Corneal Repair Research Articles

Molecular basis of Mitomycin C enhanced corneal sensory nerve repair after debridement wounding

The ocular surface is covered by stratified squamous corneal epithelial cells that are in cell:cell contact with the axonal membranes of a dense collection of sensory nerve fibers that act as sentinels to detect chemical and mechanical injuries which could lead to blindness. The sheerness of the cornea makes it susceptible to superficial abrasions and recurrent erosions which demand continuous regrowth of the axons throughout life. We showed previously that topical application of the antibiotic and anticancer drug Mitomycin C (MMC) enhances reinnervation of the corneal nerves and reduces recurrent erosions in mice via an unknown mechanism. Here we show using RNA-seq and confocal imaging that wounding the corneal epithelium by debridement upregulates proteases and protease inhibitors within the epithelium and leads to stromal nerve disruption. MMC attenuates these effects after debridement injury by increasing serpine1 gene and protein expression preserving L1CAM on axon surfaces of reinnervating sensory nerves. These data demonstrate at the molecular level that gene expression changes in the corneal epithelium and stroma modulate sensory axon integrity. By preserving the ability of axons to adhere to corneal epithelial cells, MMC enhances sensory nerve recovery after mechanical debridement injury.

Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice

Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2′-deoxyuridine (IdU), 5-chloro-2′-deoxyuridine (CldU) and 5-ethynyl-2′-deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ± 0.2 days) compared to 10 week old mice (4.97 ± 0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ± 1.4% of LESCs were shown to enter S phase in a 24 h period and were estimated to divide every 2–3 weeks. Within 24 h of corneal injury this rose significantly to 32.8 ± 10.0% of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24–48 h period after wounding in young adults, LESC activation continued to increase (86.5 ± 8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ± 9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.

Universal Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells for Cellularization of a Corneal Scaffold.

PurposeWe generated universal corneal epithelial cells (CEC) from human embryonic stem cells (hESC) by genetically removing human leukocyte antigens (HLA) class I from the cell surface.MethodsThe serum-free, growth factor-free, and defined medium E6 was used to differentiate hESC to CEC. Decellularized murine corneas were recellularized with hESC-derived CEC. Using CRISPR/Cas9, β-2-microglobulin (B2M) was deleted in hESC to block the assembly of HLA class-I antigens on the cell surface to generate B2M−/− CEC.ResultsE6 alone was sufficient to allow hESC differentiation to CEC. A time-course analysis of the global gene expression of the differentiating cells indicates that the differentiation closely resembles the corneal development in vivo. The hESC-CEC were highly proliferative, and could form multilayer epithelium in decellularized murine cornea, retain its transparency, and form intact tight junctions on its surface. As reported before, B2M knockout led to the absence of HLA class-I on the cell surface of hESC and subsequently derived CEC following stimulation with inflammatory factors. Moreover, B2M−/− CEC, following transplantation into mouse eyes, caused less T-cell infiltration in the limbal region of the eye than the wild-type control.ConclusionsCEC can be derived from hESC via a novel and simple protocol free of any proteins, hESC-CEC seeded on decellularized animal cornea form tight junctions and allow light transmittance, and B2M−/− CEC are hypoimmunogenic both in vitro and in vivo.Translational RelevanceB2M−/− hESC-CEC can be an unlimited and universal therapy for corneal repair in patients of any HLA type.

A collagen film with micro-rough surface can promote the corneal epithelization process for corneal repair

Corneal disease is a major cause of blindness and keratoplasty is an effective treatment method. A freeze-dried collagen film (FD-Col) with micro-rough surface structure for corneal epithelial repair was reported in our previous studies. In this research, we conducted a more comprehensive study on the FD-Col film. Besides with excellent mechanical property and optical transparency, the FD-Col film also has good penetrating ability in nutrient solutions. The permeability coefficient of the FD-Col in NaCl and tryptophan solution is (2.58 ± 0.47) × 10−6 cm2/s and (2.67 ± 0.13) × 10−7 cm2/s, respectively. In addition, the morphology change of the FD-Col film before and after water absorption is relatively stable suggesting that this film can be fabricated with various dimensions easily. Moreover, corneal lamellar keratoplasty shows that the FD-Col film can be sutured in rabbit's ocular surface and the re-epithelization process in vivo is complete in about 12 days, and the transparency is restored quickly in the first month. Corneal rejection reaction, neovascularization and keratoconus are not observed within 2 months. This FD-Col film, which can be prepared in large quantities and at low cost, should have potential application in corneal repair in the future.

Nidogen-2: Location and expression during corneal wound healing

Nidogen-2 is a basement membrane (BM) glycoprotein that could be a key to understanding why defects in BM regeneration occur after severe trauma to the cornea. We monitored the location and expression of nidogen-2 during corneal repair after alkali burn in rabbits.In rabbits that received both general and ocular topical anaesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. Right corneas were used as controls. The eyes were evaluated at 2, 7, 15, and 30 days after burning and analysed by immunohistochemistry for nidogen-2 and α-smooth muscle actin, a myofibroblast marker. Nidogen-2 mRNA expression levels were determined by quantitative real-time polymerase chain reaction.In control corneas, nidogen-2-positive cells were in all epithelial layers, the endothelium, and the anterior and posterior stromal regions. At Day 2 after the alkali burn, the wound area epithelium and the peripheral epithelium were made up of only 1 to 2 cell layers, all of them nidogen-2 positive. At Day 7 in the wound area, the epithelium consisted of two cell layers, and the basally located cells were mostly nidogen-2 positive. The greatest change was observed at Day 30. At this time, the ulcer prevalence in the alkali-burned corneas was approximately 50% and the central epithelial defects remained. In unepithelialized corneas, frequent epithelial detachments were present, in which almost of the epithelial cells were nidogen-2 negative.The injured stroma was repopulated by activated stromal cells that synthesized nidogen-2. The nidogen-2 was retained in the newly secreted, but disordered, matrix produced mainly by the myofibroblasts localized in the stroma at 7, 15, and 30 days after burning. Thus, even though nidogen-2 was present, it was unable to contribute to the effective regeneration of the BM.

Anti-Inflammatory and Anti-Fibrotic Effects of Human Amniotic Membrane Mesenchymal Stem Cells and Their Potential in Corneal Repair.

Acute ocular chemical burns are ophthalmic emergencies requiring immediate diagnosis and treatment as they may lead to permanent impairment of vision. The clinical manifestations of such burns are produced by exacerbated innate immune response via the infiltration of inflammatory cells and activation of stromal fibroblasts. New therapies are emerging that are dedicated to repair mechanisms that improve the ocular surface after damage; for example, transplantation of stem cells (SC) has been successfully reported for this purpose. The pursuit of easily accessible, noninvasive procedures to obtain SC has led researchers to focus on human tissues such as amniotic membrane. Human amniotic mesenchymal SC (hAM‐MSC) inhibits proinflammatory and fibrotic processes in different diseases. hAM‐MSC expresses low levels of classical MHC‐I and they do not express MHC‐II, making them suitable for regenerative medicine. The aim of this study was to evaluate the effect of intracameral injection of hAM‐MSC on the clinical manifestations, the infiltration of inflammatory cells, and the activation of stromal fibroblasts in a corneal alkali‐burn model. We also determined the in vitro effect of hAM‐MSC conditioned medium (CM) on α‐SMA+ human limbal myofibroblast (HLM) frequency and on release of neutrophil extracellular traps (NETs). Our results show that intracameral hAM‐MSC injection reduces neovascularization, opacity, stromal inflammatory cell infiltrate, and stromal α‐SMA+ cells in our model. Moreover, in in vitro assays, CM from hAM‐MSC decreased the quantity of α‐SMA+ HLM and the release of NETs. These results suggest that intracameral hAM‐MSC injection induces an anti‐inflammatory and anti‐fibrotic environment that promotes corneal wound healing. Stem Cells Translational Medicine 2018;7:906–917

Corneal Neurotization Improves Ocular Surface Health in a Novel Rat Model of Neurotrophic Keratopathy and Corneal Neurotization

Corneal neurotization is a novel surgical procedure to reinnervate the cornea in patients with neurotrophic keratopathy (NK). We developed a rat model of NK and corneal neurotization to further investigate corneal neurotization as a treatment to improve maintenance and healing of the corneal epithelium. Thy1-GFP+ Sprague Dawley rats were used to develop the model. Corneal denervation was performed via stereotactic electrocautery of the ophthalmomaxillary branch of the trigeminal nerve. Corneal neurotization was performed by guiding donor sensory axons from the contralateral infraorbital nerve into the cornea via two nerve grafts. Corneal imaging, including nerve density measurements and retrograde labeling were performed to validate the model. In vivo assays of corneal maintenance and repair were used to examine whether treatment with corneal neurotization improved healing in rats with NK. Corneal neurotization significantly increased corneal axon density in rats with NK (P < 0.01). Retrograde labeling of the cornea in rats with corneal neurotization labeled 206 ± 82 neurons in the contralateral trigeminal ganglion, confirming axons reinnervating the cornea derived from the contralateral infraorbital nerve. Corneal reinnervation after corneal neurotization improved corneal epithelial maintenance and corneal healing after injury (P < 0.01). Donor nerve fibers reinnervate the insensate cornea after corneal neurotization and significantly improve corneal maintenance and repair. This model can be used to further investigate how corneal neurotization influences epithelial maintenance and repair in the context of NK.

Cell-laden and orthogonal-multilayer tissue-engineered corneal stroma induced by a mechanical collagen microenvironment and transplantation in a rabbit model

The development of functional therapies for corneal repair and regeneration is a pressing issue. Corneal stroma provides the principal functions of the cornea. However, because of the highly organized nature of the stromal matrix, the attempts to reproduce corneal stroma might follow a scar model. Here, we have developed a protocol for the efficient generation of a cell-laden and orthogonal-multilayer tissue-engineered (TE) corneal stroma, which is induced by the mechanical effects of compressed collagen (CC) or stretched compressed collagen (SCC). Within SCC, with applied compression and force extension, collagen microfibres and corneal stromal cells (CSCs) are arranged orderly, while collagen fibres and CSCs in CC are randomly arranged. Dehydrated SCC has higher tensile strength than dehydrated CC. Hydrated SCC has similar transparency with hydrated native corneal stroma. Compared with those cultured on tissue culture plates (TCP), down-regulation of the genes and proteins of cytoskeleton, activation, proliferation, collagen and TRPV4, up-regulation of proteoglycans, gap junction proteins and TRPA1 are in CSCs of CC and SCC. Moreover, SCC and CC grafts displayed biocompatibility and integration with host corneal tissue after rabbit intra-corneal stromal transplantation by wk 6 under slit lamp microscopy, in vivo confocal microscopy and histological examination. The SCC model facilitates the construction of physiological feature TE corneal stroma, which serves as a foundation for physiological TE construction of other tissues. Statement of SignificanceThe development of functional therapies for corneal repair and regeneration is a pressing issue. Corneal stroma provides the principal functions of the cornea. Here, we have developed a protocol for the efficient generation of a cell-laden and orthogonal-multilayer tissue-engineered (TE) corneal stroma, which is induced by the mechanical effects of compressed collagen (CC) or stretched compressed collagen (SCC). These models facilitate the construction of physiological feature TE corneal stroma, which serves as a foundation for physiological TE construction of other tissues and helps to reverse fibrosis pathologies in general.

Natural Biomaterials for Corneal Tissue Engineering, Repair, and Regeneration.

Corneal blindness is a major cause of vision loss, estimated to affect over 10 million people worldwide. Once impaired through clouding or shape change, the best treatment option for restoring vision is corneal transplantation using full or partial thickness cadaveric grafts. However, donor corneas are globally limited and face rejection and graft failure, similar to other transplanted organs. Thus, there is a need for viable alternatives to donor corneas in order to increase supply, reduce rejection, and to minimize variability in tissue quality. To address this, researchers have developed new materials and strategies to tissue engineer full or partial thickness cornea grafts in order to repair, regenerate, or replace the diseased cornea. This progress report first reviews the anatomy and physiology of the cornea to frame the biological requirements and discuss the injuries and diseases that necessitate the need fortransplantation, as well as the requirements for a suitable donor tissue alternative. This is followed by recent progress using naturally derived biomaterials including silk, collagen, amniotic membranes, and decellularized corneas. Finally, remaining challenges in the field as they relate to the biomaterials discussed are identified, and the future research directions that should result in further advances in restoring corneal vision are highlighted.

Observation on the effect of applying mitomycin C in transepithelial photorefractive keratectomy

Objective To observe the efficacy of applying mitomycin C(MMC)in transepithelial photorefractive keratectomy(TPRK )surgery. Methods A retrospective nonrandomized controlled study. The data of 122 eyes of 61 cases received TPRK surgery were collected and analyzed.All cases were divided into two groups.Group A, 60 eyes of 30 patients, 0.02% MMC sponge was put on to the cornea ablation area during operation for 20-40 s; group B, 62 eyes of 31 cases received surgery without MMC. The follow-up time was 6 months.The incidence of haze formation, cornea epithelium healing and visual acuity of the two groups were observed. Results The eye irritation symptoms of group B was lighter than that of group A, the difference was statistically significant between the two groups (t=7.582, P=0.000). At 3 and 10 days postoperatively corneal epithelium repair were faster, the difference was statistically significant between the two groups(P=0.002, 0.007). The visual acuity at 3, 10 days, 1 and 3 months after surgery was same with that before surgery.The difference was not statistically significant(χ2=0.439, P=0.508; χ2=2.367, P=0.124; χ2=0.370, P=0.543; χ2=0.919, P=0.338). The difference in incidence of haze was not statistically significant between the two groups(χ2=0.726, P=0.190). Conclusion During TPRK surgery without MMC can reduce postoperative eye irritation, and repair corneal epithelium relatively fast, without influence on visual recovery and the incidence of haze. Key words: Keratectomy, transepithelial, photorefractive; Mitomycin C, intraoperative application; Haze, corneal; Visual acuity

The effect of PRK on corneal epithelium thickness and pachmetry in treatment of myopia.

In order to facilitate the corneal stromal ablation in photorefractive keratectomy the epithelium is removed so corneal repair associated with changes in epithelium and stroma. To study the corneal epithelial thickness and pachymetry profile changes after photorefractive keratec­tomy (PRK) for myopia. Retrospective analysis of the postoperative corneal epithelial thickness and pachymetry profile changes in 22 eyes of 12 patients treated with PRK for myopia or myopic astigmatism. Corneal and epithelial thickness maps within the central 6 mm were obtained by anterior segment spectral-domain optical coherence tomography (SD-OCT) preop­eratively and at 3 months postoperatively. Correlations between pachymetry,epithelial thickness changes and the amount of correction,were analyzed.Compared to preoperative values,the central 2 mm and the paracentral 2 to 5 mm zone epithelium was 1 ± 2.85 and 1 ± 3.11 μm thicker,respec­tively,at 3 months postoperatively (P &lt;.05). The spheri­cal equivalent (SE) changed from-2.80 ± 2.028 diop­ters (D) preoperatively to -0.40 ± 0.42 D at 3 months postoperatively. Females show greater postoperative epithelial thickening, 2.6 ± 3.77 μm,than males,0.34 ± 1.98 μm. There was a trend toward greater epithelial thickening with a larger amount of programmed SE correction, and thinner preoperative epithelium. No correlation between epithelial thickness change and postoperative change in refraction was detected.Negative correlation between between age, refractive error,with the pre and post-operative pachymetry. In general female pachymetry reading is higher than it in male.The corneal epithelial thickness in­creased after PRK up to 3 months postoperatively. It was affected by the amount of myopia treated, gender, and preoperative epithelial thickness. The refractive outcomes did not affected by the postoperative epithelial thickening. Negative correlation between between age, refractive error, with the pre and post-operative pachymetry. In general female pachymetry reading is higher than it in male.

Human Conjunctival Epithelial Sheets Grown on Poly(Lactic-Co-Glycolic) Acid Membranes and Cocultured With Human Tenon's Fibroblasts for Corneal Repair.

We determine the feasibility of human conjunctival epithelial cells (hCjECs) on poly(lactic-co-glycolic) acid (PLGA) membranes for corneal epithelium regeneration. hCjECs on PLGA or polyester (PET) membranes with or without coculture of human Tenon's fibroblasts (hTFs) were compared in vitro, and to determine whether epithelial sheets grown on PLGA membranes can repair injured rabbit corneal epithelium by transplantation for 2 weeks in vivo. Primary hCjECs were cultured on PLGA or the original PET membrane-based transwell inserts with or without coculture of hTFs on the floor of the culture plate. Cell behaviors, such as proliferation and differentiation, were compared. For in vivo assessment, the corneas of rabbits were burned, and PLGA-based epithelial sheets then were transplanted for 2 weeks before histologic staining was conducted and analyzed to determine the effectiveness of the repair. Primary human epithelial cells on the PLGA membrane showed an increased proliferation when cocultured with fibroblasts, which was confirmed by CCK-8 analysis, and upregulation of Ki67, with the expression of the epithelial marker CK19. The stratified squamous cell marker MUC1 and conjunctival cell marker MUC5AC also were expressed in the epithelial sheet. The epithelial defect in the burned corneas was decreased in the PLGA-based epithelial sheet treatment group (6.1% ± 1.6% of the area) compared to that in the no-treatment group (30.5% ± 6.3%) 2 weeks postoperatively. We developed a coculture system using a human feeder cell layer and PLGA membrane-based transwell inserts to create human conjunctival epithelial sheets. This system represents a promising strategy to regenerate corneal epithelium by transplantation.

Collagen-Hydroxypropyl Methylcellulose Membranes for Corneal Regeneration.

To improve intraocular transparency of collagen matrices, hydroxypropyl methylcellulose (HPMC) was introduced for the first time into cross-linked collagen to form collagen–HPMC composite membranes. Light transmittance and refractive indices of the membranes are enhanced by incorporation of HPMC in comparison to the control of cross-linked collagen membranes. Maximum light transmittance of the collagen–HPMC membrane was up to 92%. In addition, their permeability of nutrients such as glucose, tryptophan, and NaCl was superior or comparable to that of human corneas. In vitro results demonstrated that the collagen–HPMC membrane supported adhesion and proliferation of human corneal epithelial cells (HCECs), showing good cytocompatibility to HCECs. The corneas maintained a smooth surface and clear stroma postoperatively after 7 months of implantation of collagen–HPMC membranes into the corneas of rabbits. The good intraocular biocompatibility was verified by maintaining a high optical clarity for over 6 months after transplantation. Hematoxylin and eosin staining results showed the growth of stromal keratocytes into the collagen–HPMC implants, indicating the ability of the collagen–HPMC membrane to induce corneal cell regeneration. Taken together, the collagen–HPMC membrane might be a promising candidate for use in corneal repair and regeneration.

Meta analysis of therapeutic effects of domestic deproteinized calf blood extract eye gel on corneal epithelial repair after laser refractive surgery

Meta analysis of therapeutic effects of domestic deproteinized calf blood extract eye gel on corneal epithelial repair after laser refractive surgery

Modified conjunctival flap as a primary procedure for nontraumatic acute corneal perforation.

Objective:Appropriate management of nontraumatic acute corneal perforation is always a challenge even with the many advances in surgical materials and techniques. We reported the outcomes of a case series of acute corneal perforation repair using a newly modified conjunctival flap with amniotic membrane transplant (AMT), fibrin glue, and a bandage soft contact lens (BCL).Materials and Methods:A total of 13 consecutive referral cases with nontraumatic acute corneal perforation at the University of Washington were reviewed. All open globes were repaired by a combined surgery with a modified conjunctival flap, AMT, fibrin glue, and BCL. Visual acuity, a slit lamp examination, and the patient-reported pain level were collected preoperatively and postoperatively. Subsequent corneal surgeries to improve visual function were also reviewed.Results:All ocular surfaces of the 13 eyes were stable at postoperative follow-up. Eleven of the 13 patients had the same or worse visual acuity 1 week postoperatively. Nine of the 13 patients achieved better vision 6 months postoperatively. None of the patients developed perioperative or postoperative complications. Five patients with good visual potential underwent further corneal surgeries, including Boston keratoprosthesis and penetrating keratoplasty. The average referral distance was 217 miles and the median number of follow-up visits within 6 months was 4.Conclusions:The combination of a modified conjunctival flap, AMT, fibrin glue, and a BCL could provide a temporary method to stabilize and secure the integrity of the globe as well as the ocular surface after a nontraumatic acute corneal perforation. This approach allows easy follow-up and preserves the eye for future corneal surgery under optimal conditions.

An antibacterial collagen membrane crosslinked by the inclusion complex of β-cyclodextrin dialdehyde and ofloxacin for bacterial keratitis.

Infectious keratitis is one of the leading causes of blindness in the world, especially in developing countries. Corneal transplant surgery is a feasible treatment for bacterial keratitis when drug therapy cannot be effective. However, the amount of corneal donors is far from requirements of clinical treatment and thus, the prognosis of bacterial keratitis is not satisfactory. In this study, we developed a novel antibacterial corneal repair material (β-CD-DA/OFLX-Col) for bacterial keratitis. The inclusion complex of β-cyclodextrin dialdehyde (β-CD-DA) with ofloxacin formed by host–guest interaction was used as both a drug vector and a crosslinker for further reaction with the amino groups on lysine of collagen chains. Physical properties, cytocompatibility, and antibacterial property of β-CD-DA/OFLX-Col film were characterized. The results indicated that the film was mainly transparent and possessed superior mechanical properties. Moreover, human corneal epithelial cells could adhere to the film and proliferate normally, indicating that the β-CD-DA/OFLX-Col film was non-cytotoxic and had good biocompatibility. Most importantly, the β-CD-DA/OFLX-Col film exhibited prominent antibacterial effects against E. coli and S. aureus in vitro, which could minimize the risks of infection. The prepared β-CD-DA/OFLX-Col film could greatly increase bioavailability of drugs and reduce toxic side effects, thus displaying great potential in bacterial keratitis treatment.

Corneal matrix repair therapy with the regenerating agent in neurotrophic persistent epithelial defects.

Corneal matrix repair therapy with the regenerating agent in neurotrophic persistent epithelial defects.

Nerve Growth Factor Changes and Corneal Nerve Repair after Keratoplasty.

SIGNIFICANCEMeasured tear concentration of nerve growth factor is correlated with postoperative corneal reinnervation among patients who undergo keratoplasty. This may be a future therapeutic target for post-keratoplasty corneal nerve regeneration.PURPOSETo determine the relationship between changes in the content of nerve growth factor (NGF) in tear fluid and corneal subepithelial nerve regeneration in patients after keratoplasty.METHODSIn this retrospective study, 30 eyes of 28 patients (15 males, 13 females; mean age 42.8 [range 16–73] years) who underwent primary keratoplasty for the first time were recruited through the clinics of the Department of Ophthalmology, Jilin University affiliated First Hospital, between May and December 2015. All patients underwent a complete ophthalmic examination preoperatively. Tear fluid samples were collected to detect the content of NFG at different time points in the follow-up period (day 1 preoperatively and days 1, 7, 30, and 90 postoperatively) and analyzed correlations between NFG content and age, infective factors, and variables of the surgical procedure as well as with subepithelial nerve repair at 30 and 90 days postoperatively.RESULTSThe NFG content in tear fluid on day 1 postoperatively was lower than that on the day preceding surgery; however, it was higher than the preoperative value on postoperative days 7, 30, and 90 (F = 5.046, P < 0.05). Further, the NFG content of tear fluid at 30 days postoperatively correlated with the surgical procedure (coefficient = −2.775, P = 0.010); however, no significant correlation was found on postoperative day 1 (coefficient = −1.315, P > 0.05). At all study time points, the NFG content of tear fluid had no correlation with infective factors or age (P > 0.05). Postoperatively, at day 30, small nerve buds were observed in the periphery of the corneal graft in 13 eyes (43.3% of cases) but not in 17 eyes (56.7% of cases), which showed a significant correlation with the NFG content of tear fluid (coefficient = −3.370, P = 0.010). By postoperative day 90, small nerve buds were observed in the periphery of the corneal graft in 24 eyes (80.0% of cases) and showed a significant correlation with the NFG content of tear fluid (coefficient = −2.750, P = 0.006).CONCLUSIONSThe NFG content in tear fluid increases with the increasing ratio of small nerve buds indicating corneal nerve regeneration. NFG promotes subepithelial nerve regeneration in patients after keratoplasty.

Adipose Derived Stem Cells for Corneal Wound Healing after Laser Induced Corneal Lesions in Mice.

The aim of our study was to assess the clinical effectiveness of topical adipose derived stem cell (ADSC) treatment in laser induced corneal wounds in mice by comparing epithelial repair, inflammation, and histological analysis between treatment arms. Corneal lesions were performed on both eyes of 40 mice by laser induced photorefractive keratectomy. All eyes were treated with topical azythromycin bid for three days. Mice were divided in three treatment groups (n = 20), which included: control, stem cells and basic serum; which received topical treatment three times daily for five consecutive days. Biomicroscope assessments and digital imaging were performed by two masked graders at 30, 54, 78, 100, and 172 h to analyze extent of fluorescein positive epithelial defect, corneal inflammation, etc. Immunohistochemical techniques were used in fixed eyes to assess corneal repair markers Ki67, α Smooth Muscle Actin (α-SMA) and E-Cadherin. The fluorescein positive corneal lesion areas were significantly smaller in the stem cells group on days 1 (p < 0.05), 2 (p < 0.02) and 3. The stem cell treated group had slightly better and faster re-epithelization than the serum treated group in the initial phases. Comparative histological data showed signs of earlier and better corneal repair in epithelium and stromal layers in stem cell treated eyes, which showed more epithelial layers and enhanced wound healing performance of Ki67, E-Cadherin, and α-SMA. Our study shows the potential clinical and histological advantages in the topical ADSC treatment for corneal lesions in mice.

Topical dimethyl sulfoxide inhibits corneal neovascularization and stimulates corneal repair in rabbits following acid burn

Neovascularization of the cornea is characterized by the growth of blood vessels caused by imbalances between angiogenic and anti-angiogenic factors. We investigated whether the expression of Vascular endothelial growth factor (VEGF), Vascular endothelial growth factor receptor (VEGF), Vascular endothelial growth inhibitor (VEGI) receptors, as well as topical drug treatments, participate in regulating corneal neovascularization after corneal damage and remodeling. We used 72 mature male New Zealand rabbits. Corneal burns were induced by hydrofluoric acid under general anesthesia. The rabbits then were treated with indomethacin or dimethyl sulfoxide (DMSO). The animals were euthanized on days 2, 7 and 14 after injury. Each cornea was fixed with 10% neutral formalin. On days 2, 7 and 14, VEGF, flk1/KDR and flt1/fms were strongly expressed in the epithelial, stromal and inflammatory cells, but not in the corneal endothelial cells. On day 7, newly formed blood vessels were observed growing toward the center of the cornea. In the control, indomethacin treated, DMSO–treated, and indomethacin + DMSO–treated animals, VEGI, VEGF, and the receptors, flk1/KDR, flt1/fms and flt4, were expressed at different densities in the neovascular regions. This was particularly evident in the indomethacin- and indomethacin + DMSO–treated groups on days 7 and 14, compared to day 2. Treatment with VEGF and DMSO stimulated repair of corneal damage. We suggest that VEGI in the endothelial cells of neovascularized cornea may act as a signaling protein that promotes balance between cell proliferation and apoptosis. Topical administration of DMSO inhibited corneal neovascularization more effectively than indomethacin.