## 1 Introduction  
Glaucoma is the major irreversible blinding disease worldwide, which is characterized by optic nerve atrophy and visual field defects. It is predicted that 111.8 million people will develop glaucoma by 2040 [1, 2]. Primary open-angle glaucoma (POAG) is the most common subtype of glaucoma, and its pathogenesis remains unclear. The homeostasis of AH production and drainage is essential for the maintenance of IOP. Increased AH outflow resistance can lead to increased IOP, which is a significant risk factor for POAG [3]. Lowering IOP is the primary treatment for POAG [4].  
The TM is an avascular, structurally complex connective tissue located at the angle between the cornea and the iris [5]. It is the main pathway for AH drainage and provides approximately 50-75% of the resistance to AH outflow [6], so it plays an essential role in regulating the resistance to AH outflow and modulating IOP. The ability of cells to resist stress and maintain shape, which relies on the cytoskeleton [7], is essential for tissue function. Cellular dysfunction in TM can lead to \"extra\" resistance, resulting in OHT [5]. In this process, cells actively remod and reorient their cytoskeleton in response to cyclic forces [8]. A previous study has shown that the TM tissue in glaucomatous patients is 20-fold stiffer than that of normal people, and this significant distinction may be associated with alterations in the cytoskeleton [9]. It has been revealed that the cytoskeletal arrangement of TM cells in human glaucomatous eyes is more random and disordered than in control eyes [10]. The cytoskeleton consists mainly of F-actin, microtubules, and intermediate filaments (IFs). Vimentin is one of the major proteins in IFs, which can confer mechanical flexibility to the cell [7]. Therefore, studies of cell mechanics, cytoskeleton, and associated proteins can help to elucidate the interaction mechanisms between TM cells and OHT.  
The response of cells to mechanical stimuli is essential for their biological function. Mechanobiology studies how the mechanical forces and mechanical properties of proteins, cells, and tissues exert an influence on physiology and disease [11]. Due to the special location of TM, TM cells are exposed to various mechanical stimuli [5] and are pressure-sensitive. Studies of TM mechanobiology may provide a theoretical basis for the understanding of the development and progression of OHT. To simulate the mechanical changes induced by OHT, researchers applied mechanical stretch, compression, or shear forces to the TM cells cultured in vitro using Flexcell or self-constructed equipment [12]. However, most of the published studies on TM response to mechanical stimuli are performed on a single mechanical factor in vitro, such as stretch, compression, or shear, which is different from the combination of mechanical stimuli TM experience in vivo. Studies of the TM cells extracted from the eyes of the OHT animal model will be helpful to further understand the mechanobiological response of TM to OHT and may contribute to the discovery of new and improved treatments for POAG.  
Rodents are the most favored animals in glaucoma models, and the anterior segment of the rat has a high degree of anatomical and developmental similarity to the human eye. The method of inducing OHT by microsphere injection was initially applied to mammals [13], but was later successfully applied to rats and mice [14]. Microspheres accumulate in the TM and Schlemm's canal (SC) and physically block the AH outflow pathway [15]. To understand the effect of OHT on TM cells, this study first established an OHT rat model by microsphere injection. Then, TM cells were extracted from the OHT and control eyes respectively. Finally, the mechanobiological alterations of these cells, including cytoskeletal morphology, cell elastic modulus, and vimentin expression level, were detected.