Intra-stromal injection of honey-treated keratocytes as a cell-based therapy for experimental corneal laceration.

OBJECTIVES: This study aimed to investigate the potential of honey-supplemented medium (HSM) for expanding corneal keratocytes and its transplantation in a model of corneal laceration. METHODS: Keratocytes were cultured in 1 % HSM- or 10 % fetal bovine serum (FBS)-supplemented medium for 24 h. The effect of HSM on keratocyte proliferation was evaluated using the MTT assay. The relative expression of Lum, Kera, and ALDH3A1, known markers of native keratocytes, was quantified by real-time PCR. The safety and efficacy of HSM-treated keratocyte intrastromal injection in a rabbit model of corneal laceration were also evaluated. RESULTS: The MTT assay showed that HSM treatment did not significantly affect cell viability compared to FBS-supplemented medium (84.71 ± 2.38 vs. 100.08 ± 10.92, respectively; p=0.076). Moreover, HSM-treated keratocytes had significantly increased expression of Lum, Kera, and ALDH3A1 compared to cells treated with FBS, while the expression of the proliferation biomarker Thy-1 did not significantly differ between the two treatments. Intrastromal injection of HSM-treated keratocytes in the laceration animal model was safe and uneventful, resulting in less stromal inflammation and neovascularization, and consequently, better final architecture with less residual haze compared to the group injected with FBS-treated keratocytes. CONCLUSIONS: These findings suggest that honey is a suitable supplement for keratocyte treatment and corneal cell therapy. The use of HSM may have potential applications in the treatment of corneal injuries and diseases.

Corneal Neuro-Regenerative Effect of Transcutaneous Electrical Stimulation in Rabbit Lamellar Keratectomy Model.

Purpose: This study aimed to evaluate the effect of transcutaneous electrical stimulation (TES) on corneal nerve regeneration in rabbits injured from superficial lamellar keratectomy (SLK). Methods: New Zealand White rabbits were used in this experimental study. To induce corneal nerve damage, SLK was performed using a 7.0-mm trephine. TES was applied for 28 days after the corneal nerve injury. Corneal sensitivity, Western blotting, real-time polymerase chain reaction (PCR), and immunofluorescence were performed to observe changes in the corneal tissue. Results: In the 2-Hz and 20-Hz electrical stimulation groups, the degree of corneal wound healing increased by more than 10% compared to the control group, but no significant difference was observed. Conversely, the electrical stimulation (2-Hz or 20-Hz) group showed significantly increased corneal sensitivity compared to the control group. Western blot analysis revealed that small proline-rich protein 1A (SPRR1a), a regeneration-associated protein was significantly increased in the 2-Hz group on days 1 and 7 compared to that in the other groups. Once again, nerve regeneration in the 2-Hz group was supported by the results of PCR, in which a significant increase in the nerve growth factor (NGF) on day 1 was observed compared with the other groups. Moreover, immunofluorescence after 28 days of electrical stimulation showed significant nerve regeneration in the 2-Hz group. Conclusions: TES promoted corneal nerve regeneration in rabbit SLK model. The application of electrical stimulation of 2-Hz frequency was more effective than the 20-Hz frequency, showing potential clinical applications for corneal diseases. Translational Relevance: This study shows how application of TES to the eyes that exhibit corneal nerve damage can improve corneal nerve regeneration examined by histologic analysis.

Wireless-Powered Electrical Bandage Contact Lens for Facilitating Corneal Wound Healing.

Corneal injury can lead to severe vision impairment or even blindness. Although numerous methods are developed to accelerate corneal wound healing, most of them are passive treatments that rarely participate in controlling endogenous cell behaviors or are incompatible with nontransparent bandage. In this work, a wireless-powered electrical bandage contact lens (EBCL) is developed to generate a localized external electric field to accelerate corneal wound healing and vision recovery. The wireless electrical stimulation circuit employed a flower-shaped layout design that can be compactly integrated on bandage contact lens without blocking the vision. The role of the external electric field in promoting corneal wound healing is examined in vitro, where the responses of directional migration and corneal cells alignment to the electric field are observed. The RNA sequencing (RNA-seq) analysis indicates that the electrical stimulation can participate in controlling cell division, proliferation, and migration. Furthermore, the wireless EBCL is demonstrated to accelerate the completed recovery of corneal wounds on rabbits' eyes by electrical stimulation, while the control group exhibits delayed recovery and obvious corneal defects. As a new generation of intelligent device, the wireless and patient-friendly EBCL can provide a promising therapeutic strategy for ocular diseases.

Effect of topical bovine colostrum in wound healing of corneal surface after acute ocular alkali burn in mice.

The purpose of this study was to evaluate the effect of bovine colostrum (BC) in the regeneration of corneal epithelial cells on an ocular alkali burn model. Twenty-four C57BL/6 mice were categorized into two gender/age-matched groups for treatment. Two days after inducing a corneal alkali burn in all left eyes with 4 µl of sodium hydroxide 0.15 mol/l, both eyes of group 1 were treated with BC 4 times per day, and both eyes of group 2 were treated with isotonic saline solution (SS). The epithelial defect was photographed and measured by fluorescein staining on days two, four, seven, and ten. Ocular burn damage was assessed with a pre-established classification in clock hours from the limbus. After 10 days both eyes were processed, half of the group's corneas were assessed histopathologically, and the other half was used for pro/anti-inflammatory cytokine quantification using ELISA. BC treated (Group 1) corneas revealed significantly improved fluorescein staining score for limbal involvement when compared to SS treated (Group 2) corneas at days 4 (p = 0.013), 7 (p < 0.001), and 10 (p < 0.001), respectively. No differences were noted in limbal involvement at day 2 between the two groups (p > 0.99). The overall change (difference in slope) in fluorescein staining for limbal involvement between days 2 and 10 was -0.1669 (p = 0.006). Histologic examinations and cytokine measurements of group 2 demonstrated a strong inflammatory component compared to group 1. Our data indicates that topical application of BC facilitates corneal re-epithelialization and wound healing by suppressing the inflammatory process in an ocular alkali burn model.

Determining the efficacy of the bovine amniotic membrane homogenate during the healing process in rabbits' ex vivo corneas.

OBJECTIVE: To evaluate the efficacy of bovine amniotic membrane homogenate (BAMH) on wounded ex vivo rabbit corneas. PROCEDURE: Eighteen corneas obtained from normal rabbit eyes were wounded equally using a 6 mm trephine and cultured into an air-liquid interface model. Corneas were treated with phosphate-buffered saline (PBS) (n = 6, control group), 0.2% ethylenediaminetetraacetic acid (EDTA; n = 6), or BAMH (n = 6). All treatments were applied topically 6 times/day. Each cornea was macrophotographed daily with and without fluorescein stain to assess epithelialization and haziness. After 7 days, corneal transparency was evaluated, and the tissues prepared for histologic analysis of viability, and total and epithelial thickness. RESULTS: The mean epithelialization time was 6.2 ± 0.82 days for the control group, 6.2 ± 0.75 days for the EDTA-treated group, and 5.1 ± 0.40 days for the BAMH-treated group, demonstrating a significant difference between the BAMH and the other groups. The corneas that received EDTA had better transparency compared with the other groups. Histologically, all corneas had adequate morphology and architecture after healing. Analysis of corneal and epithelial thickness revealed no significant difference among groups. CONCLUSION: Bovine amniotic membrane homogenate is an effective and promising treatment for stromal and epithelial ulcers.

PLGA nanoparticles containing Lingzhi extracts rescue corneal epithelial cells from oxidative damage.

Oxidative stress-related ocular surface epithelial damage can be initiated by ambient oxygen, UV radiation, and chemical burns. The oxidative damage to cornea can lead to inflammation and even vision loss. Lingzhi (Ganoderma lucidum) is a Chinese herbal drug and has been shown to prevent chronic diseases in clinical practices and has been proven to possess anti-oxidative and anti-inflammatory properties. In the study, we prepared poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as a sustained drug release system of Lingzhi (LZH) to improve bioavailability. The particle size of developed NPs containing LZH (LZH-NPs) was ~184 nm with narrow size distribution. The results of cellular uptake revealed that using NPs as a drug delivery system could significantly increases the intracellular retention time. The results of the cell viability and chemiluminescence assay revealed that 5 µg/ml of LZH-NPs might be the threshold concentration for cultivation of corneal epithelial cells. After treating LZH-NPs in oxidative damaged cells, the results showed that the inflammation-related gene expression and DNA fragmentation level were both significantly decreased. Post-treatment of LZH-NPs in damaged corneal epithelial cells could increase the cell survival rate. In the rabbit corneal alkali burn model, topical instillation of LZH-NPs could promote corneal wound healing and decrease the inflammation. These results suggest that LZH-NPs may have the potential to treat ocular surface diseases caused by oxidative stress.

Topical cell-free conditioned media harvested from adipose tissue-derived stem cells promote recovery from corneal epithelial defects caused by chemical burns.

Corneal chemical burns can lead to blindness following serious complications. As most of these complications are caused by failure of reepithelization during the acute phase, treatment at this stage is critical. Although there have been some studies on corneal injury recovery using adipose tissue-derived stem cells (ADSCs), none has reported the effect of topical cell-free conditioned culture media (CM) derived from ADSCs on corneal epithelial regeneration. Here, the best conditions for CM were selected and used for in vitro and in vivo experiments. Corneal burn in rats was induced using 100% alcohol. The chosen CM was administered to corneal burn rats (CM-treated [CT] group) four times a day for three days and this group was compared with the normal control and corneal burn (CB) groups. Biomicroscopic fluorescence images and the actual physical corneas were taken over time and used for analysis. mRNA levels of hepatocyte growth factor and epidermal growth factor (EGF) were significantly increased, whereas those of vascular endothelial growth factor, interleukin (IL)-1ß, IL-6, IL-10, and matrix metalloproteinase-9 were significantly decreased in the CT group compared with those in the CB group. The numbers of proliferating cell nuclear antigen- and zonular occludens-1-positive cells in the CT group were significantly higher than those in the CB group. The macrophage-infiltrating corneas in the CT group expressed significantly more of the M2 marker arginase than corneas in the CB group. Optimal CM (× 0.5 concentration) treatment significantly accelerated the migration of corneal epithelial cells and induced upregulation of the expression of IL-6, EGF, and C-X-C chemokine receptor type 4 mRNAs. Overall, in this study, topical administration of cell-free CM promoted regeneration of the corneal epithelium after induction of chemical burns.

AAV vector-meditated expression of HLA-G reduces injury-induced corneal vascularization, immune cell infiltration, and fibrosis.

Over 1.5 million individuals suffer from cornea vascularization due to genetic and/or environmental factors, compromising visual acuity and often resulting in blindness. Current treatments of corneal vascularization are limited in efficacy and elicit undesirable effects including, ironically, vision loss. To develop a safe and effective therapy for corneal vascularization, adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by human leukocyte antigen G (HLA-G), was investigated. Self-complementary AAV cassettes containing codon optimized HLA-G1 (transmembrane) or HLA-G5 (soluble) isoforms were validated in vitro. Then, following a corneal intrastromal injection, AAV vector transduction kinetics, using a chimeric AAV capsid, were determined in rabbits. One week following corneal trauma, a single intrastromal injection of scAAV8G9-optHLA-G1 + G5 prevented corneal vascularization, inhibited trauma-induced T-lymphocyte infiltration (some of which were CD8+), and dramatically reduced myofibroblast formation compared to control treated eyes. Biodistribution analyses suggested AAV vectors persisted only in the trauma-induced corneas; however, a neutralizing antibody response to the vector capsid was observed inconsistently. The collective data demonstrate the clinical potential of scAAV8G9-optHLA-G to safely and effectively treat corneal vascularization and inhibit fibrosis while alluding to broader roles in ocular surface immunity and allogenic organ transplantation.

Short-term effects of extremely low-frequency pulsed electromagnetic field and pulsed low-level laser therapy on rabbit model of corneal alkali burn.

This study was conducted to investigate the effect of combining extremely low frequency-pulsed electromagnetic field (ELF-PEMF) and low-level laser therapy (LLLT) on alkali-burned rabbit corneas. Fifty alkali-burned corneas of 50 rabbits were categorized into five groups: ELF-PEMF therapy with 2 mT intensity (ELF 2) for 2 h daily; LLLT for 30 min twice daily; combined ELF-PEMF and LLLT (ELF + LLLT); medical therapy (MT); and control (i.e., no treatment). Clinical examination and digital photography of the corneas were performed on days 0, 2, 7, and 14. After euthanizing the rabbits, the affected eyes were evaluated by histopathology. The clinical and histopathologic results were compared between the groups. On days 7 and 14, no significant difference in the corneal defect area was evident between the ELF, LLLT, ELF + LLLT, and MT groups. Excluding the controls, none of the study groups demonstrated a significant corneal neovascularization in both routine histopathology and immunohistochemistry for CD31. Keratocyte loss was significantly higher in the MT group than in the ELF, LLLT, and ELF + LLLT groups. Moderate to severe stromal inflammation in the LLLT group was comparable with that in the MT group and was significantly lower than that in the other groups. In conclusion, combining LLLT and ELF was not superior to ELF alone or LLLT alone in healing corneal alkali burns. However, given the lower intensity of corneal inflammation and the lower rate of keratocytes loss with LLLT, this treatment may be superior to other proposed treatment modalities for healing alkali-burned corneas.