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Inducible nitric oxide synthase, Nos2, does not mediate optic neuropathy and retinopathy in the DBA/2J glaucoma model.

Libby RT, Howell GR, Pang IH, Savinova OV, Mehalow AK, Barter JW, Smith RS, Clark AF, John SW - BMC Neurosci (2007)

Bottom Line: The expression of Nos2 in the optic nerve head was analyzed at both the RNA and protein levels at different stages of disease pathogenesis.Optic nerve head Nos2 RNA levels varied and increased during moderate but decreased at early and severe stages of disease.Additionally, neither Nos2 deficiency nor aminoguanidine had any detectable affect on the glaucomatous optic nerve damage.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, ME 04609, USA. Richard_Libby@URMC.rochester.edu

ABSTRACT

Background: Nitric oxide synthase 2 (NOS2) contributes to neural death in some settings, but its role in glaucoma remains controversial. NOS2 is implicated in retinal ganglion cell degeneration in a rat glaucoma model in which intraocular pressure (IOP) is experimentally elevated by blood vessel cauterization, but not in a rat glaucoma model where IOP was elevated by injection of hypertonic saline. To test the importance of NOS2 for an inherited glaucoma, in this study we both genetically and pharmacologically decreased NOS2 activity in the DBA/2J mouse glaucoma model.

Methods: The expression of Nos2 in the optic nerve head was analyzed at both the RNA and protein levels at different stages of disease pathogenesis. To test the involvement of Nos2 in glaucomatous neurodegeneration, a allele of Nos2 was backcrossed into DBA/2J mice and the incidence and severity of glaucoma was assessed in mice of each Nos2 genotype. Additionally, DBA/2J mice were treated with the NOS2 inhibitor aminoguanidine and the disease compared to untreated mice.

Results: Optic nerve head Nos2 RNA levels varied and increased during moderate but decreased at early and severe stages of disease. Despite the presence of a few NOS2 positive cells in the optic nerve head, NOS2 protein was not substantially increased during the glaucoma. Genetic deficiency of Nos2 or aminoguanidine treatment did not alter the IOP profile of DBA/2J mice. Additionally, neither Nos2 deficiency nor aminoguanidine had any detectable affect on the glaucomatous optic nerve damage.

Conclusion: Glaucomatous neurodegeneration in DBA/2J mice does not require NOS2 activity. Further experiments involving various models are needed to assess the general importance of Nos2 in glaucoma.

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NOS2 expression during glaucoma progression. (A) Optic nerve head Nos2 RNA expression. Relative expression levels of Nos2 at different stages of glaucoma were determined using ΔCT ratios (see Methods). 'Control' eyes from 4 months old DBA/2J mice were considered to be pre-glaucomatous and showed no axon damage. 'Sample' eyes from 10.5 months old DBA/2J mice were assessed for glaucomatous axon damage and grouped into the no or early, moderate or severe stages of damage (see Methods). Values given are relative to pre-glaucoma controls. Nos2 expression was variable within each disease group (indicated by the SEM bars). Overall it decreased comparing pre-glaucoma with no or early glaucoma and increased modestly in eyes with moderate glaucoma (p < 0.05). (B) NOS2 protein localization in LPS-treated kidney (above) and no primary control (below). The NOS2 antibody shows strong and specific NOS2 staining (Red) in LPS-treated kidneys, with no non-specific binding of the secondary antibody. (C) NOS2 protein localization in the optic nerve heads of 10.5 months old DBA/2J mice, with the lower panels showing higher magnification of the boxed regions. There was no obvious correlation between NOS2 localization and glaucoma progression, although rare NOS2 positive cells were seen in eyes with severe glaucoma (arrow). All sections were processed, stained and imaged under identical conditions. The images shown here are representative of three different sections from three different eyes with the three different stages of glaucoma. Bars = 75 μm.
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Figure 1: NOS2 expression during glaucoma progression. (A) Optic nerve head Nos2 RNA expression. Relative expression levels of Nos2 at different stages of glaucoma were determined using ΔCT ratios (see Methods). 'Control' eyes from 4 months old DBA/2J mice were considered to be pre-glaucomatous and showed no axon damage. 'Sample' eyes from 10.5 months old DBA/2J mice were assessed for glaucomatous axon damage and grouped into the no or early, moderate or severe stages of damage (see Methods). Values given are relative to pre-glaucoma controls. Nos2 expression was variable within each disease group (indicated by the SEM bars). Overall it decreased comparing pre-glaucoma with no or early glaucoma and increased modestly in eyes with moderate glaucoma (p < 0.05). (B) NOS2 protein localization in LPS-treated kidney (above) and no primary control (below). The NOS2 antibody shows strong and specific NOS2 staining (Red) in LPS-treated kidneys, with no non-specific binding of the secondary antibody. (C) NOS2 protein localization in the optic nerve heads of 10.5 months old DBA/2J mice, with the lower panels showing higher magnification of the boxed regions. There was no obvious correlation between NOS2 localization and glaucoma progression, although rare NOS2 positive cells were seen in eyes with severe glaucoma (arrow). All sections were processed, stained and imaged under identical conditions. The images shown here are representative of three different sections from three different eyes with the three different stages of glaucoma. Bars = 75 μm.

Mentions: In some [19-22,32] but not all studies [35,36,47], increased expression of Nos2 is reported in glaucoma patients and rat models of glaucoma. To determine the temporal and spatial expression pattern of Nos2 in DBA/2J mice, we examined RNA and protein levels in the optic nerve head at different stages of glaucoma. The optic nerve head extending from the vitreal surface to approximately the non-myelinated-myelin junction of the optic nerve was dissected and used for RNA preparation. There was a modest increase of Nos2 RNA by quantitative real time PCR analysis in eyes with moderate axon damage, but Nos2 levels appeared to be lower in eyes with early or severe damage (Fig. 1A). Immunohistochemical analysis of sagittal sections did not detect NOS2 protein in either the retina or optic nerve head in the majority of eyes at each stage of disease. As a positive control, kidney sections from mice treated with lipopolysaccahride (LPS) were immunoreacted with the NOS2 antibody and NOS2 positive cells were detected (Fig. 1B). Rare cells that were NOS2 positive were identified in the optic nerve head of two severely affected eyes (Fig. 1C). Although these expression studies provided no clear association between NOS2 and glaucoma, NOS2 is a powerful modulator of cell functions that may be transiently expressed by single cells. Therefore, we continued to test the role of NOS2 in this glaucoma by both genetically and pharmacologically inhibiting its function.


Inducible nitric oxide synthase, Nos2, does not mediate optic neuropathy and retinopathy in the DBA/2J glaucoma model.

Libby RT, Howell GR, Pang IH, Savinova OV, Mehalow AK, Barter JW, Smith RS, Clark AF, John SW - BMC Neurosci (2007)

NOS2 expression during glaucoma progression. (A) Optic nerve head Nos2 RNA expression. Relative expression levels of Nos2 at different stages of glaucoma were determined using ΔCT ratios (see Methods). 'Control' eyes from 4 months old DBA/2J mice were considered to be pre-glaucomatous and showed no axon damage. 'Sample' eyes from 10.5 months old DBA/2J mice were assessed for glaucomatous axon damage and grouped into the no or early, moderate or severe stages of damage (see Methods). Values given are relative to pre-glaucoma controls. Nos2 expression was variable within each disease group (indicated by the SEM bars). Overall it decreased comparing pre-glaucoma with no or early glaucoma and increased modestly in eyes with moderate glaucoma (p < 0.05). (B) NOS2 protein localization in LPS-treated kidney (above) and no primary control (below). The NOS2 antibody shows strong and specific NOS2 staining (Red) in LPS-treated kidneys, with no non-specific binding of the secondary antibody. (C) NOS2 protein localization in the optic nerve heads of 10.5 months old DBA/2J mice, with the lower panels showing higher magnification of the boxed regions. There was no obvious correlation between NOS2 localization and glaucoma progression, although rare NOS2 positive cells were seen in eyes with severe glaucoma (arrow). All sections were processed, stained and imaged under identical conditions. The images shown here are representative of three different sections from three different eyes with the three different stages of glaucoma. Bars = 75 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2211487&req=5

Figure 1: NOS2 expression during glaucoma progression. (A) Optic nerve head Nos2 RNA expression. Relative expression levels of Nos2 at different stages of glaucoma were determined using ΔCT ratios (see Methods). 'Control' eyes from 4 months old DBA/2J mice were considered to be pre-glaucomatous and showed no axon damage. 'Sample' eyes from 10.5 months old DBA/2J mice were assessed for glaucomatous axon damage and grouped into the no or early, moderate or severe stages of damage (see Methods). Values given are relative to pre-glaucoma controls. Nos2 expression was variable within each disease group (indicated by the SEM bars). Overall it decreased comparing pre-glaucoma with no or early glaucoma and increased modestly in eyes with moderate glaucoma (p < 0.05). (B) NOS2 protein localization in LPS-treated kidney (above) and no primary control (below). The NOS2 antibody shows strong and specific NOS2 staining (Red) in LPS-treated kidneys, with no non-specific binding of the secondary antibody. (C) NOS2 protein localization in the optic nerve heads of 10.5 months old DBA/2J mice, with the lower panels showing higher magnification of the boxed regions. There was no obvious correlation between NOS2 localization and glaucoma progression, although rare NOS2 positive cells were seen in eyes with severe glaucoma (arrow). All sections were processed, stained and imaged under identical conditions. The images shown here are representative of three different sections from three different eyes with the three different stages of glaucoma. Bars = 75 μm.
Mentions: In some [19-22,32] but not all studies [35,36,47], increased expression of Nos2 is reported in glaucoma patients and rat models of glaucoma. To determine the temporal and spatial expression pattern of Nos2 in DBA/2J mice, we examined RNA and protein levels in the optic nerve head at different stages of glaucoma. The optic nerve head extending from the vitreal surface to approximately the non-myelinated-myelin junction of the optic nerve was dissected and used for RNA preparation. There was a modest increase of Nos2 RNA by quantitative real time PCR analysis in eyes with moderate axon damage, but Nos2 levels appeared to be lower in eyes with early or severe damage (Fig. 1A). Immunohistochemical analysis of sagittal sections did not detect NOS2 protein in either the retina or optic nerve head in the majority of eyes at each stage of disease. As a positive control, kidney sections from mice treated with lipopolysaccahride (LPS) were immunoreacted with the NOS2 antibody and NOS2 positive cells were detected (Fig. 1B). Rare cells that were NOS2 positive were identified in the optic nerve head of two severely affected eyes (Fig. 1C). Although these expression studies provided no clear association between NOS2 and glaucoma, NOS2 is a powerful modulator of cell functions that may be transiently expressed by single cells. Therefore, we continued to test the role of NOS2 in this glaucoma by both genetically and pharmacologically inhibiting its function.

Bottom Line: The expression of Nos2 in the optic nerve head was analyzed at both the RNA and protein levels at different stages of disease pathogenesis.Optic nerve head Nos2 RNA levels varied and increased during moderate but decreased at early and severe stages of disease.Additionally, neither Nos2 deficiency nor aminoguanidine had any detectable affect on the glaucomatous optic nerve damage.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, ME 04609, USA. Richard_Libby@URMC.rochester.edu

ABSTRACT

Background: Nitric oxide synthase 2 (NOS2) contributes to neural death in some settings, but its role in glaucoma remains controversial. NOS2 is implicated in retinal ganglion cell degeneration in a rat glaucoma model in which intraocular pressure (IOP) is experimentally elevated by blood vessel cauterization, but not in a rat glaucoma model where IOP was elevated by injection of hypertonic saline. To test the importance of NOS2 for an inherited glaucoma, in this study we both genetically and pharmacologically decreased NOS2 activity in the DBA/2J mouse glaucoma model.

Methods: The expression of Nos2 in the optic nerve head was analyzed at both the RNA and protein levels at different stages of disease pathogenesis. To test the involvement of Nos2 in glaucomatous neurodegeneration, a allele of Nos2 was backcrossed into DBA/2J mice and the incidence and severity of glaucoma was assessed in mice of each Nos2 genotype. Additionally, DBA/2J mice were treated with the NOS2 inhibitor aminoguanidine and the disease compared to untreated mice.

Results: Optic nerve head Nos2 RNA levels varied and increased during moderate but decreased at early and severe stages of disease. Despite the presence of a few NOS2 positive cells in the optic nerve head, NOS2 protein was not substantially increased during the glaucoma. Genetic deficiency of Nos2 or aminoguanidine treatment did not alter the IOP profile of DBA/2J mice. Additionally, neither Nos2 deficiency nor aminoguanidine had any detectable affect on the glaucomatous optic nerve damage.

Conclusion: Glaucomatous neurodegeneration in DBA/2J mice does not require NOS2 activity. Further experiments involving various models are needed to assess the general importance of Nos2 in glaucoma.

Show MeSH
Related in: MedlinePlus