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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

Intermittent intraocular hypertension and analysis of the optic nerve axon density. a An exemplary profile of 12 iOHT eyes conducted at the ninth day of the experiment shows the effective mean IOP (ordinate) during simulation of a 1-h wavelike IOP profile (abscissa). IOP reduces to 10 mmHg after anesthesia, and IOP is raised to 35 mmHg for 20 min (interval I), followed by 45 mmHg for another 20 min (interval II). The loop was released to simulate a pressure drop. IOP was reset to 35 mmHg for 20 min (interval III), and IOP decreased to 12.5 mmHg during recovery phase. Arrows indicate important pressure changes before and after iOHT. b The survival of optic nerve axons was evaluated as the number of axons in an area of 0.05 mm2. Injured eyes of iOHT group showed a significant loss of optic nerve axons compared with fellow eyes in the same group (gray bar, p < 0.01) and control eyes (black bar, p < 0.01). Additionally, axon densities of injured eyes of iOHT group were significantly reduced compared with fellow eyes of iOHT + Belimumab group (grey bar, p < 0.01) but not to injured eyes of the iOHT + Belimumab group (horizontal lines, p = 0.05). Note that no significant loss of axonal density was observed for injured eyes of the Belimumab-treated group compared with fellow eyes (p = 0.5) and with control eyes (p = 0.07). Significances are indicated as follows: **p < 0.01. c A representative p-phenylenediamine-stained ultrathin transverse cross-sections (×10 magnification) shows the area where 15 high-resolution pictures were taken for the optic nerve axon density analysis. In total, 32 % of the optic nerve was analyzed for axon density survival. Scale bar represents 200 μm. d–h Representative high-resolution images (×100 magnification) show different stages of degeneration in optic nerve cross-sections. Healthy axons were present in control optic nerves (ON, d), while fellow eyes of iOHT ON (e) and iOHT + Belimumab (g) showed a slight damage, including swollen and collapsing axons indicated by arrows. Major axon degeneration with bulged myelin sheaths around axons (arrows) and gliotic areas (triangle) were observed in injured ONs of iOHT group (f) and iOHT + Belimumab group (h). Scale bar represents 20 μm
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Fig2: Intermittent intraocular hypertension and analysis of the optic nerve axon density. a An exemplary profile of 12 iOHT eyes conducted at the ninth day of the experiment shows the effective mean IOP (ordinate) during simulation of a 1-h wavelike IOP profile (abscissa). IOP reduces to 10 mmHg after anesthesia, and IOP is raised to 35 mmHg for 20 min (interval I), followed by 45 mmHg for another 20 min (interval II). The loop was released to simulate a pressure drop. IOP was reset to 35 mmHg for 20 min (interval III), and IOP decreased to 12.5 mmHg during recovery phase. Arrows indicate important pressure changes before and after iOHT. b The survival of optic nerve axons was evaluated as the number of axons in an area of 0.05 mm2. Injured eyes of iOHT group showed a significant loss of optic nerve axons compared with fellow eyes in the same group (gray bar, p < 0.01) and control eyes (black bar, p < 0.01). Additionally, axon densities of injured eyes of iOHT group were significantly reduced compared with fellow eyes of iOHT + Belimumab group (grey bar, p < 0.01) but not to injured eyes of the iOHT + Belimumab group (horizontal lines, p = 0.05). Note that no significant loss of axonal density was observed for injured eyes of the Belimumab-treated group compared with fellow eyes (p = 0.5) and with control eyes (p = 0.07). Significances are indicated as follows: **p < 0.01. c A representative p-phenylenediamine-stained ultrathin transverse cross-sections (×10 magnification) shows the area where 15 high-resolution pictures were taken for the optic nerve axon density analysis. In total, 32 % of the optic nerve was analyzed for axon density survival. Scale bar represents 200 μm. d–h Representative high-resolution images (×100 magnification) show different stages of degeneration in optic nerve cross-sections. Healthy axons were present in control optic nerves (ON, d), while fellow eyes of iOHT ON (e) and iOHT + Belimumab (g) showed a slight damage, including swollen and collapsing axons indicated by arrows. Major axon degeneration with bulged myelin sheaths around axons (arrows) and gliotic areas (triangle) were observed in injured ONs of iOHT group (f) and iOHT + Belimumab group (h). Scale bar represents 20 μm

Mentions: In accordance with the previously described technique for minimally invasive short-term IOP elevations [19, 20], iOHT was conducted in a wavelike profile. Adjusting a silicone loop around the limbus of the right eye (Fig. 1a), it is possible to narrow the anterior iridocorneal angle without modifying the shape of the eye or inducing subsequent structural injuries to the eye, which was monitored by regular funduscopy. In contrast to the first description of this method by Joos [19], the loop was not placed posterior to the limbus, but around the limbus. The loop slightly compresses Schlemm’s canal and the episcleral veins (Fig. 1b’) and provokes a marginal relocation of the iridocorneal angle, resulting in an increase of aqueous humor outflow resistance. Rats were anesthetized using 0.185 ml/kg body weight medetomidine (Dorbene vet., Pfizer, New York, NY, intramuscular administration), and eyes received oxybuprocain (Novesine 0.4 %, OmniVision, Puchheim, Germany) prior to, and regularly during, the procedure. Fellow eyes were covered with dexpanthenol to avoid corneal dehydration and prevent superficial irritations (Bepanthen, Bayer, Leverkusen, Germany). Once the loop is attached around the eye, the IOP rises immediately but returns to baseline levels within minutes when no adjustment of the loop occurs [20]. If the mean IOP was lower than the desired level, the diameter of the loop was slightly decreased. Conversely, the diameter was increased by gentle release of the loop when the mean IOP was too high. To mimic IOP fluctuations, which is a common observation in glaucoma patients, a wavelike IOP profile was applied to the animal’s eyes. This profile consists of three intervals of 20 min to 35 mmHg (interval I), 45 mmHg (interval II), and again to 35 mmHg (interval III). The loop was removed for 10 min between intervals II and III to simulate a pressure drop to a physiologic IOP level (Fig. 2a). The IOP elevation was performed on five consecutive days followed by 2 days of rest for 5 weeks. In week 6, IOP was elevated on two consecutive days and animals were sacrificed afterwards. Thus, each animal received a total of 27 unilateral IOP elevations. One group received IOP elevation (iOHT), while another group received 10 mg/kg of the B lymphocyte inhibitor Belimumab (Benlysta, GlaxoSmithKline, Parma, Italy, intravenous administration), initially 3 days before the first iOHT and weekly throughout the study in addition to IOP elevations as described above (iOHT + Belimumab). Control animals underwent the same examinations in a corresponding time frame but were not subjected to iOHT injury (CTRL).Fig. 1


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)

Intermittent intraocular hypertension and analysis of the optic nerve axon density. a An exemplary profile of 12 iOHT eyes conducted at the ninth day of the experiment shows the effective mean IOP (ordinate) during simulation of a 1-h wavelike IOP profile (abscissa). IOP reduces to 10 mmHg after anesthesia, and IOP is raised to 35 mmHg for 20 min (interval I), followed by 45 mmHg for another 20 min (interval II). The loop was released to simulate a pressure drop. IOP was reset to 35 mmHg for 20 min (interval III), and IOP decreased to 12.5 mmHg during recovery phase. Arrows indicate important pressure changes before and after iOHT. b The survival of optic nerve axons was evaluated as the number of axons in an area of 0.05 mm2. Injured eyes of iOHT group showed a significant loss of optic nerve axons compared with fellow eyes in the same group (gray bar, p < 0.01) and control eyes (black bar, p < 0.01). Additionally, axon densities of injured eyes of iOHT group were significantly reduced compared with fellow eyes of iOHT + Belimumab group (grey bar, p < 0.01) but not to injured eyes of the iOHT + Belimumab group (horizontal lines, p = 0.05). Note that no significant loss of axonal density was observed for injured eyes of the Belimumab-treated group compared with fellow eyes (p = 0.5) and with control eyes (p = 0.07). Significances are indicated as follows: **p < 0.01. c A representative p-phenylenediamine-stained ultrathin transverse cross-sections (×10 magnification) shows the area where 15 high-resolution pictures were taken for the optic nerve axon density analysis. In total, 32 % of the optic nerve was analyzed for axon density survival. Scale bar represents 200 μm. d–h Representative high-resolution images (×100 magnification) show different stages of degeneration in optic nerve cross-sections. Healthy axons were present in control optic nerves (ON, d), while fellow eyes of iOHT ON (e) and iOHT + Belimumab (g) showed a slight damage, including swollen and collapsing axons indicated by arrows. Major axon degeneration with bulged myelin sheaths around axons (arrows) and gliotic areas (triangle) were observed in injured ONs of iOHT group (f) and iOHT + Belimumab group (h). Scale bar represents 20 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836145&req=5

Fig2: Intermittent intraocular hypertension and analysis of the optic nerve axon density. a An exemplary profile of 12 iOHT eyes conducted at the ninth day of the experiment shows the effective mean IOP (ordinate) during simulation of a 1-h wavelike IOP profile (abscissa). IOP reduces to 10 mmHg after anesthesia, and IOP is raised to 35 mmHg for 20 min (interval I), followed by 45 mmHg for another 20 min (interval II). The loop was released to simulate a pressure drop. IOP was reset to 35 mmHg for 20 min (interval III), and IOP decreased to 12.5 mmHg during recovery phase. Arrows indicate important pressure changes before and after iOHT. b The survival of optic nerve axons was evaluated as the number of axons in an area of 0.05 mm2. Injured eyes of iOHT group showed a significant loss of optic nerve axons compared with fellow eyes in the same group (gray bar, p < 0.01) and control eyes (black bar, p < 0.01). Additionally, axon densities of injured eyes of iOHT group were significantly reduced compared with fellow eyes of iOHT + Belimumab group (grey bar, p < 0.01) but not to injured eyes of the iOHT + Belimumab group (horizontal lines, p = 0.05). Note that no significant loss of axonal density was observed for injured eyes of the Belimumab-treated group compared with fellow eyes (p = 0.5) and with control eyes (p = 0.07). Significances are indicated as follows: **p < 0.01. c A representative p-phenylenediamine-stained ultrathin transverse cross-sections (×10 magnification) shows the area where 15 high-resolution pictures were taken for the optic nerve axon density analysis. In total, 32 % of the optic nerve was analyzed for axon density survival. Scale bar represents 200 μm. d–h Representative high-resolution images (×100 magnification) show different stages of degeneration in optic nerve cross-sections. Healthy axons were present in control optic nerves (ON, d), while fellow eyes of iOHT ON (e) and iOHT + Belimumab (g) showed a slight damage, including swollen and collapsing axons indicated by arrows. Major axon degeneration with bulged myelin sheaths around axons (arrows) and gliotic areas (triangle) were observed in injured ONs of iOHT group (f) and iOHT + Belimumab group (h). Scale bar represents 20 μm
Mentions: In accordance with the previously described technique for minimally invasive short-term IOP elevations [19, 20], iOHT was conducted in a wavelike profile. Adjusting a silicone loop around the limbus of the right eye (Fig. 1a), it is possible to narrow the anterior iridocorneal angle without modifying the shape of the eye or inducing subsequent structural injuries to the eye, which was monitored by regular funduscopy. In contrast to the first description of this method by Joos [19], the loop was not placed posterior to the limbus, but around the limbus. The loop slightly compresses Schlemm’s canal and the episcleral veins (Fig. 1b’) and provokes a marginal relocation of the iridocorneal angle, resulting in an increase of aqueous humor outflow resistance. Rats were anesthetized using 0.185 ml/kg body weight medetomidine (Dorbene vet., Pfizer, New York, NY, intramuscular administration), and eyes received oxybuprocain (Novesine 0.4 %, OmniVision, Puchheim, Germany) prior to, and regularly during, the procedure. Fellow eyes were covered with dexpanthenol to avoid corneal dehydration and prevent superficial irritations (Bepanthen, Bayer, Leverkusen, Germany). Once the loop is attached around the eye, the IOP rises immediately but returns to baseline levels within minutes when no adjustment of the loop occurs [20]. If the mean IOP was lower than the desired level, the diameter of the loop was slightly decreased. Conversely, the diameter was increased by gentle release of the loop when the mean IOP was too high. To mimic IOP fluctuations, which is a common observation in glaucoma patients, a wavelike IOP profile was applied to the animal’s eyes. This profile consists of three intervals of 20 min to 35 mmHg (interval I), 45 mmHg (interval II), and again to 35 mmHg (interval III). The loop was removed for 10 min between intervals II and III to simulate a pressure drop to a physiologic IOP level (Fig. 2a). The IOP elevation was performed on five consecutive days followed by 2 days of rest for 5 weeks. In week 6, IOP was elevated on two consecutive days and animals were sacrificed afterwards. Thus, each animal received a total of 27 unilateral IOP elevations. One group received IOP elevation (iOHT), while another group received 10 mg/kg of the B lymphocyte inhibitor Belimumab (Benlysta, GlaxoSmithKline, Parma, Italy, intravenous administration), initially 3 days before the first iOHT and weekly throughout the study in addition to IOP elevations as described above (iOHT + Belimumab). Control animals underwent the same examinations in a corresponding time frame but were not subjected to iOHT injury (CTRL).Fig. 1

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