Characterization of Macroglia Response during Tissue Repair in a Laser-Induced Model of Retinal Degeneration.

Reactive gliosis is a hallmark of chronic degenerative diseases of the retina. As gliosis involves macroglia, we investigated their gliotic response to determine the role of S100ß and intermediate filaments (IFs) GFAP, vimentin, and nestin during tissue repair in a laser-induced model of retinal degeneration. We validated the results with human retinal donor samples. Experiments were performed in zebrafish and mice using an argon laser (532 nm) to induce focal lesions in the outer retina. At different time points following injury induction, the kinetics of retinal degeneration and regeneration were assessed using hematoxylin and eosin staining (H&E). Immunofluorescence was performed to evaluate Müller cell (GS) and astrocyte (GFAP) injury response and to distinguish between both cell types. Additionally, staining was performed in human retinal sections containing drusen. Focal laser treatment elevated the expression of gliotic markers in the area of the damage, which was associated with increased expression of S100ß, GFAP, vimentin, and nestin in mice and humans. In zebrafish, we detected S100ß at the first time point, but not GFAP or nestin. Double-positive cells with the selected glia markers were detected in all models. However, in zebrafish, no double-positive GFAP/GS cells were found on days 10 and 17, nor were S100ß/GS double-positive cells found on day 12. Macroglia cells showed a different pattern in the expression of IFs in degenerative and regenerative models. In particular, S100ß may prove to be a target for suppressing chronic gliosis in retinal degeneration.

Diverse Signaling by TGFß Isoforms in Response to Focal Injury is Associated with Either Retinal Regeneration or Reactive Gliosis.

Müller cells may have stem cell-like capability as they regenerate photoreceptor loss upon injury in some vertebrates, but not in mammals. Indeed, mammalian Müller cells undergo major cellular and molecular changes summarized as reactive gliosis. Transforming growth factor beta (TGFß) isoforms are multifunctional cytokines that play a central role, both in wound healing and in tissue repair. Here, we studied the role of TGFß isoforms and their signaling pathways in response to injury induction during tissue regeneration in zebrafish and scar formation in mouse. Our transcriptome analysis showed a different activation of canonical and non-canonical signaling pathways and how they shaped the injury response. In particular, TGFß3 promotes retinal regeneration via Smad-dependent canonical pathway upon regulation of junb gene family and mycb in zebrafish Müller cells. However, in mice, TGFß1 and TGFß2 evoke the p38MAPK signaling pathway. The activation of this non-canonical pathway leads to retinal gliosis. Thus, the regenerative versus reparative effect of the TGFß pathway observed may rely on the activation of different signaling cascades. This provides one explanation of the different injury response in zebrafish and mouse retina.

Retinal microglia signaling affects Müller cell behavior in the zebrafish following laser injury induction.

Microglia are the resident tissue macrophages of the central nervous system including the retina. Under pathophysiological conditions, microglia can signal to Müller cells, the major glial component of the retina, affecting their morphological, molecular, and functional responses. Microglia-Müller cell interactions appear to be bidirectional shaping the overall injury response in the retina. Hence, microglia and Müller cell responses to disease and injury have been ascribed both positive and negative outcomes. However, Müller cell reactivity and survival in the absence of immune cells after injury have not been investigated in detail in adult zebrafish. Here, we develop a model of focal retinal injury combined with pharmacological treatments for immune cell depletion in zebrafish. The retinal injury was induced by a diode laser to damage photoreceptors. Two pharmacological treatments were used to deplete either macrophage-microglia (PLX3397) or selectively eliminate peripheral macrophages (clodronate liposomes). We show that PLX3397 treatment hinders retinal regeneration in zebrafish, which is reversed by microglial repopulation. On the other hand, selective macrophage elimination did not affect the kinetics of retinal regeneration. The absence of retinal microglia and macrophages leads to dysregulated Müller cell behavior. In the untreated fish, Müller cells react after injury induction showing glial fibrillary acidic protein (GFAP), Phospho-p44/42 MAPK (Erk1/2), and PCNA upregulation. However, in the immunosuppressed animals, GFAP and phospho-p44/42 MAPK (Erk1/2) expression was not upregulated overtime and the reentry in the cell cycle was not affected. Thus, microglia and Müller cell signaling is pivotal to unlock the regenerative potential of Müller cells in order to repair the damaged retina.

Modulation of Extracellular Matrix Composition and Chronic Inflammation with Pirfenidone Promotes Scar Reduction in Retinal Wound Repair.

Wound repair in the retina is a complex mechanism, and a deeper understanding of it is necessary for the development of effective treatments to slow down or even prevent degenerative processes leading to photoreceptor loss. In this study, we harnessed a laser-induced retinal degeneration model (532-nm laser photocoagulation with 300 µm spot size, 60 ms duration and 60 mV pulse), enabling a profound molecular elucidation and a comprehensive, prolonged observation of the wound healing sequence in a murine laser-induced degeneration model (C57BL/6J mice, 6-12 weeks) until day 49 post-laser. Our observations included the expression of specific extracellular matrix proteins and myofibroblast activity, along with an analysis of gene expression related to extracellular matrix and adhesion molecules through RNA measurements. Furthermore, the administration of pirfenidone (10 mg/kg via drinking water), an anti-inflammatory and anti-fibrotic compound, was used to modulate scar formation after laser treatment. Our data revealed upregulated collagen expression in late regenerative phases and sustained inflammation in the damaged tissue. Notably, treatment with pirfenidone was found to mitigate scar tissue formation, effectively downregulating collagen production and diminishing the presence of inflammatory markers. However, it did not lead to the regeneration of the photoreceptor layer.

The TGFß/Notch axis facilitates Müller cell-to-epithelial transition to ultimately form a chronic glial scar.

BACKGROUND: Contrasting with zebrafish, retinal regeneration from Müller cells (MCs) is largely limited in mammals, where they undergo reactive gliosis that consist of a hypertrophic response and ultimately results in vision loss. Transforming growth factor ß (TGFß) is essential for wound healing, including both scar formation and regeneration. However, targeting TGFß may affect other physiological mechanisms, owing its pleiotropic nature. The regulation of various cellular activities by TGFß relies on its interaction with other pathways including Notch. Here, we explore the interplay of TGFß with Notch and how this regulates MC response to injury in zebrafish and mice. Furthermore, we aimed to characterize potential similarities between murine and human MCs during chronic reactive gliosis. METHODS: Focal damage to photoreceptors was induced with a 532 nm diode laser in TgBAC (gfap:gfap-GFP) zebrafish (ZF) and B6-Tg (Rlbp1-GFP) mice. Transcriptomics, immunofluorescence, and flow cytometry were employed for a comparative analysis of MC response to laser-induced injury between ZF and mouse. The laser-induced injury was paired with pharmacological treatments to inhibit either Notch (DAPT) or TGFß (Pirfenidone) or TGFß/Notch interplay (SIS3). To determine if the murine laser-induced injury model translates to the human system, we compared the ensuing MC response to human donors with early retinal degeneration. RESULTS: Investigations into injury-induced changes in murine MCs revealed TGFß/Notch interplay during reactive gliosis. We found that TGFß1/2 and Notch1/2 interact via Smad3 to reprogram murine MCs towards an epithelial lineage and ultimately to form a glial scar. Similar to what we observed in mice, we confirmed the epithelial phenotype of human Müller cells during gliotic response. CONCLUSION: The study indicates a pivotal role for TGFß/Notch interplay in tuning MC stemness during injury response and provides novel insights into the remodeling mechanism during retinal degenerative diseases.