Glaucoma Model

Glaucoma

Glaucoma is a disease that leads to a chronic loss of retinal ganglion cells and their axons and is associated with damage to the papilla and the visual field. Some risk factors for glaucoma, such as increased intraocular pressure, are known, but the exact pathogenesis remains unclear. Mechanical processes, ischemic damage, excitotoxicity or immunological processes are discussed as disease triggers.

EXPERIMENTAL AUTOIMMUNE GLAUKOMA MODEL

In order to investigate these theses, our group works with a so-called autoimmune glaucoma model, in which a loss of retinal ganglion cells is induced by immunization with ocular antigens. We are concerned with various components of the autoimmune system that can induce cell death via different apoptosis mechanisms. These investigations are carried out with various cell biological and protein biochemical methods, such asiImmunohistology, Western Blot, RT-qPCR, cell culture, and fluorescence activated cell sorter (FACS).
We were able to show that immunization with various antigens leads to a loss of retinal ganglion cells and degeneration of optic nerves. A homogenate of ocular antigen (ONA) (Laspas et al. 2011) leads to cell loss, as well as the administration of purified antigens such as heat shock protein 27 (Wax et al. 2008, Joachim et al. 2009), S100B (Casola et al. 2015, Reinehr / Kuehn et al. 2018) and GDNF (Casola et al. 2016). In initial studies, autoantibody deposits were found in both the retina and the tendon (Laspas et al. 2011, Joachim et al. 2012).
The question now arises to what extent these deposits are related to cell loss. These autoantibodies can, for example, activate the complement system. The complement system is part of the innate immune system and also plays a decisive role in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (McGeer and McGeer 2004, Fonseca et al. 2011). The complement system can be activated in three different ways. All routes ultimately lead to protein C3 and the formation of the membrane attack complex (MAC), which lyses the target cells. Analyzes of the autoimmune glaucoma model in the retinae and optic nerves were able to demonstrate an increased formation of MAC (Reinehr et al. 2016). The activation of the complement cascade seems to take place in the model via the lectin path (Reinehr et al. 2016; 2018).

Figure 1: Activation of the terminal membrane attack complex in the retina and optic nerve

A) The membrane attack complex (MAC) was marked in green on retinal cross-sections. The cell nuclei (DAPI) are labeled blue. B) 7 days after immunization with ONA, a first significant increase in signals of MAC was detectable in the retina compared to the control (p = 0.3). While no differences could be detected after 14 days, there was a renewed increase in the MAC signals after 28 days (p = 0.003). C) MAC was also stained (green) on longitudinal sections of the optic nerve and cell nuclei were shown in blue with DAPI. D) Simultaneously with the retina there was a significant increase in MAC + cells in the ONA optic nerve 7 days after immunization (p <0.001). After 3 and 14 days, no significant differences between the groups could be detected. GCL = ganglion cell layer. Scale: 20 µm. * p <0.05; *** p <0.001. (Reinehr et al., 2016).

In addition to the complement system, knowledge about glia cells could also be generated. The glial cells are responsible for various tasks in the central nervous system (CNS), and thus also in the eye. The microglia, as one of three glial cell types, are assigned to the mononuclear-phagocytic system and serve the immune defense in the CNS. Interestingly, we were able to detect an increase in these cells in the retina and optic nerve after immunization, even before retinal ganglion cells die (Noristani / Kuehn et al. 2016). In addition to the microglia, macroglia cells can also be found in the retina. We were able to show that these too react after immunization and that astrogliosis occurs (Casola et al. 2015).

In order to expand the research possibilities within this model, the model, which was previously only established in rats, has now been transferred to mice in collaboration with the Department of Cell Morphology and Molecular Neurobiology (Ruhr University Bochum). In contrast to rats, however, due to their genome, mice offer the advantage of so-called gene targeting, the targeted modification or inhibition of the genome ,in order to be able to identify the role of a certain protein. This could result in new pharmaceutical therapeutic approaches in glaucoma therapy.
The findings from studies on glaucoma models can lead to a better understanding of the pathomechanism. With a more precise knowledge of the triggers of ganglion cell death, it will hopefully be possible in the future to develop new, targeted therapy options for the treatment of glaucoma.
Publications:
Casola, C, Schiwek, JE, Reinehr, S, Kuehn, S, Grus, FH, Kramer, M, Dick, HB and Joachim, SC (2015). "S100 Alone Has the Same Destructive Effect on Retinal Ganglion Cells as in Combination with HSP 27 in an Autoimmune Glaucoma Model." J Mol Neurosci 56 (1): 228-236.
C.J. Gassel, S. Reinehr, S.C. Gomes, H.B. Dick, S.C. Joachim (2020) “Preservation of optic nerve structure by complement inhibition in experimental glaucoma”. Cell Tissue Res. Jul 17.
S. Reinehr, V. Buschhorn, A.M. Mueller-Buehl, T. Goldmann, F.H. Grus, U. Wolfrum, H.B. Dick, S.C. Joachim (2020) “Occurrence of Retinal Ganglion Cell Loss via Autophagy and Apoptotic Pathways in an Autoimmune Glaucoma Model.” Curr Eye Res. Sep; 45 (9): 1124-1135.
P. Grotegut, S. Kuehn, W. Meißner, H.B. Dick, S.C. Joachim (2020) “Intravitreal S100B Injection Triggers a Time-Dependent Microglia Response in a Pro-Inflammatory Manner in Retina and Optic Nerve”. Mol Neurobiol. Feb; 57 (2): 1186-1202.
P. Grotegut, S. Kuehn, H.B. Dick, S.C. Joachim (2020) "Destructive Effect of Intravitreal Heat Shock Protein 27 Application on Retinal Ganglion Cells and Neurofilament." Int J Mol Sci. Jan 15; 21 (2): 549.
S. Reinehr S, S.C. Gomes, C.J. Gassel, M.A. Asaad, G. Stute, M. Schargus, H.B. Dick, S.C. Joachim. (2019) "Intravitreal Therapy Against the Complement Factor C5 Prevents Retinal Degeneration in an Experimental Autoimmune Glaucoma Model." Front Pharmacol. Dec 2; 10: 1381.
S. Reinehr, J. Reinhard, S. Wiemann, K. Hesse, C. Voss, M. Gandej, H.B. Dick, A. Faissner *, S.C. Joachim * (2019) "Transfer of the experimental autoimmune glaucoma model from rats to mice - new options to study glaucoma." International Journal of Molecular S.

PRIMARY OPEN ANGLE GLAUCOMA (betaB1-CTGF)

The main risk factor for developing glaucoma is increased intraoclar pressure. An animal model that makes it possible to get to the bottom of this pathomechanism is the so-called βB1-CTGF high pressure glaucoma model. CTGF (= Connective Tissue Growth Factor) is a protein that influences a number of important processes, such as the mediation of cell adhesion, migration and proliferation, as well as angiogenesis. In the mice of the model, CTGF is increasingly formed, which leads to a compression of the trabecular meshwork and thus to an obstruction of the aqueous humor outflow. One advantage of this model is that it increases intraocular pressure without external surgical manipulation. This makes it possible to rule out the possibility of inflammatory or immunological changes being triggered by the invasive interventions. Significant loss of optic nerve fibers can already be seen at 4 weeks of age (Junglas et al. 2012). In our working group we were also able to show that after 15 weeks the intraocular pressure of the animals is also increased and the number of retinal ganglion cells is significantly reduced (Reinehr et al. 2019; Fig. 1 A-C). This new model therefore seems to be well suited to better analyze possible mechanisms that also occur in human glaucoma.
In further studies, different cell death mechanisms as well as components of the immune system and their connection with cell loss are to be investigated in this animal model.

Figure 1: Loss of retinal ganglion cells

A) After 15 weeks, retinal flamounts were stained (green) with Brn-3a, a specific marker for retinal ganglion cells, and the cell nuclei were depicted with DAPI (blue). Fewer signals were observed in the CTGF group. B) After 15 weeks showed a significant decrease in the retinal ganglion cells in the CTGF animals (p = 0.02). C) The mRNA expression of the specific ganglion cell marker Pou4f1 was also significantly downregulated in the retinas of the CTGF animals (p = 0.2). Scale: 20 µm. * p <0.05. (Reinehr et al., 2019)