## 4 Discussion
In addition, our results demonstrated that OHT induced a significant increase in TM cell stiffness compared with the control cells. To the authors' knowledge, there are few reports on changes in TM cell mechanical properties under the effect of OHT. Previous studies have revealed that mammalian cells rely on the cytoskeleton to modify cell shape, while the cytoskeletal structure is correlated with cellular mechanical properties [7, 41, 42, 43]. Latrunculin B (Lat-B), a cytoskeletal disrupting agent, can obviously reduce the stiffness of human TM cells [44]. These results suggest that the OHT-induced increase in the elastic modulus of TM cells is probably due to the changes in cytoskeleton structure. Researchers have found that the mechanical properties of cells are closely linked to the cell function and development of disease [45, 46]. The senescent human TM cells showed increased stiffness [47]. DEX-treated human TM cells showed increased elastic moduli and increased cellular fibrotic activity [40]. In addition, there is a relationship between cell stiffness and tissue stiffness [48]. Therefore, the OHT-induced increase in TM cell stiffness in this study may also indicate the increase in TM tissue stiffness. It was found that the elastic modulus of TM tissue in glaucomatous patients was about 20-fold higher than that of normal people [9]. Increased stiffness of the TM would cause higher resistance to AH outflow [49], causing IOP to be elevated. By mathematical modeling, the researchers also found that the resistance to flow increases as the TM elastic modulus increases [9]. The function of the cytoskeleton relies on its three main components, actin filaments, microtubules, and intermediate filaments (IFs). IFs are critical in establishing and maintaining the structural and mechanical properties of cells [7]. Vimentin, an abundant cytoskeletal protein, is a major member of the IF family [50]. We observed that OHT downregulated the vimentin expression level compared with the control group. Studies have demonstrated that decreased expression levels of vimentin lead to increased assembly and contractility of stress fibers [51]. In addition, several studies have shown that the expression of vimentin is related to cell stiffness [52, 53, 54, 55]. Loss of vimentin increases contractile stress 3-fold [56]. Therefore, decreased expression of vimentin in TM cells may be one of the reasons for the increased cell stiffness induced by OHT. Actin filaments, microtubules, and IFs, as three distinct cytoskeletal filaments, are interconnected by protein-protein interactions, and if one of these filaments changes, the others are likely to change as well [57, 58]. This interaction provides flexibility for the entire cytoskeleton, allowing it to be continually rearranged to fulfill the needs of the cell under different conditions [59]. Consequently, the change in vimentin expression in this study may be the cause of the OHT-induced cytoskeletal rearrangement. Since vimentin IFs are tightly connected to the cell nucleus, forming a cage-like network surrounding the nucleus [60,61], vimentin is a key protein that connects the actin cytoskeleton to the nucleus and transduces mechanical signals to the nucleus [7]. This study found that vimentin is sensitive to OHT, which may be a key component of TM mechanotransduction, and its detailed function deserves further investigation. One limitation of this study is that we established an OHT model lasting only 2 weeks. In future studies, more animals can be involved to increase the OHT duration time points and explore the changes in TM cells' mechanobiological characteristics with OHT duration time. The other limitation is that the TM cells were extracted from an OHT rat model, and then cultured and tested in vitro. The differences between cells in vivo and cultured in vitro need to be further explored.