Oliver W. Gramlich

Retinal Ganglion Cell Loss Is Accompanied by Antibody Depositions and Increased Levels of Microglia after Immunization with Retinal Antigens

Background Antibodies against retinal and optic nerve antigens are detectable in glaucoma patients. Recent studies using a model of experimental autoimmune glaucoma demonstrated that immunization with certain ocular antigens causes an immun-mediated retinal ganglion cell loss in rats. Methodology/Principal Findings Rats immunized with a retinal ganglion cell layer homogenate (RGA) had a reduced retinal ganglion cell density on retinal flatmounts (p = 0.007) and a lower number of Brn3+retinal ganglion cells (p = 0.0001) after six weeks. The autoreactive antibody development against retina and optic nerve was examined throughout the study. The levels of autoreactive antibodies continuously increased up to 6 weeks (retina: p = 0.004; optic nerve: p = 0.000003). Additionally, antibody deposits were detected in the retina (p = 0.02). After 6 weeks a reactive gliosis (GFAP density: RGA: 174.7±41.9; CO: 137.6±36.8, p = 0.0006; %GFAP+ area: RGA: 8.5±3.4; CO: 5.9±3.6, p = 0.006) as well as elevated level of Iba1+ microglia cells (p = 0.003) was observed in retinas of RGA animals. Conclusions/Significance Our findings suggest that these antibodies play a substantial role in mechanisms leading to retinal ganglion cell death. This seems to lead to glia cell activation as well as the invasion of microglia, which might be associated with debris clearance.

Enhanced Insight into the Autoimmune Component of Glaucoma: IgG Autoantibody Accumulation and Pro-Inflammatory Conditions in Human Glaucomatous Retina

Background There is accumulating evidence that autoimmune components, such as autoantibodies and autoantibody depositions, play a role in the pathogenesis of neurodegenerative diseases like Alzheimeŕs disease or Multiple Sclerosis. Due to alterations of autoantibody patterns in sera and aqueous humor, an autoimmune component is also assumed in the pathogenesis of glaucoma, a common reason for irreversible blindness worldwide. So far there has been no convincing evidence that autoantibodies are accumulated in the retina of glaucoma patients and that the local immune homeostasis might be affected. Methods and Results Six human glaucomatous donor eyes and nine samples from donors with no recorded ocular disease were included. Antibody microarrays were used to examine the patterns of pro-inflammatory proteins and complement proteins. Analysis of TNF-α and interleukin levels revealed a slight up-regulation exclusively in the glaucomatous group, while complement protein levels were not altered. IgG autoantibody accumulations and/or cellular components were determined by immunohistology (n = 4 per group). A significantly reduced number of retinal ganglion cells was found in the glaucomatous group (healthy: 104±7 nuclei/mm, glaucoma: 67±9 nuclei/mm; p = 0.0007). Cell loss was accompanied by strong retinal IgG autoantibody accumulations, which were at least twice as high as in healthy subjects (healthy: 5.0±0.5 IgG deposits/100 cells, glaucoma: 9.4±1.9 IgG deposits/100 cells; p = 0.004). CD27+ cells and CD27+/IgG+ plasma cells were observed in all glaucomatous subjects, but not in controls. Conclusion This work provides serious evidence for the occurrence of IgG antibody deposition and plasma cells in human glaucomatous retina. Moreover, the results suggest that these IgG deposits occurred in a pro-inflammatory environment which seems to be maintained locally by immune-competent cells like microglia. Thereby, glaucoma features an immunological involvement comparable to other neurodegenerative diseases, but also shows a multifactorial pathomechanism, which diverges and might be linked to the specific nature of both eye and retina.

Autoreactive antibodies and loss of retinal ganglion cells in rats induced by immunization with ocular antigens.

PURPOSE In an experimental autoimmune animal model, retinal ganglion cell (RGC) loss was induced through immunization with glaucoma-related antigens. The target of this study was to investigate the pathomechanism behind this decline and the serum antibody reactivity against ocular and neuronal tissues after immunization with glaucoma- and non-glaucoma-associated antigens. METHODS Rats immunized with optic nerve antigen homogenate (ONA) or keratin (KER) were compared to control rats (CO). Intraocular pressure (IOP) was measured, and the fundi were examined regularly. Four weeks afterward, cells were counted in retinal flat mounts. Retina, optic nerve, and brain sections from healthy animals and optic nerve sections from immunized animals were incubated with serum collected at different time points. The occurrence of autoreactive antibodies was examined. Signs of antibody deposits, microglia activation, and demyelination were sought in optic nerves of immunized animals. Brain sections were examined for abnormalities. RESULTS No IOP or fundus changes were observed. Animals immunized with ONA showed a significant cell loss compared with the CO group. Elevated autoreactive antibodies against retina, optic nerve, and brain were observed. Animals immunized with KER, despite their immunologic response against KER, demonstrated neither RGC loss, nor increased development of autoreactive antibodies. Optic nerve from animals immunized with ONA demonstrated antibody accumulation, glia activation, and demyelination. No such observations were made in the KER or CO groups. Brain sections were without pathologic findings. CONCLUSIONS Systemic autoimmunity against ocular and neuronal epitopes, mediated by accordant autoreactive antibodies, is involved in the inflammatory processes that cause RGC degeneration in this experimental animal model.

Does autoimmunity play a part in the pathogenesis of glaucoma

Glaucoma is a chronic neurodegenerative disease and one of the leading causes of blindness. Several risk factors have been described, e.g. an elevated intraocular pressure (IOP), oxidative stress or mitochondrial dysfunction. Additionally, alterations in serum antibody profiles of glaucoma patients, upregulation (e.g. anti-HSP60, anti-MBP) and downregulation (e.g. anti-14-3-3), have been described, but it still remains elusive if the autoantibodies seen in glaucoma are an epiphenomenon or causative. However, it is known that elicited autoimmunity causes retinal ganglion cell loss resulting in glaucomatous-like damage and according to the autoaggressive nature of some autoantibodies we found antibody deposits in human glaucomatous retinae in a pro-inflammatory environment. Furthermore, glaucomatous serum has the potential to influence neuroretinal cell regulatory processes. Importantly, we demonstrate that some autoantibodies hold neuroprotective potential for neuroretinal cells. The protective nature of autoantibodies and the molecular mechanisms underlying the very sensitive equilibrium between autoaggression and protection remain subject of future examinations and offer promising target sites for new therapeutic approaches. Additionally, the changes in antibody profiles could be used as highly sensitive and specific marker for diagnostics purposes. Early diagnosis and intervention in risk patients would offer the chance of early treatment and to slow down the progression of glaucoma and delay the resulting blindness.

Autoimmune biomarkers in glaucoma patients

There is growing evidence showing an autoimmune involvement in the pathogenesis of glaucoma, and that alterations in natural occurring autoantibody levels play a key role. The upregulation of autoantibodies can be associated with fatal conditions, but several studies demonstrate that natural autoantibodies entail also protective characteristics and influence the protein expression of neuroretinal cells. A disbalance of natural occurring autoantibodies may shift the physiological equilibrium of protective immunity leading to a predisposition for developing glaucoma. This article highlights recent advances in understanding of autoimmune mechanisms in the pathogenesis of glaucoma.

Glaucoma related Proteomic Alterations in Human Retina Samples

Glaucoma related proteomic changes have been documented in cell and animal models. However, proteomic studies investigating on human retina samples are still rare. In the present work, retina samples of glaucoma and non-glaucoma control donors have been examined by a state-of-the-art mass spectrometry (MS) workflow to uncover glaucoma related proteomic changes. More than 600 proteins could be identified with high confidence (FDR < 1%) in human retina samples. Distinct proteomic changes have been observed in 10% of proteins encircling mitochondrial and nucleus species. Numerous proteins showed a significant glaucoma related level change (p < 0.05) or distinct tendency of alteration (p < 0.1). Candidates were documented to be involved in cellular development, stress and cell death. Increase of stress related proteins and decrease of new glaucoma related candidates, ADP/ATP translocase 3 (ANT3), PC4 and SRFS1-interacting protein 1 (DFS70) and methyl-CpG-binding protein 2 (MeCp2) could be documented by MS. Moreover, candidates could be validated by Accurate Inclusion Mass Screening (AIMS) and immunostaining and supported for the retinal ganglion cell layer (GCL) by laser capture microdissection (LCM) in porcine and human eye cryosections. The workflow allowed a detailed view into the human retina proteome highlighting new molecular players ANT3, DFS70 and MeCp2 associated to glaucoma.

Effect of ischemia duration on autoantibody response in rats undergoing retinal ischemia-reperfusion.

Both the innate and the adaptive immune systems are involved in the pathogenic processes following ischemia-reperfusion injury. We analyzed the possible correlation between the duration of ischemia and autoantibody diversification in a model of ocular ischemia. Rats were subjected to 30, 45, or 90 min of ischemia, and retinal ganglion cell (RGC) density and antibody reactivity were analyzed via customized protein microarray slides. After ocular ischemia, significant alterations in antibody response were observed, while increasing exposure caused more severe RGC damage. Distinct antibody responses after ischemia were detected; these alterations comprised decreased reactivities against cyclophilin A and glyceraldehyde-3-phosphate dehydrogenase, possibly due to increased binding of circulating antibodies to debris material. Other antibodies, like those against α5β1-integrin or β2-adrenergic receptor, were upregulated after ischemia.

PE-11, a peptide derived from chromogranin B, in the rat eye

The aim of the study was to investigate the presence and distribution of PE-11, a peptide derived from chromogranin B, in the rat eye. For this purpose, newborn rats were injected with a single dosage of 50mg/kg capsaicin subcutaneously under the neck fold and after three months, particular eye tissues were dissected and the concentration of PE-11-like immunoreactivity was determined by radioimmunoassay. Furthermore, PE-11-like immunoreactivities were characterized in an extract of the rat eye by reversed phase HPLC. Then, the distribution pattern of PE-11 was investigated in the rat eye and rat trigeminal ganglion by immunofluorescence. As a result, PE-11 was present in each tissue of the rat eye and capsaicin pretreatment led to a 88.05% (±7.07) and a 64.26% (±14.17) decrease of the levels of PE-11 in the cornea and choroid/sclera, respectively, and to a complete loss in the iris/ciliary body complex. Approximately 70% of immunoreactivities detected by the PE-11 antiserum have been found to represent authentic PE-11. Sparse nerve fibers were visualized in the corneal and uveal stroma, surrounding blood vessels at the limbus, ciliary body and choroid and in association with the dilator and sphincter muscle. Furthermore, immunoreactivity was present in the corneal endothelium. In the retina and optic nerve, glia was labeled. In the rat trigeminal ganglion, PE-11-immunoreactivity was visualized in small and medium sized ganglion cells with a diameter of up to 30μm. In conclusion, there is unequivocal evidence that PE-11 is a constituent of capsaicin-sensitive sensory neurons innervating the rat eye and the distribution pattern is typically peptidergic in the peripheral innervation but in the retina completely atypical for neuropeptides and unique.

GE-25-like immunoreactivity in the rat eye.

This study aimed to investigate the presence and distribution of the chromogranin A-derived peptide GE-25 in the rat eye. The molecular form detected by the GE-25 antiserum was evaluated in the rat trigeminal ganglion, retina and remaining tissues of the rat eye by means of Western blots and the distribution pattern of GE-25-like immunoreactivity was studied in the rat eye and rat trigeminal ganglion by immunofluorescence. One single band of approximately 70kDa was stained in the trigeminal ganglion and retina which represents the uncleaved intact chromogranin A indicating that the proteolytic processing of chromogranin A to GE-25 is limited in these tissues. Sparse GE-25-like immunoreactive nerve fibers were visualized in the corneal stroma, at the limbus around blood vessels, in the sphincter and dilator muscle and stroma of the iris, in the stroma of the ciliary body and ciliary processes and in the stroma and around blood vessels in the choroid. This distribution pattern is characteristic for neuropeptides whereas the presence of immunoreactivity in the corneal endothelium and in Müller glia in the retina is atypical. GE-25-like immunoreactivity was found in small to medium-sized ganglion cells in the rat trigeminal ganglion clearly indicating that the nerve fibers in the rat eye are of sensory origin. The colocalization of GE-25-immunoreactivity with SP-immunoreactivity in the rat ciliary body is in agreement with the presumption of the sensory nature of the innervation of the anterior segment of the eye by GE-25.

Repeated Intraocular Pressure Measurement in Awake Lewis Rats Does Not Bias Retinal Ganglion Cell Survival

Purpose: The TonoPen applanation tonometry is an established method for intraocular pressure (IOP) measurement. The IOP is one of the main variables affecting retinal ganglion cell (RGC) loss in experimental animal models in ophthalmology and the main risk factor for human glaucoma. In this study, we examined if IOP measurements with the TonoPen itself lead to retinal ganglion cell loss or any other possible retina damages, such as intraocular bleedings or ablation, in Lewis rats. Methods: Three groups of rats (n = 5 each) were formed. IOP monitoring, using a TonoPen XL, was performed on groups 1 and 3. Animals in groups 1 and 2 received funduscopies before and after one and two weeks of the study, in order to detect possible abnormalities. After two weeks, retinal flatmounts were stained to detect ganglion cells. RGCs were manually counted in eight predefined areas to compare mean RGC densities between groups 1 and 2 (IOP readings vs. no readings), using student t-test. Results: No significant difference in RGC density between animals that underwent IOP readings and controls could be observed (p = 0.8). As expected, no IOP alterations were monitored in groups 1 and 3 throughout the study. No retinal abnormalities, such as bleeding or retina ablation, were detectable. Conclusion: We could detect no effects on retinal ganglion cell survival in Lewis rats or any other damages to the retina caused by IOP measurements using a TonoPen XL. This study proposes that repeated applanation tonometry does not affect RGC numbers, one of the main monitored variables in most glaucoma model studies. Therefore, the use of a TonoPen XL for repeated IOP monitoring in Lewis rats can be considered harmless.