Preserved Amniotic Membrane Transplantation for Conjunctival Surface Reconstruction

Objective. To study the efficacy of preserved human amniotic membrane in the reconstruction of conjunctival defect created during surgical removal of conjunctival lesions or symblepharon lysis. Methods. Preserved human amniotic membrane transplantation was performed in 93 eyes of 85 patients for reconstruction of various conjunctival surface problems. The indications for surgery were (1) pterygium excision (54 eyes), (2) conjunctival tumors excision (23 eyes), lysis of symblepharon (13 eyes), and covering a scleral graft (three eyes). Results. Success was noted in 69.9% (65/93) eyes, partially success in 22.6% (21/93) eyes, and failure in 7.5% (7/93) eyes with a mean follow-up of 8.9 months (1–28 months). In pterygium, conjunctival tumor, symblepharon, and scleral graft group, the success rate in each group was 70.3%, 78.3%, 53.8%, and 66.7% respectively. No serious immediate post-operative complications or graft rejection occurred. Conclusion. Amniotic membrane transplantation can be considered an alternative treatment for difficult ocular surface problems, and is effective in promoting epithelial healing, and reducing inflammation and scarring.     

Expression of vascular endothelial growth factor C in human pterygium

Vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 mediate lymphangiogenesis. In this study, we analyzed the expression of VEGF-C and VEGFR-3 as well as lymphatic vessels in the pterygium and normal conjunctiva of humans. Fifteen primary nasal pterygia and three normal bulbar conjunctivas, surgically removed, were examined in this study. The lymphatic vessel density (LVD) and blood vessel density were determined by the immunolabeling of D2-40 and CD31, markers for lymphatic and blood vessels, respectively. VEGF-C and VEGFR-3 expression in pterygial and conjunctival tissue proteins was detected by Western blotting and were evaluated using immunohistochemistry. The LVD was significantly higher in the pterygium than normal conjunctiva (p < 0.05). Western blot demonstrated high-level expression of VEGF-C and VEGFR-3 in the pterygium compared with normal conjunctiva. VEGF-C immunoreactivity was detected in the cytoplasm of pterygial and normal conjunctival epithelial cells. The number of VEGF-C-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival cells (p < 0.05). VEGFR-3 immunoreactivity was localized in the D2-40-positive lymphatic endothelial cells. The present findings suggest the potential role of VEGF-C in the pathogenesis and development of a pterygium through lymphangiogenesis and the VEGF-C/VEGFR-3 pathway as a novel therapeutic target for the human pterygium.     

Etiologic theories,methods of treatment,and results

Although it has been postulated that a pterygium develops from the ordinary pinguecula which is a phenomenon of aging, the average age of patients in a Veterans Administration hospital who were operated upon for removal of pterygia was 34.9 years. Mexicans appeared to be particularly susceptible to the disease. Analysis of the literature and review of observations in the present series of cases indicated that long-continued exposure to the elements, particularly the sun's rays, is a prime etiologic factor and that dust and other irritants are of less importance. In a study of 285 patients who had pterygia, no correlation of this disease with other local or general disease was observed. An operation which brings previously unexposed conjunctiva into the area from which a pterygium is removed, and which does not bury the epithelium, was used successfully.     

Human bites of the eyelid

Five patients with traumatic colobomas of the eyelid secondary to human bites were surgically repaired with retrieved autogenous tissue. All patients were treated with prophylactic intravenous antibiotics. Surgical repair consisted of debridement of the autograft, meticulous layered closure of the autograft to the wound, and placement of a lid margin suture. In two of the patients, mild upper eyelid retraction was noted, and two patients had loss of cilia.     

Ultraviolet light and pterygium

The purpose of this study was to evaluate the contribution of ultraviolet light (UV) as a causal factor of primary and pterygium recurrence. A conjuctival autograft transplantation was a surgical method of pterygium treatment. In the first group (38 eyes) were patients with primary and recurrent pterygium exposed to sun (worked outdoors), evaluating geodemographic status, and in the second group (20 eyes) were patients who were not. During 6-12 months of follow up recurrence rate after surgical removal was 27% in the first group and 10% in the second one. UV light seems to have an important role in cause of primary and recurrent pterygium.     

Does glaucoma medication influence the diameter of the retinal arteriole in the human eye? (A pilot study using the retinal vessel analyser)

PURPOSE: To investigate the potential in vivo influence of different topical glaucoma medications on the diameter of the retinal arterioles of healthy volunteers and glaucoma patients. METHODS: The diameter of one pre-selected retinal arteriole per eye was measured using the Retinal Vessel Analyser (RVA), an instrument developed for non-invasive clinical measurement of the diameter of the main retinal vessels. The instrument contains a video system, and the integrated software recognises the boundaries of the retinal vessels by detecting their light-transmission profile. The vessel diameter (in arbitrary units) is plotted against time (seconds) on a separate display screen. In Study I the vessel diameter was measured in 12 eyes of six healthy volunteers (age 21-26 years, mean age 24.0 years) on six occasions each separated by 14 days. In a double-masked fashion, each subject's right eye was treated with one of 5 glaucoma medications (brinzolamide 1%, timolol 0.5%, betaxolol 0.5%, brimonidine 0.2% or latanoprost 0.005%) and the left eye always received balanced salt solution. In Study II, one randomly selected eye of 16 patients (age 50-79 years, mean age 65.2 years) suffering from primary open-angle glaucoma controlled with topical monotherapy was investigated, in an unmasked fashion. Four patients were on betaxolol 0.5% treatment, six subjects were receiving non-selective topical beta receptor blockers and six subjects were being treated with once daily latanoprost 0.005%. RESULTS: The coefficient of variation for the arteriole diameter in the healthy volunteers was less than 12% in each case. No significant post-treatment change of the diameter of the pre-selected arteriole was found for any topical medication investigated, either in the healthy volunteers (Study I) or in the patients suffering from glaucoma (Study II) (p>0.05, paired t-test). In addition, in Study I no difference was observed in the alteration of the arteriole diameter between the baseline and the hour 2 measurements when the values from the drug-treated and placebo treated eyes were compared (p>0.05, two-way ANOVA). CONCLUSION: In the present investigations it was not possible to detect any statistically meaningful change of the arteriole diameter at two hours after the instillation of any of several topical antiglaucoma drugs widely used in clinical practice. Further investigations are necessary to clarify whether the lack of observed change is due to the lack of retinal vascular effects of the drugs investigated, or is due to an inability of the RVA instrument in practice to detect alterations between time-points separated by several hours.     

Histopathology of the pterygium in population on Croatian Island Rab

In the 1975-2004 period, 130 cases of pterygium were estimated, 83 males and 47 females, on a small island Rab. Island Rab is located in the north part of the Adriatic sea, which has very high index of sun radiation. Pterygium is usually histopathologically defined as a hyperplasia of conjunctival tissue, elastoid degeneration of subepithelieum and fragmentation of Bowman's membrane. Our histopathological findings in 73 eyes are following: conjunctiva with neovascularisation, leukocyte margination and subepithelial basophilic degeneration, proliferation of conjunctival tissue, acanthosis and squamous metaplasia, and mucinous elements, focaly present plasma cells, focuses of increased pigmentisation in basal epithelial layers, tenon capsule with edema, diffuse neovascularisation and fragmentation of fibrils. These findings suggest that in severe cases of pterygium histopathologically exists precancerosis. In conclusion, on the basis of these histopathological findings, especially in the Tenon capsule, we suggest that for surgical procedure of pterygium the excision of the Tenon capsule, extensively under pterygium, is necessary.     

"Staying power"--absorbable vs. nonabsorbable

An experiment was designed to compare the "holding power" or "staying power" of absorbable (polyglycolic acid and polyglactin 910) and nonabsorbable (nylon) suture. The aim of this experiment was to determine what provides the lasting strength of the bond between soft tissues that are approximated or plicated. When correcting the rectus diastasis during abdominoplasty, we used nylon sutures in 15 patients and absorbable synthetic sutures in 15 other patients. We then marked the closed folds of the rectus sheath with small metal vascular clips. Two days later and approximately 6 months after operation an upright anteroposterior abdominal x-ray was taken and the position of the metal clips was compared in the test groups. Although there was usually slight separation of the clips after 6 months, no significant difference between the two groups was noted, thereby indicating that holding power is not related to type of suture material but more likely to fibroplasia.     

Expression profiles of collagens,HSP47, TGF-beta1, MMPs and TIMPs in epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a chronic, uncommon, sub-epidermal blistering disease involving the skin and mucous membranes that heals with scar formation and milia. Collagens, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are important components that play an essential role(s) in matrix remodeling during scar formation. However, the possible involvement of these components in EBA-induced scarring is not yet known. In the present study, we examined the expression profile of collagens, collagen-binding heat shock protein 47 (HSP47), MMPs and their inhibitory enzymes, TIMPs, in matrix remodeling during conjunctival scarring. The involvement of TGF-beta1, a fibrogenic factor, was also studied. Compared to the controls, an increased expression of type I collagen, type III collagen and HSP47 was detected in conjunctival biopsy sections of patient with EBA using immunohistochemistry. Similar increase in the expression of type I collagen, type III collagen and HSP47 was noted in conjunctival fibroblasts obtained from the patient with EBA. Up-regulation in the expression of MMP-1 and MMP-14 was also noted in conjunctival fibroblasts isolated from the patient with EBA, while no significant changes in the expression of MMP-3, MMP-8, MMP-9 and MMP-13 were seen. As for TIMPs, conjunctival fibroblasts isolated from the patient with EBA, grown in vitro, exhibited increased expression of TIMP-1, TIMP-2 and TIMP-3, when compared with fibroblasts grown from control conjunctival tissues, although the expression level varies with different molecules of the same family. Additionally, compared to the control conjunctival fibroblasts, an increased expression of TGF-beta1 was detected in fibroblasts isolated from the conjunctival tissues of patient with EBA.This study suggests that there is up-regulation in the production of collagens (type I and III), collagen-binding protein (HSP47), matrix degrading collagenases (MMP-1 and 14), and their inhibitory enzymes (TIMP-1, 2 and 3) during the process of conjunctival matrix remodeling in the patient with EBA. The presented data is preliminary and could serve as a basis for further studies to enhance our understanding about the molecular mechanisms of conjunctival scarring in patients with EBA.     

Non-Invasive Monitoring of Temporal and Spatial Blood Flow during Bone Graft Healing Using Diffuse Correlation Spectroscopy

Vascular infiltration and associated alterations in microvascular blood flow are critical for complete bone graft healing. Therefore, real-time, longitudinal measurement of blood flow has the potential to successfully predict graft healing outcomes. Herein, we non-invasively measure longitudinal blood flow changes in bone autografts and allografts using diffuse correlation spectroscopy in a murine femoral segmental defect model. Blood flow was measured at several positions proximal and distal to the graft site before implantation and every week post-implantation for a total of 9 weeks (autograft n = 7 and allograft n = 10). Measurements of the ipsilateral leg with the graft were compared with those of the intact contralateral control leg. Both autografts and allografts exhibited an initial increase in blood flow followed by a gradual return to baseline levels. Blood flow elevation lasted up to 2 weeks in autografts, but this duration varied from 2 to 6 weeks in allografts depending on the spatial location of the measurement. Intact contralateral control leg blood flow remained at baseline levels throughout the 9 weeks in the autograft group; however, in the allograft group, blood flow followed a similar trend to the graft leg. Blood flow difference between the graft and contralateral legs (ΔrBF), a parameter defined to estimate graft-specific changes, was elevated at 1–2 weeks for the autograft group, and at 2–4 weeks for the allograft group at the proximal and the central locations. However, distal to the graft, the allograft group exhibited significantly greater ΔrBF than the autograft group at 3 weeks post-surgery (p < 0.05). These spatial and temporal differences in blood flow supports established trends of delayed healing in allografts versus autografts.     

Very long-term radiographic and bone scan results of frozen autograft and allograft bone grafting in 17 patients (20 grafts) a 30- to 35-year follow-up

In the early 1950s, 48 patients received bone implants from a bone bank in Tel-Hashomer Hospital that stored frozen autograft and allograft bones at temperatures less than -17 degrees C. Seventeen (35%) of these patients (20 implants), 10 men and 7 women, with a mean age of 52.4 (34-69) years were available for follow-up after a mean period of 32.5 (30-35) years. They underwent clinical examination, radiographs and bone scans to evaluate their surgical results. Fracture healing, non-union, graft resorption, osteoporosis and bone sclerosis were used as radiographic criteria for bone incorporation, and normal, increased and decreased uptake served to assess the bone scan. Based on the above criteria, the results were satisfactory in 17 (85%) and poor in 3 (15%). The three failures were after shelf operation for hip dysplasia that used two allografts and one autograft. Allogenous or a combination of allogenous with autogenous frozen bone grafts proved to be a satisfactory and durable method for filling bone defects.     

Low-dose strontium-90 irradiation is effective in preventing the recurrence of pterygia: a ten-year study

Background

To study the long-term effects of low-dosage strontium-90 (Sr90) irradiation on the recurrence of pterygium.

Methodology/Principal Findings

One hundred twenty eyes from 104 patients with primary or recurrent pterygia were treated with surgery followed by Sr90 irradiation. In brief, starting on the sixth day after surgery, patients were treated with irradiation three times every other day at a total combined dosage of 2000 cGy to 3000 cGy. Corneal topography was used to evaluate ocular surface regularity before and after treatment. Patient follow-up was performed 2 days, 5 days, 2 weeks, 1 month, 3 months, 1 year, 5 years, and 10 years after surgery. Recurrence of pterygium was not observed in any of the patients in this study. Obvious cataract progression was observed in 6 eyes, which may be due to aging. During follow-up studies, only one eye was reported with dryness and foreign-body sensation. Significant pterygium-induced astigmatism was observed in corneal topography, which decreased after surgery.

Conclusions/Significance

Sr90 irradiation is effective in preventing the recurrence of primary and recurrent pterygia. We recommend delivering a total combined dosage of 2000 cGy to 3000 cGy of Sr90 irradiation administered in three batches every other day starting from the sixth day after surgery. Surgery is important in the rapid recovery of the cornea from pterygium-induced astigmatism.     

Effect of bovine fetal growth plate as a new xenograft in experimental bone defect healing: radiological,histopathological and biomechanical evaluation

Bone grafting is used to enhance healing in osteotomies, arthrodesis, and multifragmentary fractures and to replace bony loss resulting from neoplasia or cysts. They are source of osteoprogenitor cells and induce bone formation and provide mechanical support for vascular and bone ingrowth. Autografts are used commonly but quantity of retrieved bone is limit. This study was designed to evaluate autograft and new xenograft (Bovine fetal growth plate) effects on bone healing process. Twenty male White New Zealand rabbits were used in this study. In autograft group the defect was filled by fresh autogenous cortical graft, in xenograft group the defect was filled by a segment of bovine fetal growth plate and was fixed by cercelage wire. Radiological, histopathological and biomechanical evaluations were performed blindly and results scored and analyzed statistically. Statistical tests did not support significant differences between two groups at the 14th and 28th postoperative day radiographically (P > 0.05). There was a significant difference for remodeling at the 42nd postoperative radiologically (P < 0.05). Xenograft was superior to autograft at the 56th postoperative day for radiological bone formation (P < 0.03). Histopathological and biomechanical evaluation revealed no significant differences between two groups. The results of this study indicate that satisfactory healing occurred in rabbit radius defect filled with calf fetal growth plate. Complications were not identified and healing was faster than cortical autogenous grafting. It was concluded that the use of calf fetal growth plate as a new xenograft is an acceptable alternative to cortical autogenous graft and could reduce the morbidity associated with harvesting autogenous graft during surgery.     

Carbonic anhydrase inhibitors: N-(p-sulfamoylphenyl)-alpha-D-glycopyranosylamines as topically acting antiglaucoma agents in hypertensive rabbits

A series of N-(p-sulfamoylphenyl)-alpha-D-glycopyranosylamines was prepared by reaction of sulfanilamide with different monosaccharides in the presence of ammonium chloride. The new compounds were investigated for inhibition of the metallo-enzyme carbonic anhydrase (CA, EC 4.2.1.1), involved in aqueous humor secretion within the mammalian eye. Isozymes CA I and CA II were strongly inhibited by some of these compounds, which showed inhibition constants in the range of 510-1200 nM against CA I and 10-25 nM against CA II, similarly to clinically used sulfonamides, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide and brinzolamide. The presence of sugar moieties in these molecules induced an enhanced water solubility as compared to other sulfonamides. In hypertensive rabbits (a widely used animal model of glaucoma), two of the new compounds showed strong and long-lasting intraocular pressure (IOP) lowering, being more effective than dorzolamide and brinzolamide, the two clinically used, topically acting antiglaucoma sulfonamides with CA inhibitory properties.     

Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy

Structural allograft healing is limited because of a lack of vascularization and remodeling. To study this we developed a mouse model that recapitulates the clinical aspects of live autograft and processed allograft healing. Gene expression analyses showed that there is a substantial decrease in the genes encoding RANKL and VEGF during allograft healing. Loss-of-function studies showed that both factors are required for autograft healing. To determine whether addition of these signals could stimulate allograft vascularization and remodeling, we developed a new approach in which rAAV can be freeze-dried onto the cortical surface without losing infectivity. We show that combination rAAV-RANKL- and rAAV-VEGF-coated allografts show marked remodeling and vascularization, which leads to a new bone collar around the graft. In conclusion, we find that RANKL and VEGF are necessary and sufficient for efficient autograft remodeling and can be transferred using rAAV to revitalize structural allografts.     

Trabeculectomy with mitomycin C in glaucoma associated with uveitis

The patients with uveitic glaucoma are at high risk for failure following drainage surgery because of young age of these patients, preoperative long-term control of inflammation and postoperative complications. Twenty-two trabeculectomies performed in 22 patients with uveitic glaucoma were retrospectively evaluated to analyze the effect of intraoperative application of mitomycin C (MMC). Success rates, postoperative levels of intraocular pressure (IOP) and postoperative complications were studied. After a mean follow-up of 10.6 months (range, 5-28 months), 15 patients (68.2%) achieved IOP of 21mmHg or less without antiglaucoma medications. There were statistically significant reduction in IOP postoperatively during the period studied (p < 0.001). Early postoperative complications included chorioidal detachment (9.1%), shallow anterior chamber (9.1%), hyphema (13.6%), macular edema (4.5%) and raised IOP (27.3%). Late postoperative complications included exacerbation of uveitis (4.5%), macular edema (4.5%), cataract (22.7%) and raised IOP (31.8%). The eyes with raised IOP needed additional antiglaucoma medication. The results of this retrospective and uncontrolled study suggest that intraoperative application of MMC may be a good option for enhancement of short-term trabeculectomy success rates in patients with uveitic glaucoma.     

Amniotic membrane transplantation for ocular surface reconstruction

The purpose of this study is to analyze the clinical experience and the effect of human amniotic membrane transplantation on pterygium excision and bullous keratopathy. From January 1999 to January 2001 at University Hospital "Sestre milosrdnice" amniotic membrane transplantation was performed consecutively in 21 eyes: 11 eyes with bullous keratopathy and 10 with recurrent pterygia. In the group with bullous keratopathy epithelization took place in 19.6 days in 72.7% and the reduction of pain was satisfactory. Recurrence rate in group with recurrent pterygia was 20%. Based on the presented results it could be concluded that amniotic membrane transplantation can be considered as an effective alternative for treating severe ocular surface diseases and as an alternative for penetrating keratoplasty if there is a lack of grafts.     

Distinct gene subsets in pterygia formation and recurrence: dissecting complex biological phenomenon using genome wide expression data

Background

Pterygium is a common ocular surface disease characterized by fibrovascular invasion of the cornea and is sight-threatening due to astigmatism, tear film disturbance, or occlusion of the visual axis. However, the mechanisms for formation and post-surgical recurrence of pterygium are not understood, and a valid animal model does not exist. Here, we investigated the possible mechanisms of pterygium pathogenesis and recurrence.

Methods

First we performed a genome wide expression analysis (human Affymetrix Genechip, >22000 genes) with principal component analysis and clustering techniques, and validated expression of key molecules with PCR. The controls for this study were the un-involved conjunctival tissue of the same eye obtained during the surgical resection of the lesions. Interesting molecules were further investigated with immunohistochemistry, Western blots, and comparison with tear proteins from pterygium patients.

Results

Principal component analysis in pterygium indicated a signature of matrix-related structural proteins, including fibronectin-1 (both splice-forms), collagen-1A2, keratin-12 and small proline rich protein-1. Immunofluorescence showed strong expression of keratin-6A in all layers, especially the superficial layers, of pterygium epithelium, but absent in the control, with up-regulation and nuclear accumulation of the cell adhesion molecule CD24 in the pterygium epithelium. Western blot shows increased protein expression of beta-microseminoprotein, a protein up-regulated in human cutaneous squamous cell carcinoma. Gene products of 22 up-regulated genes in pterygium have also been found by us in human tears using nano-electrospray-liquid chromatography/mass spectrometry after pterygium surgery. Recurrent disease was associated with up-regulation of sialophorin, a negative regulator of cell adhesion, and never in mitosis a-5, known to be involved in cell motility.

Conclusion

Aberrant wound healing is therefore a key process in this disease, and strategies in wound remodeling may be appropriate in halting pterygium or its recurrence. For patients demonstrating a profile of 'recurrence', it may be necessary to manage as a poorer prognostic case and perhaps, more adjunctive treatment after resection of the primary lesion.