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Immune response after intermittent minimally invasive intraocular pressure elevations in an experimental animal model of glaucoma.

Gramlich OW, Teister J, Neumann M, Tao X, Beck S, von Pein HD, Pfeiffer N, Grus FH - J Neuroinflammation (2016)

Bottom Line: A wavelike IOP profile led to a significant neurodegeneration of optic nerve axons (-10.6 %, p < 0.001) and RGC (-19.5 %, p = 0.02) in iOHT eyes compared with fellow eyes.Belimumab-treated animals only showed slightly higher axonal survival and reduced serum IgG concentration (-29 %) after iOHT.Neuroinflammatory events, indicated by significantly upregulated microglia activation and IgG autoantibody depositions, were shown in all injured retinas.

View Article: PubMed Central - PubMed

Affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131, Mainz, Germany.

ABSTRACT

Background: Elevated intraocular pressure (IOP), as well as fluctuations in IOP, is a main risk factor for glaucoma, but its pathogenic effect has not yet been clarified. Beyond the multifactorial pathology of the disease, autoimmune mechanisms seem to be linked to retinal ganglion cell (RGC) death. This study aimed to identify if intermittent IOP elevations in vivo (i) elicit neurodegeneration, (ii) provokes an immune response and (iii) whether progression of RGC loss can be attenuated by the B lymphocyte inhibitor Belimumab.

Methods: Using an intermittent ocular hypertension model (iOHT), Long Evans rats (n = 21) underwent 27 unilateral simulations of a fluctuating pressure profile. Nine of these animals received Belimumab, and additional seven rats served as normotensive controls. Axonal density was analyzed in PPD-stained optic nerve cross-sections. Retinal cross-sections were immunostained against Brn3a, Iba1, and IgG autoantibody depositions. Serum IgG concentration and IgG reactivities were determined using ELISA and protein microarrays. Data was analyzed using ANOVA and Tukey HSD test (unequal N) or student's independent t test by groups.

Results: A wavelike IOP profile led to a significant neurodegeneration of optic nerve axons (-10.6 %, p < 0.001) and RGC (-19.5 %, p = 0.02) in iOHT eyes compared with fellow eyes. Belimumab-treated animals only showed slightly higher axonal survival and reduced serum IgG concentration (-29 %) after iOHT. Neuroinflammatory events, indicated by significantly upregulated microglia activation and IgG autoantibody depositions, were shown in all injured retinas. Significantly elevated serum autoantibody immunoreactivities against glutathione-S-transferase, spectrin, and transferrin were observed after iOHT and were negatively correlated to the axon density.

Conclusions: Intermittent IOP elevations are sufficient to provoke neurodegeneration in the optic nerve and the retina and elicit changes of IgG autoantibody reactivities. Although the inhibition of B lymphocyte activation failed to ameliorate axonal survival, the correlation between damage and changes in the autoantibody reactivity suggests that autoantibody profiling could be useful as a biomarker for glaucoma.

No MeSH data available.


Related in: MedlinePlus

Activation of microglia and analysis of the distribution in the retina. a The activation of microglia was analyzed as Iba1-positive cells per mm retina in naso-temporal cross-sections of the eye. After iOHT, a significant increase of microglia occurred in fellow and injured eyes (both p < 0.05) compared with control eyes. No significant changes were observed for Belimumab-treated animals. Significant values are indicated as follows: *p < 0.05. b–f Representative images of retinal cross-sections (×20 magnification) show the numbers and distribution of Iba1-positive cells (arrows) in diverse retinal layers in control (CTRL, b), fellow and injured eyes of iOHT group (c and d, respectively), and fellow and injured eyes of iOHT + Belimumab group (e and f, respectively). Magnifications of representative microglia show a transformation from non-activated ramified phenotype with long ramifications in controls (b) to an activated, amoeboid phenotype in injured eyes (d, f). g Percentile distribution of microglia cells in retinal layers are given for all groups. Data were normalized in relation to the difference of the total number of microglia between the groups. Significant values are indicated as follows: *p < 0.05, **p < 0.01. Abbreviations: GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, CHO choroid. Scale bar represents 100 μm
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Fig4: Activation of microglia and analysis of the distribution in the retina. a The activation of microglia was analyzed as Iba1-positive cells per mm retina in naso-temporal cross-sections of the eye. After iOHT, a significant increase of microglia occurred in fellow and injured eyes (both p < 0.05) compared with control eyes. No significant changes were observed for Belimumab-treated animals. Significant values are indicated as follows: *p < 0.05. b–f Representative images of retinal cross-sections (×20 magnification) show the numbers and distribution of Iba1-positive cells (arrows) in diverse retinal layers in control (CTRL, b), fellow and injured eyes of iOHT group (c and d, respectively), and fellow and injured eyes of iOHT + Belimumab group (e and f, respectively). Magnifications of representative microglia show a transformation from non-activated ramified phenotype with long ramifications in controls (b) to an activated, amoeboid phenotype in injured eyes (d, f). g Percentile distribution of microglia cells in retinal layers are given for all groups. Data were normalized in relation to the difference of the total number of microglia between the groups. Significant values are indicated as follows: *p < 0.05, **p < 0.01. Abbreviations: GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, CHO choroid. Scale bar represents 100 μm

Mentions: To detect activation of retinal microglia, immunohistological staining of retinal cross-sections was performed and showed the occurrence of Iba1-positive cells in almost all retinal layers. In comparison to the control group, the amount of Iba1-positive cells was significantly higher in the eyes of the iOHT group (p < 0.03) and virtually identical to numbers found in animals which received iOHT + Belimumab treatment (p = 0.99, Fig. 4a, Table 1). The majority of microglia cells in the eyes of the iOHT groups that were located in the ganglion cell layer displayed an amoeboid phenotype (Fig. 4c–f, inset), while microglia in controls showed a ramified morphology with high numbers of long and thin processes (Fig. 4b, inset). Likewise, microglia cells of the inner plexiform layers and outer retinal layers exhibited a uniform, ramified morphology in all groups.Fig. 4


Immune response after intermittent minimally invasive intraocular pressure elevations in an experimental animal model of glaucoma.

Gramlich OW, Teister J, Neumann M, Tao X, Beck S, von Pein HD, Pfeiffer N, Grus FH - J Neuroinflammation (2016)

Activation of microglia and analysis of the distribution in the retina. a The activation of microglia was analyzed as Iba1-positive cells per mm retina in naso-temporal cross-sections of the eye. After iOHT, a significant increase of microglia occurred in fellow and injured eyes (both p < 0.05) compared with control eyes. No significant changes were observed for Belimumab-treated animals. Significant values are indicated as follows: *p < 0.05. b–f Representative images of retinal cross-sections (×20 magnification) show the numbers and distribution of Iba1-positive cells (arrows) in diverse retinal layers in control (CTRL, b), fellow and injured eyes of iOHT group (c and d, respectively), and fellow and injured eyes of iOHT + Belimumab group (e and f, respectively). Magnifications of representative microglia show a transformation from non-activated ramified phenotype with long ramifications in controls (b) to an activated, amoeboid phenotype in injured eyes (d, f). g Percentile distribution of microglia cells in retinal layers are given for all groups. Data were normalized in relation to the difference of the total number of microglia between the groups. Significant values are indicated as follows: *p < 0.05, **p < 0.01. Abbreviations: GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, CHO choroid. Scale bar represents 100 μm
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Fig4: Activation of microglia and analysis of the distribution in the retina. a The activation of microglia was analyzed as Iba1-positive cells per mm retina in naso-temporal cross-sections of the eye. After iOHT, a significant increase of microglia occurred in fellow and injured eyes (both p < 0.05) compared with control eyes. No significant changes were observed for Belimumab-treated animals. Significant values are indicated as follows: *p < 0.05. b–f Representative images of retinal cross-sections (×20 magnification) show the numbers and distribution of Iba1-positive cells (arrows) in diverse retinal layers in control (CTRL, b), fellow and injured eyes of iOHT group (c and d, respectively), and fellow and injured eyes of iOHT + Belimumab group (e and f, respectively). Magnifications of representative microglia show a transformation from non-activated ramified phenotype with long ramifications in controls (b) to an activated, amoeboid phenotype in injured eyes (d, f). g Percentile distribution of microglia cells in retinal layers are given for all groups. Data were normalized in relation to the difference of the total number of microglia between the groups. Significant values are indicated as follows: *p < 0.05, **p < 0.01. Abbreviations: GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, CHO choroid. Scale bar represents 100 μm
Mentions: To detect activation of retinal microglia, immunohistological staining of retinal cross-sections was performed and showed the occurrence of Iba1-positive cells in almost all retinal layers. In comparison to the control group, the amount of Iba1-positive cells was significantly higher in the eyes of the iOHT group (p < 0.03) and virtually identical to numbers found in animals which received iOHT + Belimumab treatment (p = 0.99, Fig. 4a, Table 1). The majority of microglia cells in the eyes of the iOHT groups that were located in the ganglion cell layer displayed an amoeboid phenotype (Fig. 4c–f, inset), while microglia in controls showed a ramified morphology with high numbers of long and thin processes (Fig. 4b, inset). Likewise, microglia cells of the inner plexiform layers and outer retinal layers exhibited a uniform, ramified morphology in all groups.Fig. 4

Bottom Line: A wavelike IOP profile led to a significant neurodegeneration of optic nerve axons (-10.6 %, p < 0.001) and RGC (-19.5 %, p = 0.02) in iOHT eyes compared with fellow eyes.Belimumab-treated animals only showed slightly higher axonal survival and reduced serum IgG concentration (-29 %) after iOHT.Neuroinflammatory events, indicated by significantly upregulated microglia activation and IgG autoantibody depositions, were shown in all injured retinas.

View Article: PubMed Central - PubMed

Affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131, Mainz, Germany.

ABSTRACT

Background: Elevated intraocular pressure (IOP), as well as fluctuations in IOP, is a main risk factor for glaucoma, but its pathogenic effect has not yet been clarified. Beyond the multifactorial pathology of the disease, autoimmune mechanisms seem to be linked to retinal ganglion cell (RGC) death. This study aimed to identify if intermittent IOP elevations in vivo (i) elicit neurodegeneration, (ii) provokes an immune response and (iii) whether progression of RGC loss can be attenuated by the B lymphocyte inhibitor Belimumab.

Methods: Using an intermittent ocular hypertension model (iOHT), Long Evans rats (n = 21) underwent 27 unilateral simulations of a fluctuating pressure profile. Nine of these animals received Belimumab, and additional seven rats served as normotensive controls. Axonal density was analyzed in PPD-stained optic nerve cross-sections. Retinal cross-sections were immunostained against Brn3a, Iba1, and IgG autoantibody depositions. Serum IgG concentration and IgG reactivities were determined using ELISA and protein microarrays. Data was analyzed using ANOVA and Tukey HSD test (unequal N) or student's independent t test by groups.

Results: A wavelike IOP profile led to a significant neurodegeneration of optic nerve axons (-10.6 %, p < 0.001) and RGC (-19.5 %, p = 0.02) in iOHT eyes compared with fellow eyes. Belimumab-treated animals only showed slightly higher axonal survival and reduced serum IgG concentration (-29 %) after iOHT. Neuroinflammatory events, indicated by significantly upregulated microglia activation and IgG autoantibody depositions, were shown in all injured retinas. Significantly elevated serum autoantibody immunoreactivities against glutathione-S-transferase, spectrin, and transferrin were observed after iOHT and were negatively correlated to the axon density.

Conclusions: Intermittent IOP elevations are sufficient to provoke neurodegeneration in the optic nerve and the retina and elicit changes of IgG autoantibody reactivities. Although the inhibition of B lymphocyte activation failed to ameliorate axonal survival, the correlation between damage and changes in the autoantibody reactivity suggests that autoantibody profiling could be useful as a biomarker for glaucoma.

No MeSH data available.


Related in: MedlinePlus