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The Presence of the Neuronal Nitric Oxide Synthase Isoform in the Intervertebral Disk

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Abstract

Intervertebral disk degeneration is a progressive and debilitating disease with multifactorial causes. Nitric oxide (NO) might contribute to the cell death pathway. We evaluated the presence of the constitutive form of the neuronal NOS (nNOS) in both health and degenerated intervertebral disk through qPCR and immunohistochemistry. We also analyzed the potential role of nNOS modulation in the tail needle puncture model of intervertebral disk degeneration. Male Wistar rats were submitted to percutaneous disk puncture with a 21-gauge needle of coccygeal vertebras. The selective nNOS pharmacological inhibitor N (ω)-propyl-l-arginine (NPLA) or a nNOS-target siRNA (siRNAnNOShum_4400) was injected immediately after the intervertebral disk puncture with a 30-gauge needle. Signs of disk degeneration were analyzed by in vivo magnetic resonance imaging and histological score. We found that intact intervertebral disks express low levels of nNOS mRNA. Disk injury caused a 4 fold increase in nNOS mRNA content at 5 h post disk lesion. However, NPLA or nNOS-target siRNA slight mitigate the intervertebral disk degenerative progress. Our data show evidence of the nNOS presence in the intervertebral disk and its upregulation during degeneration. Further studies would disclose the nNOS role and its potential therapeutical value in the intervertebral disk degeneration.

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Acknowledgments

We thank Célia Ap. da Silva (FORP, USP) and Vani Maria Alves (FMRP, USP) for their excellent technical assistance. This research was supported by CAPES/PNPD, Brazil. The equipment and drugs used in this work were acquired from FAPESP, CNPq, and CAPES, Brazil. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Morphology, Physiology and Basic Pathology, Dental School, University of São Paulo (USP), Ribeirão Prêto, SP, 14049-904, Brazil

    Vitor Castania, Ana Carolina Issy, João Walter Silveira, Nádia Rubia Ferreira & Elaine Del Bel

  2. Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil

    Frederico Rogério Ferreira

  3. Technology for Gene Therapy Laboratory, University of Brasilia - UnB/FAV, Brasília, DF, Brazil

    Simoneide S. Titze-de-Almeida, Fernando F. B. Resende & Ricardo Titze-de-Almeida

  4. Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo (USP), São Paulo, Brazil

    Ana Carolina Issy, Frederico Rogério Ferreira & Elaine Del Bel

  5. Department of Biomechanics, Medicine and Rehabilitation of the Locomotor Apparatus, School of Medicine, University of São Paulo (USP), Ribeirão Prêto, Brazil

    Helton L. A. Defino

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  1. Vitor Castania
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  2. Ana Carolina Issy
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Correspondence to Elaine Del Bel.

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12640_2016_9676_MOESM1_ESM.tif

Supplementary material 1 (TIFF 5633 kb). Figure 1 Representative histological specimens of rat intervertebral disc 2 days after 21-gauge needle puncture stained with Alcian Blue. A Intact control (×2.5) showing preserved nucleus pulposus, which cells are presented in detail in panel A′ (×10), and well organized annulus fibrosus fibers. B Lesioned disc showing absence of nucleus pulposus cells, and a fragmentation of annulus fibrosus collagen fibers, showed in detail in B′ (×10). C Lesioned disc injected with NPLA also showing an obliteration of nucleus pulposus central cavity through a disorganized annulus fibrosus fibers, showed in C′ (×10)

12640_2016_9676_MOESM2_ESM.tif

Supplementary material 2 (TIFF 3787 kb). Figure 2 Representative histologic specimens of rat intervertebral disc (×2.5) 2 days after 21-gauge needle puncture. Normal control (A), lesioned disc treated with vehicle (B), and lesioned disc treated with NPLA (300 nmol; C). Note, in C, that annulus fibrosus seems more regular, and the increased cellularity of nucleus pulposus compared with B

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Castania, V., Issy, A.C., Silveira, J.W. et al. The Presence of the Neuronal Nitric Oxide Synthase Isoform in the Intervertebral Disk. Neurotox Res 31, 148–161 (2017). https://doi.org/10.1007/s12640-016-9676-7

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