Nitric Oxide In Multiple Sclerosis

Diseases such as Multiple Sclerosis and Guillain-Barre Syndrome are characterized not only by widespread loss of Myelin from Nerve Fibers, but also by widespread Inflammation in the Central and Peripheral Nervous Systems, respectively.

While the DeMyelination alone is sufficient to block Conduction and thereby cause symptoms, there is increasing evidence that the Inflammation may also contribute significantly to the Conduction Block, although the mechanisms are not understood.

Nitric Oxide is an important Inflammatory Mediator which is elevated within the Central Nervous System in Multiple Sclerosis and which can be experimentally applied to tissues using Nitric Oxide Donors.

We report that such compounds cause Reversible Conduction Block in both Normal and DeMyelinated Axons of the Central and Peripheral Nervous Systems.

Notably, Conduction in DeMyelinated and early ReMyelinated Axons is particularly sensitive to block by Nitric Oxide, so that at lower concentrations, including those expected at sites of Inflammation, DeMyelinated Axons are selectively affected.

We therefore propose that inflammation may directly cause symptoms via Nitric Oxide release, and that the inhibition of such release may open a new therapeutic avenue for DeMyelinating Diseases.

Inducible Nitric Oxide Synthase (iNOS) is expressed by Astrocytes in DeMyelinating regions of Multiple Sclerosis (MS) Brain plaques, suggesting that NO contributes to MS pathology.

Since the ImmunoSuppressive Cytokine IFN-ß ameliorates MS disease activity, it is of interest to assess the modulatory role of IFN-ß on NO production.

We studied the effects of IFN-ß, as well as Dexamethasone, IL-10, and Transforming Growth Factor-ß (TGF-ß), on Cytokine-induced NO production by the human Astrocytoma cell line, A172.

L-NMMA and Aminoguanidine, competitive inhibitors of iNOS suppressed NO production as measured by the NO byproduct, Nitrite, as did Interferon-ß.

Dexamethasone enhanced NO production, and IFN-ß decreased the amount of the enhancement. Neither IL-10 nor TGF-ß inhibited Nitrite production.

The therapeutic effect of IFN-ß in MS may be partly due to suppression of Pathogenic NO production.

We investigated Nitric Oxide (NO) production and the presence of Nitric Oxide Synthase (NOS) in the Mammary Gland by use of an organ culture system of rat Mammary Glands.

Mammary Glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17ß-Estradiol and Progesterone and were diced into approximately 3-mm cubes.

Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days.

The amount of NO produced spontaneously by the cultured Mammary Glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS.

This enhancement of NO production was completely eliminated by addition of HydroCortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyzes with specific AntiSera against NOS isoforms such as iNOS.

Endothelial NOS (eNOS), and Brain NOS (bNOS) showed ImmunoReactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the Mammary Glands in the culture without LPS.

The ImmunoReactive band of iNOS was highly intense after the treatment of Mammary Glands with LPS, whereas the corresponding eNOS Immunoreactive band was faded.

The ImmunoHistoChemical study of anti-iNOS AntiSerum on frozen sections of the cultured Mammary Glands showed that an ImmunoReactive substance with the AntiSerum was localized to the basal layer (composed of MyoEpithelial Cells of Alveoli and Lactiferous Ducts) of the Mammary Epithelia.

And to the Endothelium of blood vessels that penetrated into the Interstitium of the Mammary Glands.

HistoChemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the Mammary Glands.

Similar observations were noted in the ImmunoHistoChemistry of eNOS. In contrast, the ImmunoReactive signal with the bNOS AntiSerum was barely detected in the Epithelial parts of Alveoli and Lactiferous Ducts of the Mammary Glands.

These observations demonstrate that three isoforms of NOS are present not only in the Endothelium of blood vessels, but also in the Parenchymal Cells (the glandular Epithelium) of the rat Mammary Gland.