Open Access
Glaucoma  |   October 2024
Low Intraocular Pressure Induces Fibrotic Changes in the Trabecular Meshwork and Schlemm's Canal of Sprague Dawley Rats
Author Affiliations & Notes
  • Lijuan Xu
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
    Glaucoma Research Institute of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Yin Zhao
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Xinyao Zhang
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Xiaorui Gang
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Jialing Han
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Tao Zhou
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Binyan Qi
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Shuning Song
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Ruiyi Ren
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
    Glaucoma Research Institute of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Yuanbo Liang
    State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China
    The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
    Glaucoma Research Institute of Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Correspondence: Yuanbo Liang, The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China. e-mail: [email protected] 
  • Ruiyi Ren, The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China. e-mail: [email protected] 
Translational Vision Science & Technology October 2024, Vol.13, 10. doi:https://doi.org/10.1167/tvst.13.10.10
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      Lijuan Xu, Yin Zhao, Xinyao Zhang, Xiaorui Gang, Jialing Han, Tao Zhou, Binyan Qi, Shuning Song, Ruiyi Ren, Yuanbo Liang; Low Intraocular Pressure Induces Fibrotic Changes in the Trabecular Meshwork and Schlemm's Canal of Sprague Dawley Rats. Trans. Vis. Sci. Tech. 2024;13(10):10. https://doi.org/10.1167/tvst.13.10.10.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Continuous artificial aqueous humor drainage in the eyes of patients with glaucoma undergoing trabeculectomy likely exerts abnormal shear stress. However, it remains unknown how changes in intraocular pressure (IOP) can affect aqueous humor outflow (AHO).

Methods: Here, we induced and maintained low intraocular pressure (L-IOP) in healthy Sprague Dawley (SD) rats by puncturing their eyes using a tube (200-µm diameter) for 2 weeks. After the rats were euthanized, their eyes were removed, fixed, embedded, stained, and scanned to analyze the physiological and pathological changes in the trabecular meshwork (TM) and Schlemm's canal (SC). We measured SC parameters using ImageJ software and assessed the expression of various markers related to flow shear stress (KLF4), fibrosis (TGF-β1, TGF-β2, α-SMA, pSmad1/5, pSmad2/3, and fibronectin), cytoskeleton (integrin β1 and F-actin), diastolic function (nitric oxide synthase and endothelial nitric oxide synthase [eNOS]), apoptosis (cleaved caspase-3), and proliferation (Ki-67) using immunofluorescence or immunohistochemistry.

Results: L-IOP eyes showed a larger SC area, higher eNOS expression, and lower KLF4 and F-actin expression in the TM and SC (both P < 0.05) than control eyes. The aqueous humor of L-IOP eyes had a higher abundance of fibrotic proteins and apoptotic cells than that of control eyes, with significantly higher TGF-β1, α-SMA, fibronectin, and cleaved caspase-3 expression (all P < 0.05).

Conclusions: In conclusion, a persistence of L-IOP for 2 weeks may contribute to fibrosis in the TM and SC and might be detrimental to conventional AHO in SD rat eyes.

Translational Relevance: Clinicians should consider that aberrant shear force induced by aqueous humor fluctuation may damage AHO outflow channel when treating patients.

Introduction
Glaucoma is the leading cause of irreversible blindness worldwide and poses a great burden on both patients and society.1 Several studies have shown that the trabecular meshwork (TM) and Schlemm's canal (SC) play crucial roles in generating predominant resistance to trabecular aqueous humor outflow (AHO). Damage to these structures initiates an intraocular pressure (IOP) rise in primary open-angle glaucoma.2 
TM cells (TMCs) and SC endothelial cells (SCECs) reside in a dynamic environment under constantly reciprocating pulsatile flow-dependent shear stress (FSS), which is responsive to IOP and influenced by thermal gradients, velocity, aqueous humor (AqH) density, and canal diameter.3,4 FSS, which physically fluctuates within a certain range, provides sensory signals that initiate mechanotransduction responses and are essential for maintaining AHO function.5,6 Although some researchers have studied the structural changes of the TM and SC in glaucoma,7,8 detailed information on the AHO components and the molecular mechanisms of IOP regulation remains elusive. 
In glaucomatous eyes, the FSS may be beyond the normal limits following persistent IOP elevation, it can drop below its physiologic set point after external drainage surgeries, or it can be disturbed by insufficient nutrition supply or metabolism imbalance.9,10 Several studies have shown that an increase in IOP can lead to narrowing or even collapse of the SC lumen in certain regions,11,12 ultimately leading to outflow facility dysfunction.5 This phenomenon occurs when the AqH flow slows down or stops, resulting in a dramatic decrease in FSS across the TM and SC. McDonnell et al.13 found that high FSS promotes the activation of nitric oxide in the SC to maintain IOP homeostasis, similar to the modulation patterns in blood circulation, in which blood vessels under high FSS are protected against atherosclerosis compared to those under low shear stress.14 Gijsen et al.15 reported that vascular endothelial cells undergo fibrotic changes in response to alterations in the magnitude or direction of the FSS. Park et al.16 confirmed this finding in hypoperfused AHO of C57BL/6J mice. These studies suggest that endothelial cells in circulating canals or vessels are flow dependent, and high FSS within a certain range is a protective factor but low FSS may be detrimental. However, the influence of abnormal IOP and subsequent FSS on the physiological and pathological changes in the TM and SC remains elusive. 
Mechanical signals of FSS can be converted into biochemical signals, regulate gene expression, and manifest as biological effects such as proliferation, apoptosis, extracellular matrix (ECM) secretion, and permeability.17,18 Integrins are transmembrane proteins that receive mechanical signals,19 and integrin β1 plays a critical role in binding collagen and fibronectin (FN)-related signals.20 Krüppel-like factors (KLFs) play key roles in maintaining canal homeostasis; among these, KLF2 and KLF4 receive signals from FSS, regulate the expression of key vasoactive genes (such as Nos3 and Thbd), and are highly expressed in endothelial cells.21 Deng et al.22 found that high FSS promotes the expression of KLF2 by activating ALK1 and inhibiting the phosphorylation of Smad2/3, whereas low FSS has the opposite effect and leads to fibrosis. 
Typical low FSS conditions, such as TM outflow facility dysfunction or eyes with functional external drainage orifices, commonly occur in glaucoma. Schultz et al.23 reported a novel device (Beacon Aqueous Microshunt [BAM]) for the treatment of glaucoma, which continuously guides AqH from the anterior chamber to the surface of the eye. They achieved an IOP of 8 to 12 mm Hg with no severe complications; however, little is known about the long-term effects of such devices on AHO. In this study, we aimed to explore the changes in the TM and SC in response to low intraocular pressure (L-IOP) induced by external drainage puncture in vivo. 
Materials and Methods
Animals and Ethical Approval
Healthy female Sprague Dawley (SD) rats (6–8 weeks old; Charles River Laboratories, Wilmington, MA) were used in this study. The rats were housed and bred in clear cages placed in housing rooms at 21°C with a 12:12-hour light/dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University (wydw2022-0201). The rats were treated in accordance with the principles of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Animal Model of L-IOP
Previous studies have shown that an ocular puncture causes most of the AqH to flow through the artificial orifice,2426 leaving the residual traditional TM pathway hypoperfused. In this study, we created a rat model of L-IOP SD following the protocol described in a previous study,16 with a few modifications. Briefly, an artificial 2- to 3-mm tube (200-µm diameter) was prepared for puncturing. During surgery, healthy SD rats were anesthetized using gaseous isoflurane, and the eyes were punctured at the temporal side of the sclera, located approximately 1 mm away from the sclerocorneal junction. Ofloxacin ointment (0.3%) was prescribed immediately after surgery. To prevent spontaneous closure of the canal, the tube was rechecked and recanalized and the pupil was dilated with tropicamide every other day for 14 days. For recanalization, we removed tube obstructions using a needle and opened the conjunctiva or fibrous membrane that wraps the opening of the tube. Twenty-five eyes from 25 SD rats were used for L-IOP modeling, and 12 eyes from 12 untreated rats were used as the control. L-IOP was defined as IOP values (measured at different days within 1 week postoperatively, usually at postoperative days 1, 3, 5, and 7) less than 3 standard deviations of the mean at three or more time points. 
IOP Measurements
IOP measurements were conducted under anesthesia (gaseous isoflurane for approximately 2 minutes) with a rebound tonometer (iCare TONOVET, Vantaa, Finland) every 2 to 3 days (Monday, Wednesday, and Friday between 2 PM and 3 PM), based on a previous study.27 Each recorded IOP was the average of five measurements, and three IOP readings were recorded in the same eye to calculate the mean value. 
Histology and Immunostaining
The eyes collected from the SD rats were fixed in 4% paraformaldehyde overnight. The areas beyond the surgical sites (opposite areas of the surgical sites, approximately 12–6 o'clock) were embedded in paraffin or optimal cutting temperature compound in the sagittal axis. The 5-µm slices were serially numbered from the maximum diameter direction. The same- or similar-numbered slices were used for antibody staining. One section per eye was randomly selected for further analysis. 
For immunofluorescence (IF) staining, the sections were incubated overnight with primary antibodies specific to KLF4, pSmad2/3, pSmad1/5, endothelial nitric oxide synthase (eNOS), cleaved caspase-3, Ki-67, FN, F-actin, and TGF-β1 at 4°C according to the manufacturers’ instructions and a previous study.28 The secondary antibodies used were goat anti-rabbit or anti-mouse (Cell Signaling Technology, Danvers, MA) at a 1:500 dilution. Sections were subsequently incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI dihydrochloride; Beyotime Institute of Biotechnology, Shanghai, China) for 5 minutes to stain the nuclei; they were then washed and mounted. 
Hematoxylin and eosin (H&E) staining was performed according to an established protocol.29 For immunohistochemical (IHC) staining, the sections were incubated overnight with primary antibodies specific for integrin β1 and α-SMA at 4°C. After incubation with secondary antibodies at room temperature (20°–25°C) for 30 minutes, the sections were incubated with 3-3′-diaminobenzidine tetrahydrochloride (Genentech Shanghai Company Limited; Shanghai, China) for 30 minutes at room temperature and counterstained with hematoxylin (Beyotime Institute of Biotechnology). Detailed information on the antibodies used is provided in the Table
Table.
 
Antibodies Used in This Study
Table.
 
Antibodies Used in This Study
Fluorescing Sodium and Fluorescing Isothiocyanate–Bovine Serum Albumin Angiography in AHO
Fluorescing sodium (International Medication Systems, South El Monte, CA) and fluorescein isothiocyanate–bovine serum albumin (FITC-BSA, Solarbio, Beijing, China) angiography were performed according to a previous study.30 Briefly, fluorescent sodium was injected through the anterior chamber, and the fluorescence intensity of the subconjunctival AqH veins was observed under cobalt blue light. For fluorescing sodium, images were captured in situ, whereas for FITC-BSA staining the eyes were obtained 2 hours after dye injection for further evaluation of AHO opening by fluorescence microscopy. 
AqH Turnover Assessment
AqH turnover was measured following the protocols described in previous studies,31,32 with a few modifications. Briefly, 1 mL of benzalkonium chloride (BAC, 0.05%) (Shanghai Macklin Biochemical, Shanghai, China) was applied to the eye of an anesthetized SD rat to permeabilize the cornea to fluorescein for 10 minutes. Then, the BAC was thoroughly washed with plenty of saline. Subsequently, 1 mL of 0.025% sodium fluorescein (Alcon, Geneva, Switzerland) was applied to the eye for another 10 minutes. Images were acquired using a microscope (SZX2-ILLTS; Olympus Life Science, Waltham, MA) equipped with an Olympus DP23 camera and a green fluorescent protein (GFP) filter and were analyzed using in-built software (Olympus cellSens 4.1). The average pixel intensity in the green channel was determined in the area of interest using ImageJ (National Institutes of Health, Bethesda, MD). AqH clearance was determined by the decay constant calculated from the relative fluorescent intensity measured at 10-minute intervals for 60 minutes after a single fluorescein treatment. The artificial external drainage tube was blocked before AqH turnover measurement. To eliminate the influence of surgical procedures on the AqH, we analyzed the whole cornea and the 180° contralateral area of the surgical site (the nasal part). 
Enzyme-Linked Immunosorbent Assay
The concentrations of collagen-I (COL-I, cat. no. SU-B30413) and FN (cat. no. SU-B30659) in AqH were measured using commercially available kits (Konodi Biotechnology Company, Fujian, China) according to a previous study.33 Briefly, the enzyme conjugate was added to the sample and incubated for 60 minutes at 37°C. After incubating with substrates A and B for 15 minutes, the optical density was read at 450 nm using a microplate reader (SpectraMax 190, version 7.1.0; Molecular Devices, San Jose, CA) within 15 minutes. The concentrations of COL-I or FN were determined by comparing the optic density of the samples to the standard curve. The mean proportional inter-assay and intra-assay coefficients of variation were both <15%. The assay ranges were as follows: 1.25 to 40 ng/mL in the COL-I kit and 31.25 to 1000 µg/mL in the FN kit. The sensitivities of the tests were 0.1 ng/mL in the COL-I kit and 1.0 µg/mL in the FN kit. All assays were performed according to the manufacturer's instructions. 
Image Analysis
HE and IHC images were captured using a three-dimensional (3D) pathological section scanner (PANNORAMIC SCAN digital slide scanner; The Digital Pathology Company, Budapest, Hungary). The IF images were obtained using a microscope (ECLIPSE 80i; Nikon, Tokyo, Japan). The SC is defined as a thin, lucent, lymphatic-like vessel in the anterior chamber composed of a single layer of endothelial cells, within which the nuclei of cells in the inner wall usually protrude into the lumen. The TM is defined as the area between the ciliary body and the inner wall of the SC, with anterior and posterior endpoints of the Schwalbe’s line and scleral spur, respectively.34 Cells in the TM and SC that stained with antibodies (with the merged color of blue from DAPI and red or green from the secondary antibody) were considered positive. The SC parameters were measured by manually delineating the lumen cross-sectional area, anterior-to-posterior distance (the largest distance), and width (a straight line was drawn between the inner and outer walls of the SC and the maximum width was chosen), according to a previous study.35 For quantification, high-power fields (400× magnification) of the AHO (including the TM and SC) were captured from each model. ImageJ 1.52a was used to quantify positively stained cells. The experiments and image analyses were performed in a blinded manner. 
Statistical Analysis
Data analysis was conducted using the SPSS Statistics 22.0 (IBM, Chicago, IL) and Prism 7.0 (GraphPad, Boston, MA). Animal-based experiments included at least three biological and technical replicates. Values are presented as mean ± standard deviation. Statistical differences were determined by unpaired two-tailed Student's t-tests or analysis of variance. A paired t-test was used to compare IOP changes from baseline. Statistical significance was set at P < 0.05. 
Results
Decreased AqH Outflow in L-IOP Models
All modeled eyes reached the standard of L-IOP definition. IOPs were significantly decreased in these eyes from the baseline: 14.3 ± 1.7 mm Hg to 9.7 ± 1.9 mm Hg on day 2 and 10.1 ± 1.7 mm Hg on day 14 (P < 0.01) (Figs. 1A, 1B; Supplementary Fig. S1). The mean reduction on day 14 was 31.2% (95% confidence interval [CI], 22.4–40.1) of the baseline values. 
Figure 1.
 
Comparisons of IOP and fluorescent dye accumulation in AHO between groups. (A) Representative figures of the eyes of SD rats at different follow-ups. (B) IOP at all postoperative visits within 14 days. (C) Comparison of AHO angiography between the two groups. (D) FITC-BSA accumulation in AHO after 2 hours of injection into the anterior chamber. The dotted line represents the accumulated fluorescent dyes. **P < 0.01. Scale bar: 100 µm.
Figure 1.
 
Comparisons of IOP and fluorescent dye accumulation in AHO between groups. (A) Representative figures of the eyes of SD rats at different follow-ups. (B) IOP at all postoperative visits within 14 days. (C) Comparison of AHO angiography between the two groups. (D) FITC-BSA accumulation in AHO after 2 hours of injection into the anterior chamber. The dotted line represents the accumulated fluorescent dyes. **P < 0.01. Scale bar: 100 µm.
Next, to confirm the direction of AqH flow, we injected a fluorescent dye into the anterior chamber and recorded the perfusion pattern, which revealed significantly slower AqH flow through the traditional TM pathway in eyes with L-IOP than that in control eyes (Fig. 1C). These results were further confirmed using FITC-BSA accumulation (Fig. 1D), suggesting the hypoperfusion of AHO in L-IOP models. 
The AqH outflow rate assessed using fluorophotometry tended to be lower in L-IOP eyes than in the control from 40 minutes onward in whole eyes, although the differences were not statistically significant (P = 0.09 to 0.10) (Fig. 2A). In contrast, AqH clearance was significantly delayed in the L-IOP eyes from 40 minutes onward in the nasal part of the eyes as indicated by a significantly reduced exponential decay constant and elevated fluorescent intensities (all P < 0.05) (Figs. 2B, 2C). These results indicated a decay of fluorescence clearance in L-IOP eyes. 
Figure 2.
 
Decreased AqH outflow rates in the L-IOP SD rats. (A, B) Comparison of the rate of AqH outflow between the L-IOP and control eyes in the whole eye and the nasal parts. (C) Representative series of images captured at 10-minute intervals after the application of fluorescein in SD rats. *P < 0.05. CON, control; T, time.
Figure 2.
 
Decreased AqH outflow rates in the L-IOP SD rats. (A, B) Comparison of the rate of AqH outflow between the L-IOP and control eyes in the whole eye and the nasal parts. (C) Representative series of images captured at 10-minute intervals after the application of fluorescein in SD rats. *P < 0.05. CON, control; T, time.
SC is Enlarged in SD Rat Eyes With L-IOP
The cross-sectional area of the SC in the L-IOP group (n = 5) was significantly larger than that in the control group (n = 10), with an average expansion of 65.1% over the baseline (P < 0.05) (Figs. 3A–C). However, the anterior-to-posterior distance and width of the SC between the two groups did not differ significantly (Figs. 3D, 3E). Additionally, eNOS, which was mostly expressed in the inner wall of the SC, was significantly increased in modeled eyes (P < 0.05) (Fig. 4). These results indicate that the SC expanded after L-IOP modeling. 
Figure 3.
 
(A, B) Representative H&E images of SD rat eyes in the control and L-IOP groups. (CE) Comparison of SC parameters between the two groups: area (C), anterior to posterior distance (D), width (E). *P < 0.05. Scale bar: 50 µm. ns, no difference.
Figure 3.
 
(A, B) Representative H&E images of SD rat eyes in the control and L-IOP groups. (CE) Comparison of SC parameters between the two groups: area (C), anterior to posterior distance (D), width (E). *P < 0.05. Scale bar: 50 µm. ns, no difference.
Figure 4.
 
(A) Representative IF images of eNOS expression in L-IOP and control eyes. (B) Quantitative comparison between the two groups. The white star in (A) represents the SC, and white arrows indicate significantly positive cells. **P < 0.01. Scale bars: 50 µm in the first column; 20 µm in the second and third columns. CB, ciliary body.
Figure 4.
 
(A) Representative IF images of eNOS expression in L-IOP and control eyes. (B) Quantitative comparison between the two groups. The white star in (A) represents the SC, and white arrows indicate significantly positive cells. **P < 0.01. Scale bars: 50 µm in the first column; 20 µm in the second and third columns. CB, ciliary body.
L-IOP Induces Fibrotic Changes in the TM and SC of SD Rats
The expression of TGF-β1 and TGF-β2 was significantly higher in the TM and SC of L-IOP eyes than in the control eyes (both P < 0.05) (Figs. 5A, 5B; Supplementary Fig. S2). However, the expression of pSmad1/5 and pSmad2/3 was not statistically different between them (Figs. 5A, 5C, 5D). The expression of FN increased significantly in L-IOP eyes (Figs. 5A, 5E). As shown in Figure 6, the expression of ɑ-SMA (P < 0.05) was significantly increased, whereas that of integrin β1 showed a decreasing trend (P = 0.12) in L-IOP eyes compared to control eyes. On day 14, the concentration of COL-I in the AqH did not change, whereas that in FN was significantly higher in L-IOP eyes than in the control (Figs. 7A, 7B). These results confirmed the fibrotic changes in the TM and SC of eyes with L-IOP. 
Figure 5.
 
(A) Representative images of TGF-β1, pSmad1/5, pSmad2/3, and FN in L-IOP and control eyes. (Be) Quantitative comparison of fibrotic marker expression between the two groups: TGF-β1 (B), pSmad1/5 (C), pSmad2/3 (D), FN (E). White arrows identify significantly positive cells. **P < 0.01. Scale bar: 20 µm.
Figure 5.
 
(A) Representative images of TGF-β1, pSmad1/5, pSmad2/3, and FN in L-IOP and control eyes. (Be) Quantitative comparison of fibrotic marker expression between the two groups: TGF-β1 (B), pSmad1/5 (C), pSmad2/3 (D), FN (E). White arrows identify significantly positive cells. **P < 0.01. Scale bar: 20 µm.
Figure 6.
 
(A) Representative images. (B) Comparison of integrin β1 and α-SMA expression between L-IOP and control groups: integrin β1 (B), α-SMA (C). *P < 0.05. Scale bar: 50 µm.
Figure 6.
 
(A) Representative images. (B) Comparison of integrin β1 and α-SMA expression between L-IOP and control groups: integrin β1 (B), α-SMA (C). *P < 0.05. Scale bar: 50 µm.
Figure 7.
 
(A, B) Quantitative comparison of COL-I (A) and FN (B) concentrations assessed using enzyme-linked immunosorbent assays between the L-IOP and the control eyes. *P < 0.05.
Figure 7.
 
(A, B) Quantitative comparison of COL-I (A) and FN (B) concentrations assessed using enzyme-linked immunosorbent assays between the L-IOP and the control eyes. *P < 0.05.
L-IOP Promotes Apoptosis of TMCs and SCECs in SD Rats
The expression of the downstream effector marker of apoptosis, cleaved caspase-3, was significantly increased in TMCs and SCECs of L-IOP eyes (P < 0.05) (Figs. 8A, 8B), whereas that of the proliferative marker Ki-67 did not change (P > 0.05) (Fig. 8C), suggesting that L-IOP modeling induced apoptosis in TMCs and SCECs. 
Figure 8.
 
(A) Representative images of cleaved caspase-3 and Ki-67 in L-IOP eyes. (B) Quantitative comparison of their expression between the groups: cleaved caspase-3 (B), Ki-67 (C). White arrows identify significantly positive cells. *P < 0.05. Scale bar: 20 µm.
Figure 8.
 
(A) Representative images of cleaved caspase-3 and Ki-67 in L-IOP eyes. (B) Quantitative comparison of their expression between the groups: cleaved caspase-3 (B), Ki-67 (C). White arrows identify significantly positive cells. *P < 0.05. Scale bar: 20 µm.
L-IOP Inhibits Integrin β1/KLF4 Signaling in SD Rats
The expression of KLF4 in the TM and SC of L-IOP eyes significantly decreased compared to that in the TM and SC of control eyes (P < 0.05) (Figs. 9A, 9B), suggesting a lower FSS sensed by TMCs and SCECs in eyes with L-IOP. Moreover, the L-IOP group showed slightly lower integrin β1 levels than the control group (Fig. 6). These results indicate that L-IOP inhibited the integrin β1–KLF4 pathway. Additionally, eyes with L-IOP had significantly lower F-actin levels than control eyes (P < 0.05) (Figs. 9A, 9C), indicating destruction of the cytoskeleton in TMCs and SCECs. 
Figure 9.
 
(A) Representative images of KLF4 and F-actin in the L-IOP and control groups. (B) Quantitative comparison of their expression between the two groups: KLF4 (B), F-actin (C). White arrows identify significantly positive cells. *P < 0.05. Scale bar: 20 µm.
Figure 9.
 
(A) Representative images of KLF4 and F-actin in the L-IOP and control groups. (B) Quantitative comparison of their expression between the two groups: KLF4 (B), F-actin (C). White arrows identify significantly positive cells. *P < 0.05. Scale bar: 20 µm.
Discussion
Abnormal fluid dynamics play a crucial role in the development of fibrosis within systemic circulation.36 Continuous AqH drainage through an artificial pathway in the eyes of patients with glaucoma who undergo trabeculectomy is likely to exert abnormal shear stress and trigger the overexpression of fibrotic factors, such as TGF-β.37 With regard to AqH-related TGF-β, studies have mainly focused on conjunctiva fibrosis, but little is known about the relationship among IOP, FSS, and TGF-β and their influence on AHO. Moreover, most studies on glaucoma have concentrated on high IOP, a characteristic feature of glaucoma. In contrast, in the present study, we focused on the influence of low IOP and the resulting low FSS on the TM and SC. L-IOP conditions, such as sudden AqH loss during internal ocular surgeries, strong external filtration after anti-glaucoma surgeries, and corneal ulcers, are common occurrences that lead to hypoperfusion of the TM and SC. However, despite its prevalence, the effects of L-IOP on the TM and SC and on AqH drainage from the perspective of long-term efficacy have remained unknown. 
In the present study, we explored the effects of L-IOP and thus low FSS on the TM and SC in detail. We hypothesized that low FSS across TMCs and SCECs induces the release of profibrotic factors and activates them in the circulation, leading to progressive fibrosis of the TM and SC. To test this hypothesis, we used an animal model of L-IOP with AqH external drainage surgery that closely mimicked the dynamics of patients with glaucoma after trabeculectomy. Healthy eyes possess an automatic inflow and outflow regulation mechanism to maintain equilibrium: When the IOP deviates from the normal value, the eye automatically adjusts the outflow rate through the TM to gradually return the IOP to normal levels. Our experimental model demonstrated a pronounced decrease in IOP and relatively low FSS, as indicated by the expression of KLF4, confirming successful establishment of the low FSS model. Overall, we observed a spectrum of anatomical and molecular alterations in the TM and SC driven by L-IOP lasting for 14 days. Based on the in vivo data, dilation of the SC after L-IOP modeling revealed a complex relationship between the TM and SC fibrosis and shear stress in a surgical L-IOP SD rat model. Furthermore, the findings also showed cytoskeletal degradation in TMCs and SCECs in this model. 
One of the major findings of the present study is the significantly enlarged area of the SC along with overexpressed eNOS in the inner wall of the SC in the L-IOP model. This finding aligns with those of previous studies.38,39 For example, Zhang et al.39 demonstrated an average 123% increase in SC volume by visible light OCT when IOP decreased by 10 mm Hg from the baseline. FSS plays a crucial role in initiating wall responses that optimize the elasticity, compliance, and lumen size of the canal wall, thereby maintaining intrinsic pressure homeostasis through a feedback loop. In normal vasculature of the canal, low FSS elicits an adaptive response by the wall, leading to constrictive remodeling and restoration of the FSS to physiological baseline levels.40,41 However, the response to low FSS in the SC is complex. We speculate that the dramatic and persistent decrease in FSS across the TM and SC in our study promoted vasoactive factors, such as nitric oxide or prostaglandin, which elicited compensatory expansive remodeling. However, factors determining whether the expansive remodeling response to low FSS becomes compensatory or excessive have not been studied previously and therefore warrant further elucidation. 
Another novel aspect of our model is the histopathological confirmation of fibrosis of the TM and SC. Although the causative role of low FSS in fibrosis has been previously studied in vivo and in vitro,42,43 it is unclear how low FSS orchestrates fibrosis in the TM and SC. A recent study,42 using a three-dimensional liver sinusoidal model, showed that a lower than normal range of FSS was associated with decreased functionality and increased cell damage, eventually leading to fibrosis. In our study, we observed activation of TGF-β1 and TGF-β2 in the TM and SC of L-IOP eyes. Furthermore, we demonstrated upregulation of the downstream markers FN and α-SMA, indicating the activation of canonical fibrosis pathways. These findings suggest that TGF-β may act as a signal for fibrosis under conditions of low shear stress. These findings are consistent with those of previous studies.44,45 Due to the circulating nature of the AqH, it has been postulated that shear force may activate TGF-β in a paracrine fashion through tissue stretching and mechanical, traction-based cellular activation.46 However, shear force may additionally lead to the activation of circulating TGF-β. 
The mean difference between the IOP at baseline and 14 days after low FSS modeling was 31.2%, demonstrating substantial changes in FSS strength. Ishibazawa et al.47 reported that relatively low shear stress (1.5 dyn/cm2) significantly upregulated the mRNA expression of adhesion molecules, proinflammatory cytokines, and procoagulant factors in vascular endothelial cells. Low FSS can also lead to cell depolarization, disorganization of cell–cell junctions,48 inflammation,49 and enhanced epithelial–mesenchymal transition in endothelial cells.43 Accordingly, we speculate that, in our low-FSS model, the disintegration of intercellular junctions and the underlying basement membrane may also participate in the initiation of excessive ECM accumulation in the TM and SC. Furthermore, severe junction and basement membrane disintegration provides a gateway for inflammatory cells to enter the AHO, where they promote enzymatic degradation of collagen and elastin fibers, disturb the cellular environment, and ultimately induce excessive ECM production. However, this hypothesis warrants further investigation. 
In addition to the above findings, we observed that L-IOP induced apoptosis and cytoskeletal destruction in TMCs and SCECs, as confirmed by the increased levels of cleaved caspase-3 and decreased levels of F-actin. We suggest that TMCs and SCECs, like vascular endothelial cells, sense shear stresses and transmit these stresses into biochemical signals that not only induce excessive ECM production but also mediate cell morphology and survival. 
Shear force plays a crucial role in vascular homeostasis, and its destabilization can initiate disease pathogenesis. Endothelial cells lining the inner wall of blood vessels can sense changes in shear flow and subsequently align their cytoskeleton to modulate their traction forces. Long et al.43 reported that cell junctions, such as focal adhesion, tight junction, and adhesion junction, are involved in mechanotransduction induced by shear stresses. Furthermore, several signals, including PIEZO1 and PECAM1 interactions,49 plexin D1,50 FOX2,51 and IGPR-1,52 participate in cell junction remodeling. However, the exact mechanisms underlying shear-force pulsatile oscillation and TM and SC responses require further study. 
A major limitation of our study pertains to the experimental model used. The uncertainty of the tube blockage time and the functional outflow facility, as well as interference from recanalization, resulted in an unstable experimental condition. Ideally, animal models exposed to controlled FSS with detailed flow parameters are preferable. However, achieving such conditions is challenging due to technical limitations, which hinder the direct extrapolation of our results to humans. Furthermore, our experimental low FSS lasted for only 14 days, as prolonged low FSS could result in canal stagnation due to fibrosis. This time frame may not have been sufficient to investigate the impact of severely low FSS on AHO changes. Although the observed AHO behavior supports the concept that low FSS leads to excessive ECM production, animal growth during the period of our investigation may have contributed to the observed TM and SC remodeling patterns. Another limitation is that considering that TMCs and SCECs have the same function as endothelial cells and that both underwent shear force induced by AqH pumping, we treated and analyzed the TM and SC as a unit. However, this approach may have masked some information due to the different nature of these two cell types. Additionally, the fibrotic-like phenotype in AHO of our L-IOP models was apparently similar to glaucoma eyes with high IOP; although the molecular injury mechanisms and components (e.g., collagen subtypes) between them may differ; we did not compare these aspects in the current study. Finally, we studied a limited number of animals, which might have reduced the statistical power of our findings. 
In conclusion, our study demonstrates that L-IOP and thus FSS interference induce expansion of the SC and promote the progression of fibrosis in the TM and SC. However, a direct demonstration showing that the prevention of FSS changes or the neutralization of the effects prevents pathological TM and SC changes is required to establish a causal relationship. Our findings expand our current understanding of the correlation between IOP reduction and trabecular AHO and suggest that controlling IOP levels within a certain stable and safe range is essential in glaucoma management. Nevertheless, further research is warranted to determine the exact range of IOPs that are considered stable and safe. 
Acknowledgments
Supported by grants from the National Nature Science Foundation of China (82201177), Health Commission of Zhejiang Province (2023KY914), Key Research and Development Projects of Zhejiang Province (2022C03112), Program for Zhejiang Leading Talent of S&T Innovation (2021R52012), National Key Research and Development Project of China (2020YFC2008200), Zhejiang Provincial National Science Foundation of China (LQ18H120010), Key Innovation and Guidance Program of the Eye Hospital, School of Ophthalmology & Optometry at Wenzhou Medical University (YNZD 2201903), Wenzhou Municipal Technological Innovation Program of High-Level Talents (604090352/577), and High-Level Innovation Team of Wenzhou (2022). 
Disclosure: L. Xu, None; Y. Zhao, None; X. Zhang, None; X. Gang, None; J. Han, None; T. Zhou, None; B. Qi, None; S. Song, None; R. Ren, None; Y. Liang, None 
References
Tham YC, Li X, Wong TY, Quigley HA, Aung T, Cheng C-Y. Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology. 2014; 121(11): 2081–2090. [CrossRef] [PubMed]
Wang L-Y, Su G-Y, Wei Z-Y, Zhang Z-J, Liang Q-F. Progress in the basic and clinical research on the Schlemm's canal. Int J Ophthalmol. 2020; 13(5): 816–821. [CrossRef] [PubMed]
Guo J-M, Chen Z-Q, Chen W, Yan X-Q, Zhang H, Wang J-M. Numerical simulation of the flow of aqueous humor in the Schlemm's canal. Med Eng Phys. 2021; 88: 25–31. [CrossRef] [PubMed]
Qin Z, Meng L, Yang F, Zhang C, Wen B. Aqueous humor dynamics in human eye: a lattice Boltzmann study. Math Biosci Eng. 2021; 18(5): 5006–5028. [CrossRef] [PubMed]
Johnstone M, Xin C, Tan J, Martin E, Wen J, Wang RK. Aqueous outflow regulation - 21st century concepts. Prog Retin Eye Res. 2021; 83: 100917. [CrossRef] [PubMed]
Johnstone MA. Intraocular pressure regulation: findings of pulse-dependent trabecular meshwork motion lead to unifying concepts of intraocular pressure homeostasis. J Ocul Pharmacol Ther. 2014; 30(2-3): 88–93. [CrossRef] [PubMed]
Tektas OY, Lutjen-Drecoll E. Structural changes of the trabecular meshwork in different kinds of glaucoma. Exp Eye Res. 2009; 88(4): 769–775. [CrossRef] [PubMed]
Chung HW, Park JH, Yoo C, Kim YY. Effects of trabecular meshwork width and Schlemm's canal area on intraocular pressure reduction in glaucoma patients. Korean J Ophthalmol. 2021; 35(4): 311–317. [CrossRef] [PubMed]
Leidl MC, Choi CJ, Syed ZA, Melki SA. Intraocular pressure fluctuation and glaucoma progression: what do we know? Br J Ophthalmol. 2014; 98(10): 1315–1319. [CrossRef] [PubMed]
Yang H, Song H, Mei X, et al. Experimental research on intraocular aqueous flow by PIV method. Biomed Eng Online. 2013; 12: 108. [CrossRef] [PubMed]
Li G, Farsiu S, Chiu SJ, et al. Pilocarpine-induced dilation of Schlemm's canal and prevention of lumen collapse at elevated intraocular pressures in living mice visualized by OCT. Invest Ophthalmol Vis Sci. 2014; 55(6): 3737–3746. [CrossRef] [PubMed]
Johnstone MA, Grant WG. Pressure-dependent changes in structures of the aqueous outflow system of human and monkey eyes. Am J Ophthalmol. 1973; 75(3): 365–383. [CrossRef] [PubMed]
McDonnell F, Perkumas KM, Ashpole NE, et al. Shear stress in Schlemm's canal as a sensor of intraocular pressure. Sci Rep. 2020; 10(1): 5804. [CrossRef] [PubMed]
Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991; 43(2): 109–142. [PubMed]
Gijsen F, Katagiri Y, Barlis P, et al. Expert recommendations on the assessment of wall shear stress in human coronary arteries: existing methodologies, technical considerations, and clinical applications. Eur Heart J. 2019; 40(41): 3421–3433. [CrossRef] [PubMed]
Park DY, Lee J, Park I, et al. Lymphatic regulator PROX1 determines Schlemm's canal integrity and identity. J Clin Invest. 2014; 124(9): 3960–3974. [CrossRef] [PubMed]
Culver JC, Dickinson ME. The effects of hemodynamic force on embryonic development. Microcirculation. 2010; 17(3): 164–178. [CrossRef] [PubMed]
Baeyens N, Bandyopadhyay C, Coon BG, Yun S, Schwartz MA. Endothelial fluid shear stress sensing in vascular health and disease. J Clin Invest. 2016; 126(3): 821–828. [CrossRef] [PubMed]
Chen J, Green J, Yurdagul AJ, Albert P, McInnis MC, Orr AW. αvβ3 integrins mediate flow-induced NF-κB activation, proinflammatory gene expression, and early atherogenic inflammation. Am J Pathol. 2015; 185(9): 2575–2589. [CrossRef] [PubMed]
Takada Y, Ye X, Simon S. The integrins. Genome Biol. 2007; 8(5): 215. [CrossRef] [PubMed]
Sangwung P, Zhou G, Nayak L, et al. KLF2 and KLF4 control endothelial identity and vascular integrity. JCI Insight. 2017; 2(4): e91700. [CrossRef] [PubMed]
Deng H, Min E, Baeyens N, et al. Activation of Smad2/3 signaling by low fluid shear stress mediates artery inward remodeling. Proc Natl Acad Sci USA. 2021; 118(37): e2105339118. [CrossRef] [PubMed]
Schultz T, Schojai M, Kersten-Gomez I, Matthias E, Boecker J, Dick HB. Ab externo device for the treatment of glaucoma: direct flow from the anterior chamber to the ocular surface. J Cataract Refract Surg. 2020; 46(7): 941–943. [CrossRef] [PubMed]
Villamarin A, Roy S, Hasballa R, Vardoulis O, Reymond P, Stergiopulos N. 3D simulation of the aqueous flow in the human eye. Med Eng Phys. 2012; 34(10): 1462–1470. [CrossRef] [PubMed]
Lutjen-Drecoll E, Barany EH. Functional and electron microscopic changes in the trabecular meshwork remaining after trabeculectomy in cynomolgus monkeys. Invest Ophthalmol. 1974; 13(7): 511–524. [PubMed]
Kotliar KE, Kozlova TV, Lanzl IM. Postoperative aqueous outflow in the human eye after glaucoma filtration surgery: biofluidmechanical considerations. Biomed Tech (Berl). 2009; 54(1): 14–22. [CrossRef] [PubMed]
McDowell CM, Kizhatil K, Elliott MH, et al. Consensus recommendation for mouse models of ocular hypertension to study aqueous humor outflow and its mechanisms. Invest Ophthalmol Vis Sci. 2022; 63(2): 12. [CrossRef] [PubMed]
Xu LJ, Rong SS, Xu YS, et al. Anti-fibrosis potential of pirarubicin via inducing apoptotic and autophagic cell death in rabbit conjunctiva. Exp Eye Res. 2020; 200: 108215. [CrossRef] [PubMed]
Maslin JS, Chen PP, Sinard J, Nguyen AT, Noecker R. Histopathologic changes in cadaver eyes after MicroPulse and continuous wave transscleral cyclophotocoagulation. Can J Ophthalmol. 2020; 55(4): 330–335. [CrossRef] [PubMed]
Yang J, Sun N, Xiong Q, Yang R. Effect of moxonidine on the uveoscleral outflow: role of α2-adrenoceptors or I1 imidazoline receptors. Curr Eye Res. 2009; 34(4): 287–296. [CrossRef] [PubMed]
Avila MY, Mitchell CH, Stone RA, Civan MM. Noninvasive assessment of aqueous humor turnover in the mouse eye. Invest Ophthalmol Vis Sci. 2003; 44(2): 722–727. [CrossRef] [PubMed]
Buys ES, Ko YC, Alt C, et al. Soluble guanylate cyclase a1-deficient mice: a novel murine model for primary open angle glaucoma. Ann Neurosci. 2013; 20(2): 65–66. [PubMed]
Koçak N, Can E, Yeter V, et al. Aqueous humor and serum levels of 4-hydroxynonenal and 8-hydroxy-2′deoxyguanosine in pseudoexfoliation syndrome and glaucoma. Int Ophthalmol. 2023; 43(4): 1395–1404. [CrossRef] [PubMed]
Yan X, Li M, Wang J, Zhang H, Zhou X, Chen Z. Morphology of the trabecular meshwork and Schlemm's canal in Posner–Schlossman syndrome. Invest Ophthalmol Vis Sci. 2022; 63(1): 1. [CrossRef] [PubMed]
Park JH, Chung HW, Yoon EG, Ji MJ, Yoo C, Kim YY. Morphological changes in the trabecular meshwork and Schlemm's canal after treatment with topical intraocular pressure-lowering agents. Sci Rep. 2021; 11(1): 18169. [CrossRef] [PubMed]
Paliwal N, Ali RL, Salvador M, et al. Presence of left atrial fibrosis may contribute to aberrant hemodynamics and increased risk of stroke in atrial fibrillation patients. Front Physiol. 2021; 12: 657452. [CrossRef] [PubMed]
Gater R, Ipek T, Sadiq S, et al. Investigation of conjunctival fibrosis response using a 3D glaucoma Tenon's capsule + conjunctival model. Invest Ophthalmol Vis Sci. 2019; 60(2): 605–614. [CrossRef] [PubMed]
Hann CR, Vercnocke AJ, Bentley MD, Jorgensen SM, Fautsch MP. Anatomic changes in Schlemm's canal and collector channels in normal and primary open-angle glaucoma eyes using low and high perfusion pressures. Invest Ophthalmol Vis Sci. 2014; 55(9): 5834–5841. [CrossRef] [PubMed]
Zhang X, Beckmann L, Miller DA, et al. In vivo imaging of Schlemm's canal and limbal vascular network in mouse using visible-light OCT. Invest Ophthalmol Vis Sci. 2020; 61(2): 23. [CrossRef]
Fisher AB, Chien S, Barakat AI, Nerem RM. Endothelial cellular response to altered shear stress. Am J Physiol Lung Cell Mol Physiol. 2001; 281(3): L529–L533. [CrossRef] [PubMed]
Gulino-Debrac D. Mechanotransduction at the basis of endothelial barrier function. Tissue Barriers. 2013; 1(2): e24180. [CrossRef] [PubMed]
Li W, Li P, Li N, et al. Matrix stiffness and shear stresses modulate hepatocyte functions in a fibrotic liver sinusoidal model. Am J Physiol Gastrointest Liver Physiol. 2021; 320(3): G272–G282. [CrossRef] [PubMed]
Long Y, Niu Y, Liang K, Du Y. Mechanical communication in fibrosis progression. Trends Cell Biol. 2022; 32(1): 70–90. [CrossRef] [PubMed]
Leask A, Abraham DJ. TGF-β signaling and the fibrotic response. FASEB J. 2004; 18(7): 816–827. [CrossRef] [PubMed]
Varshney R, Murphy B, Woolington S, et al. Inactivation of platelet-derived TGF-β1 attenuates aortic stenosis progression in a robust murine model. Blood Adv. 2019; 3(5): 777–788. [CrossRef] [PubMed]
Gould ST, Srigunapalan S, Simmons CA, Anseth KS. Hemodynamic and cellular response feedback in calcific aortic valve disease. Circ Res. 2013; 113(2): 186–197. [CrossRef] [PubMed]
Ishibazawa A, Nagaoka T, Yokota H, Ono S, Yoshida A. Low shear stress up-regulation of proinflammatory gene expression in human retinal microvascular endothelial cells. Exp Eye Res. 2013; 116: 308–311. [CrossRef] [PubMed]
Bougaran P, Bats ML, Delobel V, et al. ROR2/PCP a new pathway controlling endothelial cell polarity under flow conditions. Arterioscler Thromb Vasc Biol. 2023; 43(7): 1199–1218. [CrossRef] [PubMed]
Chuntharpursat-Bon E, Povstyan OV, Ludlow MJ, et al. PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing. Commun Biol. 2023; 6(1): 358. [CrossRef] [PubMed]
Mehta V, Pang KL, Rozbesky D, et al. The guidance receptor plexin D1 is a mechanosensor in endothelial cells. Nature. 2020; 578(7794): 290–295. [CrossRef] [PubMed]
Sabine A, Bovay E, Demir CS, et al. FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature. J Clin Invest. 2015; 125(10): 3861–3877. [CrossRef] [PubMed]
Ho RX, Tahboub R, Amraei R, et al. The cell adhesion molecule IGPR-1 is activated by and regulates responses of endothelial cells to shear stress. J Biol Chem. 2019; 294(37): 13671–13680. [CrossRef] [PubMed]
Figure 1.
 
Comparisons of IOP and fluorescent dye accumulation in AHO between groups. (A) Representative figures of the eyes of SD rats at different follow-ups. (B) IOP at all postoperative visits within 14 days. (C) Comparison of AHO angiography between the two groups. (D) FITC-BSA accumulation in AHO after 2 hours of injection into the anterior chamber. The dotted line represents the accumulated fluorescent dyes. **P < 0.01. Scale bar: 100 µm.
Figure 1.
 
Comparisons of IOP and fluorescent dye accumulation in AHO between groups. (A) Representative figures of the eyes of SD rats at different follow-ups. (B) IOP at all postoperative visits within 14 days. (C) Comparison of AHO angiography between the two groups. (D) FITC-BSA accumulation in AHO after 2 hours of injection into the anterior chamber. The dotted line represents the accumulated fluorescent dyes. **P < 0.01. Scale bar: 100 µm.
Figure 2.
 
Decreased AqH outflow rates in the L-IOP SD rats. (A, B) Comparison of the rate of AqH outflow between the L-IOP and control eyes in the whole eye and the nasal parts. (C) Representative series of images captured at 10-minute intervals after the application of fluorescein in SD rats. *P < 0.05. CON, control; T, time.
Figure 2.
 
Decreased AqH outflow rates in the L-IOP SD rats. (A, B) Comparison of the rate of AqH outflow between the L-IOP and control eyes in the whole eye and the nasal parts. (C) Representative series of images captured at 10-minute intervals after the application of fluorescein in SD rats. *P < 0.05. CON, control; T, time.
Figure 3.
 
(A, B) Representative H&E images of SD rat eyes in the control and L-IOP groups. (CE) Comparison of SC parameters between the two groups: area (C), anterior to posterior distance (D), width (E). *P < 0.05. Scale bar: 50 µm. ns, no difference.
Figure 3.
 
(A, B) Representative H&E images of SD rat eyes in the control and L-IOP groups. (CE) Comparison of SC parameters between the two groups: area (C), anterior to posterior distance (D), width (E). *P < 0.05. Scale bar: 50 µm. ns, no difference.
Figure 4.
 
(A) Representative IF images of eNOS expression in L-IOP and control eyes. (B) Quantitative comparison between the two groups. The white star in (A) represents the SC, and white arrows indicate significantly positive cells. **P < 0.01. Scale bars: 50 µm in the first column; 20 µm in the second and third columns. CB, ciliary body.
Figure 4.
 
(A) Representative IF images of eNOS expression in L-IOP and control eyes. (B) Quantitative comparison between the two groups. The white star in (A) represents the SC, and white arrows indicate significantly positive cells. **P < 0.01. Scale bars: 50 µm in the first column; 20 µm in the second and third columns. CB, ciliary body.
Figure 5.
 
(A) Representative images of TGF-β1, pSmad1/5, pSmad2/3, and FN in L-IOP and control eyes. (Be) Quantitative comparison of fibrotic marker expression between the two groups: TGF-β1 (B), pSmad1/5 (C), pSmad2/3 (D), FN (E). White arrows identify significantly positive cells. **P < 0.01. Scale bar: 20 µm.
Figure 5.
 
(A) Representative images of TGF-β1, pSmad1/5, pSmad2/3, and FN in L-IOP and control eyes. (Be) Quantitative comparison of fibrotic marker expression between the two groups: TGF-β1 (B), pSmad1/5 (C), pSmad2/3 (D), FN (E). White arrows identify significantly positive cells. **P < 0.01. Scale bar: 20 µm.
Figure 6.
 
(A) Representative images. (B) Comparison of integrin β1 and α-SMA expression between L-IOP and control groups: integrin β1 (B), α-SMA (C). *P < 0.05. Scale bar: 50 µm.
Figure 6.
 
(A) Representative images. (B) Comparison of integrin β1 and α-SMA expression between L-IOP and control groups: integrin β1 (B), α-SMA (C). *P < 0.05. Scale bar: 50 µm.
Figure 7.
 
(A, B) Quantitative comparison of COL-I (A) and FN (B) concentrations assessed using enzyme-linked immunosorbent assays between the L-IOP and the control eyes. *P < 0.05.
Figure 7.
 
(A, B) Quantitative comparison of COL-I (A) and FN (B) concentrations assessed using enzyme-linked immunosorbent assays between the L-IOP and the control eyes. *P < 0.05.
Figure 8.
 
(A) Representative images of cleaved caspase-3 and Ki-67 in L-IOP eyes. (B) Quantitative comparison of their expression between the groups: cleaved caspase-3 (B), Ki-67 (C). White arrows identify significantly positive cells. *P < 0.05. Scale bar: 20 µm.
Figure 8.
 
(A) Representative images of cleaved caspase-3 and Ki-67 in L-IOP eyes. (B) Quantitative comparison of their expression between the groups: cleaved caspase-3 (B), Ki-67 (C). White arrows identify significantly positive cells. *P < 0.05. Scale bar: 20 µm.
Figure 9.
 
(A) Representative images of KLF4 and F-actin in the L-IOP and control groups. (B) Quantitative comparison of their expression between the two groups: KLF4 (B), F-actin (C). White arrows identify significantly positive cells. *P < 0.05. Scale bar: 20 µm.
Figure 9.
 
(A) Representative images of KLF4 and F-actin in the L-IOP and control groups. (B) Quantitative comparison of their expression between the two groups: KLF4 (B), F-actin (C). White arrows identify significantly positive cells. *P < 0.05. Scale bar: 20 µm.
Table.
 
Antibodies Used in This Study
Table.
 
Antibodies Used in This Study
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