GNAO1 is an alpha subunit of the G-protein complex involved in signal transduction in neurons. The G203R mutation in the GNAO1 gene arises recurrently de novo and causes epileptic encephalopathy and movement disorder. GNAO1 has two main isoforms, GNAO1-A and GNAO1-B, but their functional or expression differences are poorly understood. Molecular functions of GNAO1 are mainly studied in neurons, yet glial cells also express GNAO1 and participate in the pathogenesis of epilepsy. Here, we used human-induced pluripotent stem cell-based models to investigate the localization and expression of GNAO1 isoforms in astrocytes. We showed that in astrocytes, almost 100% of GNAO1 transcripts encoded GNAO1-B with very low GNAO1-A expression. We showed that there were no differences in localization between GNAO1-A and GNAO1-B, both in WT and G203R states. We also showed that GNAO1 localized in astrocytic retraction fibers and migrasomes, structures not previously described in this cell type. We showed that GNAO1-positive retraction fibers of neighboring cells provided cell-to-cell contacts and also provided calcium waves during astrocytic excitation. Overexpression of both GNAO1-A and GNAO1-B tends to lower calcium activity in astrocytes, with GNAO1-A providing the most severe impairment of activity. Our results demonstrate that astrocytes, in addition to neurons, should be used as a model for studying GNAO1-related disorders and that GNAO1 mutations should be evaluated in the context of both the GNAO1-A and GNAO1-B isoforms.

The main complication of glaucoma surgery is excessive scarring of newly created aqueous humor outflow pathways. To prevent fibrotic tissue formation, medications with different mechanisms of action are used intraoperatively and during the early postoperative period.The primary group of anti-inflammatory drugs used in glaucoma surgery are topical steroids. A particularly high antiinflammatory efficacy has been demonstrated with a slowrelease dexamethasone implant placed under the scleral flap.Nonsteroidal anti-inflammatory drugs, in addition to their direct effect, can potentiate the hypotensive action of prostaglandin analogs and reduce the severity of their local side effects. The use of antimetabolites such as mitomycin C and 5-fluorouracil is justified by their antifibrotic activity, which inhibits DNA synthesis and, consequently, the proliferation of cells including myofibroblasts. However, they are associated with a high risk of hypotonic complications. Angiogenesis inhibitors suppress vascular endothelial growth factor production and thus block proliferation.The use of ranibizumab, bevacizumab, and infliximab as adjuncts to standard therapy can enhance the longterm effectiveness of glaucoma surgery. However, their use remains limited and not fully explored.

The main complication of glaucoma surgery is cicatricial changes in newly formed outflow tracts. This process leads to a repeated increase in intraocular pressure (IOP) and is the reason for a decrease in the effectiveness of glaucoma surgeries. In the cascade of successive stages of surgical wound healing, the inflammation phase is considered the most important. Reducing its severity and duration is the main goal of postoperative therapy, since excessive inflammation leads to the formation of scar tissues. In turn, its excessive suppression leads to the development of "ineffective resolution". In this case, the inflammation becomes chronic, and the next healing phase does not occur. For complete restoration of the damaged tissue architecture, the healing process must consistently go through all phases. The use of pro-resolving mediators can contribute to the correct completion of inflammation.This review explores the physiological processes of restoration of the damaged tissue architecture, as well as the causes of cicatricial changes in the newly formed intraocular fluid outflow tracts.

Excessive production of extracellular matrix is a key component in the pathogenesis of Salzmann’s nodular degeneration (SND). In vitro studies of drugs that suppress excessive fibroblast activity may become crucial in developing pathogenetically oriented treatments for SND. Purpose. This study evaluates the antifibrotic properties of pirfenidone and cyclosporine A (CsA) on cell cultures obtained from patients with SND. Material and methods. Cell cultures were derived from corneal tissue samples obtained during keratectomy in patients with confirmed SND. Nodular samples were also examined histologically. Fibroblasts were treated with various concentrations of CsA (5, 25, 50 µg/mL) and pirfenidone (100, 500, 1000 µg/mL). Effects were assessed at 24 and 48 hours using metabolic and migration assays, measurement of doubling time, evaluation of proliferation activity, assessment of cell death, and analysis of α-smooth muscle actin (α-SMA) expression. Results. Histological examination of nodular tissue revealed pathological remodeling of the subepithelial stroma. The phenotypic characteristics of cell cultures derived from biopsies indicated the presence of myofibroblasts. According to the MTT assay, pirfenidone at concentrations of 500 and 1000 µg/mL reduced metabolic activity within 24 hours; similar effects were observed with CsA at concentrations of 25 and 50 µg/mL. Proliferation activity decreased after 24 hours with pirfenidone at 500 and 1000 µg/mL and CsA at 5, 25, and 50 µg/mL. Pirfenidone at 100 and 500 µg/mL did not cause cytotoxic effects. Both drugs reduced α-SMA expression, indicating suppression of excessive myofibroblast activation. Conclusion. Myofibroblast activity markers decreased under the influence of pirfenidone and CsA, suggesting their potential for developing new therapeutic approaches for SND and possibly other diseases associated with pathological corneal fibrosis.

Objective. The study tested a method for 3D culture of human lacrimal gland (LG) cells with the formation of structures suitable for subsequent investigation of the pathogenetic mechanisms underlying its diseases. Material and methods. Material for microbiological culture was obtained during a diagnostic biopsy performed as part of surgical correction of recurrent LG prolapse. No morphological changes were detected in the LG tissue. Cells were isolated from the tissue specimen by sequential mechanical and enzymatic dissociation. The resulting cell populations were separated by differential trypsinization. Spheroids were formed by embedding the cell suspension in a dome of concentrated Matrigel solution. Cell viability was assessed using double staining with calcein-AM and ethidium homodimer, and expression of specific markers (Muc5AC, Na/K-ATPase, panCK) was analyzed by immunocytochemistry. Results. A sufficient number of viable cells was cultured from the postoperative material to form spheroids in Matrigel. Spheroids derived from epithelial and fibroblast-like human LG cells were obtained and characterized. Epithelial cells expressed Muc5AC and Na/K-ATPase, while fibroblasts formed characteristic reticular structures in the matrix. Conclusion. The tested protocol allows for the formation of viable spheroid structures that retain the morphofunctional properties of native LG tissue. The developed in vitro model of the human LG can be used to study the pathogenetic mechanisms of its diseases, particularly chronic nonspecific dacryoadenitis.