Ocular Surface Surgery Research Articles

Effect of trapping vascular endothelial growth factor-A in a murine model of dry eye with inflammatory neovascularization.

To evaluate whether trapping vascular endothelial growth factor A (VEGF-A) would suppress angiogenesis and inflammation in dry eye corneas in a murine corneal suture model. We established two groups of animals, one with non-dry eyes and the other with induced dry eyes. In both groups, a corneal suture model was used to induce inflammation and neovascularization. Each of two groups was again divided into three subgroups according to the treatment; subgroup I (aflibercept), subgroup II (dexamethasone) and subgroup III (phosphate buffered saline, PBS). Corneas were harvested and immunohistochemical staining was performed to compare the extents of neovascularization and CD11b+ cell infiltration. Real-time polymerase chain reaction was performed to quantify the expression of inflammatory cytokines and VEGF-A in the corneas. Trapping VEGF-A with aflibercept resulted in significantly decreased angiogenesis and inflammation compared with the dexamethasone and PBS treatments in the dry eye corneas (all P<0.05), but with no such effects in non-dry eyes. The anti-inflammatory and anti-angiogenic effects of VEGF-A trapping were stronger than those of dexamethasone in both dry eye and non-dry eye corneas (all P<0.05). The levels of RNA expression of VEGF-A, TNF-alpha, and IL-6 in the aflibercept subgroup were significantly decreased compared with those in the PBS subgroup in the dry eye group. Compared with non-dry eye corneas, dry eye corneas have greater amounts of inflammation and neovascularization and also have a more robust response to anti-inflammatory and anti-angiogenic agents after ocular surface surgery. Trapping VEGF-A is effective in decreasing both angiogenesis and inflammation in dry eye corneas after ocular surface surgery.

What Causes Eye Pain?

Eye pain is an unpleasant sensory and emotional experience including sensory-discriminative, emotional, cognitive, and behavioral components and supported by distinct, interconnected peripheral and central nervous system elements. Normal or physiological pain results of the stimulation by noxious stimuli of sensory axons of trigeminal ganglion (TG) neurons innervating the eye. These are functionally heterogeneous. Mechano-nociceptors are only excited by noxious mechanical forces. Polymodal nociceptors also respond to heat, exogenous irritants, and endogenous inflammatory mediators, whereas cold thermoreceptors detect moderate temperature changes. Their distinct sensitivity to stimulating forces is determined by the expression of specific classes of ion channels: Piezo2 for mechanical forces, TRPV1 and TRPA1 for heat and chemical agents, and TRPM8 for cold. Pricking pain is evoked by mechano-nociceptors, while polymodal nociceptors are responsible of burning and stinging eye pain; sensations of dryness appear to be mainly evoked by cold thermoreceptors. Mediators released by local inflammation, increase the excitability of eye polymodal nociceptors causing their sensitization and the augmented pain sensations. During chronic inflammation, additional, long-lasting changes in the expression and function of stimulus-transducing and voltage-sensitive ion channels develop, thereby altering polymodal terminal’s excitability and evoking chronic inflammatory pain. When trauma, infections, or metabolic processes directly damage eye nerve terminals, these display aberrant impulse firing due to an abnormal expression of transducing and excitability-modulating ion channels. This malfunction evokes ‘neuropathic pain’ which may also result from abnormal function of higher brain structures where ocular TG neurons project. Eye diseases or ocular surface surgery cause different levels of inflammation and/or nerve injury, which in turn activate sensory fibers of the eye in a variable degree. When inflammation dominates (allergic or actinic kerato-conjunctivitis), polymodal nociceptors are primarily stimulated and sensitized, causing pain. In uncomplicated photorefractive surgery and moderate dry eye, cold thermoreceptors appear to be mainly affected, evoking predominant sensations of unpleasant dryness.

Elevated expression of matrix metalloproteinase-9 and inflammatory cytokines in keratoconus patients is inhibited by cyclosporine A.

The present study was designed to understand the role of inflammatory cytokines secreted by corneal epithelial cells in keratoconus (KC) and the response to treatment with cyclosporine A (CyA). The study involved 129 Indian KC patients clinically graded according to Amsler-Krumeich classification and 20 healthy, nonectatic subjects as controls. Tear levels of matrix metalloproteinase-9 (MMP9), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) were measured using ELISA kits. Gene expression was measured by qPCR in corneal epithelial cells obtained by debridement from subjects undergoing ocular surface surgeries. In addition, epithelial cells were stimulated with TNFα and treated with CyA to study its role on MMP9 expression. Finally, 20 KC patients (27 eyes) with inflammatory symptoms were treated with topical CyA application. We observed that MMP9, TNFα, and IL6 levels were strongly upregulated at the mRNA level in KC patient epithelia. Similarly, tears collected from KC patients exhibited high levels of MMP9 and IL6 protein. Cyclosporine A treatment significantly reduced the mRNA expression levels of IL6 and TNFα in both short- and long-term treatments; however, it reduced MMP9 levels only in long-term treatment in cultured corneal epithelial cells. Subsequent treatment of KC patients with CyA for approximately 6 months reduced tear MMP9 levels and led to local reduction in corneal curvatures as determined by corneal topography maps. The data indicate that corneal epithelium contributes to elevated MMP9 and inflammatory cytokine expression in tears of KC patients. Cyclosporine A treatment reduced MMP9 and inflammatory cytokine levels in an in vitro inflammation model system. In KC patients, CyA treatment reduced MMP9 levels measured in tears with concomitant arrest of disease progression. Therefore, CyA might be a novel treatment strategy in KC patients but requires additional evaluation in larger cohorts. (ClinicalTrials.gov number, NCT01746823.).

Robot-Assisted Pterygium Surgery: Feasibility Study in a Nonliving Porcine Model.

This study aims to investigate the feasibility of pterygium surgery using the DaVinci Si HD robotic surgical system, and to describe a porcine model for pterygium surgery and evaluate its usefulness. The pterygium models were constructed using enucleated pig eyes and cold cuts. Robotically-assisted pterygium surgeries in nonliving biological pterygium models were performed using the DaVinci Si HD robotic surgical system. Twelve models were prepared, and 12 pterygium excision and conjunctival autografts were performed. The DaVinci system provided the necessary dexterity to perform delicate ocular surface surgery and robotic tools were safe for the tissues. The mean duration of the surgical procedures was 36 minutes. There were no intraoperative complications and no unexpected events. Robotic-assisted pterygium surgery is technically feasible for porcine eyes using the DaVinci Si HD robotic surgical system. The pterygium model that we describe could be of interest for surgical training. Little research has been done in robotic microsurgery. Animal experimentation will allow the advantages of robotic-assisted microsurgery to be identified, while underlining the improvements and innovations necessary for clinical use.

Characterization of the modified chitosan membrane cross-linked with genipin for the cultured corneal epithelial cells

ObjectivesTo modify a chitosan membrane (CM) by cross-linking the chitosan with genipin, a naturally occurring cross-linker extracted from Gardenia jasminoides fructus, with the aim of developing a new cell culture support and to observe the phenotypes of cultured human corneal epithelial cells (HCECs) on genipin-cross-linked chitosan membrane (GCM). MethodsWe tested the cross-linking characteristics and mechanical strength of the GCM. CMs modified by cross-linking with different concentrations of genipin were prepared to investigate the rate of membrane degradation. The biocompatibility of the GCMs was investigated by determining the viability of HCECs cultured on them in vitro. The morphology of the HCECs cultured on CM or GCM was analyzed by confocal microscopy and scanning electron microscopy (SEM). Immunocytochemical staining was conducted to determine the phenotypes of the cultured cells. ResultsThe fixation index of the GCM was 31±3% after treatment of CM with 0.5mM genipin. A stress–strain test showed that the GCM could tolerate three times the mechanical force of noncross-linked CM. The biodegradation rate of GCM was much slower than for CM. A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that cell viability was not affected by cross-linking with 5.0mM genipin. SEM showed that the cultured HCECs adhered to and grew well on the surface of the GCM. Immunocytochemical staining showed keratin 3 (K3) and connexin 43 (Cx-43) immunoreactive HCECs on the GCM and their proliferative ability was not significantly affected by strong immunoreactivity of Ki-67 and p63 markers. ConclusionsGCM has potential as a scaffold for corneal epithelium in ocular surface surgery and greater mechanical strength and slower degradation than unmodified CM.

Corneal Surgery (with DVD-ROM), Zuberbuhler B, Tuft S, Gartry D, Spokes D, (2013), ISBN: 978-3-642-12501-0, Springer

Corneal Surgery is a joint venture by British cornea specialists from Moorfields Eye Hospital, St. Thomas Hospital in London, and St. James University Hospital in Leeds. The authors provide a portable and condensed overview of the current surgical techniques in cornea and external eye disease. They do not aim to provide an extended textbook and also their aim is not to present cutting-edge new procedures but rather commonly available and established surgical techniques. The authors succeeded in providing a very condensed overview of all commonly performed surgical techniques in corneal surgery. The omission of all references and exhaustive discussions allows them to keep the textbook in an acceptable and portable format. The book starts with an introduction into anatomy and common surgical procedures as well as corneal examination techniques. All modern examination techniques such as OCT, topography, and wavefront systems are well covered. They then describe all common surgical techniques for ocular surface and reconstructive surgery such as amniotic membrane transplantation, superficial keratectomy, and PTK. The next large chapter covers discussions on eye banking, penetrating, as well as all modern lamellar surgical transplant approaches. Here, DSAEK, DMEK, as well as DALK are well covered. The last large chapter covers corneal refractive surgical procedures focusing on LASIK, LASEK, PRK, and also introducing all the common laser platforms available. Very helpful for the budding corneal surgeon is the last section of the book, which is a wet laboratory introduction and description, which is accompanied by a DVD explaining the essential surgical techniques in corneal surgery. In my opinion, this handy textbook is a very well condensed and concise overview of the most commonly performed surgical techniques in corneal and refractive surgery. It will be very helpful for the budding corneal surgeon, the corneal and refractive surgery fellow, and also as a short reference book for corneal surgeons. Since references are missing, the presented evidence is somewhat subjective, and also in certain aspects reflecting more the common practice pattern in the UK. These are not always representative for other European countries, which seems to be especially true for the modern surgical procedures such as a corneal crosslinking, DMEK, and DALK. In summary, I think this condensed and well-pictured essential surgical technique guide will be helpful for budding corneal surgeons and is a quick reference guide for more experienced ophthalmic surgeons with a cornea focus.

Corneoplastique: art of vision surgery.

Corneoplastique incorporates the entire spectrum of Vision corrective surgery including Lasik, premium cataract surgery, corneal surgery, ocular surface surgery and the full range of anterior segment surgery itself in manipulating the optics of every eye towards unaided emmetropia to define each and every eye surgeon as a “Vision Corrective Surgeon”. This concept of approaching each case individually and designing vision therewith enables surgeons to correct not only virgin eyes but also approach complex cases and complications with the goal of 20/20 vision. Armed with this holistic approach, eye surgeons can use minimally invasive, aesthetically pleasing and visually focused surgery in single or staged process aiming for each patient's Best Vision Potential (BVP) raising eye surgery itself then to an Art!

Delayed Epithelial Closure After PRK Associated With Topical Besifloxacin Use

To report the observation of prolonged reepithelialization after photorefractive keratectomy (PRK) associated with the use of besifloxacin 0.6% (Besivance; Bausch & Lomb, Rochester, NY) underneath bandage contact lenses (BCLs) placed during surgery. An office-based private practice and retrospective chart review. The healing parameters examined included epithelial healing time, haze formation, discomfort, and visual recovery of 4 patients (7 eyes) treated with besifloxacin 0.6% under BCLs placed after the PRK was performed. All the eyes had delayed epithelial closure (mean, 8.8 days; range 5-13 days). All the patients experienced a delayed visual recovery and significant pain after the surgery, and 2 of 4 patients experienced recurrent corneal erosions for weeks to months after they underwent the PRK. All but 1 eye developed corneal haze persisting for 1 year or more after the surgery. Only 1 eye among the 7 eyes treated with besifloxacin 0.6% under the BCL had 20/20 or better uncorrected visual acuity 3 months postoperatively. All the patients treated with besifloxacin 0.6% on the stromal bed exhibited significant problems with corneal epithelial healing and delayed visual recovery. We caution the use of besifloxacin 0.6% underneath a BCL during a PRK or other ocular surface surgeries requiring corneal epithelial debridement.

Analysis of Ethanol Effects on Corneal Epithelium

Ethanol is widely used in ocular surface surgeries and for the treatment of corneal diseases. However, ethanol is a toxic agent that is related to the development of a number of alcohol-related diseases. Despite the common use of ethanol for therapeutic purposes in ophthalmology, effects of ethanol on the ocular surface have been poorly defined. Hence, we performed this study to investigate effects of ethanol on corneal epithelium from various aspects. We exposed corneal epithelial cells in culture to different concentrations of ethanol for 30 seconds and evaluated the cells for toxicity, survival, and expression of cell-specific markers and inflammatory cytokines at 24, 48, and 72 hours after ethanol exposure. We found that ethanol markedly decreased the viability of cells in a concentration-dependent manner by causing cell lysis, suppressing proliferation, and inducing apoptosis. Also, expression of corneal epithelial cell-specific markers, both stem cell and differentiation markers, was significantly reduced by ethanol exposure. Expression of proinflammatory cytokines and chemokines was highly increased in corneal epithelial and stromal cells that were exposed to ethanol. Together, data suggest that brief exposure of the corneal surface to ethanol may have long-term effects by disrupting the integrity of corneal epithelium and generating inflammation, both of which are precursors to a number of ocular surface diseases.

Aktuelle und zukünftige Therapieoptionen bei Erkrankungen der Limbusstammzellen

Therapy for limbal stem cell insufficiency (LSCI) is one of the most challenging tasks in ocular surface surgery. Partial and unilateral LSCI can be treated by fractionated abrasion or autologous limbal stem cell transplantation from the fellow eye. In cases of advanced bilateral and partial LSCI, transplantation of ex vivo expanded sheets of limbal stem cells on amniotic membrane or fibrin may be performed. All patients with complete bilateral LSCI must rely on allogenic limbal stem cell transplantation comprising higher immunological risks. Progress using this technique has been achieved by the application of mitomycin C and amniotic membrane. Alternatively, transplantation of ex vivo expanded oral mucosal sheets may be used in bilateral LSCI, but only few long-term data are available on this technique as yet. While experimental attempts to use transdifferentiated bone marrow stem cells as a substitute for corneal epithelial cells have been without success, transdifferentiation of hair follicle stem cells to corneal epithelial cells may be more promising due to the identical lineage. But these experiments have not gone beyond the in vitro stage yet.

Time-Dependent Stability of Growth Factors and Endostatin in Human Amniotic Membrane Eye Drops

Purpose: Amniotic Membrane (AM) is commonly used as a graft for reconstruction in ocular surface surgery. It has been demonstrated that the beneficial effects of the AM graft are due largely to its content in several growth factors. For this reason, we have measured the levels of growth factors in lyophilized AM eye-drops and their variation over time.Results: The levels of bFGF and endostatin in 20% and 30% AM eye drops were higher than those of HGF, NGF and EGF. The concentration of all these growth factors and of total protein remained constant over 6 weeks, independent of the dilution factor. Conclusion: The growth factor and endostatin composition of AM eye drops is stable, at least over 6 weeks, further supporting the viability and potential therapeutic utility of this method for the treatment of ocular surface diseases and warranting its application in clinical trials.

Light-Initiated Bonding of Amniotic Membrane to Cornea

Suturing amniotic membrane to cornea during surgery is time consuming, and sutures may further damage the eye. The authors introduce a novel sutureless, light-activated technique that securely attaches amnion to cornea through protein-protein crosslinks. Cryopreserved human amniotic membrane, stained with Rose Bengal (RB), was placed over a full-thickness wound in deepithelialized rabbit cornea and was treated with green laser. The intraocular pressure that broke the seal (IOP(L)) was measured, and adhesion was measured with a peel test. The influences on bonding strength of fluence, irradiance, RB concentration, and amnion surface bonded were measured. Epithelial cell migration on treated amnion and keratocyte viability after bonding were also measured. The involvement in the bonding mechanism of oxygen, singlet oxygen, and association of RB with stromal collagen was investigated. Sealing amniotic membrane over cornea using 0.1% RB and 150 J/cm(2) at 532 nm produced an IOP(L) of 261 ± 77 mm Hg ex vivo and 448 mm ± 212 mm Hg in vivo. The ex vivo IOP(L) increased with increasing fluence (50-150 J/cm(2)). Equivalent IOP(L) was produced for bonding basement membrane or stromal amnion surfaces. The bonding treatment was not toxic to keratocytes but slightly reduced the migration of corneal epithelial cells on amnion ex vivo. Mechanism studies indicated that RB forms two complexes with amnion stromal collagen, that bonding requires oxygen, and that singlet oxygen mediates protein crosslinking. A rapid, light-activated technique produces strong, immediate bonding between amnion and cornea and merits further evaluation for ocular surface surgeries.

Ocular surface and refractive surgery

AbstractPurpose Dry eye represents nowadays the most common complication after PRK and even more frequent after lasik. Preoperative dry eye constitutes a risk factor for postoperative severe dryness; that it why it appears crucial to detect and treat surface disorder before the surgery.Methods We will remind the list and hierarchy of tests available for the assessment of ocular surface. Unfortunately, values of the defined criteria are variable and there are a lot of variations in the methods. Moreover, the correlation between clinical symptoms and tests results is very poor. The access to tear osmolarity (TearLab©) could be an interesting new biomarker. A study will be presented showing that 44% of patients, candidates to Lasik have an asymptomatic dryness before surgery (mild to moderate stage.Results Related to the principle of these two procedures, the cause of ocular surface deregulation lies on neurotrophic disturbances (secondary to nerves section) and inflammation ( wound healing process). In addition, changes in corneal shape can affect the tear dynamics with an increase in ocular surface dessication. All these phenomenon are systematically observed after photoablation for a transitory period of 1 to 3 months. But, sometimes, they do persist beyond one year (20%), becoming chronic and source of a great discomfort and dissatisfaction (10%). The strategy of treatment will be discussed, in graduating the medications according the growing severity.Conclusion Is there a difference between surface PRK and Lasik ? Hinged flap induced transection of large number of afferent sensory nerve fibers during lamellar cut, that impacts the integrated lacrymal gland OS functional unit. Finally, Lasik is responsible of more dry eye syndrome that PRK, that should be prefered in case of preoperative Ocular surface disorder.

Biological and Ultrastructural Properties of Acelagraft, a Freeze-Dried γ-Irradiated Human Amniotic Membrane

To compare the biological and ultrastructural properties of a commercially available decellularized and dehydrated human amniotic membrane (DDHAM) product with cryopreserved human amniotic membrane (CHAM) and to demonstrate the feasibility of DDHAM transplant in a case of chronic ocular surface disease. Histologic examination, immunohistochemical examination for extracellular matrix molecules and growth factors, transmission and scanning electron microscopy, and atomic force microscopy were performed on both DDHAM and CHAM specimens. A DDHAM transplant was performed in a patient with chronic bullous keratopathy and ocular surface instability. Histologic examination and transmission electron microscopy revealed the disruption of the trilaminar structure of the basement membrane compared with CHAM, and immunohistochemical examination confirmed the loss of collagen IV and VII, laminin, and fibronectin in DDHAM. Lower levels of several growth factors were also seen in DDHAM compared with CHAM. Clinical transplant of DDHAM was, however, successful, with rapid reepithelialization. Significant differences in composition and ultrastructure exist between DDHAM and CHAM but do not appear to compromise cell survival in vivo. The ease of storage and handling of DDHAM make it potentially valuable in ocular surface surgery, but its biological properties have not been well characterized, and there are also few data on its clinical application.

Donor age and gestational age influence on growth factor levels in human amniotic membrane

Amniotic membrane (AM) is used as a biomaterial for reconstruction in ocular surface surgery. This study investigated the influence of interdonor variations and processing and preservation procedures applied to the AM on growth factors and protein levels. Samples of human AM from thirteen donors were analysed. Collected donor data were age, parity and gestational age. Total protein amount was measured in extracts of intact AM nonpreserved, lyophilized and cryopreserved, at -80°C and in liquid nitrogen. An enzyme-linked immunosorbent assay (ELISA) was used to assay growth factors protein levels for epidermal growth factor, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), transforming growth factor beta1 (TFG-β(1) ) and nerve growth factor (NGF). Univariate and multivariate statistical analyses were used to study the influence of the preservation method applied and interdonor variations on growth factors levels. We detected important variations in growth factors and protein concentrations between samples from different donors. Total protein amount, bFGF, HGF, KGF and TGF-β(1) showed lower levels in samples from donors with higher gestational ages and donor ages, for all groups. The variability in the biochemical composition of AM from different donors is considerable, and it is related with donor factors as donor age and gestational age. As AM biochemical composition has a role in its therapeutic effects, these variations could affect the clinical results of amniotic membrane transplantation and must be taken into account in donor selection processes.

This issue of Acta

This issue of <i>Acta</i>

Sutureless and glue-free conjunctival autograft in pterygium surgery: a case series

Foreign materials used in ocular surface surgery may lead to local complications such as discomfort, scarring, or infection. Plasma-derived products such as fibrin glue may produce possible hypersensitivity reactions whereas the risk of viral transmission remains. We describe a simple method of achieving conjunctival autograft adherence during pterygium surgery avoiding potential complications associated with the use of fibrin glue or sutures. After pterygium excision and fashioning of the autologous conjunctival graft, the recipient bed is encouraged to achieve natural haemostasis and relative dessication before graft placement. Excessive haemorrhage in the graft bed is tamponaded. Graft adherence and positioning is examined 20 min after surgery. A total of 15 eyes of 12 patients (mean (SD) age 73.7 (11.2) years), 8 females underwent SGF autologous conjunctival graft post-pterygium excision. Mean graft area was 24(1.5)mm(2). Mean follow-up time was 9.2 (2.2) months. Cosmesis was excellent in all cases and visual acuity improved in one patient. There were no intra- or post-operative complications requiring further treatment. This simple technique for pterygium surgery may prevent potential adverse reactions encountered with the use of foreign materials and in this small series provided safe and comparable results to current methods.

Clinical Features of Korean Patients with Congenital Aniridia

PurposeTo investigate the clinical features of Korean patients with congenital aniridia.MethodsThis retrospective study focused on 60 eyes from 31 patients who were diagnosed with congenital aniridia at Kangnam St. Mary's Hospital from 1996 to 2007. Patient age, gender, visual acuity (VA), family history, and previous ocular history were recorded. The presence of keratopathy, glaucoma, cataract, foveal hypoplasia, and other ocular or systemic anomalies were evaluated for each patient.ResultsThe proportion of sporadic cases was 29.0%. Cataract (82.5%), glaucoma (51.6%), keratopathy (71.6%), and foveal hypoplasia (81.8%) commonly accompanied aniridia. Thirty-four (60.7%) eyes had VAs less than 20/200 and 20 eyes (35.7%) had VAs between 20/200 and 20/60. In patients without a past history of ocular surgery, the mean central corneal thickness was 643.05 ± 37.67 µm and the mean endothelial cell count was 3,349.44 ± 408.17 cells/mm2. Ocular surface surgeries were performed in 6 eyes. The clarity of the transplanted corneal graft vanished in 5 eyes with the progression of peripheral neovascularization and subepithelial fibrosis. The mean age of cataract surgery in 8 eyes was 29.8 ± 5.9 years. Postoperative worsening of corneal clouding and glaucomatous damage were observed in 4 eyes. Two infants had bilateral congenital glaucoma. Two children with sporadic aniridia were identified to have Wilm's tumors.ConclusionsCongenital aniridia is a progressive congenital disorder that is commonly accompanied by complications that can lead to impaired vision. Regular, careful examinations for these accompanying complications should be performed in all patients with congenital aniridia.

Inflammatory Molecules in Aqueous Humour and on Ocular Surface and Glaucoma Surgery Outcome

Purpose. To investigate the influence of inflammatory molecules in the aqueous humour and on the ocular surface on the outcome of glaucoma surgery. Methods. Thirty patients who needed antiglaucomatous surgery were included. The interleukin- (IL-) 8, IL-1β, IL-6, IL-10, tumour necrosis factor- (TNF-) α; and IL-12 were determined from aqueous humour preoperatively and the imprints of conjunctiva were analysed for expression of human leukocyte antigen- (HLA-)-DR after surgery by flow cytometry. The success of trabeculectomy was defined as intraocular pressure less than 21 mmHg without antiglaucoma medication. Results. Eyes with trabeculectomy failure at 3 months showed significantly higher TNF-α and IL-6 levels in the aqueous than eyes with successful surgery. Increased expression of HLA-DR on epithelial cells and antigen-presenting cells was not associated with the trabeculectomy outcome. Conclusions. Higher preoperative levels of TNF-α and IL-6 in aqueous humour may contribute to the development of inflammatory milieu and were associated with worse outcome of glaucoma surgery.

Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications

Cryopreserved amniotic membrane (Cryo-AM) is widely used in ocular surface surgery because of its positive effect on wound healing and its anti-inflammatory properties. A new peracetic acid/ethanol sterilized air-dried amniotic membrane (AD-AM) recently became available which might be an alternative to Cryo-AM. Our aim was to compare AM preserved with both methods with regard to the release of wound-healing modulating proteins, the preservation of basement membrane components, and the ability to serve as a substrate for the cultivation of human limbal epithelial cells (HLECs). Pieces of Cryo-AM and AD-AM from three different donors were incubated in DMEM for five days. The culture supernatant was collected after an incubation period of 0.1, 24, 48, 72 and 120 h; in the case of AD-AM, this period was extended up to 14 days. TIMP-1, IL-1ra, CTGF and TGF-beta1 were detected in the culture supernatant using Western blotting. Twenty human limbal epithelial cultures were initiated on both AD- and Cryo-AM. The cultures were analyzed morphologically, and the outgrowth area was measured in 3-day intervals. Cryosections of Cryo- and AD-AM from three different donors were analyzed histochemically to detect the basement membrane components collagen IV, collagen VII, laminin, laminin 5 and fibronectin. The release of TIMP-1, IL-1ra and TGF-beta1 from Cryo-AM was constant for the studied period. CTGF showed a stronger signal after 120 h. None of the analyzed proteins, except for a small amount of IL-1ra, could be detected in the supernatant of AD-AM. An outgrowth of HLEC was observed in all cultures on Cryo-AM, but in only 30% of cultures on AD-AM. The outgrowth area on Cryo-AM was at all time points significantly higher than on AD-AM (p < 0.0001). Collagen IV, -VII, laminins and fibronectin were detectable in the basement membrane of Cryo-AM, but only collagen IV and fibronectin in AD-AM. Cryo-AM is a more suitable substrate for the cultivation of HLECs than AD-AM. The higher outgrowth rate of cultured limbal epithelium, release of intact soluble wound-healing modulating factors and a better preservation of basement membrane components suggest the superiority of Cryo-AM for use in ophthalmology in comparison to AD-AM.