Complement activation and extracellular matrix remodeling in an autoimmune glaucoma model

Das Glaukom ist eine multifaktorielle Erkrankung und führt zu einem Untergang retinaler Ganglienzellen (RGZ) und einer Sehnervdegeneration. Ein erhöhter Augeninnendruck ist immer noch der Hauptrisikofaktor, es scheinen aber auch andere Mechanismen beteiligt zu sein. Veränderte Antikörpermuster im Patienten, lassen auf eine Beteiligung des Komplementsystems schließen. Daher analysierten wir das Komplementsystem sowohl in einem Augeninnendruck unabhängigen Experimentellen Autoimmunen Glaukom Modell (EAG-Modell) als auch in einem okulären Hypertension Modell (OHT-Modell). Im EAG-Modell konnte eine frühe Komplementaktivierung über den Lektin-Weg detektiert werden. Im OHT-Modell kam es zu einem moderaten Anstieg des IODs und einen Verlust von RGZ. Auch hier kam es zu einer Aktivierung der Komplementsystem Komponenten. Ebenfalls im EAG-Modell lagen die Komponenten der extrazellulären Matrix, Tenascin-C und Phosphacan, signifikant erhöht vor, noch bevor degenerative Prozesse detektiert wurden

Role of heat shock proteins in glaucoma

Glaucoma, one of the most common causes of blindness worldwide, is a multifactorial neurodegenerative disease characterized by damage of retinal ganglion cells and optic nerve degeneration. However, the exact mechanism leading to glaucoma is still not understood. Evidences suggest an immunological involvement in the pathogenesis. Among other immune responses, altered autoantibody patterns were found in glaucoma patients. Especially elevated antibody levels against heat shock proteins (HSPs), like HSP27 or HSP60, were identified. In an animal model, an immunization with these HSPs induced a pressure-independent retinal ganglion cell degeneration and axon loss, hence mimicking glaucoma-like damage. In addition, development of autoreactive antibodies, as well as a glia and T-cell activation, were described in these animals. Recently, we noted that intravitreal HSP27 injection likewise led to a degeneration of retinal ganglion cells and their axons. Therefore, HSP27 might have a direct damaging effect on retinal cells, and might play a key role in glaucoma

Immune mediated degeneration and possible protection in glaucoma

The underlying pathomechanisms for glaucoma, one of the most common causes of blindness worldwide, are still not identified. In addition to increased intraocular pressure (IOP), oxidative stress, excitotoxicity, and immunological processes seem to play a role. Several pharmacological or molecular/genetic methods are currently investigated as treatment options for this disease. Altered autoantibody levels were detected in serum, aqueous humor, and tissue sections of glaucoma patients. To further analyze the role of the immune system, an IOP-independent, experimental autoimmune glaucoma (EAG) animal model was developed. In this model, immunization with ocular antigens leads to antibody depositions, misdirected T-cells, retinal ganglion cell death and degeneration of the optic nerve, similar to glaucomatous degeneration in patients. Moreover, an activation of the complement system and microglia alterations were identified in the EAG as well as in ocular hypertension models. The inhibition of these factors can alleviate degeneration in glaucoma models with and without high IOP. Currently, several neuroprotective approaches are tested in distinct models. It is necessary to have systems that cover underlying pathomechanisms, but also allow for the screening of new drugs. In vitro models are commonly used, including single cell lines, mixed-cultures, and even organoids. In ex vivo organ cultures, pathomechanisms as well as therapeutics can be investigated in the whole retina. Furthermore, animal models reveal insights in the in vivo situation. With all these models, several possible new drugs and therapy strategies were tested in the last years. For example, hypothermia treatment, neurotrophic factors or the blockage of excitotoxity. However, further studies are required to reveal the pressure independent pathomechanisms behind glaucoma. There is still an open issue whether immune mechanisms directly or indirectly trigger cell death pathways. Hence, it might be an imbalance between protective and destructive immune mechanisms. Moreover, identified therapy options have to be evaluated in more detail, since deeper insights could lead to better treatment options for glaucoma patients

HSP27 induced glaucomatous damage in mice of young and advanced age

$\bf Introduction:$ Age-related diseases such as glaucoma, a leading cause of blindness, are having an upward trend due to an aging society. In glaucoma, some patients display altered antibody profiles and increased antibody titers, for example against heat shock protein 27 ($\it HSP27$). An intravitreal injection of $\it HSP27$ leads to glaucoma-like damage in rats. We now aimed to investigate if aged mice are more prone to this damage than younger ones. $\bf Methods:$ We intravitreally injected $\it HSP27$ into young (1–2 months) and aged (7–8 months) mice to compare glaucomatous damage. Respective age-matched controls received PBS. Not injected eyes served as naive controls. $\bf Results:$ Optical coherence tomography 4 weeks after injection showed no changes in retinal thickness in all groups at both ages. Cell counts and RT-qPCR revealed a significant reduction in RGC numbers in $\it HSP27$ mice at both ages. Comparing aged and young $\it HSP27$ mice, no differences in $\it Rbpms$ and $\it Pou4f1$ (RGCs) expression was detected, while the $\it Tubb3$ expression (neuronal cells) was significantly upregulated in aged $\it HSP27$ animals. Neither microglia/macrophages nor (resident) microglia counts revealed significant differences in $\it HSP27$ mice at both ages. Nevertheless, increased relative $\it Iba1$ and $\it Tmem119$ expression was detected in young and aged $\it HSP27$ mice. Aged $\it HSP27$ mice displayed a significantly lower $\it Iba1$ expression than young ones, whereas $\it Cd68$ levels were upregulated. A larger $\it GFAP+$ area and an upregulation of $\it GFAP$ expression in $\it HSP27$ animals of both ages indicated a macrogliosis. Also, elevated $\it Il1b$ and $\it Nos2$ expression levels were observed in young and aged $\it HSP27$ mice. However, only $\it Il1b$ levels were upregulated when comparing 7–8 months to 1–2 months old animals. A larger $\it HSP25+$ area was seen in aged $\it HSP27$ animals, while $\it Hspb2$ expression levels were downregulated in both $\it HSP27$ groups. The aged $\it HSP27$ group displayed an upregulated $\it Hspb2$ expression compared to young mice. Furthermore, a higher optic nerve degeneration score was noted in young and aged $\it HSP27$ groups. $\bf Discussion:$ These findings indicate that an intravitreal injection of $\it HSP27$ led to $\it RGC$ loss accompanied by inflammation. Age-dependent effects (7–8 months vs. 1–2 months) were not very prominent. The results suggest a potential role of extracellular $\it HSP27$ in the development of glaucoma

Proteomic analysis of retinal tissue in an S100B autoimmune glaucoma model

Glaucoma is a neurodegenerative disease that leads to damage of retinal ganglion cells and the optic nerve. Patients display altered antibody profiles and increased antibody titer, e.g., against S100B. To identify the meaning of these antibodies, animals were immunized with S100B. Retinal ganglion cell loss, optic nerve degeneration, and increased glial cell activity were noted. Here, we aimed to gain more insights into the pathophysiology from a proteomic point of view. Hence, rats were immunized with S100B, while controls received sodium chloride. After 7 and 14 days, retinae were analyzed through mass spectrometry and immunohistology. Using data-independent acquisition-based mass spectrometry, we identified more than 1700 proteins on a high confidence level for both study groups, respectively. Of these 1700, 43 proteins were significantly altered in retinae after 7 days and 67 proteins revealed significant alterations at 14 days. For example, $\alpha$2-macroglobulin was found significantly increased not only by mass spectrometry analysis, but also with immunohistological staining in S100B retinae at 7 and 14 days. All in all, the identified proteins are often associated with the immune system, such as heat shock protein 60. Once more, these data underline the important role of immunological factors in glaucoma pathogenesis

Heat shock protein upregulation supplemental to complex mRNA alterations in autoimmune glaucoma

Glaucomatous optic neuropathy is a common cause for blindness. An elevated intraocular pressure is the main risk factor, but also a contribution of the immune system seems likely. In the experimental autoimmune glaucoma model used here, systemic immunization with an optic nerve homogenate antigen (ONA) leads to retinal ganglion cell (RGC) and optic nerve degeneration. We processed retinae for quantitative real-time PCR and immunohistology 28 days after immunization. Furthermore, we performed mRNA profiling in this model for the first time. We detected a significant RGC loss in the ONA retinae. This was accompanied by an upregulation of mRNA expression of genes belonging to the heat shock protein family. Furthermore, mRNA expression levels of the genes of the immune system, such as $\textit {C1qa, C1qb, Il18,}$ and $\it Nfkb1$, were upregulated in ONA animals. After laser microdissection, inner retinal layers were used for mRNA microarrays. Nine of these probes were significantly upregulated in ONA animals ($\it p$ < 0.05), including $\it Hba-a1$ and $\it Cxcl10$, while fifteen probes were significantly downregulated in ONA animals ($\it p$ < 0.05), such as $\it Gdf15$ and $\it Wwox$. Taken together, these findings provide further insights into the pivotal role of the immune response in glaucomatous optic neuropathy and could help to identify novel diagnostic or therapeutic strategies

Novel porcine retina cultivation techniques provide improved photoreceptor preservation

Age-related macular degeneration (AMD) is the leading cause of blindness in industrialized countries among people over 60 years. It has multiple triggers and risk factors, but despite intense research efforts, its pathomechanisms are currently not completely understood. AMD pathogenesis is characterized by soft drusen in Bruch’s membrane and involves the retinal pigment epithelium–Bruch’s membrane-choroid complex and adjacent structures, like photoreceptors. This study explores the potential of novel cultivation techniques to preserve photoreceptors in retinal explants to gain better insights in AMD pathology. The porcine retina explants were cultured for 4 and 8 days using three different explantation techniques, namely, control (photoreceptors facing down, touching the filter), filter (photoreceptors facing up, turned sample using a filter), and tweezers (photoreceptors facing up, turned sample using tweezers). Optical coherence tomography revealed that the tweezers method had the best capacity to limit thinning of the retinal explants. Both novel methods displayed advantages in maintaining outer segment thickness. Additionally, immunofluorescence evaluation revealed a better preservation of opsin$^{+}$ cells and rhodopsin signal intensity in both novel methods, especially the tweezers method. Furthermore, RT-qPCR analysis demonstrated an upregulation of $\it OPSIN$ and $\it RHODOPSIN$ mRNA expression in tweezers samples at 8 days. Amacrine and bipolar cell numbers were not altered at day 4 of cultivation, while cultivation until 8 days led to reduced bipolar cell numbers. At 4 days, $\it CALRETININ$ mRNA was upregulated in filter samples, but $\textit {protein kinase C alpha}$ expression was downregulated. Retinal ganglion cells were diminished in both novel techniques due to a direct physical contact with the insert. Remarkably, no difference in $\it TUBB3$ mRNA expression was detected among the techniques. Nevertheless, both novel methods exhibited an improved retention of photoreceptor cells. In conclusion, the tweezers technique was the most promising one. Due to the high homology of the porcine to the human retina, it provides a reasonable alternative to $\it in vivo$ rodent models. Consequently, an adapted coculture system based on the current findings may serve as an $\it ex vivo$ model suitable to analyze AMD pathomechanisms and novel therapeutic approaches

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced

$\bf Background:$ The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. $\bf Methods:$ Six-week-old wildtype (WT+ONA) and $\beta$B1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. $\bf Results:$ We noticed elevated numbers of $C1q^{+}$ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of $\it C1qa$, $\it C1qb$, and $\it C1qc$ mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor $\alpha$ were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. $\bf Conclusion:$ These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future

Investigation of inter- and intra-day variability of tear fluid regarding flow rate, protein concentration as well as protein composition

$\bf Purpose:$ The purpose of this study was to present the determination of inter- and intra-day variations in tear flow rate, and tear fluid protein concentration, as well as protein composition regarding their impact for future biomarker studies. $\bf Methods:$ Tear fluid was collected noninvasively from 18 healthy subjects by performing Schirmer tests at 4 different time points repetitive in a period of 2 days. The tear flow rate on the Schirmer test strips was measured. Proteins were extracted from strips and quantified using amino acid analysis. Protein composition was analyzed by the strips data-independent (DIA) based mass spectrometry. To exclude any impairments to health, volunteers underwent a detailed neurological as well as an ophthalmological examination. $\bf Results:$ Whether tear fluid was collected from oculus sinister or oculus dexter did not affect the tear flow rate ($\it P$ $\thickapprox$ 0.63) or protein concentration ($\it P$ $\thickapprox$ 0.97) of individual subjects. Moreover, protein concentration was independent from the tear volume, so that a change in volume may only influence the total protein amount. When the examination days were compared, investigation of tear flow rate ($\it P$ $\thickapprox$ 0.001) and protein concentration ($\it P$ $\thickapprox$ 0.0003) indicated significant differences. Further, mass spectrometric analysis of tear fluid revealed 11 differentially regulated proteins when comparing both examination days. $\bf Conclusions:$ Our findings provide evidence of inter-day variation in tear flow rate, tear proteome concentration, and composition in healthy subjects, suggesting that inter-day variation needs to be taken into consideration in biomarker research of tear fluid. Identified proteins were assigned to functions in the immune response, oxidative and reducing processes, as well as mannose metabolism

Simultaneous complement response via lectin pathway in retina and optic nerve in an experimental autoimmune glaucoma model

Glaucoma is a multifactorial disease and especially mechanisms occurring independently from an elevated intraocular pressure (IOP) are still unknown. Likely, the immune system contributes to the glaucoma pathogenesis. Previously, IgG antibody depositions and retinal ganglion cell (RGC) loss were found in an IOP-independent autoimmune glaucoma model. Therefore, we investigated the possible participation of the complement system in this model. Here, rats were immunized with bovine optic nerve homogenate antigen (ONA), while controls (Co) received sodium chloride ($\it n$ = 5–6/group). After 14 days, RGC density was quantified on flatmounts. No changes in the number of RGCs could be observed at this point in time. Longitudinal optic nerve sections were stained against the myelin basic protein (MBP). We could note few signs of degeneration processes. In order to detect distinct complement components, retinas and optic nerves were labeled with complement markers at 3, 7, 14, and 28 days and analyzed. Significantly more C3 and MAC depositions were found in retinas and optic nerves of the ONA group. These were already present at day 7, before RGC loss and demyelination occurred. Additionally, an upregulation of C3 protein was noted via Western Blot at this time. After 14 days, quantitative real-time PCR revealed significantly more $\it C3$ mRNA in the ONA retinas. An upregulation of the lectin pathway-associated mannose-serine-protease-2 (MASP2) was observed in the retinas as well as in the optic nerves of the ONA group after 7 days. Significantly more MASP2 in retinas could also be observed via Western Blot analyses at this point in time. No effect was noted in regard to C1q. Therefore, we assume that the immunization led to an activation of the complement system via the lectin pathway in retinas and optic nerves at an early stage in this glaucoma model. This activation seems to be an early response, which then triggers degeneration. These findings can help to develop novel therapy strategies for glaucoma patients