The role of TLR4/NF-κB signaling pathway in activated microglia of rats with chronic high intraocular pressure and vitro scratch injury-induced microglia.

Glaucoma is a kind of blind-causing disease with structural damages of optic nerve and defection of visual field. It is believed that the death of retinal ganglion cell (RGC) is a consequential event of over-reactive immune orchestral cells such as microglia. Previous evidences in animal and clinical studies show the innate immunity plays a pivotal role in neuro-inflammation of glaucoma. Toll-like receptor 4 (TLR4) is expressed on microglia and mediates many neuroinflammatory diseases. We aimed to explore the impacts of high intraocular pressure (IOP) on rat microglia in retina and the regulation of TLR4/NF-κB signaling pathway in scratched microglia cells. In our study, we successfully established chronic high IOP rat model by episcleral vein cauterization (EVC) which behaved like the chronic glaucoma. Besides, we set up an in vitro scratch-induced injury model in rat microglia cells. We found the level of activated microglia cells were significantly increased in the retina of chronic high IOP groups. Moreover, the inhibition of TLR4/NF-κB signaling pathway suppressed the expression of TLR4 protein and mRNA levels of P50, IL-6 and TNF-α. Our original study provided a theoretical basis on targeting TLR4/NF-κB to suppress pro-inflammatory factors releasing in activated microglia and it might be a good treatment target to prevent glaucoma from progressing.

Agonistic Autoantibodies to the ß2-Adrenergic Receptor Involved in the Pathogenesis of Open-Angle Glaucoma.

Glaucoma is a frequent ocular disease that may lead to blindness. Primary open-angle glaucoma (POAG) and ocular hypertension (OHT) are common diseases with increased intraocular pressure (IOP), which are mainly responsible for these disorders. Their pathogenesis is widely unknown. We screened the sera of patients with POAG and OHT for the prevalence of autoantibodies (AAb) against G protein-coupled receptors (GPCRs) in comparison to controls. Employing frequency modulation of spontaneously contracting neonatal rat cardiomyocytes in vitro, agonistic GPCR AAb were to be detected in roughly 75% of the patients with POAG and OHT, however, not in controls. Using inhibitory peptides the AAb' target was identified as ß2 adrenergic receptor (ß2AR). The AAb interact with the second extracellular loop of ß2AR. The peptides 181-187 and 186-192 were identified as binding sites of the AAb within the extracellular loop II. The binding of the AAb to ß2ARs was verified by surface-plasmon-resonance analysis. The isotype of the AAb was (immunoglobulin) IgG3. In an additional pilot principal-of-proof study, including four patients with POAG, the removal of the AAb against the ß2AR and other immunoglobulins G by immunoadsorption resulted in a transient reduction of IOP. These findings might indicate a possible role of agonistic AAb directed against ß2ARs in the dynamics of aqueous humor and might support a contribution of adaptive autoimmunity in the etiopathogenesis of POAG and OHT.

[Determination of cytomegalovirus IgG synthesis by the albumin correction in the aqueous humor of posner-schlossmann syndrome].

Objective: To evaluate the usefulness of albumin correction in determination of cytomegalovirus IgG in the aqueous humor of Posner-Schlossman syndrome (PSS) patients. Methods: Cases series studies. Forty-two patients (26 men and 16 women) who were diagnosed as PSS were enrolled from Oct. 2009 to Oct. 2015 at the Eye and ENT Hospital. During the same period, 20 patients with primary open-angle glaucoma (POAG) and 30 patients with bacterial endophthalmitis or retinal necrosis were enrolled as negative control group and inflammatory disease control group, respectively. Aqueous humor and serum samples were assayed to detect CMV IgG by enzyme-linked immunosorbent assay (ELISA), and albumin by scattering immunonephelometry. CMV DNA in aqueous humor was assayed by polymerase chain reaction (PCR). The ratio which was calculated as the (aqueous humor CMV IgG/serum CMV IgG)/(aqueous humor concentration of albumin/serum albumin concentration) over 0.6 was considered as intraocular antibody formation. Performance of differentiating control eyes from eyes with CMV-positive PSS was evaluated by the receiver operating characteristic curve. The ANOVA test, Mann-Whitney test and Chi-square test were performed to compare the differences among groups. Results: The detectable rate of CMV IgG antibody in the aqueous humor was 76.2%, 100.0% and 10.0% in PSS, inflammatory disease control and POAG groups, respectively. The levels of CMV IgG antibody in the PSS groups were significantly higher than that of POAG groups (Z=4.23, P<0.001).The positive rate corrected by the albumin was 71.4%, 3.3% and 0.0%.The corrected positive rate in PSS groups was significantly higher than that of the inflammatory disease control and POAG groups (χ(2)=30.38, P<0.01; χ(2)= 24.89, P<0.01), with a sensitivity of 75.0% and a specificity of 98.0%. The area under the curve for calibrated ratio was 0.942 (95%CI: 0.859 to 0.984) which was higher than that of CMV IgG (Z=6.19, P<0.001).The corrected positive rate of CMV IgG antibody (71.4%) was higher than that of CMV DNA (47.6%, χ(2)=4.003, P=0.045). Conclusions: CMV IgG antibody ratio which was corrected by aqueous humor and serum albumin could effectively improve aqueous antibody specificity in PSS patients. Furthermore, CMV IgG antibody ratio combined with PCR could improve the sensitivity of CMV detection. All of which help clarify the CMV infection in PSS in CMV DNA negative eyes. (Chin J Ophthalmol, 2017, 53: 104-108).

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

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.

Immune protective mechanism of rMOMP protein ophthalmic vaccine regarding intraocular hypertension and retinal optic nerve injury in rats.

The aim of this study was to explore the immune protective mechanism of rMOMP protein vaccine in intraocular hypertension and retinal optic nerve injury in rats. The rMOMP protein ophthalmic vaccine was prepared and quality-controlled. Sixty normal adult SD rats were randomly divided into two groups to establish a chronic ocular hypertension model and an optic nerve injury model. The model rats were vaccinated with rMOMP-CS ophthalmic vaccine. Fluorogold retrograde tracing was used to observe retinal ganglion cells, and an immunofluorescence method to determine the expression of retinal GAP43, CD3, BDNF, and GDNF. rMOMP protein ophthalmic vaccine met the requirements for medicinal use. The number of retinal ganglion cells (RGCs) of the rMOMP-CS group in the chronic ocular hypertension model was significantly higher than that of the CS group (P < 0.05). The count of RGCs of the rMOMP-CS group in the optic nerve clamping injury model was significantly higher than that of the CS group (P < 0.01). Thus, rMOMP protein ophthalmic vaccine can induce an increase in the expression of retinal neurotrophic factors, thereby exerting a protective effect on damaged retinal optic nerve.

[Complement activation after induction of ocular hypertension in an animal model]. / Komplementaktivierung nach Induktion einer okulären Hypertension im Tiermodell.

BACKGROUND: Although an elevated intraocular pressure (IOP) is known as the main risk factor for glaucoma, many studies also showed an involvement of the immune system in this disease. In this study we investigated if a moderate increase in IOP leads to activation of the complement system. METHODS: The IOP was elevated experimentally in the left eye of rats, whereas the fellow eye served as the control. The IOP was measured at regular intervals. The number of retinal ganglion cells (RGC) was quantified via NeuN staining. To evaluate the activation of the complement system staining for C3, membrane attack complex (MAC), and mannose-binding lectin (MBL) was performed. Furthermore, we investigated possible glia activation (GFAP and vimentin) and apoptosis (Bax). RESULTS: A moderate elevation of the IOP was noted from day 11 after induction of ocular hypertension (OHT) until the end of the study (28 days, p = 0.0005). In the OHT-group significantly fewer RGCs (p = 0.02) were detected. Additionally, we noted significant C3 and MAC activation in the ganglion cell layer (C3, p = 0.001 and MAC, p = 0.02) as well as in the total retina (C3, p = 0.002 and MAC, p = 0.012). An activation via the lectin pathway by MBL staining could not be detected (p = 0.40). At this point in time no alterations with regard to glia cells were noted (GFAP, p = 0.97 and vimentin, p = 0.99). No apoptosis via Bax pathway could be observed (p = 0.90). CONCLUSION: The results suggest that the complement system is involved in the loss of RGCs even by a moderate IOP elevation which was indicated by significantly more C3 and MAC depositions in the OHT group.

Serum and antibodies of glaucoma patients lead to changes in the proteome, especially cell regulatory proteins, in retinal cells.

PURPOSE: Previous studies show significantly specifically changed autoantibody reactions against retinal antigens in the serum of glaucoma and ocular hypertension (OHT) patients in comparison to healthy people. As pathogenesis of glaucoma still is unknown the aim of this study was to analyze if the serum and antibodies of glaucoma patients interact with neuroretinal cells. METHODS: R28 cells were incubated with serum of patients suffering from primary open angle glaucoma (POAG), normal tension glaucoma (NTG) or OHT, POAG serum after antibody removal and serum from healthy people for 48 h under a normal or an elevated pressure of 15000 Pa (112 mmHg). RGC5 cells were additionally incubated with POAG antibodies under a normal pressure. Protein profiles of the R28 cells were measured with Seldi-Tof-MS, protein identification was performed with Maldi-TofTof-MS. Protein analysis of the RGC5 cells was performed with ESI-Orbitrap MS. Statistical analysis including multivariate statistics, variance component analysis as well as calculating Mahalanobis distances was performed. RESULTS: Highly significant changes of the complex protein profiles after incubation with glaucoma and OHT serum in comparison to healthy serum were detected, showing specific changes in the cells (e.g. Protein at 9192 Da (p<0.001)). The variance component analysis showed an effect of the serum of 59% on the cells. The pressure had an effect of 11% on the cells. Antibody removal led to significantly changed cell reactions (p<0.03). Furthermore, the incubation with POAG serum and its antibodies led to pro-apoptotic changes of proteins in the cells. CONCLUSIONS: These studies show that the serum and the antibodies of glaucoma patients significantly change protein expressions involved in cell regulatory processes in neuroretinal cells. These could lead to a higher vulnerability of retinal cells towards stress factors such as an elevated IOP and eventually could lead to an increased apoptosis of the cells as in glaucoma.

New insights into autoantibody profiles from immune privileged sites in the eye: a glaucoma study.

Glaucoma is a chronic neurodegenerative disease and one of the leading causes of blindness. Autoantibody based immune processes are assumed to be involved in its pathogenesis. However, it is still unclear to what extent autoantibody patterns found in the eye (aqueous humor) are congruent to systemic autoantibodies (blood). Consistency would underline the specificity of known serum antibody markers for glaucoma. In this study we used antigen microarrays to analyze autoantibody reactivities in sera and corresponding aqueous humor samples of primary open-angle glaucoma patients (N=37) and non-glaucomatous controls (N=31). Compared to control subjects several divergent immunoreactivities were identified for the glaucoma group in both body fluids. Interestingly, 20% of the tested antigens revealed increased immunoreactivities (e.g., against HSP27, MBP, and α-1-antitrypsin) and 7.5% decreased immunoreactivities (e.g., against GFAP and ß-L-crystallin), thus demonstrating a significant alteration of the autoantibody profiles in glaucoma patients. Using an artificial neural network in combination with a unique serum autoantibody pattern on prospective sera we were able to detect glaucoma with a specificity and sensitivity of approximately 93%. The intraindividual comparison revealed a strong correlation of detected immunoreactivities in sera and comparative aqueous humor samples in both study groups. These results emphasize the specificity of immunoreactions found in blood samples of glaucoma patients. Furthermore they indicate the necessity of analyzing not only up-regulated but also down-regulated antibody reactivities, which might be likewise relevant for the understanding of other diseases.

Roles of PI3K and JAK pathways in viability of retinal ganglion cells after acute elevation of intraocular pressure in rats with different autoimmune backgrounds.

BACKGROUND: We recently showed that whereas inhibition of PI3K/akt or JAK/STAT pathway promoted retinal ganglion cell (RGC) survival after optic nerve (ON) injury in Fischer 344 (F344) rats, the same inhibition resulted in aggravated RGC loss after acute intraocular pressure (IOP) elevation in Sprague Dawley (SPD) rats. In addition, the responses of macrophages to ON injury and acute IOP elevation were different between F344 and Lewis rats, i.e., different autoimmune profiles. Using an acute IOP elevation paradigm in this study, we investigated 1) whether autoimmune background influences PI3K/akt and JAK/STAT functions by examining the effect of PI3K/akt and JAK/STAT pathway inhibition on RGC survival in F344 and Lewis rats, and 2) whether differential actions of macrophages occur in PI3K/akt and JAK/STAT pathways-dependent modulation of RGC survival. IOP elevation was performed at 110 mmHg for 2 hours. PI3K/akt and JAK/STAT pathway inhibitors were applied intravitreally to block their respective pathway signaling transduction. Because macrophage invasion was seen in the eye after the pathway inhibition, to examine the role of these pathways independent of macrophages, macrophages in the retina were removed by intravitreal application of clodronate liposomes. Viable RGCs were retrogradely labelled by FluoroGold 40 hours before animal sacrifice. RESULTS: Similar to what was previously observed, significantly more RGCs were lost in Lewis than F344 rats 3 weeks after acute IOP elevation. As in SPD rats, inhibition of the PI3K/akt or JAK/STAT pathway increased the loss of RGCs in both F344 and Lewis rats. Removal of macrophages in the eye by clodronate liposomes reduced RGC loss due to pathway inhibition in both strains. CONCLUSION: This study demonstrates that following acute IOP elevation 1) PI3K/akt and JAK/STAT pathways mediate RGC survival in both F344 and Lewis rats, 2) autoimmune responses do not influence the functions of these two pathways, and 3) PI3K/akt and JAK/STAT pathway inhibition-dependent activation of macrophages is detrimental to RGCs.

Analysis of autoantibodies against human retinal antigens in sera of patients with glaucoma and ocular hypertension.

PURPOSE: The aim of this study was to show that complex antibody patterns against retinal antigens in sera of patients with glaucoma, found in previous studies, are autoantibodies against human antigens. METHODS: Sera of 179 patients were collected at the Department of Ophthalmology (University of Mainz, Germany): non-glaucomatous control patients (n=45), primary open-angle glaucoma (n=45), ocular hypertension (n=44), and normal tension glaucoma patients (n=45). The sera were tested against Western blots of human retinal antigens. IgG antibody patterns were analyzed by multivariate statistical techniques, and some significant antigens were identified by mass spectrometry. RESULTS: All subjects, even healthy ones, showed different and complex banding patterns. Glaucoma groups showed up- and down-regulations of antibody reactivities compared to the control group. The multivariate analysis of discriminance found significant differences (p<0.05) in IgG antibody profiles between glaucoma groups, ocular hypertension, and healthy subjects against human retinal antigens. The antigen band at 12 kDa was identified as Histone H4 via mass spectrometry, the 29 kDa band as cellular retinaldehyde-binding protein, and one at 49 kDa as retinal S-antigen. CONCLUSIONS: Using human retinal antigen, we demonstrated that complex autoantibody patterns exist in sera of patients with glaucoma. Large correlations with previous studies using bovine retinal antigens could be seen.

Different responses of macrophages in retinal ganglion cell survival after acute ocular hypertension in rats with different autoimmune backgrounds.

Recently macrophages were shown to play a protective role in retinal ganglion cells (RGCs) after optic nerve (ON) injury. In the present study, we investigated how macrophages responded after acute intraocular pressure (IOP) elevation in experimental autoimmune encephalomyelitis (EAE)-resistant Fischer 344 (F344) and Sprague Dawley (SD) rats and EAE-vulnerable Lewis rats. Acute IOP elevation was performed at 110mmHg for 2h to mimic acute glaucoma. Phagocytic cells in the eye were removed by intravitreal application of clodronate liposomes whereas macrophage activation was achieved by intravitreal injection of zymosan, a yeast wall preparation. Fluorescence dye, FluoroGold, was applied behind the eyeballs to retrogradely label surviving RGCs 40h before animal sacrifice. Macrophages in the retina were identified by ED1 immunostaining. Loss of 25% RGCs in F344 but over 90% in Lewis rats was seen 2 weeks after acute IOP elevation. Significant increase in the number of macrophages in the retina was seen to accompany the great RGC loss in Lewis rats; removal of these macrophages reduced the extent of RGC loss, suggesting the involvement of macrophages in RGC death in Lewis strain. Low numbers of macrophages were seen in F344 retinas after acute IOP elevation, and removal of macrophages did not show clear effect on RGC viability. Whereas macrophage activation by zymosan protected RGCs after ON axotomy in F344 rats, the same macrophage activation became detrimental to RGCs after acute IOP elevation. The extent of RGC loss 3 weeks after acute IOP elevation or after macrophage activation by zymosan in EAE-resistant SD rats was similar to that in F344 rats. We thus demonstrate that macrophages in rats with different autoimmune backgrounds react differently to acute IOP elevation and differentially modulate RGC loss, a phenomenon contrary to the protective action in RGCs after ON axotomy. These data suggest that autoimmune background plays a role in modulating vulnerability of RGCs to acute IOP elevation.

Retinal synthesis and deposition of complement components induced by ocular hypertension.

Inappropriate activity of the complement cascade contributes to the pathophysiology of several neurodegenerative conditions. This study sought to determine if components of the complement cascade are synthesized in the retina following the development of ocular hypertension (OHT) and if complement accumulates in association with retinal ganglion cells. Toward this goal the gene expression levels of complement components 1qb (C1qb) and 3 (C3) were determined in the retina by quantitative polymerase chain reaction in human eyes with elevated intraocular pressure (IOP) and healthy retinal tissue as well as in a rat model of OHT induced by laser cauterization of the trabecular meshwork and episcleral veins. Immunohistochemical methods were employed to determine the sites of complement deposition in the retina and optic nerve head. Our data demonstrate that transcript levels for C1q and C3 are significantly elevated in retinae subjected to OHT, both in the animal model as well as in human eyes. Immunohistochemical analyses indicate that C1q and C3 accumulate specifically in the retinal ganglion cell layer and the nerve fiber layer. In addition, we demonstrate that the terminal complement complex, or membrane attack complex, is formed both in the human and rat model as a consequence of OHT. Complement activation, particularly formation of membrane attack complexes, has the potential to exacerbate ganglion cell death through bystander lysis or glial cell activation. The results show that complement activation occurs in the retina that has been subjected to elevated IOP, and may have implications in pathophysiology of glaucoma.

Complex autoantibody repertoires in patients with glaucoma.

PURPOSE: Glaucoma is one of the leading causes for blindness in the world. It is characterized by a progressive loss of retinal ganglion cells. An elevated intraocular pressure cannot explain the disease in all subjects. Autoimmune mechanisms maybe involvemed in the pathogenesis of the disease. The aim of our study was to analyze the IgG autoantibody repertoires in sera of glaucomatous subjects against optic nerve antigens. METHODS: Ninety-four subjects were included in this comparative cross-sectional study of healthy (CTRL), primary-open-angle-glaucoma (POAG), ocular-hypertension (OHT), and normal-tension-glaucoma (NTG) volunteers. Sera of subjects were tested against western blots of optic nerve antigens. For each western blot, a densitograph was built by digital image analysis and subsequently a multivariate analysis of discriminance was performed. RESULTS: Complex IgG autoantibody repertoires could be found in all subjects, even in healthy subjects. The multivariate analysis of discriminance can test for statistical differences between the groups using the whole complex staining pattern for the calculation. A significant difference between all groups against optic nerve antigens was found. The NTG group had the highest variance from controls (p<0.01). CONCLUSIONS: This study demonstrates immunological effects in POAG and NTG and may provide further evidence for an involvement of autoantibodies in the pathogenesis of both NTG and POAG. Using the techniques presented in this study, the differences in the complex autoantibody repertoires were assessed by means of statistical analysis. Further studies are needed to determine whether these changes in autoantibodies could be helpful in the diagnosis of glaucoma.

Analysis of autoantibody repertoires in sera of patients with glaucoma.

PURPOSE: Glaucoma is the second cause of blindness worldwide. It is usually considered a neurodegenerative disease. There is evidence that an autoimmune mechanism is involved in the development of glaucoma in some patients. The aim of this study was to analyze the IgG autoantibody repertoires in sera of glaucoma patients and healthy subjects. METHODS: A total of 82 patients were divided into four groups: healthy volunteers without any ocular disorders (CO, n = 30), patients with primary open-angle glaucoma (POAG, n = 19), ocular hypertension (OHT, n = 16), and normal tension glaucoma (NTG, n = 17). All groups were matched for age and gender. The sera of these patients were tested against Western blots of retinal antigens. Immunodetection was done using 4-chloro-1-naphthol staining. The autoantibody patterns were digitized and subsequently analyzed by multivariate statistical techniques. RESULTS: All patients showed different, complex staining patterns of autoantibodies against retinal antigens. There was an increase in the number of peaks in sera of patients with primary open-angle glaucoma (POAG) compared to healthy subjects (CO). Including all peaks the analysis of discriminance revealed a statistically significant difference between the patterns of POAG compared to all other groups (p < 0.01). Sera of normal tension glaucoma (NTG) had no statistically different autoantibody pattern compared to those of control subjects. CONCLUSIONS: In this study, we demonstrated a difference in the IgG autoantibody patterns of primary open-angle glaucoma patients compared to healthy subjects. However, the patterns were not significantly different in normal tension glaucoma compared to control subjects.

Resistance of retinal ganglion cells to an increase in intraocular pressure is immune-dependent.

PURPOSE: Glaucoma is widely accepted as a neurodegenerative disease in which retinal ganglion cell (RGC) loss is initiated by a primary insult to the optic nerve head, caused, for example, by increased intraocular pressure (IOP). In some cases, the surviving RGCs, despite adequate IOP control, may continue to degenerate as a result of their heightened susceptibility to self-destructive processes evoked by the initial damage. In animal models of mechanical or biochemical injury to the optic nerve or retina, a T-cell-mediated immune response evoked by the insult helps to reduce this ongoing loss. The current study was conducted to find out whether the ability to resist the IOP-induced loss of RGCs in a rat model is affected by the immune system. METHODS: The ocular veins and limbal plexus of rats of two strains differing in their resistance to experimental autoimmune encephalomyelitis (EAE) and in their ability to manifest a beneficial autoimmune response were laser irradiated twice to induce an increase in IOP. The pressure was measured weekly, and RGC losses were assessed 3 and 6 weeks after the first irradiation. To verify the existence of a relationship between the immune system and RGC survival, we assessed neuronal survival in Sprague-Dawley (SPD) rats devoid of mature T cells as well as after transferring splenocytes from Fisher rats, an EAE-resistant rat strain capable of manifesting T-cell-mediated neuroprotection, to rats of a major histocompatibility complex (MHC)-matched EAE-susceptible strain (Lewis), in which the ability to manifest such protective immunity is limited. RESULTS: Both 3 and 6 weeks after the increase in IOP was initiated, the number of surviving RGCs in SPD rats, a strain in which a beneficial autoimmune response can be evoked spontaneously, was significantly higher than in Lewis rats. Moreover, in SPD rats that were thymectomized at birth, the number of surviving RGCs after an increase in IOP as adults was significantly diminished. Passive transfer of splenocytes from Fisher rats to Lewis rats significantly reduced the IOP-induced loss of RGCs in the latter. CONCLUSIONS: In rats of different strains, a similar increase in IOP results in differing amounts of RGC loss. This disparity was found to correlate with immune potency. These findings may explain why patients with glaucoma experience different degrees of visual loss after pressure reduction, even when the severity of the disease at the time of diagnosis is similar. The results have far-reaching prognostic and therapeutic implications.

Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: implications for glaucoma.

Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freund's adjuvant than in mice injected with PBS in the same adjuvant (2,133 +/- 270 and 1,329 +/- 121, respectively, mean +/- SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 +/- 101 compared with 1,414 +/- 36; P < 0.05), but not when they were immunized 48 h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8% +/- 6.8% to 4.3% +/- 1.6%, without affecting the intraocular pressure. This study may point the way to a therapy for glaucoma, a neurodegenerative disease of the optic nerve often associated with increased intraocular pressure, as well as for acute and chronic degenerative disorders in which glutamate is a prominent participant.

Retinal ganglion cells recognized by serum autoantibody against gamma-enolase found in glaucoma patients.

PURPOSE: To study pathologic roles of the presence of serum autoantibodies against retinal ganglion cells in patients with glaucoma. METHODS: Serum autoantibody reactions were detected by Western blot analysis using retinal soluble fractions in 79 patients with glaucoma (normal-tension glaucoma [NTG], 23 cases; primary open-angle glaucoma [POAG], 56 cases) and 60 age-matched healthy subjects. Clinical characteristics including visual acuity, visual field, intraocular pressure (IOP), and optic disc features were compared between the serum autoantibody-positive and -negative patients. The retinal autoantigen recognized by patients' sera was identified by a combination of in-gel digestion and Edman sequencing. RESULTS: Western blot analysis revealed that serum autoantibody against retinal 50-kDa antigen was recognized in 20 out of 79 glaucoma patients (25.3%; 14 POAG and 6 NTG patients) and 60 age-matched control subjects (11.7%), respectively. Immunocytochemistry revealed that labeling of the ganglion cell layer (GCL) by IgG from glaucoma patients (POAG: 13/56, 23.2%; NTG: 6/23, 26%) existed at a significantly higher rate than that by IgG from control subjects (2/60, 3.3%; P < 0.05). In POAG, maximum IOP in the serum antibody positive-patients was significantly lower than that in the antibody-negative patients (P < 0.05). However, no statistical differences were observed in visual field loss, disc cupping, and other clinical factors between the antibody-positive and -negative groups in POAG and NTG. In-gel digestion of the 50-kDa band in two-dimensional polyacrylamide gels and Edman sequence analysis of the high-performance liquid chromatography-purified peptides identified the 50-kDa protein as gamma-enolase. Injection of the 50-kDa IgG from glaucoma patients or anti-gamma-enolase serum into the vitreous cavity of Lewis rats caused reduction of the b-wave of the electroretinogram and TdT-dUTP terminal nick-end labeling (TUNEL)-positive staining within the GCL. CONCLUSIONS: In the current study, serum autoantibody against 50-kDa protein identified as gamma-enolase in 25% of glaucoma patients.

[The constitutional prerequisites for the development of glaucoma]. / Konstitutsional'nye predposylki razvitiia glaukomy.

Information on the existence of the interspecific constitutional polymorphism in rabbits of chinchilla [correction of "Shinshilla"] and "white giant" to ophthalmic hypertension and glaucoma in abundance of catecholamine was presented. Experiences show that rabbits of chinchilla [correction of "shinshilla"] under the abundance of catecholamine in difference of "albino" rabbits do not have glaucoma. Albinos are not protected from the abundance of POL under the influence of catecholamine.

HLA-Bw54 and glaucomatocyclitic crisis.

Twenty-two Japanese patients with glaucomatocyclitic crisis were typed for HLA-A, HLA-B, and HLA-C antigens to detect the immunogenetic factors in this disease. It was shown that HLA-Bw54 was present in nine (41%) of 22 patients, a significant increase even after correcting the P value for the number of antigens studied. The haplotype frequency of HLA-Bw54-Cw1 showed a significant association, and it was not due to linkage disequilibrium in the Japanese. Our results suggest that immunogenetic factors closely associated with the major histocompatibility complex may play an important role in the pathogenesis of glaucomatocyclitic crisis.