## 4 Discussion  
Optic nerve head (ONH) damage and progressive visual field loss are common features of all types of glaucoma [14]. POAG is the most prevalent subtype of glaucoma. Pathological IOP elevation due to increased AH outflow resistance is a significant risk factor for POAG blindness, but the mechanism is still unclear. This research aimed to explain the effect of OHT on TM at the cellular level. To reveal the effects of OHT on TM tissues and cells, researchers have applied stretch, compression, and shear stresses to normal TM cells cultured in vitro [25; 26; 27], or cultured the cells in 3D scaffolds [28; 29]. However, this does not comprehensively mimic the actual mechanical stimuli effects in vivo, making it difficult to obtain a complete understanding of the TM cell response to biomechanical stimuli. There are also studies based on TM cells from glaucomatous patients, wherein the cells are affected by many factors such as age, medications, and the course of the disease [30], so there may be some limitations in using the TM cells of glaucomatous patients directly for research. Therefore, establishing a proper animal model is essential to investigating the biomechanical effects of OHT on the structure and function of TM cells. Due to the similarity in structures of human and rat TMs [31; 32; 33] and the damage of the laser method to the TM tissue, we established rat OHT models  
Figure 5: The stiffness of the cells was tested by AFM. The Hertz equation was applied to fit the curve to obtain the elastic modulus. Elastic moduli of TM cells in the OHT group were greater than in the control group (***** * * \(p<0.0001\), \(n=30\)). Figure 6: Effects of OHT on the expression of vimentin in TM cells. (**A**) The bands of vimentin. Analysis of the bands was performed using Image J. (**B**) Quantification analysis of the expression level of vimentin. TM cells in the OHT group showed significantly decreased level of vimentin compared with control cells based on western blot analyses. * \(p<0.05\). using the microsphere injection method [17, 18] to investigate the mechanical properties, cytoskeletal changes, and protein expression of TM cells in this study. The average IOP of the experimental group remained above 24 mmHg from the 8 th day to the end of the experiment. On the 22 nd day after injection of the microspheres, the rats were executed, and TM cells were extracted for subsequent studies. We observed that the actin fibers of the control TM cells were regularly arranged in the same direction. In contrast, the actin fiber arrangement of the TM cells in the OHT group was significantly changed. These rearranged fibers showed a disordered morphology. The cytoskeletal network's structure is crucial for transmitting stresses and perceiving the mechanical microenvironment cues [34]. TM cells respond to mechanics by remodeling the cytoskeleton and extracellular matrix (ECM) pathways, thereby inhibiting their ability to drain AH [35]. Furthermore, we observed that OHT induced TM cells to exhibit affluent thin filamentous pseudopods and to form CLANs. In previous studies, CLAN formation has been found to lead to the suppression of TM cell functions, including proliferation, migration, and phagocytosis [36, 37, 38]. At the molecular level, changes in the cytoskeleton structure induce the activation of related proteins and pathways. For example, increased ECM stiffness (mimicking the glaucomatous TM matrix) induces YAP/TAZ activation by rearranging the cytoskeleton in TM cells [39], and YAP/TAZ is associated with increased TM cell fibrotic activity [40], which may lead to impaired AH outflow. We believe that the OHT-induced cytoskeletal rearrangement of TM cells is not only the result of OHT effects, but may also induce a series of intracellular mechanotransduction leading to a sustained increase in IOP. Then, we measured the elastic moduli of TM cells using AFM. The results showed that the elastic modulus in the control group was 1.02 \(\pm\) 0.10 KPa, which was consistent with the reported results [40].