribution pattern of Nitric Oxide synthesizing neurons in the lumbar (L1–L7) and sacral (S1–S3) dorsal root ganglia of the dog. Nitric Oxide Synthase immunolabelling was present in a large number of small- (area 2000 μm^2) neurons. Although neuronal Nitric Oxide Synthase immunolabelling and histochemical staining provided intense staining of multiple small- and medium-sized neurons in all lumbar and sacral dorsal root ganglia, immunolabelled or histochemically stained somata exhibited little topographic distribution in individual dorsal root ganglia. Great heterogeneity was noticed in the immunolabelling of medium-sized Nitric Oxide Synthase immunopositive neurons ranging from lightly immunolabelled somata to heavily immunoreactive ones with completely obscured nuclei. Both staining procedures proved to be highly effective in visualizing intraganglionic fibers of various diameters. In general, the largest fibers revealed at the peripheral end of lumbar and sacral dorsal root ganglia were larger, 6.49–9.35 μm in diameter, while those running centrally and proceeding into the dorsal roots were about 30% reduced, ranging between 5.32 and 8.67 μm in diameter. Peripherally, the occurrence of Nitric Oxide Synthase detected in axonal profiles, and confirmed histochemically, in the specimens of the femoral and sciatic nerves, is the first indication of the presence of Nitric Oxide Synthase in the peripheral processes of somata located in L4–S2 dorsal root ganglia. Large and thin central Nitric Oxide Synthase immunoreactive processes of L1–S3 dorsal root ganglion neurons segregate shortly before entering the spinal cord, the former making a massive medial bundle in the dorsal root accompanied by a slim lateral bundle penetrating Lissauer's tract. Quantitative assessment of the distribution of bNOS-IR and/or NADPHd-stained neurons showed a peculiar pattern in relation to spinal levels. Apparent incongruity was found in the total number of NADPHd-stained versus bNOS-IR neurons, demonstrating a clear prevalence of small bNOS-IR somata in all lumbar ganglia, while medium-sized NADPHd-stained somata clearly prevailed all along the rostrocaudal axis with a peak in L5 ganglion. While the number of small bNOS-IR neurons clearly outnumbered NADPHd-stained and NADPHd-unstained somata in S1–S3 ganglia, an inverse relation appeared comparing the total number of medium-sized NADPHd-stained and NADPHd-unstained somata compared with the number of moderate and intense bNOS-IR neurons. Densitometry of bNOS-IR and NADPHd-stained neurons in lumbar and sacral ganglia revealed two distinct subsets of densitometric profiles, one relating to more often found medium-sized bNOS immunolabelled and the other, characteristic for moderately bNOS immunoreactive somata of the same cell size. Considerable differences in catalytic Nitric Oxide Synthase activity, determined by conversion of [^3H]arginine to [^3H]citrulline were obtained in lumbosacral dorsal root ganglia all along the lumbosacral intumescence, the lowest (0.898± 0.2 dpm/min/μg protein) being in the L4 dorsal root ganglion and the highest (4.194± 0.2 dpm/min/μg protein) in the S2 dorsal root ganglion.

  • the evidence for Nitric Oxide Synthase immunopositivity in the monosynaptic ia motoneuron pathway of the dog
    Experimental Neurology, 2005
    Co-Authors: Jozef Maršala, Nadežda Lukacova, Igor Sulla, Peter Wohlfahrt, Martin Marsala
    Abstract:

    In this study, Nitric Oxide Synthase immunohistochemistry supported by nicotinamide adenine dinucleotide phosphate diaphorase histochemistry was used to demonstrate the Nitric Oxide Synthase immunoreactivity in the monosynaptic Ia-motoneuron pathway exemplified by structural components of the afferent limb of the soleus H-reflex in the dog. A noticeable number of medium-sized intensely Nitric Oxide Synthase immunoreactive somata (1000-2000 microm(2) square area) and large intraganglionic Nitric Oxide Synthase immunoreactive fibers, presumed to be Ia axons, was found in the L7 and S1 dorsal root ganglia. The existence of Nitric Oxide Synthase immunoreactive fibers (6-8 microm in diameter, not counting the myelin sheath) was confirmed in L7 and S1 dorsal roots and in the medial bundle of both dorsal roots before entering the dorsal root entry zone. By virtue of the funicular organization of Nitric Oxide Synthase immunoreactive fibers in the dorsal funiculus, the largest Nitric Oxide Synthase immunoreactive fibers represent stem Ia axons located in the deep portion of the dorsal funiculus close to the dorsomedial margin of the dorsal horn. Upon entering the gray matter of L7 and S1 segments and passing through the medial half of the dorsal horn, tapered Nitric Oxide Synthase immunoreactive collaterals of the stem Ia fibers pass through the deep layers of the dorsal horn and intermediate zone, and terminate in the group of homonymous motoneurons in L7 and S1 segments innervating the gastrocnemius-soleus muscles. Terminal fibers issued in the ventral horn intensely Nitric Oxide Synthase immunoreactive terminals with long axis ranging from 0.7 to >or=15.1 microm presumed to be Ia bNOS-IR boutons. This finding is unique in that it focuses directly on Nitric Oxide Synthase immunopositivity in the signalling transmitted by proprioceptive Ia fibers. Nitric Oxide Synthase immunoreactive boutons were found in the neuropil of Clarke's column of L4 segment, varying greatly in size from 0.7 to >or=15.1 microm in length x 0.7 to 4.8 microm wide. Subsequent to identification of the afferent Nitric Oxide Synthase immunoreactive limb of the monosynaptic Ia-motoneuron pathway on control sections, intramuscular injections of the retrograde tracer Fluorogold into the gastrocnemius-soleus muscles, combined with Nitric Oxide Synthase immunohistochemistry of L7 and S1 dorsal root ganglia, confirmed the existence of a number of medium-sized Nitric Oxide Synthase immunoreactive somata (1000-2000 microm(2) square area) in the dorsolateral part of both dorsal root ganglia, presumed to be proprioceptive Ia neurons. Concurrently, large Nitric Oxide Synthase immunoreactive fibers were detected at the input and output side of both dorsal root ganglia. S1 and S2 dorsal rhizotomy caused a marked depletion of Nitric Oxide Synthase immunoreactivity in the medial bundle of S1 and S2 dorsal roots and in the dorsal funiculus of S1, S2 and lower lumbar segments. In addition, anterograde degeneration of large Nitric Oxide Synthase immunoreactive Ia fibers in the dorsal funiculus of L7-S2 segments produces direct evidence that the afferent limb of the soleus H-reflex is Nitric Oxide Synthase immunoreactive and presents new immunohistochemical characteristics of the monosynaptic Ia-motoneuron pathway, unseparably coupled with the performance of the stretch reflex.

  • Premotor Nitric Oxide Synthase immunoreactive pathway connecting lumbar segments with the ventral motor nucleus of the cervical enlargement in the dog
    Journal of Chemical Neuroanatomy, 2004
    Co-Authors: Jozef Maršala, Imrich Lukáč, Dáša Čížková, Nadežda Lukacova, Karolina Kucharova, Martin Marsala
    Abstract:

    Abstract In this study we investigate the occurrence and origin of punctate Nitric Oxide Synthase immunoreactivity in the neuropil of the ventral motor nucleus in C7-Th1 segments of the dog spine, which are supposed to be the terminal field of an ascending premotor propriospinal Nitric Oxide Synthase-immunoreactive pathway. As the first step, Nitric Oxide Synthase immunohistochemistry was used to distinguish Nitric Oxide Synthase-immunoreactive staining of the ventral motor nucleus. Dense, punctate Nitric Oxide Synthase immunoreactivity was found on control sections in the neuropil of the ventral motor nucleus. After hemisection at Th10–11, axotomy-induced retrograde changes consisting in a strong upregulation of Nitric Oxide Synthase-containing neurons were found mostly unilaterally in lamina VIII, the medial part of lamina VII and in the pericentral region in all segments of the lumbosacral enlargement. Concurrently, a strong depletion of the punctate Nitric Oxide Synthase immunopositivity in the neuropil of the ventral motor nucleus ipsilaterally with the hemisection was detected, thus revealing that an uncrossed ascending premotor propriospinal pathway containing a fairly high number of Nitric Oxide Synthase-immunoreactive fibers terminates in the ventral motor nucleus. Application of the retrograde fluorescent tracer Fluorogold injected into the ventral motor nucleus and analysis of alternate sections processed for Nitric Oxide Synthase immunocytochemistry revealed the presence of Fluorogold-labeled and Nitric Oxide Synthase-immunoreactive axons in the ventrolateral funiculus and in the lateral and medial portions of the ventral column throughout the thoracic and upper lumbar segments. A noticeable number of Fluorogold-labeled and Nitric Oxide Synthase-immunoreactive somata detected on consecutive sections were found in the lumbosacral enlargement, mainly in laminae VIII–IX, the medial part of lamina VII and in the pericentral region (lamina X), ipsilaterally with the injection of Fluorogold into the ventral motor nucleus. In summary, the present study provides evidence for a hitherto unknown ascending premotor propriospinal Nitric Oxide Synthase-immunoreactive pathway connecting the lumbosacral enlargement with the motoneurons of the ventral motor nucleus in the dog.

  • segmental and laminar distributions of nicotinamide adenine dinucleotide phosphate diaphorase expressing and neuronal Nitric Oxide Synthase immunoreactive neurons versus radioassay detection of catalytic Nitric Oxide Synthase activity in the rabbit spinal cord
    Neuroscience, 1999
    Co-Authors: Nadežda Lukacova, Martin Marsala, D Cižkova, P Jalc, J Marsala
    Abstract:

    Abstract The distributions of neuronal Nitric Oxide Synthase-immunoreactive neurons and of nicotinamide adenine dinucleotide phosphate-diaphorase activity were studied in the C6, Th2, L1, L5, S2 and S3 segments and laminae in the rabbit spinal cord and compared with the catalytic Nitric Oxide Synthase activity, determined by monitoring the conversion of [ 3 H]arginine to [ 3 H]citrulline in the same segments and laminae. Morphologically, a heterogeneous population of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal Nitric Oxide Synthase-immunoreactive neurons was detected in the superficial and deep dorsal horn and the pericentral region in all segments studied, and in the intermediolateral cell column of the thoracic and lumbosacral segments. A disproportionate distribution of both neuronal categories which had a significantly higher number of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing rather than neuronal Nitric Oxide Synthase-immunoreactive cell bodies was found in all segments. The catalytic Nitric Oxide Synthase activity was distributed unequally in the C6, Th2, L1, L5, S2 and S3 segments, with a comparatively low value in the Th2 segment (70±5.1 d.p.m./μg protein) in comparison with the S3 segment, where the highest level (140±5.5 d.p.m./μg protein) was found. A close correlation between the number of neuronal Nitric Oxide Synthase-immunoreactive somata and catalytic Nitric Oxide Synthase activity was revealed in the dorsal horn (laminae I–VI). Whereas a low number of neuronal Nitric Oxide Synthase-immunoreactive somata in laminae VII–X was found in the L5, S2 and S3 segments, the values of catalytic Nitric Oxide Synthase activity in the same laminae and segments were found to be exceedingly high. These findings indicate that the occurrence of many neuronal Nitric Oxide Synthase-immunoreactive fibers (mainly axons), and dense, punctate, non-somatic neuronal Nitric Oxide Synthase immunopositivity in the neuropil staining of the same laminae and segments, can substantially enhance catalytic Nitric Oxide Synthase activity.

Nadežda Lukacova - One of the best experts on this subject based on the ideXlab platform.

  • the effect of a spinal cord hemisection on changes in Nitric Oxide Synthase pools in the site of injury and in regions located far away from the injured site
    Cellular and Molecular Neurobiology, 2006
    Co-Authors: Nadežda Lukacova, Maria Kolesarova, Karolina Kucharova, Jaroslav Pavel, Dalibor Kolesar, Jozef Radoňak, Martin Marsala, Malgorzata Chalimoniuk, Józef Langfort, J Marsala
    Abstract:

    : 1. The present study was designed to examine the Nitric Oxide Synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury. 2. A radioassay detection was used to determine Ca(2+)-dependent and inducible Nitric Oxide Synthase activities. Somal, axonal and neuropil neuronal Nitric Oxide Synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal Nitric Oxide Synthase immunoreactivity was made by an image analyser. The level of neuronal Nitric Oxide Synthase protein was measured by the Western blot analysis. 3. Our data show the increase of inducible Nitric Oxide Synthase activity and a decrease of Ca(2+)-dependent Nitric Oxide Synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible Nitric Oxide Synthase activity was increased at both time points. Ca(2+)-dependent Nitric Oxide Synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the Nitric Oxide Synthase activities and in the level of neuronal Nitric Oxide Synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal Nitric Oxide Synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal Nitric Oxide Synthase immunoreactive staining in the ventral part of rostrally oriented segments. 4. Our findings provide evidence that the changes in Nitric Oxide Synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of Nitric Oxide Synthase activity and neuronal Nitric Oxide Synthase immunoreactivity, measured segment by segment shows that Nitric Oxide may play a significant role in the stepping cycle in the quadrupeds.

  • Immunohistochemical, Histochemical and Radioassay Analysis of Nitric Oxide Synthase Immunoreactivity in the Lumbar and Sacral Dorsal Root Ganglia of the Dog
    Cellular and Molecular Neurobiology, 2006
    Co-Authors: Nadežda Lukacova, Dalibor Kolesar, Martin Marsala, Jozef Maršala
    Abstract:

    In this study, immunohistochemistry for neuronal Nitric Oxide Synthase (bNOS-IR), nicotinamide adenine dinucleotide phosphate diaphorase histochemistry (NADPHd) and Nitric Oxide Synthase radioassay were used to study the occurrence, number and distribution pattern of Nitric Oxide synthesizing neurons in the lumbar (L1–L7) and sacral (S1–S3) dorsal root ganglia of the dog. Nitric Oxide Synthase immunolabelling was present in a large number of small- (area 2000 μm^2) neurons. Although neuronal Nitric Oxide Synthase immunolabelling and histochemical staining provided intense staining of multiple small- and medium-sized neurons in all lumbar and sacral dorsal root ganglia, immunolabelled or histochemically stained somata exhibited little topographic distribution in individual dorsal root ganglia. Great heterogeneity was noticed in the immunolabelling of medium-sized Nitric Oxide Synthase immunopositive neurons ranging from lightly immunolabelled somata to heavily immunoreactive ones with completely obscured nuclei. Both staining procedures proved to be highly effective in visualizing intraganglionic fibers of various diameters. In general, the largest fibers revealed at the peripheral end of lumbar and sacral dorsal root ganglia were larger, 6.49–9.35 μm in diameter, while those running centrally and proceeding into the dorsal roots were about 30% reduced, ranging between 5.32 and 8.67 μm in diameter. Peripherally, the occurrence of Nitric Oxide Synthase detected in axonal profiles, and confirmed histochemically, in the specimens of the femoral and sciatic nerves, is the first indication of the presence of Nitric Oxide Synthase in the peripheral processes of somata located in L4–S2 dorsal root ganglia. Large and thin central Nitric Oxide Synthase immunoreactive processes of L1–S3 dorsal root ganglion neurons segregate shortly before entering the spinal cord, the former making a massive medial bundle in the dorsal root accompanied by a slim lateral bundle penetrating Lissauer's tract. Quantitative assessment of the distribution of bNOS-IR and/or NADPHd-stained neurons showed a peculiar pattern in relation to spinal levels. Apparent incongruity was found in the total number of NADPHd-stained versus bNOS-IR neurons, demonstrating a clear prevalence of small bNOS-IR somata in all lumbar ganglia, while medium-sized NADPHd-stained somata clearly prevailed all along the rostrocaudal axis with a peak in L5 ganglion. While the number of small bNOS-IR neurons clearly outnumbered NADPHd-stained and NADPHd-unstained somata in S1–S3 ganglia, an inverse relation appeared comparing the total number of medium-sized NADPHd-stained and NADPHd-unstained somata compared with the number of moderate and intense bNOS-IR neurons. Densitometry of bNOS-IR and NADPHd-stained neurons in lumbar and sacral ganglia revealed two distinct subsets of densitometric profiles, one relating to more often found medium-sized bNOS immunolabelled and the other, characteristic for moderately bNOS immunoreactive somata of the same cell size. Considerable differences in catalytic Nitric Oxide Synthase activity, determined by conversion of [^3H]arginine to [^3H]citrulline were obtained in lumbosacral dorsal root ganglia all along the lumbosacral intumescence, the lowest (0.898± 0.2 dpm/min/μg protein) being in the L4 dorsal root ganglion and the highest (4.194± 0.2 dpm/min/μg protein) in the S2 dorsal root ganglion.

  • the evidence for Nitric Oxide Synthase immunopositivity in the monosynaptic ia motoneuron pathway of the dog
    Experimental Neurology, 2005
    Co-Authors: Jozef Maršala, Nadežda Lukacova, Igor Sulla, Peter Wohlfahrt, Martin Marsala
    Abstract:

    In this study, Nitric Oxide Synthase immunohistochemistry supported by nicotinamide adenine dinucleotide phosphate diaphorase histochemistry was used to demonstrate the Nitric Oxide Synthase immunoreactivity in the monosynaptic Ia-motoneuron pathway exemplified by structural components of the afferent limb of the soleus H-reflex in the dog. A noticeable number of medium-sized intensely Nitric Oxide Synthase immunoreactive somata (1000-2000 microm(2) square area) and large intraganglionic Nitric Oxide Synthase immunoreactive fibers, presumed to be Ia axons, was found in the L7 and S1 dorsal root ganglia. The existence of Nitric Oxide Synthase immunoreactive fibers (6-8 microm in diameter, not counting the myelin sheath) was confirmed in L7 and S1 dorsal roots and in the medial bundle of both dorsal roots before entering the dorsal root entry zone. By virtue of the funicular organization of Nitric Oxide Synthase immunoreactive fibers in the dorsal funiculus, the largest Nitric Oxide Synthase immunoreactive fibers represent stem Ia axons located in the deep portion of the dorsal funiculus close to the dorsomedial margin of the dorsal horn. Upon entering the gray matter of L7 and S1 segments and passing through the medial half of the dorsal horn, tapered Nitric Oxide Synthase immunoreactive collaterals of the stem Ia fibers pass through the deep layers of the dorsal horn and intermediate zone, and terminate in the group of homonymous motoneurons in L7 and S1 segments innervating the gastrocnemius-soleus muscles. Terminal fibers issued in the ventral horn intensely Nitric Oxide Synthase immunoreactive terminals with long axis ranging from 0.7 to >or=15.1 microm presumed to be Ia bNOS-IR boutons. This finding is unique in that it focuses directly on Nitric Oxide Synthase immunopositivity in the signalling transmitted by proprioceptive Ia fibers. Nitric Oxide Synthase immunoreactive boutons were found in the neuropil of Clarke's column of L4 segment, varying greatly in size from 0.7 to >or=15.1 microm in length x 0.7 to 4.8 microm wide. Subsequent to identification of the afferent Nitric Oxide Synthase immunoreactive limb of the monosynaptic Ia-motoneuron pathway on control sections, intramuscular injections of the retrograde tracer Fluorogold into the gastrocnemius-soleus muscles, combined with Nitric Oxide Synthase immunohistochemistry of L7 and S1 dorsal root ganglia, confirmed the existence of a number of medium-sized Nitric Oxide Synthase immunoreactive somata (1000-2000 microm(2) square area) in the dorsolateral part of both dorsal root ganglia, presumed to be proprioceptive Ia neurons. Concurrently, large Nitric Oxide Synthase immunoreactive fibers were detected at the input and output side of both dorsal root ganglia. S1 and S2 dorsal rhizotomy caused a marked depletion of Nitric Oxide Synthase immunoreactivity in the medial bundle of S1 and S2 dorsal roots and in the dorsal funiculus of S1, S2 and lower lumbar segments. In addition, anterograde degeneration of large Nitric Oxide Synthase immunoreactive Ia fibers in the dorsal funiculus of L7-S2 segments produces direct evidence that the afferent limb of the soleus H-reflex is Nitric Oxide Synthase immunoreactive and presents new immunohistochemical characteristics of the monosynaptic Ia-motoneuron pathway, unseparably coupled with the performance of the stretch reflex.

  • Premotor Nitric Oxide Synthase immunoreactive pathway connecting lumbar segments with the ventral motor nucleus of the cervical enlargement in the dog
    Journal of Chemical Neuroanatomy, 2004
    Co-Authors: Jozef Maršala, Imrich Lukáč, Dáša Čížková, Nadežda Lukacova, Karolina Kucharova, Martin Marsala
    Abstract:

    Abstract In this study we investigate the occurrence and origin of punctate Nitric Oxide Synthase immunoreactivity in the neuropil of the ventral motor nucleus in C7-Th1 segments of the dog spine, which are supposed to be the terminal field of an ascending premotor propriospinal Nitric Oxide Synthase-immunoreactive pathway. As the first step, Nitric Oxide Synthase immunohistochemistry was used to distinguish Nitric Oxide Synthase-immunoreactive staining of the ventral motor nucleus. Dense, punctate Nitric Oxide Synthase immunoreactivity was found on control sections in the neuropil of the ventral motor nucleus. After hemisection at Th10–11, axotomy-induced retrograde changes consisting in a strong upregulation of Nitric Oxide Synthase-containing neurons were found mostly unilaterally in lamina VIII, the medial part of lamina VII and in the pericentral region in all segments of the lumbosacral enlargement. Concurrently, a strong depletion of the punctate Nitric Oxide Synthase immunopositivity in the neuropil of the ventral motor nucleus ipsilaterally with the hemisection was detected, thus revealing that an uncrossed ascending premotor propriospinal pathway containing a fairly high number of Nitric Oxide Synthase-immunoreactive fibers terminates in the ventral motor nucleus. Application of the retrograde fluorescent tracer Fluorogold injected into the ventral motor nucleus and analysis of alternate sections processed for Nitric Oxide Synthase immunocytochemistry revealed the presence of Fluorogold-labeled and Nitric Oxide Synthase-immunoreactive axons in the ventrolateral funiculus and in the lateral and medial portions of the ventral column throughout the thoracic and upper lumbar segments. A noticeable number of Fluorogold-labeled and Nitric Oxide Synthase-immunoreactive somata detected on consecutive sections were found in the lumbosacral enlargement, mainly in laminae VIII–IX, the medial part of lamina VII and in the pericentral region (lamina X), ipsilaterally with the injection of Fluorogold into the ventral motor nucleus. In summary, the present study provides evidence for a hitherto unknown ascending premotor propriospinal Nitric Oxide Synthase-immunoreactive pathway connecting the lumbosacral enlargement with the motoneurons of the ventral motor nucleus in the dog.

  • segmental and laminar distributions of nicotinamide adenine dinucleotide phosphate diaphorase expressing and neuronal Nitric Oxide Synthase immunoreactive neurons versus radioassay detection of catalytic Nitric Oxide Synthase activity in the rabbit spinal cord
    Neuroscience, 1999
    Co-Authors: Nadežda Lukacova, Martin Marsala, D Cižkova, P Jalc, J Marsala
    Abstract:

    Abstract The distributions of neuronal Nitric Oxide Synthase-immunoreactive neurons and of nicotinamide adenine dinucleotide phosphate-diaphorase activity were studied in the C6, Th2, L1, L5, S2 and S3 segments and laminae in the rabbit spinal cord and compared with the catalytic Nitric Oxide Synthase activity, determined by monitoring the conversion of [ 3 H]arginine to [ 3 H]citrulline in the same segments and laminae. Morphologically, a heterogeneous population of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal Nitric Oxide Synthase-immunoreactive neurons was detected in the superficial and deep dorsal horn and the pericentral region in all segments studied, and in the intermediolateral cell column of the thoracic and lumbosacral segments. A disproportionate distribution of both neuronal categories which had a significantly higher number of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing rather than neuronal Nitric Oxide Synthase-immunoreactive cell bodies was found in all segments. The catalytic Nitric Oxide Synthase activity was distributed unequally in the C6, Th2, L1, L5, S2 and S3 segments, with a comparatively low value in the Th2 segment (70±5.1 d.p.m./μg protein) in comparison with the S3 segment, where the highest level (140±5.5 d.p.m./μg protein) was found. A close correlation between the number of neuronal Nitric Oxide Synthase-immunoreactive somata and catalytic Nitric Oxide Synthase activity was revealed in the dorsal horn (laminae I–VI). Whereas a low number of neuronal Nitric Oxide Synthase-immunoreactive somata in laminae VII–X was found in the L5, S2 and S3 segments, the values of catalytic Nitric Oxide Synthase activity in the same laminae and segments were found to be exceedingly high. These findings indicate that the occurrence of many neuronal Nitric Oxide Synthase-immunoreactive fibers (mainly axons), and dense, punctate, non-somatic neuronal Nitric Oxide Synthase immunopositivity in the neuropil staining of the same laminae and segments, can substantially enhance catalytic Nitric Oxide Synthase activity.

J Marsala - One of the best experts on this subject based on the ideXlab platform.

  • the effect of a spinal cord hemisection on changes in Nitric Oxide Synthase pools in the site of injury and in regions located far away from the injured site
    Cellular and Molecular Neurobiology, 2006
    Co-Authors: Nadežda Lukacova, Maria Kolesarova, Karolina Kucharova, Jaroslav Pavel, Dalibor Kolesar, Jozef Radoňak, Martin Marsala, Malgorzata Chalimoniuk, Józef Langfort, J Marsala
    Abstract:

    : 1. The present study was designed to examine the Nitric Oxide Synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury. 2. A radioassay detection was used to determine Ca(2+)-dependent and inducible Nitric Oxide Synthase activities. Somal, axonal and neuropil neuronal Nitric Oxide Synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal Nitric Oxide Synthase immunoreactivity was made by an image analyser. The level of neuronal Nitric Oxide Synthase protein was measured by the Western blot analysis. 3. Our data show the increase of inducible Nitric Oxide Synthase activity and a decrease of Ca(2+)-dependent Nitric Oxide Synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible Nitric Oxide Synthase activity was increased at both time points. Ca(2+)-dependent Nitric Oxide Synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the Nitric Oxide Synthase activities and in the level of neuronal Nitric Oxide Synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal Nitric Oxide Synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal Nitric Oxide Synthase immunoreactive staining in the ventral part of rostrally oriented segments. 4. Our findings provide evidence that the changes in Nitric Oxide Synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of Nitric Oxide Synthase activity and neuronal Nitric Oxide Synthase immunoreactivity, measured segment by segment shows that Nitric Oxide may play a significant role in the stepping cycle in the quadrupeds.

  • segmental and laminar distributions of nicotinamide adenine dinucleotide phosphate diaphorase expressing and neuronal Nitric Oxide Synthase immunoreactive neurons versus radioassay detection of catalytic Nitric Oxide Synthase activity in the rabbit spinal cord
    Neuroscience, 1999
    Co-Authors: Nadežda Lukacova, Martin Marsala, D Cižkova, P Jalc, J Marsala
    Abstract:

    Abstract The distributions of neuronal Nitric Oxide Synthase-immunoreactive neurons and of nicotinamide adenine dinucleotide phosphate-diaphorase activity were studied in the C6, Th2, L1, L5, S2 and S3 segments and laminae in the rabbit spinal cord and compared with the catalytic Nitric Oxide Synthase activity, determined by monitoring the conversion of [ 3 H]arginine to [ 3 H]citrulline in the same segments and laminae. Morphologically, a heterogeneous population of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal Nitric Oxide Synthase-immunoreactive neurons was detected in the superficial and deep dorsal horn and the pericentral region in all segments studied, and in the intermediolateral cell column of the thoracic and lumbosacral segments. A disproportionate distribution of both neuronal categories which had a significantly higher number of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing rather than neuronal Nitric Oxide Synthase-immunoreactive cell bodies was found in all segments. The catalytic Nitric Oxide Synthase activity was distributed unequally in the C6, Th2, L1, L5, S2 and S3 segments, with a comparatively low value in the Th2 segment (70±5.1 d.p.m./μg protein) in comparison with the S3 segment, where the highest level (140±5.5 d.p.m./μg protein) was found. A close correlation between the number of neuronal Nitric Oxide Synthase-immunoreactive somata and catalytic Nitric Oxide Synthase activity was revealed in the dorsal horn (laminae I–VI). Whereas a low number of neuronal Nitric Oxide Synthase-immunoreactive somata in laminae VII–X was found in the L5, S2 and S3 segments, the values of catalytic Nitric Oxide Synthase activity in the same laminae and segments were found to be exceedingly high. These findings indicate that the occurrence of many neuronal Nitric Oxide Synthase-immunoreactive fibers (mainly axons), and dense, punctate, non-somatic neuronal Nitric Oxide Synthase immunopositivity in the neuropil staining of the same laminae and segments, can substantially enhance catalytic Nitric Oxide Synthase activity.

Jozef Maršala - One of the best experts on this subject based on the ideXlab platform.

  • Immunohistochemical, Histochemical and Radioassay Analysis of Nitric Oxide Synthase Immunoreactivity in the Lumbar and Sacral Dorsal Root Ganglia of the Dog
    Cellular and Molecular Neurobiology, 2006
    Co-Authors: Nadežda Lukacova, Dalibor Kolesar, Martin Marsala, Jozef Maršala
    Abstract:

    In this study, immunohistochemistry for neuronal Nitric Oxide Synthase (bNOS-IR), nicotinamide adenine dinucleotide phosphate diaphorase histochemistry (NADPHd) and Nitric Oxide Synthase radioassay were used to study the occurrence, number and distribution pattern of Nitric Oxide synthesizing neurons in the lumbar (L1–L7) and sacral (S1–S3) dorsal root ganglia of the dog. Nitric Oxide Synthase immunolabelling was present in a large number of small- (area 2000 μm^2) neurons. Although neuronal Nitric Oxide Synthase immunolabelling and histochemical staining provided intense staining of multiple small- and medium-sized neurons in all lumbar and sacral dorsal root ganglia, immunolabelled or histochemically stained somata exhibited little topographic distribution in individual dorsal root ganglia. Great heterogeneity was noticed in the immunolabelling of medium-sized Nitric Oxide Synthase immunopositive neurons ranging from lightly immunolabelled somata to heavily immunoreactive ones with completely obscured nuclei. Both staining procedures proved to be highly effective in visualizing intraganglionic fibers of various diameters. In general, the largest fibers revealed at the peripheral end of lumbar and sacral dorsal root ganglia were larger, 6.49–9.35 μm in diameter, while those running centrally and proceeding into the dorsal roots were about 30% reduced, ranging between 5.32 and 8.67 μm in diameter. Peripherally, the occurrence of Nitric Oxide Synthase detected in axonal profiles, and confirmed histochemically, in the specimens of the femoral and sciatic nerves, is the first indication of the presence of Nitric Oxide Synthase in the peripheral processes of somata located in L4–S2 dorsal root ganglia. Large and thin central Nitric Oxide Synthase immunoreactive processes of L1–S3 dorsal root ganglion neurons segregate shortly before entering the spinal cord, the former making a massive medial bundle in the dorsal root accompanied by a slim lateral bundle penetrating Lissauer's tract. Quantitative assessment of the distribution of bNOS-IR and/or NADPHd-stained neurons showed a peculiar pattern in relation to spinal levels. Apparent incongruity was found in the total number of NADPHd-stained versus bNOS-IR neurons, demonstrating a clear prevalence of small bNOS-IR somata in all lumbar ganglia, while medium-sized NADPHd-stained somata clearly prevailed all along the rostrocaudal axis with a peak in L5 ganglion. While the number of small bNOS-IR neurons clearly outnumbered NADPHd-stained and NADPHd-unstained somata in S1–S3 ganglia, an inverse relation appeared comparing the total number of medium-sized NADPHd-stained and NADPHd-unstained somata compared with the number of moderate and intense bNOS-IR neurons. Densitometry of bNOS-IR and NADPHd-stained neurons in lumbar and sacral ganglia revealed two distinct subsets of densitometric profiles, one relating to more often found medium-sized bNOS immunolabelled and the other, characteristic for moderately bNOS immunoreactive somata of the same cell size. Considerable differences in catalytic Nitric Oxide Synthase activity, determined by conversion of [^3H]arginine to [^3H]citrulline were obtained in lumbosacral dorsal root ganglia all along the lumbosacral intumescence, the lowest (0.898± 0.2 dpm/min/μg protein) being in the L4 dorsal root ganglion and the highest (4.194± 0.2 dpm/min/μg protein) in the S2 dorsal root ganglion.

  • the evidence for Nitric Oxide Synthase immunopositivity in the monosynaptic ia motoneuron pathway of the dog
    Experimental Neurology, 2005
    Co-Authors: Jozef Maršala, Nadežda Lukacova, Igor Sulla, Peter Wohlfahrt, Martin Marsala
    Abstract:

    In this study, Nitric Oxide Synthase immunohistochemistry supported by nicotinamide adenine dinucleotide phosphate diaphorase histochemistry was used to demonstrate the Nitric Oxide Synthase immunoreactivity in the monosynaptic Ia-motoneuron pathway exemplified by structural components of the afferent limb of the soleus H-reflex in the dog. A noticeable number of medium-sized intensely Nitric Oxide Synthase immunoreactive somata (1000-2000 microm(2) square area) and large intraganglionic Nitric Oxide Synthase immunoreactive fibers, presumed to be Ia axons, was found in the L7 and S1 dorsal root ganglia. The existence of Nitric Oxide Synthase immunoreactive fibers (6-8 microm in diameter, not counting the myelin sheath) was confirmed in L7 and S1 dorsal roots and in the medial bundle of both dorsal roots before entering the dorsal root entry zone. By virtue of the funicular organization of Nitric Oxide Synthase immunoreactive fibers in the dorsal funiculus, the largest Nitric Oxide Synthase immunoreactive fibers represent stem Ia axons located in the deep portion of the dorsal funiculus close to the dorsomedial margin of the dorsal horn. Upon entering the gray matter of L7 and S1 segments and passing through the medial half of the dorsal horn, tapered Nitric Oxide Synthase immunoreactive collaterals of the stem Ia fibers pass through the deep layers of the dorsal horn and intermediate zone, and terminate in the group of homonymous motoneurons in L7 and S1 segments innervating the gastrocnemius-soleus muscles. Terminal fibers issued in the ventral horn intensely Nitric Oxide Synthase immunoreactive terminals with long axis ranging from 0.7 to >or=15.1 microm presumed to be Ia bNOS-IR boutons. This finding is unique in that it focuses directly on Nitric Oxide Synthase immunopositivity in the signalling transmitted by proprioceptive Ia fibers. Nitric Oxide Synthase immunoreactive boutons were found in the neuropil of Clarke's column of L4 segment, varying greatly in size from 0.7 to >or=15.1 microm in length x 0.7 to 4.8 microm wide. Subsequent to identification of the afferent Nitric Oxide Synthase immunoreactive limb of the monosynaptic Ia-motoneuron pathway on control sections, intramuscular injections of the retrograde tracer Fluorogold into the gastrocnemius-soleus muscles, combined with Nitric Oxide Synthase immunohistochemistry of L7 and S1 dorsal root ganglia, confirmed the existence of a number of medium-sized Nitric Oxide Synthase immunoreactive somata (1000-2000 microm(2) square area) in the dorsolateral part of both dorsal root ganglia, presumed to be proprioceptive Ia neurons. Concurrently, large Nitric Oxide Synthase immunoreactive fibers were detected at the input and output side of both dorsal root ganglia. S1 and S2 dorsal rhizotomy caused a marked depletion of Nitric Oxide Synthase immunoreactivity in the medial bundle of S1 and S2 dorsal roots and in the dorsal funiculus of S1, S2 and lower lumbar segments. In addition, anterograde degeneration of large Nitric Oxide Synthase immunoreactive Ia fibers in the dorsal funiculus of L7-S2 segments produces direct evidence that the afferent limb of the soleus H-reflex is Nitric Oxide Synthase immunoreactive and presents new immunohistochemical characteristics of the monosynaptic Ia-motoneuron pathway, unseparably coupled with the performance of the stretch reflex.

  • Premotor Nitric Oxide Synthase immunoreactive pathway connecting lumbar segments with the ventral motor nucleus of the cervical enlargement in the dog
    Journal of Chemical Neuroanatomy, 2004
    Co-Authors: Jozef Maršala, Imrich Lukáč, Dáša Čížková, Nadežda Lukacova, Karolina Kucharova, Martin Marsala
    Abstract:

    Abstract In this study we investigate the occurrence and origin of punctate Nitric Oxide Synthase immunoreactivity in the neuropil of the ventral motor nucleus in C7-Th1 segments of the dog spine, which are supposed to be the terminal field of an ascending premotor propriospinal Nitric Oxide Synthase-immunoreactive pathway. As the first step, Nitric Oxide Synthase immunohistochemistry was used to distinguish Nitric Oxide Synthase-immunoreactive staining of the ventral motor nucleus. Dense, punctate Nitric Oxide Synthase immunoreactivity was found on control sections in the neuropil of the ventral motor nucleus. After hemisection at Th10–11, axotomy-induced retrograde changes consisting in a strong upregulation of Nitric Oxide Synthase-containing neurons were found mostly unilaterally in lamina VIII, the medial part of lamina VII and in the pericentral region in all segments of the lumbosacral enlargement. Concurrently, a strong depletion of the punctate Nitric Oxide Synthase immunopositivity in the neuropil of the ventral motor nucleus ipsilaterally with the hemisection was detected, thus revealing that an uncrossed ascending premotor propriospinal pathway containing a fairly high number of Nitric Oxide Synthase-immunoreactive fibers terminates in the ventral motor nucleus. Application of the retrograde fluorescent tracer Fluorogold injected into the ventral motor nucleus and analysis of alternate sections processed for Nitric Oxide Synthase immunocytochemistry revealed the presence of Fluorogold-labeled and Nitric Oxide Synthase-immunoreactive axons in the ventrolateral funiculus and in the lateral and medial portions of the ventral column throughout the thoracic and upper lumbar segments. A noticeable number of Fluorogold-labeled and Nitric Oxide Synthase-immunoreactive somata detected on consecutive sections were found in the lumbosacral enlargement, mainly in laminae VIII–IX, the medial part of lamina VII and in the pericentral region (lamina X), ipsilaterally with the injection of Fluorogold into the ventral motor nucleus. In summary, the present study provides evidence for a hitherto unknown ascending premotor propriospinal Nitric Oxide Synthase-immunoreactive pathway connecting the lumbosacral enlargement with the motoneurons of the ventral motor nucleus in the dog.

Domingos S R Souza - One of the best experts on this subject based on the ideXlab platform.

  • retaining perivascular tissue of human saphenous vein grafts protects against surgical and distension induced damage and preserves endothelial Nitric Oxide Synthase and Nitric Oxide Synthase activity
    The Journal of Thoracic and Cardiovascular Surgery, 2009
    Co-Authors: Michael R Dashwood, Kay Savage, Janice Tsui, A Dooley, Sidney Shaw, Maria Fernandez S Alfonso, Lennart Bodin, Domingos S R Souza
    Abstract:

    Objective Conventional harvesting of saphenous vein used for coronary artery bypass surgery induces a vasospasm that is overcome by high-pressure distension. Saphenous vein harvested with its cushion of perivascular tissue by a "no touch" technique does not undergo vasospasm and distension is not required, leading to an improved graft patency. The aim of this study is to investigate the effect of surgical damage and high-pressure distension on endothelial integrity and endothelial Nitric Oxide Synthase expression and activity in saphenous vein harvested with and without perivascular tissue. Methods Saphenous veins from patients (n = 26) undergoing coronary artery bypass surgery were prepared with and without perivascular tissue. We analyzed the effect of 300 mm Hg distension on morphology and endothelial Nitric Oxide Synthase/Nitric Oxide Synthase activity using a combination of immunohistochemistry, Western blot analysis, reverse transcriptase polymerase chain reaction, and enzyme assay in distended (with and without perivascular tissue) compared with nondistended (with and without perivascular tissue) segments. Results Distension induced substantial damage to the luminal endothelium (assessed by CD31 staining) and vessel wall. Endothelial Nitric Oxide Synthase expression and activity were significantly reduced by high-pressure distension and removal of, or damage to, perivascular tissue. The effect of distension was significantly less for those with perivascular tissue than for those without perivascular tissue in most cases. Conclusion The success of the saphenous vein used as a bypass graft is affected by surgical trauma and distension. Veins removed with minimal damage exhibit increased patency rates. We show that retention of perivascular tissue on saphenous vein prepared for coronary artery bypass surgery by the "no touch" technique protects against distension-induced damage, preserves vessel morphology, and maintains endothelial Nitric Oxide Synthase/Nitric Oxide Synthase activity.