The senescence difference between the central and peripheral cornea induced by sutures

Abstract Introduction Cell senescence plays a regulatory role in tissue fibrosis. Corneal scarring is usually more severe in the central cornea based on clinical observation. In this study, we attempted to explore the senescence difference between the central and peripheral cornea in an in vivo mouse model with suture-induced senescence and in an in vitro model of senescence with hydrogen peroxide (H2O2)-induced rabbit corneal fibroblasts. Methods Male Balb/c mice (6–8 weeks) received sutures in the central, superior, inferior, nasal, and temporal cornea. The sutures were removed on the 14th day. Corneal neovascularization was observed under a slit lamp microscope with a digital camera. The fibroblasts isolated from the central and peripheral rabbit cornea were induced with H2O2 to establish the senescence model in vitro. Senescence was evaluated with SA-β-gal staining and gene expression analysis of p21, p27, and p53. Results Senescent cells accumulated in the corneal stroma from the third day to the 14th day after the operation and peaked on the 14th day. More senescent keratocytes were observed in the peripheral cornea of the mouse model. In vitro, the peripheral corneal fibroblasts were more prone to senescence due to H2O2. The polymerase chain reaction results showed that the senescence-related genes p21, p27, and p53 were highly expressed in the peripheral corneal fibroblasts compared with the central corneal fibroblasts. Conclusions Senescent fibroblasts can limit tissue fibrosis; hence, the senescence difference between the central and peripheral cornea may contribute to the difference in scarring

Immunological Properties of Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells.

Transplantation of ex vivo expanded corneal limbal stem cells (LSCs) has been the main treatment for limbal stem cell deficiency, although the shortage of donor corneal tissues remains a major concern for its wide application. Due to the development of tissue engineering, embryonic stem cells (ESCs)-derived corneal epithelial-like cells (ESC-CECs) become a new direction for this issue. However, the immunogenicity of ESC-CECs is a critical matter to be solved. In the present study, we explored the immunological properties of ESC-CECs, which were differentiated from ESCs. The results showed that ESC-CECs had a similar character and function with LSCs both in vitro and in vivo. In ESC-CECs, a large number of genes related with immune response were down-regulated. The expressions of MHC-I, MHC-II, and co-stimulatory molecules were low, but the expression of HLA-G was high. The ESC-CECs were less responsible for T cell proliferation and NK cell lysis in vitro, and there was less immune cell infiltration after transplantation in vivo compared with LSCs. Moreover, the immunological properties were not affected by interferon-γ. All these results indicated a low immunogenicity of ESC-CECs, and they can be promising in clinical use

Subconjunctival Bevacizumab Injection Impairs Corneal Innervations and Epithelial Wound Healing in Mice

Citation: Dong M, Di G, Zhang X, Zhou Q, Shi W. Subconjunctival bevacizumab injection impairs corneal innervations and epithelial wound healing in mice. Invest Ophthalmol Vis Sci. 2017;58:146958: -147758: . DOI: 10.1167 PURPOSE. To investigate the effects of subconjunctival bevacizumab injection on the corneal nerve, sensitivity, and epithelial wound healing in mice. METHODS. Adult C57BL/6 mice were treated with subconjunctival injection of 1, 2, 5, or 25 mg/mL bevacizumab. The corneal nerve was observed with whole-mount anti-b3-tubulin fluorescence staining. Corneal sensitivity was measured with a Cochet-Bonnet esthesiometer. The protein levels of pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were measured by ELISA. The corneal epithelial wound-healing rate was evaluated by fluorescein staining. The recovery of impaired mouse corneal innervations and epithelial wound-healing rate following bevacizumab injection was evaluated with the co-injection of PEDF, NGF, or CNTF. RESULTS. Subconjunctival bevacizumab injection caused apparent corneal nerve degeneration, attenuated corneal sensitivity, and delayed corneal epithelial wound healing and nerve regeneration in normal mice, which was more significant with increased concentration and times of the bevacizumab injection. However, the corneal nerve and sensitivity gradually improved and recovered in mice with a single injection of 1 to 5 mg/mL bevacizumab. Moreover, the bevacizumab injection significantly decreased the corneal PEDF, NGF, and CNTF content, whereas exogenous PEDF, NGF, or CNTF supplement attenuated impairment of the corneal nerve, sensitivity, and epithelial wound healing after subconjunctival bevacizumab injection. CONCLUSIONS. Subconjunctival bevacizumab injection impairs corneal innervations, epithelial wound healing, and nerve regeneration in normal mice, which may be caused by the reduction of neurotrophic factor content in the cornea

Different Effects of Pro‐Inflammatory Factors and Hyperosmotic Stress on Corneal Epithelial Stem/Progenitor Cells and Wound Healing in Mice

Abstract Chronic inflammation and severe dry eye are two important adverse factors for the successful transplant of cultured limbal stem cells. The aim of this study was to investigate the effects of inflammation and hyperosmotic stress (a key pathological factor in dry eye) on corneal epithelial stem cells (CESCs) and corneal epithelial wound healing. We observed that the CESCs exhibited significant morphological changes when treated with interleukin‐1 beta (IL‐1β), tumor necrosis factor alpha (TNF‐α), or hyperosmotic stress. Colony‐forming efficiency or colony‐forming size was decreased with the increasing concentrations of IL‐1β, TNF‐α, or hyperosmotic stress, which was exacerbated when treated simultaneously with pro‐inflammatory factors and hyperosmotic stress. However, the colony‐forming capacity of CESCs recovered more easily from pro‐inflammatory factor treatment than from hyperosmotic stress treatment. Moreover, when compared with pro‐inflammatory factors treatment, hyperosmotic stress treatment caused a more significant increase of apoptotic and necrotic cell numbers and cell cycle arrest in the G2/M phase. Furthermore, the normal ability of corneal epithelial wound healing in the mice model was suppressed by both pro‐inflammatory factors and hyperosmotic stress treatment, and especially severely by hyperosmotic stress treatment. In addition, inflammation combined with hyperosmotic stress treatment induced more serious epithelial repair delays and apoptosis in corneal epithelium. Elevated levels of inflammatory factors were found in hyperosmotic stress‐treated cells and mice corneas, which persisted even during the recovery period. The results suggested that pro‐inflammatory factors cause transient inhibition, while hyperosmotic stress causes severe apoptosis and necrosis, persistent cell cycle arrest of CESCs, and severe corneal wound healing delay. Stem Cells Translational Medicine 2019;8:46–5

The top 10 GO terms significantly changed in ESC-CECs.

<p>The enrichment score quantified the change extend of down-regulated (A, B, C) or up-regulated (D, E, F) GO terms. Immune related GO terms were labeled by red frame.</p

Primer sequences used in real-time RT-PCR.

<p>Primer sequences used in real-time RT-PCR.</p

The clinical outcome of cell transplantation in rabbit with LSCD.

<p>Representative slit-lamp photograph and fluorescein staining revealed the ocular surface condition at different time after transplantation (A). The cornea score displayed the tendency after transplantation (n = 4, B). PAS staining, H.E. and immunofluorescence staining (C) showed a well epithelial structure. * p<0.05, ** p<0.01.</p

The flow cytometry of MHC and co-stimulatory molecules on ESC-CECs.

<p>The % expression (A) and MFI (B) was used for comparison of molecules on different cells and conditions. n = 3, * p<0.05, ** p<0.01.</p

The T cells proliferation assay and NK cells lysis assay.

<p>ESC-CECs stimulated a weaker T cells proliferation (A) and were less susceptible to NK cells lysis (C) compared with LSCs. The situation didn’t change after INF-γ treatment (B, D). n = 4, * p<0.05, ** p<0.01.</p